mTOR kinase structure, mechanism and regulation by the rapamycin-binding domain

PubMed Central

Yang, Haijuan; Rudge, Derek G.; Koos, Joseph D.; Vaidialingam, Bhamini; Yang, Hyo J.; Pavletich, Nikola P.

2015-01-01

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a truncated mTOR-mLST8 complex with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and mechanism remarkably similar to canonical protein kinases. The active site is highly recessed due to the FKBP12-Rapamycin binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR activating mutations map to the structural framework that holds these elements in place, indicating the kinase is controlled by restricted access. In vitro biochemistry indicates that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. FKBP12-rapamycin inhibits by directly blocking substrate recruitment and by further restricting active site access. The structures also reveal active site residues and conformational changes that underlie inhibitor potency and specificity. PMID:23636326

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

PubMed

Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

2017-04-11

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Electric Fields at the Active Site of an Enzyme: Direct Comparison of Experiment with Theory

NASA Astrophysics Data System (ADS)

Suydam, Ian T.; Snow, Christopher D.; Pande, Vijay S.; Boxer, Steven G.

2006-07-01

The electric fields produced in folded proteins influence nearly every aspect of protein function. We present a vibrational spectroscopy technique that measures changes in electric field at a specific site of a protein as shifts in frequency (Stark shifts) of a calibrated nitrile vibration. A nitrile-containing inhibitor is used to deliver a unique probe vibration to the active site of human aldose reductase, and the response of the nitrile stretch frequency is measured for a series of mutations in the enzyme active site. These shifts yield quantitative information on electric fields that can be directly compared with electrostatics calculations. We show that extensive molecular dynamics simulations and ensemble averaging are required to reproduce the observed changes in field.

Jack bean urease: the effect of active-site binding inhibitors on the reactivity of enzyme thiol groups.

PubMed

Krajewska, Barbara; Zaborska, Wiesława

2007-10-01

In view of the complexity of the role of the active site flap cysteine in the urease catalysis, in this work we studied how the presence of typical active-site binding inhibitors of urease, phenylphosphorodiamidate (PPD), acetohydroxamic acid (AHA), boric acid and fluoride, affects the reactivity of enzyme thiol groups, the active site flap thiol in particular. For that the inhibitor-urease complexes were prepared with excess inhibitors and had their thiol groups titrated with DTNB. The effects observed were analyzed in terms of the structures of the inhibitor-urease complexes reported in the literature. We found that the effectiveness in preventing the active site cysteine from the modification by disulfides, varied among the inhibitors studied, even though they all bind to the active site. The variations were accounted for by different extents of geometrical distortion in the active site that the inhibitors introduced upon binding, leaving the flap either open in AHA-, boric acid- and fluoride-inhibited urease, like in the native enzyme or closed in PPD-inhibited urease. Among the inhibitors, only PPD was found to be able to thoroughly protect the flap cysteines from the further reaction with disulfides, this apparently resulting from the closed conformation of the flap. Accordingly, in practical terms PPD may be regarded as the most suitable inhibitor for active-site protection experiments in inhibition studies of urease.

Two-track virtual screening approach to identify both competitive and allosteric inhibitors of human small C-terminal domain phosphatase 1

NASA Astrophysics Data System (ADS)

Park, Hwangseo; Lee, Hye Seon; Ku, Bonsu; Lee, Sang-Rae; Kim, Seung Jun

2017-08-01

Despite a wealth of persuasive evidence for the involvement of human small C-terminal domain phosphatase 1 (Scp1) in the impairment of neuronal differentiation and in Huntington's disease, small-molecule inhibitors of Scp1 have been rarely reported so far. This study aims to the discovery of both competitive and allosteric Scp1 inhibitors through the two-track virtual screening procedure. By virtue of the improvement of the scoring function by implementing a new molecular solvation energy term and by reoptimizing the atomic charges for the active-site Mg2+ ion cluster, we have been able to identify three allosteric and five competitive Scp1 inhibitors with low-micromolar inhibitory activity. Consistent with the results of kinetic studies on the inhibitory mechanisms, the allosteric inhibitors appear to be accommodated in the peripheral binding pocket through the hydrophobic interactions with the nonpolar residues whereas the competitive ones bind tightly in the active site with a direct coordination to the central Mg2+ ion. Some structural modifications to improve the biochemical potency of the newly identified inhibitors are proposed based on the binding modes estimated with docking simulations.

New insights into the interaction between pyrrolyl diketoacids and HIV-1 integrase active site and comparison with RNase H.

PubMed

Corona, Angela; di Leva, Francesco Saverio; Rigogliuso, Giuseppe; Pescatori, Luca; Madia, Valentina Noemi; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Cadeddu, Marta; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; di Santo, Roberto; Tramontano, Enzo

2016-10-01

HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg 2+ cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding. In this study, we investigated the binding mode of four pyrrolyl DKAs to the HIV-1 IN active site by molecular modeling coupled with site-directed mutagenesis studies showing that the DKA pyrrolyl scaffold primarily interacts with the IN amino residues P145, Q146 and Q148. Importantly, the tested DKAs demonstrated good effectiveness against HIV-1 Raltegravir resistant Y143A and N155H INs, thus showing an interaction pattern with relevant differences if compared with the first generation IN inhibitors. These data provide precious insights for the design of new HIV inhibitors active on clinically selected Raltegravir resistant variants. Furthermore, this study provides new structural information to modulate IN and RNase H inhibitory activities for development of dual-acting anti-HIV agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Discovery of Novel New Delhi Metallo-β-Lactamases-1 Inhibitors by Multistep Virtual Screening

PubMed Central

Wang, Xuequan; Lu, Meiling; Shi, Yang; Ou, Yu; Cheng, Xiaodong

2015-01-01

The emergence of NDM-1 containing multi-antibiotic resistant "Superbugs" necessitates the needs of developing of novel NDM-1inhibitors. In this study, we report the discovery of novel NDM-1 inhibitors by multi-step virtual screening. From a 2,800,000 virtual drug-like compound library selected from the ZINC database, we generated a focused NDM-1 inhibitor library containing 298 compounds of which 44 chemical compounds were purchased and evaluated experimentally for their ability to inhibit NDM-1 in vitro. Three novel NDM-1 inhibitors with micromolar IC50 values were validated. The most potent inhibitor, VNI-41, inhibited NDM-1 with an IC50 of 29.6 ± 1.3 μM. Molecular dynamic simulation revealed that VNI-41 interacted extensively with the active site. In particular, the sulfonamide group of VNI-41 interacts directly with the metal ion Zn1 that is critical for the catalysis. These results demonstrate the feasibility of applying virtual screening methodologies in identifying novel inhibitors for NDM-1, a metallo-β-lactamase with a malleable active site and provide a mechanism base for rational design of NDM-1 inhibitors using sulfonamide as a functional scaffold. PMID:25734558

Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

PubMed

Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

2013-06-13

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general.

Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

PubMed

García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

2016-07-01

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. Copyright © 2016 Elsevier Inc. All rights reserved.

IspE Inhibitors Identified by a Combination of In Silico and In Vitro High-Throughput Screening

PubMed Central

Tidten-Luksch, Naomi; Grimaldi, Raffaella; Torrie, Leah S.; Frearson, Julie A.; Hunter, William N.; Brenk, Ruth

2012-01-01

CDP-ME kinase (IspE) contributes to the non-mevalonate or deoxy-xylulose phosphate (DOXP) pathway for isoprenoid precursor biosynthesis found in many species of bacteria and apicomplexan parasites. IspE has been shown to be essential by genetic methods and since it is absent from humans it constitutes a promising target for antimicrobial drug development. Using in silico screening directed against the substrate binding site and in vitro high-throughput screening directed against both, the substrate and co-factor binding sites, non-substrate-like IspE inhibitors have been discovered and structure-activity relationships were derived. The best inhibitors in each series have high ligand efficiencies and favourable physico-chemical properties rendering them promising starting points for drug discovery. Putative binding modes of the ligands were suggested which are consistent with established structure-activity relationships. The applied screening methods were complementary in discovering hit compounds, and a comparison of both approaches highlights their strengths and weaknesses. It is noteworthy that compounds identified by virtual screening methods provided the controls for the biochemical screens. PMID:22563402

Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

PubMed Central

Matsuura, K; Deyashiki, Y; Sato, K; Ishida, N; Miwa, G; Hara, A

1997-01-01

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors. PMID:9173902

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

PubMed Central

Schanz, Merle; Reynell, Lucy; Günthard, Huldrych F.; Rusert, Peter; Trkola, Alexandra

2012-01-01

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo. PMID:22496655

Biological Synthesis of a Protein Analogue of Acetylcholinesterase: Monoclonal Anti-Idiotype Antibody Analogue of the Esteratic Site.

DTIC Science & Technology

1986-07-10

site- directed inhibitor DFP, the reversible site-directed inhibitors neostigmine, carbachol , edrophonium, and BW284c51; and the allosteric site...Esteratic 10-6 10 -7 - I0-2 Neostigmine Esteratic 10- 7 10- 6 - 10 5 Anionic PI Carbachol Esteratic 10- 4 10 3 10-1 Anionic PI Edrophonium Anionic 10

Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site.

PubMed

Bause, E; Wesemann, M; Bartoschek, A; Breuer, W

1997-02-15

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asu-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-alpha,gamma-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own 'suicide' inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation.

High-throughput screening (HTS) and hit validation to identify small molecule inhibitors with activity against NS3/4A proteases from multiple hepatitis C virus genotypes.

PubMed

Lee, Hyun; Zhu, Tian; Patel, Kavankumar; Zhang, Yan-Yan; Truong, Lena; Hevener, Kirk E; Gatuz, Joseph L; Subramanya, Gitanjali; Jeong, Hyun-Young; Uprichard, Susan L; Johnson, Michael E

2013-01-01

Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC50 value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.

Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

PubMed

Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

2010-01-01

Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. (c) 2010 Wiley Periodicals, Inc.

A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation.

PubMed

Kamisato, Chikako; Furugohri, Taketoshi; Morishima, Yoshiyuki

2016-05-01

We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa(307-506)) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25-600nM) and 3-30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa(307-506). In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression. Copyright © 2016 Elsevier Ltd. All rights reserved.

Steroid Sulfatase Inhibition by Aryl Sulfamates: Clinical Progress, Mechanism and Future Prospects.

PubMed

Potter, Barry V L

2018-04-04

Steroid sulfatase is an emerging drug target for the endocrine therapy of hormone-dependent diseases, catalyzing estrogen sulfate hydrolysis to estrogen. Drug discovery, developing the core aryl O-sulfamate pharmacophore, has led to steroidal and non-steroidal drugs entering numerous clinical trials, with promising results in oncology and women's health. Steroidal estrogen sulfamate derivatives were the first irreversible active-site-directed inhibitors and one was developed clinically as an oral estradiol pro-drug and for endometriosis applications. This review summarizes work leading to the therapeutic concept of sulfatase inhibition, clinical trials executed to date and new insights into the mechanism of inhibition of steroid sulfatase. To date the non-steroidal sulfatase inhibitor Irosustat has been evaluated clinically in breast cancer, alone and in combination, in endometrial cancer and in prostate cancer. The versatile core pharmacophore both imbues attractive pharmaceutical properties and functions via three distinct mechanisms of action, as a pro-drug, an enzyme active site-modifying motif, likely through direct sulfamoyl group transfer, and as a structural component augmenting activity, for example by enhancing interactions at the colchicine binding site of tubulin. Preliminary new structural data on the Pseudomonas aeruginosa arylsulfatase enzyme suggest two possible sulfamate-based adducts with active site hydrated formylglycine as candidates for the inhibition end product via sulfamoyl group transfer, and a speculative choice is suggested. The clinical status of sulfatase inhibition is surveyed and how it might develop in the future. Also discussed are dual-targeting approaches, development of 2-substituted steroidal sulfamates and nonsteroidal derivatives as multi-targeting agents for hormone-independent tumours with other emerging directions.

Dual role of Zn2+ in maintaining structural integrity and suppressing deacetylase activity of SIRT1.

PubMed

Chen, Lei; Feng, Yu; Zhou, Yinqiu; Zhu, Weiliang; Shen, Xu; Chen, Kaixian; Jiang, Hualiang; Liu, Dongxiang

2010-02-01

Zn(2+) directly participates in catalysis of histone deacetylase (HDAC) Classes I, II, IV enzymes while its role in HDAC Class III activity is not well established. Herein we investigated the effects of Zn(2+) on the deacetylase activity of sirtuin 1 (silent mating type information regulation 2 homolog 1, SIRT1). We found that the inherent Zn(2+) at the zinc-finger motif of SIRT1 is essential for the structural integrity and the deacetylase activity of SIRT1, whereas the exogenous Zn(2+) strongly inhibits the deacetylase activity with an IC(50) of 0.82muM for Zn(Gly)(2). SIRT1 activity suppressed by the exogenous Zn(2+) can be fully recovered by the metal chelator EDTA but not by the activator resveratrol. We also identified Zn(2+) as a noncompetitive inhibitor for the substrates of NAD(+) and the acetyl peptide P53-AMC. The 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence titration experiments and site-directed mutagenesis study suggested that the exogenous Zn(2+) binds to SIRT1 but not at the zinc-finger motif. These results indicate that Zn(2+) plays a dual role in SIRT1 activity. Inherent Zn(2+) at the zinc-finger motif is structurally related and essential for SIRT1 activity. On the other hand, Zn(2+) may also bind to another site different from the zinc-finger motif or the binding sites for the substrates or resveratrol and act as a potent inhibitor of SIRT1.

The binding sites of inhibitory monoclonal antibodies on acetylcholinesterase. Identification of a novel regulatory site at the putative "back door".

PubMed

Simon, S; Le Goff, A; Frobert, Y; Grassi, J; Massoulié, J

1999-09-24

We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N, N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.

Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thal, David M.; Homan, Kristoff T.; Chen, Jun

2012-08-10

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice withmore » paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.« less

Structure-based design of bacterial nitric oxide synthase inhibitors

DOE PAGES

Holden, Jeffrey K.; Kang, Soosung; Hollingsworth, Scott A.; ...

2014-12-18

Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial. Here wemore » present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Lastly, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.« less

Designing small molecules to target cryptic pockets yields both positive and negative allosteric modulators

PubMed Central

Moeder, Katelyn E.; Ho, Chris M. W.; Zimmerman, Maxwell I.; Frederick, Thomas E.; Bowman, Gregory R.

2017-01-01

Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators. PMID:28570708

Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation.

PubMed Central

O'Rourke, J.; Wang, W. P.; Donnelly, L.; Wang, E.; Kreutzer, D. L.

1987-01-01

Levels of extravascular tissue plasminogen activator activity (PA) and those of inhibitors of PA and of urokinase (UK) present within the anterior chamber of normal and inflamed feline eyes were assessed with the use of a direct PA assay of microsamples of aqueous humor. Purposes of the study were, first, to confirm prior indirect evidence that this extravascular space normally contains higher levels of uninhibited PA, but lower levels of inhibitor activity, than does plasma and, second, to determine patterns of change in these activities under in vivo conditions imposed by a chronic mycobacterial-induced uveitis (CMIU) disease model. The PA assay utilized a 125I-plasminogen substrate whose cleavage by PA contained in samples was both visualized during gel electrophoreis, and quantified by gamma counting. The results provided the first direct evidence that the higher fibrinolytic activity previously observed in normal aqueous in comparison with plasma is in fact associated with higher levels of available (uninhibited) PA (P less than 0.01) The data also indicated that normal aqueous contains a much higher level of PA inhibitor activity than previously suspected--roughly 40 times more than available PA levels. These normal values for PA and inhibitors occupied a relatively narrow, threefold range, in contrast to the wide scattering of individual values that appeared during 18-20 weeks of the chronic inflammation disease model. Despite this, however, the general pattern of observation for all individual eyes during CMIU was a significant increase in levels of both PA and inhibitors. The net effect of CMIU was thus to cause the 1:40 ratio noted above to be tilted more strongly in favor of inhibitor activity, ie, up to 1:80. Increases in local vasopermeability in this disease model were believed contributory to this change. However, local generations of PA and APA in vivo by inflammatory cells, especially monocyte-macrophages, must also be considered. Assays for UK inhibitor showed levels of activity and directions of change that closely followed those of PA inhibitor, which suggests the possibility that they may be identical. It is surmised that the above patterns, along with results of our prior studies, indicate an apparent need for a multistep, strict inhibitory control of plasmin generation and proteolysis in vivo within normal extravascular spaces such as the anterior chamber.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 2 PMID:3493701

A gratuitous β-Lactamase inducer uncovers hidden active site dynamics of the Staphylococcus aureus BlaR1 sensor domain.

PubMed

Frederick, Thomas E; Peng, Jeffrey W

2018-01-01

Increasing evidence shows that active sites of proteins have non-trivial conformational dynamics. These dynamics include active site residues sampling different local conformations that allow for multiple, and possibly novel, inhibitor binding poses. Yet, active site dynamics garner only marginal attention in most inhibitor design efforts and exert little influence on synthesis strategies. This is partly because synthesis requires a level of atomic structural detail that is frequently missing in current characterizations of conformational dynamics. In particular, while the identity of the mobile protein residues may be clear, the specific conformations they sample remain obscure. Here, we show how an appropriate choice of ligand can significantly sharpen our abilities to describe the interconverting binding poses (conformations) of protein active sites. Specifically, we show how 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) exposes otherwise hidden dynamics of a protein active site that binds β-lactam antibiotics. When CBAP acylates (binds) the active site serine of the β-lactam sensor domain of BlaR1 (BlaRS), it shifts the time scale of the active site dynamics to the slow exchange regime. Slow exchange enables direct characterization of inter-converting protein and bound ligand conformations using NMR methods. These methods include chemical shift analysis, 2-d exchange spectroscopy, off-resonance ROESY of the bound ligand, and reduced spectral density mapping. The active site architecture of BlaRS is shared by many β-lactamases of therapeutic interest, suggesting CBAP could expose functional motions in other β-lactam binding proteins. More broadly, CBAP highlights the utility of identifying chemical probes common to structurally homologous proteins to better expose functional motions of active sites.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

PubMed

Bellio, Pierangelo; Di Pietro, Letizia; Mancini, Alisia; Piovano, Marisa; Nicoletti, Marcello; Brisdelli, Fabrizia; Tondi, Donatella; Cendron, Laura; Franceschini, Nicola; Amicosante, Gianfranco; Perilli, Mariagrazia; Celenza, Giuseppe

2017-06-15

RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC 50 ) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. In this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity. Copyright © 2017 Elsevier GmbH. All rights reserved.

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Recent advances in botulinum neurotoxin inhibitor development.

PubMed

Kiris, Erkan; Burnett, James C; Kane, Christopher D; Bavari, Sina

2014-01-01

Botulinum neurotoxins (BoNTs) are endopeptidases that target motor neurons and block acetylcholine neurotransmitter release. This action results in the muscle paralysis that defines the disease botulism. To date, there are no FDA-approved therapeutics to treat BoNT-mediated paralysis after intoxication of the motor neuron. Importantly, the rationale for pursuing treatments to counter these toxins is driven by their potential misuse. Current drug discovery efforts have mainly focused on small molecules, peptides, and peptidomimetics that can directly and competitively inhibit BoNT light chain proteolytic activity. Although this is a rational approach, direct inhibition of the Zn(2+) metalloprotease activity has been elusive as demonstrated by the dearth of candidates undergoing clinical evaluation. Therefore, broadening the scope of viable targets beyond that of active site protease inhibitors represents an additional strategy that could move the field closer to the clinic. Here we review the rationale, and discuss the outcomes of earlier approaches and highlight potential new targets for BoNT inhibition. These include BoNT uptake and processing inhibitors, enzymatic inhibitors, and modulators of neuronal processes associated with toxin clearance, neurotransmitter potentiation, and other pathways geared towards neuronal recovery and repair.

SAHA-based novel HDAC inhibitor design by core hopping method.

PubMed

Zang, Lan-Lan; Wang, Xue-Jiao; Li, Xiao-Bo; Wang, Shu-Qing; Xu, Wei-Ren; Xie, Xian-Bin; Cheng, Xian-Chao; Ma, Huan; Wang, Run-Ling

2014-11-01

The catalytic activity of the histone deacetylase (HDAC) is directly relevant to the pathogenesis of cancer, and HDAC inhibitors represented a promising strategy for cancer therapy. SAHA (suberoanilide hydroxamic acid), an effective HDAC inhibitor, is an anti-cancer agent against T-cell lymphoma. However, SAHA has adverse effects such as poor pharmacokinetic properties and severe toxicities in clinical use. In order to identify better HDAC inhibitors, a compound database was established by core hopping of SAHA, which was then docked into HDAC-8 (PDB ID: 1T69) active site to select a number of candidates with higher docking score and better interaction with catalytic zinc ion. Further ADMET prediction was done to give ten compounds. Molecular dynamics simulation of the representative compound 101 was performed to study the stability of HDAC8-inhibitor system. This work provided an approach to design novel high-efficiency HDAC inhibitors with better ADMET properties. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure of the Protease Domain of Memapsin 2 (β-Secretase) Complexed with Inhibitor

NASA Astrophysics Data System (ADS)

Hong, Lin; Koelsch, Gerald; Lin, Xinli; Wu, Shili; Terzyan, Simon; Ghosh, Arun K.; Zhang, Xuenjun C.; Tang, Jordan

2000-10-01

Memapsin 2 (β-secretase) is a membrane-associated aspartic protease involved in the production of β-amyloid peptide in Alzheimer's disease and is a major target for drug design. We determined the crystal structure of the protease domain of human memapsin 2 complexed to an eight-residue inhibitor at 1.9 angstrom resolution. The active site of memapsin 2 is more open and less hydrophobic than that of other human aspartic proteases. The subsite locations from S4 to S2' are well defined. A kink of the inhibitor chain at P2' and the change of chain direction of P3' and P4' may be mimicked to provide inhibitor selectivity.

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors.

PubMed

Nongonierma, Alice B; Mooney, Catherine; Shields, Denis C; FitzGerald, Richard J

2014-07-01

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure-activity relationship modeling, rather than on docking, in computationally selecting peptides for screening. Copyright © 2014 Elsevier Inc. All rights reserved.

SERPINE1: A Molecular Switch in the Proliferation-Migration Dichotomy in Wound-“Activated” Keratinocytes

PubMed Central

Simone, Tessa M.; Higgins, Craig E.; Czekay, Ralf-Peter; Law, Brian K.; Higgins, Stephen P.; Archambeault, Jaclyn; Kutz, Stacie M.; Higgins, Paul J.

2014-01-01

Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels. PMID:24669362

Fusion proteins comprising annexin V and Kunitz protease inhibitors are highly potent thrombogenic site-directed anticoagulants

PubMed Central

Chen, Hsiu-Hui; Vicente, Cristina P.; He, Li; Tollefsen, Douglas M.; Wun, Tze-Chein

2005-01-01

The anionic phospholipid, phosphatidyl-l-serine (PS), is sequestered in the inner layer of the plasma membrane in normal cells. Upon injury, activation, and apoptosis, PS becomes exposed on the surfaces of cells and sheds microparticles, which are procoagulant. Coagulation is initiated by formation of a tissue factor/factor VIIa complex on PS-exposed membranes and propagated through the assembly of intrinsic tenase (factor VIIIa/factor IXa), prothrombinase (factor Va/factor Xa), and factor XIa complexes on PS-exposed activated platelets. We constructed a novel series of recombinant anticoagulant fusion proteins by linking annexin V (ANV), a PS-binding protein, to the Kunitz-type protease inhibitor (KPI) domain of tick anticoagulant protein, an aprotinin mutant (6L15), amyloid β-protein precursor, or tissue factor pathway inhibitor. The resulting ANV-KPI fusion proteins were 6- to 86-fold more active than recombinant tissue factor pathway inhibitor and tick anticoagulant protein in an in vitro tissue factor–initiated clotting assay. The in vivo antithrombotic activities of the most active constructs were 3- to 10-fold higher than that of ANV in a mouse arterial thrombosis model. ANV-KPI fusion proteins represent a new class of anticoagulants that specifically target the anionic membrane-associated coagulation enzyme complexes present at sites of thrombogenesis and are potentially useful as antithrombotic agents. PMID:15677561

Low Dose Sarin Leads To Murine Cardiac Dysfunction

DTIC Science & Technology

2010-03-01

work directly supported a grant held by Wright State University, grant # GW060050, from the United States Army Medical Research Acquisition Activity ...GB), acts as an irreversible AChE inhibitor. Sarin reacts with the serine hydroxyl residue in the active site of AChE to form a phosphate or...United States Air Force has become increasingly engaged with terrorist groups and other elusive enemies. As these activities increase, the potential for

Structural Analysis of Charge Discrimination in the Binding of Inhibitors to Human Carbonic Anhydrases I and II

PubMed Central

Srivastava, D. K.; Jude, Kevin M.; Banerjee, Abir L.; Haldar, Manas; Manokaran, Sumathra; Kooren, Joel; Mallik, Sanku; Christianson, David W.

2008-01-01

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively-charged, negatively-charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 Å deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively-charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively-charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in “two-prong” CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site. PMID:17407288

Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src

PubMed Central

Hari, Sanjay B.; Perera, B. Gayani K.; Ranjitkar, Pratistha; Seeliger, Markus A.; Maly, Dustin J.

2013-01-01

Over the last decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely-related tyrosine kinases, like Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases. PMID:24106839

Mechanism of Action of Thalassospiramides, A New Class of Calpain Inhibitors

PubMed Central

Lu, Liang; Meehan, Michael J.; Gu, Shuo; Chen, Zhilong; Zhang, Weipeng; Zhang, Gen; Liu, Lingli; Huang, Xuhui; Dorrestein, Pieter C.; Xu, Ying; Moore, Bradley S.; Qian, Pei-Yuan

2015-01-01

Thalassospiramides comprise a large family of lipopeptide natural products produced by Thalassospira and Tistrella marine bacteria. Here we provide further evidence of their nanomolar inhibitory activity against the human calpain 1 protease. Analysis of structure-activity relationship data supported our hypothesis that the rigid 12-membered ring containing an α,β-unsaturated carbonyl moiety is the pharmacologically active functional group, in contrast to classic electrophilic “warheads” in known calpain inhibitors. Using a combination of chemical modifications, mass spectrometric techniques, site-directed mutagenesis, and molecular modeling, we show the covalent binding of thalassospiramide's α,β-unsaturated carbonyl moiety to the thiol group of calpain's catalytic Cys115 residue by a Michael 1,4-addition reaction. As nanomolar calpain inhibitors with promising selectivity and low toxicity from natural sources are rare, we consider thalassospiramides as promising drug leads. PMID:25740631

Mechanism of action of thalassospiramides, a new class of calpain inhibitors.

PubMed

Lu, Liang; Meehan, Michael J; Gu, Shuo; Chen, Zhilong; Zhang, Weipeng; Zhang, Gen; Liu, Lingli; Huang, Xuhui; Dorrestein, Pieter C; Xu, Ying; Moore, Bradley S; Qian, Pei-Yuan

2015-03-05

Thalassospiramides comprise a large family of lipopeptide natural products produced by Thalassospira and Tistrella marine bacteria. Here we provide further evidence of their nanomolar inhibitory activity against the human calpain 1 protease. Analysis of structure-activity relationship data supported our hypothesis that the rigid 12-membered ring containing an α,β-unsaturated carbonyl moiety is the pharmacologically active functional group, in contrast to classic electrophilic "warheads" in known calpain inhibitors. Using a combination of chemical modifications, mass spectrometric techniques, site-directed mutagenesis, and molecular modeling, we show the covalent binding of thalassospiramide's α,β-unsaturated carbonyl moiety to the thiol group of calpain's catalytic Cys115 residue by a Michael 1,4-addition reaction. As nanomolar calpain inhibitors with promising selectivity and low toxicity from natural sources are rare, we consider thalassospiramides as promising drug leads.

Design and optimization of N-acylhydrazone pyrimidine derivatives as E. coli PDHc E1 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study.

PubMed

He, Haifeng; Xia, Hongying; Xia, Qin; Ren, Yanliang; He, Hongwu

2017-10-15

By targeting the thiamin diphosphate (ThDP) binding site of Escherichia coli (E. coli) pyruvate dehydrogenase multienzyme complex E1 (PDHc E1), a series of novel 'open-chain' classes of ThDP analogs A, B, and C with N-acylhydrazone moieties was designed and synthesized to explore their activities against E. coli PHDc E1 in vitro and their inhibitory activity against microbial diseases were further evaluated in vivo. As a result, A1-23 exhibited moderate to potent inhibitory activities against E. coli PDHc E1 (IC 50 =0.15-23.55μM). The potent inhibitors A13, A14, A15, C2, had strong inhibitory activities with IC 50 values of 0.60, 0.15, 0.39 and 0.34μM against E. coli PDHc E1 and with good enzyme-selective inhibition between microorganisms and mammals. Especially, the most powerful inhibitor A14 could 99.37% control Xanthimonas oryzae pv. Oryzae. Furthermore, the binding features of compound A14 within E. coli PDHc E1 were investigated to provide useful insights for the further construction of new inhibitor by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that A14 had most powerful inhibition against E. coli PDHc E1 due to the establishment of stronger interaction with Glu571, Met194, Glu522, Leu264 and Phe602 at active site of E.coli PDHc E1. It could be used as a lead compound for further optimization, and may have potential as a new microbicide. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

PubMed Central

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

2015-01-01

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.« less

Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and /sup 3/H-mazindol binding in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angel, I.; Taranger, M.A.; Claustre, Y.

1988-01-01

The mechanism of anorectic action of several serotonin uptake inhibitors was investigated by comparing their anorectic potencies with several biochemical and pharmacological properties and in reference to the novel compound SL 81.0385. The anorectic effect of the potent serotonin uptake inhibitor SL 81.0385 was potentiated by pretreatment with 5-hydroxytryptophan and blocked by the serotonin receptor antagonist metergoline. A good correlation was obtained between the ED/sub 50/ values of anorectic action and the ED/sub 50/ values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced (/sup 3/H)-mazindol frommore » its binding to the anorectic recognition site in the hypothalamus, except the pro-drug zimelidine which was inactive. Excluding zimelidine, a good correlation was obtained between the affinities of these drugs for (/sup 3/H)-mazindol binding and their anorectic action indicating that their anorectic activity may be associated with an effect mediated through this site. Taken together these results suggest that the anorectic action of serotonin uptake inhibitors is directly associated to their ability to inhibit serotonin uptake and thus increasing the synaptic levels of serotonin. The interactions of these drugs with the anorectic recognition site labelled with (/sup 3/H)-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake.« less

Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor.

PubMed

Vannini, Alessandro; Volpari, Cinzia; Filocamo, Gessica; Casavola, Elena Caroli; Brunetti, Mirko; Renzoni, Debora; Chakravarty, Prasun; Paolini, Chantal; De Francesco, Raffaele; Gallinari, Paola; Steinkühler, Christian; Di Marco, Stefania

2004-10-19

Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single alpha/beta domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Lipid raft-like liposomes used for targeted delivery of a chimeric entry-inhibitor peptide with anti-HIV-1 activity.

PubMed

Gómara, María José; Pérez-Pomeda, Ignacio; Gatell, José María; Sánchez-Merino, Victor; Yuste, Eloisa; Haro, Isabel

2017-02-01

The work reports the design and synthesis of a chimeric peptide that is composed of the peptide sequences of two entry inhibitors which target different sites of HIV-1 gp41. The chimeric peptide offers the advantage of targeting two gp41 regions simultaneously: the fusion peptide and the loop both of which are membrane active and participate in the membrane fusion process. We therefore use lipid raft-like liposomes as a tool to specifically direct the chimeric inhibitor peptide to the membrane domains where the HIV-1 envelope protein is located. Moreover, the liposomes that mimic the viral membrane composition protect the chimeric peptide against proteolytic digestion thereby increasing the stability of the peptide. The described liposome preparations are suitable nanosystems for managing hydrophobic entry-inhibitor peptides as putative therapeutics. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor*

PubMed Central

Daly, Norelle L.; Thorstholm, Louise; Greenwood, Kathryn P.; King, Gordon J.; Rosengren, K. Johan; Heras, Begoña; Martin, Jennifer L.; Craik, David J.

2013-01-01

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin. PMID:24169696

High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors and antihypertensive drugs.

PubMed

Akif, Mohd; Georgiadis, Dimitris; Mahajan, Aman; Dive, Vincent; Sturrock, Edward D; Isaac, R Elwyn; Acharya, K Ravi

2010-07-16

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE. 2010 Elsevier Ltd. All rights reserved.

Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study.

PubMed

Kopecný, David; Briozzo, Pierre; Popelková, Hana; Sebela, Marek; Koncitíková, Radka; Spíchal, Lukás; Nisler, Jaroslav; Madzak, Catherine; Frébort, Ivo; Laloue, Michel; Houba-Hérin, Nicole

2010-08-01

Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme-inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N'-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N(6)-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N'-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N'-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity. Copyright 2010 Elsevier Masson SAS. All rights reserved.

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

PubMed Central

Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

2013-01-01

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

Inhibitor-based validation of a homology model of the active-site of tripeptidyl peptidase II.

PubMed

De Winter, Hans; Breslin, Henry; Miskowski, Tamara; Kavash, Robert; Somers, Marijke

2005-04-01

A homology model of the active site region of tripeptidyl peptidase II (TPP II) was constructed based on the crystal structures of four subtilisin-like templates. The resulting model was subsequently validated by judging expectations of the model versus observed activities for a broad set of prepared TPP II inhibitors. The structure-activity relationships observed for the prepared TPP II inhibitors correlated nicely with the structural details of the TPP II active site model, supporting the validity of this model and its usefulness for structure-based drug design and pharmacophore searching experiments.

Low dielectric response in enzyme active site

PubMed Central

Mertz, Edward L.; Krishtalik, Lev I.

2000-01-01

The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

Characterization of monoclonal antibodies that strongly inhibit Electrophorus electricus acetylcholinesterase.

PubMed

Remy, M H; Frobert, Y; Grassi, J

1995-08-01

In this study, we describe three different monoclonal antibodies (mAbs Elec-403, Elec-408, and Elec-410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec-403 and Elec-410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec-403 was antagonized by 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low-molecular-mass inhibitors. The third mAb (Elec-408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec-403, Elec-410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki values less than 0.1 nM. Elec-403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o-nitrophenyl acetate) and had the characteristics of a non-competitive process. Elec-403 and Elec-410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec-408 has not been localized, but it may correspond to a new regulatory site on AChE.

Discovery of HDAC Inhibitors That Lack an Active Site Zn(2+)-Binding Functional Group.

PubMed

Vickers, Chris J; Olsen, Christian A; Leman, Luke J; Ghadiri, M Reza

2012-06-14

Natural and synthetic histone deacetylase (HDAC) inhibitors generally derive their strong binding affinity and high potency from a key functional group that binds to the Zn(2+) ion within the enzyme active site. However, this feature is also thought to carry the potential liability of undesirable off-target interactions with other metalloenzymes. As a step toward mitigating this issue, here, we describe the design, synthesis, and structure-activity characterizations of cyclic α3β-tetrapeptide HDAC inhibitors that lack the presumed indispensable Zn(2+)-binding group. The lead compounds (e.g., 15 and 26) display good potency against class 1 HDACs and are active in tissue culture against various human cancer cell lines. Importantly, enzymological analysis of 26 indicates that the cyclic α3β-tetrapeptide is a fast-on/off competitive inhibitor of HDACs 1-3 with K i values of 49, 33, and 37 nM, respectively. Our proof of principle study supports the idea that novel classes of HDAC inhibitors, which interact at the active-site opening, but not with the active site Zn(2+), can have potential in drug design.

Binding of 3,4,5,6-Tetrahydroxyazepanes to the Acid-[beta]-glucosidase Active Site: Implications for Pharmacological Chaperone Design for Gaucher Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orwig, Susan D.; Tan, Yun Lei; Grimster, Neil P.

2013-03-07

Pharmacologic chaperoning is a therapeutic strategy being developed to improve the cellular folding and trafficking defects associated with Gaucher disease, a lysosomal storage disorder caused by point mutations in the gene encoding acid-{beta}-glucosidase (GCase). In this approach, small molecules bind to and stabilize mutant folded or nearly folded GCase in the endoplasmic reticulum (ER), increasing the concentration of folded, functional GCase trafficked to the lysosome where the mutant enzyme can hydrolyze the accumulated substrate. To date, the pharmacologic chaperone (PC) candidates that have been investigated largely have been active site-directed inhibitors of GCase, usually containing five- or six-membered rings, suchmore » as modified azasugars. Here we show that a seven-membered, nitrogen-containing heterocycle (3,4,5,6-tetrahydroxyazepane) scaffold is also promising for generating PCs for GCase. Crystal structures reveal that the core azepane stabilizes GCase in a variation of its proposed active conformation, whereas binding of an analogue with an N-linked hydroxyethyl tail stabilizes GCase in a conformation in which the active site is covered, also utilizing a loop conformation not seen previously. Although both compounds preferentially stabilize GCase to thermal denaturation at pH 7.4, reflective of the pH in the ER, only the core azepane, which is a mid-micromolar competitive inhibitor, elicits a modest increase in enzyme activity for the neuronopathic G202R and the non-neuronopathic N370S mutant GCase in an intact cell assay. Our results emphasize the importance of the conformational variability of the GCase active site in the design of competitive inhibitors as PCs for Gaucher disease.« less

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

PubMed

Iwanowicz, Edwin J; Kimball, S David; Lin, James; Lau, Wan; Han, W-C; Wang, Tammy C; Roberts, Daniel G M; Schumacher, W A; Ogletree, Martin L; Seiler, Steven M

2002-11-04

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.

Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease.

PubMed

Ragland, Debra A; Nalivaika, Ellen A; Nalam, Madhavi N L; Prachanronarong, Kristina L; Cao, Hong; Bandaranayake, Rajintha M; Cai, Yufeng; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-08-27

HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE PAGES

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...

2014-12-31

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.

The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less

Synthesis and biological evaluation of 2-substituted-5-(4-nitrophenylsulfonamido)benzoxazoles as human GST P1-1 inhibitors, and description of the binding site features.

PubMed

Ertan-Bolelli, Tuğba; Musdal, Yaman; Bolelli, Kayhan; Yilmaz, Serap; Aksoy, Yasemin; Yildiz, Ilkay; Aki-Yalcin, Esin; Yalcin, Ismail

2014-05-01

Glutathione-S-transferases (GSTs) are enzymes involved in cellular detoxification by catalyzing the nucleophilic attack of glutathione (GSH) on the electrophilic center of numerous of toxic compounds and xenobiotics, including chemotherapeutic drugs. Human GST P1-1, which is known as the most prevalent isoform of the mammalian cytosolic GSTs, is overexpressed in many cancers and contributes to multidrug resistance by directly conjugating to chemotherapeutics. It is suggested that this resistance is related to the high expression of GST P1-1 in cancers, thereby contributing to resistance to chemotherapy. In addition, GSTs exhibit sulfonamidase activity, thereby catalyzing the GSH-mediated hydrolysis of sulfonamide bonds. Such reactions are of interest as potential tumor-directed prodrug activation strategies. Herein we report the design and synthesis of some novel sulfonamide-containing benzoxazoles, which are able to inhibit human GST P1-1. Among the tested compounds, 2-(4-chlorobenzyl)-5-(4-nitrophenylsulfonamido)benzoxazole (5 f) was found as the most active hGST P1-1 inhibitor, with an IC50 value of 10.2 μM, showing potency similar to that of the reference drug ethacrynic acid. Molecular docking studies performed with CDocker revealed that the newly synthesized 2-substituted-5-(4-nitrophenylsulfonamido)benzoxazoles act as catalytic inhibitors of hGST P1-1 by binding to the H-site and generating conjugates with GSH to form S-(4-nitrophenyl)GSH (GS-BN complex) via nucleophilic aromatic substitution reaction. The 4-nitrobenzenesulfonamido moiety at position 5 of the benzoxazole ring is essential for binding to the H-site and for the formation of the GST-mediated GSH conjugate. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement

PubMed Central

Esser, Lothar; Yu, Chang-An; Xia, Di

2016-01-01

The emergence of drug resistance has devastating economic and social consequences, a testimonial of which is the rise and fall of inhibitors against the respiratory component cytochrome bc1 complex, a time tested and highly effective target for disease control. Unfortunately, the mechanism of resistance is a multivariate problem, including primarily mutations in the gene of the cytochrome b subunit but also activation of alternative pathways of ubiquinol oxidation and pharmacokinetic effects. There is a considerable interest in designing new bc1 inhibitors with novel modes of binding and lower propensity to induce the development of resistance. The accumulation of crystallographic data of bc1 complexes with and without inhibitors bound provides the structural basis for rational drug design. In particular, the cytochrome b subunit offers two distinct active sites that can be targeted for inhibition - the quinol oxidation site and the quinone reduction site. This review brings together available structural information of inhibited bc1 by various quinol oxidation- and reduction-site inhibitors, the inhibitor binding modes, conformational changes upon inhibitor binding of side chains in the active site and large scale domain movements of the iron-sulfur protein subunit. Structural data analysis provides a clear understanding of where and why existing inhibitors fail and points towards promising alternatives. PMID:23688079

The 26S Proteasome Complex: An Attractive Target for Cancer Therapy

PubMed Central

Frankland-Searby, Sarah; Bhaumik, Sukesh R.

2011-01-01

The 26S proteasome complex engages in an ATP-dependent proteolytic degradation of a variety of oncoproteins, transcription factors, cell cycle specific cyclins, cyclin-dependent kinase inhibitors, ornithine decarboxylase, and other key regulatory cellular proteins. Thus, the proteasome regulates either directly or indirectly many important cellular processes. Altered regulation of these cellular events is linked to the development of cancer. Therefore, the proteasome has become an attractive target for the treatment of numerous cancers. Several proteasome inhibitors that target the proteolytic active sites of the 26S proteasome complex have been developed and tested for anti-tumor activities. These proteasome inhibitors have displayed impressive anti-tumor functions by inducing apoptosis in different tumor types. Further, the proteasome inhibitors have been shown to induce cell cycle arrest, and inhibit angiogenesis, cell-cell adhesion, cell migration, immune and inflammatory responses, and DNA repair response. A number of proteasome inhibitors are now in clinical trials to treat multiple myeloma and solid tumors. Many other proteasome inhibitors with different efficiencies are being developed and tested for anti-tumor activities. Several proteasome inhibitors currently in clinical trials have shown significantly improved anti-tumor activities when combined with other drugs such as histone deacetylase (HDAC) inhibitors, Akt (protein kinase B) inhibitors, DNA damaging agents, Hsp90 (heat shock protein 90) inhibitors, and lenalidomide. The proteasome inhibitor bortezomib is now in the clinic to treat multiple myeloma and mantle cell lymphoma. Here, we discuss the 26S proteasome complex in carcinogenesis and different proteasome inhibitors with their potential therapeutic applications in treatment of numerous cancers. PMID:22037302

Effect of MCM09, an active site-directed inhibitor of factor Xa, on B16-BL6 melanoma lung colonies in mice.

PubMed

Rossi, C; Hess, S; Eckl, R W; di Lena, A; Bruno, A; Thomas, O; Poggi, A

2006-03-01

Treatment with anticoagulant drugs has shown potential inhibitory effect on tumor invasion, although the relationship with clotting inhibition was not clear. The aim of our study was to evaluate the potential antitumor activity of MCM09, a newly developed, active site-directed, small molecule inhibitor of factor Xa (FXa) [WO0216312], and to relate the findings to anticlotting potency. MCM09 (0.1-10 mg kg(-1)) or heparin (H; 10 mg kg(-1)) was injected intravenously (i.v.), with 5 x 10(4) B16-BL6 melanoma cells, in C57BL/6 mice. Mice were killed after 18 days, to count lung colonies. Ex vivo anticoagulant activity was measured by activated partial thromboplastin time (APTT) on mouse plasma. MCM09, a selective inhibitor of FXa (IC-50 = 2.4 nm against human FXa), inhibited in a dose-dependent manner B16-BL6 melanoma lung colonies in mice. Mean lung metastasis number was 20.9 +/- 4.8 in controls (n = 10), 1.2 +/- 0.4 in mice treated with H, 10 mg kg(-1) i.v. (P < 0.01), 0.9 +/- 0.3, 9.2 +/- 2.2 and 15.5 +/- 2.6 in mice treated with MCM09, at 10 (P < 0.01), 1 (P < 0.05) and 0.1 mg kg(-1) i.v. (ns), respectively. MCM09 (10 mg kg(-1) i.v.) significantly prolonged APTT (57.1 +/- 10.2 s) 30 min after i.v. injection when compared with controls (25.3 +/- 1.6 s; P < 0.05). Lung colonies were 74.2-72.6% reduced by MCM09 (10 mg kg(-1)) given 60 or 120 min before cells, but not by MCM09 given 60 min thereafter, suggesting a direct cell interaction as a mechanism underlying antitumor activity.

Exploration of peptides that fit into the thermally vibrating active site of cathepsin K protease by alternating artificial intelligence and molecular simulation

NASA Astrophysics Data System (ADS)

Nishiyama, Katsuhiko

2017-08-01

Eighteen tripeptides that fit into the thermally vibrating active site of cathepsin K were discovered by alternating artificial intelligence and molecular simulation. The 18 tripeptides fit the active site better than the cysteine protease inhibitor E64, and a better inhibitor of cathepsin K could be designed considering these tripeptides. Among the 18 tripeptides, Phe-Arg-Asp and Tyr-Arg-Asp fit the active site the best and their structural similarity should be considered in the design process. Interesting factors emerged from the structure of the decision tree, and its structural information will guide exploration of potential inhibitor molecules for proteases.

Homology modeling and molecular dynamics simulation of N-myristoyltransferase from protozoan parasites: active site characterization and insights into rational inhibitor design

NASA Astrophysics Data System (ADS)

Sheng, Chunquan; Ji, Haitao; Miao, Zhenyuan; Che, Xiaoyin; Yao, Jianzhong; Wang, Wenya; Dong, Guoqiang; Guo, Wei; Lü, Jiaguo; Zhang, Wannian

2009-06-01

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is a cytosolic monomeric enzyme that catalyzes the transfer of the myristoyl group from myristoyl-CoA to the N-terminal glycine of a number of eukaryotic cellular and viral proteins. Recent experimental data suggest NMT from parasites could be a promising new target for the design of novel antiparasitic agents with new mode of action. However, the active site topology and inhibitor specificity of these enzymes remain unclear. In this study, three-dimensional models of NMT from Plasmodium falciparum (PfNMT), Leishmania major (LmNMT) and Trypanosoma brucei (TbNMT) were constructed on the basis of the crystal structures of fungal NMTs using homology modeling method. The models were further refined by energy minimization and molecular dynamics simulations. The active sites of PfNMT, LmNMT and TbNMT were characterized by multiple copy simultaneous search (MCSS). MCSS functional maps reveal that PfNMT, LmNMT and TbNMT share a similar active site topology, which is defined by two hydrophobic pockets, a hydrogen-bonding (HB) pocket, a negatively-charged HB pocket and a positively-charged HB pocket. Flexible docking approaches were then employed to dock known inhibitors into the active site of PfNMT. The binding mode, structure-activity relationships and selectivity of inhibitors were investigated in detail. From the results of molecular modeling, the active site architecture and certain key residues responsible for inhibitor binding were identified, which provided insights for the design of novel inhibitors of parasitic NMTs.

Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies

NASA Astrophysics Data System (ADS)

Wang, Xing; Zhang, Yuxin; Yang, Ying; Wu, Xia; Fan, Hantian; Qiao, Yanjiang

2017-03-01

Thrombin acts as a key enzyme in the blood coagulation cascade and represents a potential drug target for the treatment of several cardiovascular diseases. The aim of this study was to identify small-molecule direct thrombin inhibitors from herbs used in traditional Chinese medicine (TCM). A pharmacophore model and molecular docking were utilized to virtually screen a library of chemicals contained in compositions of traditional Chinese herbs, and these analyses were followed by in vitro bioassay validation and binding studies. Berberine (BBR) was first confirmed as a thrombin inhibitor using an enzymatic assay. The BBR IC50 value for thrombin inhibition was 2.92 μM. Direct binding studies using surface plasmon resonance demonstrated that BBR directly interacted with thrombin with a KD value of 16.39 μM. Competitive binding assay indicated that BBR could bind to the same argartroban/thrombin interaction site. A platelet aggregation assay demonstrated that BBR had the ability to inhibit thrombin-induced platelet aggregation in washed platelets samples. This study proved that BBR is a direct thrombin inhibitor that has activity in inhibiting thrombin-induced platelet aggregation. BBR may be a potential candidate for the development of safe and effective thrombin-inhibiting drugs.

Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

PubMed Central

Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

1991-01-01

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

Docking modes of BB-3497 into the PDF active site--a comparison of the pure MM and QM/MM based docking strategies.

PubMed

Kumari, Tripti; Issar, Upasana; Kakkar, Rita

2014-01-01

Peptide deformylase (PDF) has emerged as an important antibacterial drug target. Considerable effort is being directed toward developing peptidic and non-peptidic inhibitors for this metalloprotein. In this work, the known peptidic inhibitor BB-3497 and its various ionization and tautomeric states are evaluated for their inhibition efficiency against PDF using a molecular mechanics (MM) approach as well as a mixed quantum mechanics/molecular mechanics (QM/MM) approach, with an aim to understand the interactions in the binding site. The evaluated Gibbs energies of binding with the mixed QM/MM approach are shown to have the best predictive power. The experimental pose is found to have the most negative Gibbs energy of binding, and also the smallest strain energy. A quantum mechanical evaluation of the active site reveals the requirement of strong chelation by the ligand with the metal ion. The investigated ligand chelates the metal ion through the two oxygens of its reverse hydroxamate moiety, particularly the N-O(-) oxygen, forming strong covalent bonds with the metal ion, which is penta-coordinated. In the uninhibited state, the metal ion is tetrahedrally coordinated, and hence chelation with the inhibitor is associated with an increase of the metal ion coordination. Thus, the strong binding of the ligand at the binding site is accounted for.

Fragment-Based Approaches to Enhance GTP Competitive KRAS G12C Inhibitors

DTIC Science & Technology

During the current period we completed work on a series of guanine nucleotide mimetics and published results. As part of this we developed and...reported a novel method of measuring small molecule binding to KRAS G12C active site. We also published 2 additional manuscripts about KRAS G12C directed

HDAC inhibitors TSA and sodium butyrate enhanced the human IL-5 expression by altering histone acetylation status at its promoter region.

PubMed

Han, Songyan; Lu, Jun; Zhang, Yu; Cheng, Cao; Li, Lin; Han, Liping; Huang, Baiqu

2007-02-15

The expression of IL-5 correlated tightly with the maturation and differentiation of eosinophils, and is considered as a cytokine responsible for allergic inflammation. We report here that inhibition of HDAC activity by Trichostatin A (TSA) and sodium butyrate (NaBu), the two specific HDAC inhibitors, resulted in the elevation of both endogenous and exogenous activity of IL-5 promoter. We demonstrated that both the mRNA expression and protein production of IL-5 were stimulated by TSA and NaBu treatments. ChIP assays showed that treatments of TSA and NaBu caused hyperacetylation of histones H3 and H4 on IL-5 promoter in Jurkat cells, which consequently promoted the exogenous luciferase activity driven by this promoter. Moreover, site-directed mutagenesis studies showed that the binding sites for transcription factors NFAT, GATA3 and YY1 on IL-5 promoter were critical for the effects of TSA and NaBu, suggesting that the transcriptional activation of IL-5 gene by these inhibitors was achieved by affecting HDAC function on IL-5 promoter via transcription factors. These data will contribute to elucidating the unique mechanism of IL-5 transcriptional control and to the therapy of allergic disorders related to IL-5.

Structural basis of trypsin inhibition and entomotoxicity of cospin, serine protease inhibitor involved in defense of Coprinopsis cinerea fruiting bodies.

PubMed

Sabotič, Jerica; Bleuler-Martinez, Silvia; Renko, Miha; Avanzo Caglič, Petra; Kallert, Sandra; Štrukelj, Borut; Turk, Dušan; Aebi, Markus; Kos, Janko; Künzler, Markus

2012-02-03

Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a β-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the β2 and β3 strands, distinguishing cospin from other β-trefoil-fold serine protease inhibitors in which β4-β5 or β5-β6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.

The cysteine protease inhibitor, E64d, reduces brain amyloid-β and improves memory deficits in Alzheimer’s disease animal models by inhibiting cathepsin B, but not BACE1, β-secretase activity

PubMed Central

Hook, Gregory; Hook, Vivian; Kindy, Mark

2015-01-01

The cysteine protease cathepsin B is a potential drug target for reducing brain amyloid-β peptides (Aβ) and improving memory in Alzheimer’s disease (AD), because reduction of cathepsin B in transgenic mice expressing human wild-type amyloid-β protein precursor (AβPP) results in significantly decreased brain Aβ. Cathepsin B cleaves the wild-type β-secretase site sequence in AβPP to produce Aβ and cathepsin B inhibitors administered to animal models expressing AβPP containing the wild-type β-secretase site sequence reduce brain Aβ in a manner consistent with β-secretase inhibition. But such inhibitors could act either by direct inhibition of cathepsin B β-secretase activity or by off-target inhibition of the other β-secretase, the aspartyl protease BACE1. To evaluate that issue, we orally administered a cysteine protease inhibitor, E64d, to normal guinea pigs or transgenic mice expressing human AβPP, both of which express the human wild-type β-secretase site sequence. In guinea pigs, oral E64d administration caused a dose-dependent reduction of up to 92% in brain, CSF and plasma of Aβ(40) and Aβ(42), a reduction of up to 50% in the C-terminal β-secretase fragment (CTFβ), and a 91% reduction in brain cathepsin B activity but increased brain BACE1 activity by 20%. In transgenic AD mice, oral E64d administration improved memory deficits and reduced brain Aβ(40) and Aβ(42), amyloid plaque, brain CTFβ, and brain cathepsin B activity but increased brain BACE1 activity. We conclude that E64d likely reduces brain Aβ by inhibiting cathepsin B and not BACE1 β-secretase activity and that E64d therefore may have potential for treating AD patients. PMID:21613740

A binding site for non-steroidal anti-inflammatory drugs in FAAH

PubMed Central

Bertolacci, Laura; Romeo, Elisa; Veronesi, Marina; Magotti, Paola; Albani, Clara; Dionisi, Mauro; Lambruschini, Chiara; Scarpelli, Rita; Cavalli, Andrea; Vivo, Marco De; Piomelli, Daniele; Garau, Gianpiero

2013-01-01

In addition to inhibiting the cyclooxygenasemediated biosynthesis of prostanoids, various widely used non-steroidal anti-inflammatory drugs (NSAIDs) enhance endocannabinoid signaling by blocking the anandamidedegrading membrane enzyme, fatty acid amide hydrolase (FAAH). The X-ray structure of FAAH in complex with the NSAID carprofen, along with studies of site-directed mutagenesis, enzyme activity assays, and nuclear magnetic resonance, now reveal the molecular details of this interaction, providing information that may guide the design of dual FAAH-cyclooxygenase inhibitors with superior analgesic efficacy. PMID:23240907

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

PubMed Central

Jutras, Philippe V.; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Donini, Marcello

2016-01-01

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories. PMID:27893815

Designing of Protein Kinase C β-II Inhibitors against Diabetic complications: Structure Based Drug Design, Induced Fit docking and analysis of active site conformational changes

PubMed Central

Vijayakumar, Balakrishnan; Velmurugan, Devadasan

2012-01-01

Protein Kinase C β-II (PKC β-II) is an important enzyme in the development of diabetic complications like cardiomyopathy, retinopathy, neuropathy, nephropathy and angiopathy. PKC β-II is activated in vascular tissues during diabetic vascular abnormalities. Thus, PKC β-II is considered as a potent drug target and the crystal structure of the kinase domain of PKC β-II (PDB id: 2I0E) was used to design inhibitors using Structure-Based Drug Design (SBDD) approach. Sixty inhibitors structurally similar to Staurosporine were retrieved from PubChem Compound database and High Throughput Virtual screening (HTVs) was carried out with PKC β-II. Based on the HTVs results and the nature of active site residues of PKC β-II, Staurosporine inhibitors were designed using SBDD. Induced Fit Docking (IFD) studies were carried out between kinase domain of PKC β-II and the designed inhibitors. These IFD complexes showed favorable docking score, glide energy, glide emodel and hydrogen bond and hydrophobic interactions with the active site of PKC β-II. Binding free energy was calculated for IFD complexes using Prime MM-GBSA method. The conformational changes induced by the inhibitor at the active site of PKC β-II were observed for the back bone Cα atoms and side-chain chi angles. PASS prediction tool was used to analyze the biological activities for the designed inhibitors. The various physicochemical properties were calculated for the compounds. One of the designed inhibitors successively satisfied all the in silico parameters among the others and seems to be a potent inhibitor against PKC β-II. PMID:22829732

X-ray Crystallographic Analysis of [alpha]-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine

2010-11-03

Three cocrystal X-ray structures of the {alpha}-ketoheterocycle inhibitors 3-5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the {alpha}-ketoheterocycle inhibitors captured as deprotonated hemiketals mimickingmore » the tetrahedral intermediate of the enzyme-catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure-activity relationships are discussed providing important insights for future design.« less

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

PubMed Central

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J. Preben; Andreasen, Peter A.; Jensen, Jan K.

2016-01-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). PMID:27189939

Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi

2014-02-01

The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalentlymore » through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.« less

Heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.

PubMed

Taldone, Tony; Kang, Yanlong; Patel, Hardik J; Patel, Maulik R; Patel, Pallav D; Rodina, Anna; Patel, Yogita; Gozman, Alexander; Maharaj, Ronnie; Clement, Cristina C; Lu, Alvin; Young, Jason C; Chiosis, Gabriela

2014-02-27

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

Epigenetic targeting of Hedgehog pathway transcriptional output through BET bromodomain inhibition

PubMed Central

Tang, Yujie; Gholamin, Sharareh; Schubert, Simone; Willardson, Minde I.; Lee, Alex; Bandopadhayay, Pratiti; Bergthold, Guillame; Masoud, Sabran; Nguyen, Brian; Vue, Nujsaubnusi; Balansay, Brianna; Yu, Furong; Oh, Sekyung; Woo, Pamelyn; Chen, Spenser; Ponnuswami, Anitha; Monje, Michelle; Atwood, Scott X.; Whitson, Ramon J.; Mitra, Siddhartha; Cheshier, Samuel H.; Qi, Jun; Beroukhim, Rameen; Tang, Jean Y.; Wechsler-Reya, Rob; Oro, Anthony E.; Link, Brian A.; Bradner, James E.; Cho, Yoon-Jae

2014-01-01

Hedgehog signaling drives oncogenesis in several cancers and strategies targeting this pathway have been developed, most notably through inhibition of Smoothened. However, resistance to Smoothened inhibitors occurs via genetic changes of Smoothened or other downstream Hedgehog components. Here, we overcome these resistance mechanisms by modulating GLI transcription via inhibition of BET bromodomain proteins. We show the BET bromodomain protein, BRD4, regulates GLI transcription downstream of SMO and SUFU and chromatin immunoprecipitation studies reveal BRD4 directly occupies GLI1 and GLI2 promoters, with a substantial decrease in engagement of these sites upon treatment with JQ1, a small molecule inhibitor targeting BRD4. Globally, genes associated with medulloblastoma-specific GLI1 binding sites are downregulated in response to JQ1 treatment, supporting direct regulation of GLI activity by BRD4. Notably, patient- and GEMM-derived Hedgehog-driven tumors (basal cell carcinoma, medulloblastoma and atypical teratoid/rhabdoid tumor) respond to JQ1 even when harboring genetic lesions rendering them resistant to Smoothened antagonists. PMID:24973920

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites.

PubMed

Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik; Hang, Howard C

2018-05-04

S-Fatty-acylation is the covalent attachment of long chain fatty acids, predominately palmitate (C16:0, S-palmitoylation), to cysteine (Cys) residues via a thioester linkage on proteins. This post-translational and reversible lipid modification regulates protein function and localization in eukaryotes and is important in mammalian physiology and human diseases. While chemical labeling methods have improved the detection and enrichment of S-fatty-acylated proteins, mapping sites of modification and characterizing the endogenously attached fatty acids are still challenging. Here, we describe the integration and optimization of fatty acid chemical reporter labeling with hydroxylamine-mediated enrichment of S-fatty-acylated proteins and direct tagging of modified Cys residues to selectively map lipid modification sites. This afforded improved enrichment and direct identification of many protein S-fatty-acylation sites compared to previously described methods. Notably, we directly identified the S-fatty-acylation sites of IFITM3, an important interferon-stimulated inhibitor of virus entry, and we further demonstrated that the highly conserved Cys residues are primarily modified by palmitic acid. The methods described here should facilitate the direct analysis of protein S-fatty-acylation sites and their endogenously attached fatty acids in diverse cell types and activation states important for mammalian physiology and diseases.

Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization.

PubMed

Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio

2011-04-01

Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.

Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

PubMed

Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

2015-12-16

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

PubMed

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

2016-07-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Highly Potent HIV-1 Protease Inhibitors with Novel Tricyclic P2-ligands: Design, Synthesis, and Protein-ligand X-Ray Studies

PubMed Central

Ghosh, Arun K.; Parham, Garth L.; Martyr, Cuthbert D.; Nyalapatla, Prasanth R.; Osswald, Heather L.; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

2013-01-01

The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors incorporating stereochemically defined fused tricyclic P2-ligands are described. Various substituent effects were investigated in order to maximize the ligand-binding site interactions in the protease active site. Inhibitors 16a and 16f showed excellent enzyme inhibitory and antiviral activity while incorporation of sulfone functionality resulted in a decrease in potency. Both inhibitors 16a and 16f have maintained activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 16a-bound HIV-1 protease revealed important molecular insights into the ligand-binding site interactions which may account for the inhibitor’s potent antiviral activity and excellent resistance profiles. PMID:23947685

Inhibitors of nuclease and redox activity of apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1).

PubMed

Laev, Sergey S; Salakhutdinov, Nariman F; Lavrik, Olga I

2017-05-01

Human apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein which is essential in the base excision repair (BER) pathway of DNA lesions caused by oxidation and alkylation. This protein hydrolyzes DNA adjacent to the 5'-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3'-hydroxyl group and a 5'-deoxyribose phosphate moiety or activates the DNA-binding activity of certain transcription factors through its redox function. Studies have indicated a role for APE1/Ref-1 in the pathogenesis of cancer and in resistance to DNA-interactive drugs. Thus, this protein has potential as a target in cancer treatment. As a result, major efforts have been directed to identify small molecule inhibitors against APE1/Ref-1 activities. These agents have the potential to become anticancer drugs. The aim of this review is to present recent progress in studies of all published small molecule APE1/Ref-1 inhibitors. The structures and activities of APE1/Ref-1 inhibitors, that target both DNA repair and redox activities, are presented and discussed. To date, there is an urgent need for further development of the design and synthesis of APE1/Ref-1 inhibitors due to high importance of this protein target. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

PubMed Central

Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

2015-01-01

Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

Design, synthesis and antiviral evaluation of novel heteroarylcarbothioamide derivatives as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and RDDP functions.

PubMed

Corona, Angela; Onnis, Valentina; Deplano, Alessandro; Bianco, Giulia; Demurtas, Monica; Distinto, Simona; Cheng, Yung-Chi; Alcaro, Stefano; Esposito, Francesca; Tramontano, Enzo

2017-08-31

In the continuous effort to identify new HIV-1 inhibitors endowed with innovative mechanisms, the dual inhibition of different viral functions would provide a significant advantage against drug-resistant variants. The HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) is the only viral-encoded enzymatic activity that still lacks an efficient inhibitor. We synthesized a library of 3,5-diamino-N-aryl-1H-pyrazole-4-carbothioamide and 4-amino-5-benzoyl-N-phenyl-2-(substituted-amino)-1H-pyrrole-3-carbothioamide derivatives and tested them against RNase H activity. We identified the pyrazolecarbothioamide derivative A15, able to inhibit viral replication and both RNase H and RNA-dependent DNA polymerase (RDDP) RT-associated activities in the low micromolar range. Docking simulations hypothesized its binding to two RT pockets. Site-directed mutagenesis experiments showed that, with respect to wt RT, V108A substitution strongly reduced A15 IC50 values (12.6-fold for RNase H inhibition and 4.7-fold for RDDP), while substitution A502F caused a 9.0-fold increase in its IC50 value for RNase H, not affecting the RDDP inhibition, reinforcing the hypothesis of a dual-site inhibition. Moreover, A15 retained good inhibition potency against three non-nucleoside RT inhibitor (NNRTI)-resistant enzymes, confirming a mode of action unrelated to NNRTIs and suggesting its potential as a lead compound for development of new HIV-1 RT dual inhibitors active against drug-resistant viruses. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Human γ-glutamyl transpeptidase 1: Structures of the free enzyme, inhibitor-bound tetrahedral transition states, and glutamate-bound enzyme reveal novel movement within the active site during catalysis [Human gamma-glutamyl transpeptidase: Inhibitor binding and movement within the active site

DOE PAGES

Terzyan, Simon S.; Burgett, Anthony W. G.; Heroux, Annie; ...

2015-05-26

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within themore » active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less

Human γ-glutamyl transpeptidase 1: Structures of the free enzyme, inhibitor-bound tetrahedral transition states, and glutamate-bound enzyme reveal novel movement within the active site during catalysis [Human gamma-glutamyl transpeptidase: Inhibitor binding and movement within the active site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terzyan, Simon S.; Burgett, Anthony W. G.; Heroux, Annie

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within themore » active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less

STRUCTURAL CHARACTERISTICS AND ANTIHYPERTENSIVE EFFECTS OF ANGIOTENSIN-I-CONVERTING ENZYME INHIBITORY PEPTIDES IN THE RENIN-ANGIOTENSIN AND KALLIKREIN KININ SYSTEMS

PubMed Central

Manoharan, Sivananthan; Shuib, Adawiyah Suriza; Abdullah, Noorlidah

2017-01-01

Background: The commercially available synthetic angiotensin-I-converting enzyme (ACE) inhibitors are known to exert negative side effects which have driven many research groups globally to discover the novel ACE inhibitors. Method: Literature search was performed within the PubMed, ScienceDirect.com and Google Scholar. Results: The presence of proline at the C-terminal tripeptide of ACE inhibitor can competitively inhibit the ACE activity. The effects of other amino acids are less studied leading to difficulties in predicting potent peptide sequences. The broad specificity of the enzyme may be due to the dual active sites observed on the somatic ACE. The inhibitors may not necessarily competitively inhibit the enzyme which explains why some reported inhibitors do not have the common ACE inhibitor characteristics. Finally, the in vivo assay has to be carried out before the peptides as the antihypertensive agents can be claimed. The peptides must be absorbed into circulation without being degraded, which will affect their bioavailability and potency. Thus, peptides with strong in vitro IC50 values do not necessarily have the same effect in vivo and vice versa. Conclusion: The relationship between peptide amino acid sequence and inhibitory activity, in vivo studies of the active peptides and bioavailability must be studied before the peptides as antihypertensive agents can be claimed. PMID:28573254

Discovery of d-amino acid oxidase inhibitors based on virtual screening against the lid-open enzyme conformation.

PubMed

Szilágyi, Bence; Skok, Žiga; Rácz, Anita; Frlan, Rok; Ferenczy, György G; Ilaš, Janez; Keserű, György M

2018-06-01

d-Amino acid oxidase (DAAO) inhibitors are typically small polar compounds with often suboptimal pharmacokinetic properties. Features of the native binding site limit the operational freedom of further medicinal chemistry efforts. We therefore initiated a structure based virtual screening campaign based on the X-ray structures of DAAO complexes where larger ligands shifted the loop (lid opening) covering the native binding site. The virtual screening of our in-house collection followed by the in vitro test of the best ranked compounds led to the identification of a new scaffold with micromolar IC 50 . Subsequent SAR explorations enabled us to identify submicromolar inhibitors. Docking studies supported by in vitro activity measurements suggest that compounds bind to the active site with a salt-bridge characteristic to DAAO inhibitor binding. In addition, displacement of and interaction with the loop covering the active site contributes significantly to the activity of the most potent compounds. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

PubMed

Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B; Bird, Kelly E; Ginther, Patrick S; Rivalta, Ivan; Ten, Nicholas S; Batista, Victor S; Loria, J Patrick

2017-01-10

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.

Rubisco Activity: Effects of Drought Stress

PubMed Central

PARRY, MARTIN A. J.; ANDRALOJC, P. JOHN; KHAN, SHAHNAZ; LEA, PETER J.; KEYS, ALFRED J.

2002-01-01

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) activity is modulated in vivo either by reaction with CO2 and Mg2+ to carbamylate a lysine residue in the catalytic site, or by the binding of inhibitors within the catalytic site. Binding of inhibitors blocks either activity or the carbamylation of the lysine residue that is essential for activity. At night, in many species, 2‐carboxyarabinitol‐1‐phosphate (CA1P) is formed which binds tightly to Rubisco, inhibiting catalytic activity. Recent work has shown that tight‐binding inhibitors can also decrease Rubisco activity in the light and contribute to the regulation of Rubisco activity. Here we determine the influence that such inhibitors of Rubisco exert on catalytic activity during drought stress. In tobacco plants, ‘total Rubisco activity’, i.e. the activity following pre‐incubation with CO2 and Mg2+, was positively correlated with leaf relative water content. However, ‘total Rubisco activity’ in extracts from leaves with low water potential increased markedly when tightly bound inhibitors were removed, thus increasing the number of catalytic sites available. This suggests that in tobacco the decrease of Rubisco activity under drought stress is not primarily the result of changes in activation by CO2 and Mg2+ but due rather to the presence of tight‐binding inhibitors. The amounts of inhibitor present in leaves of droughted tobacco based on the decrease in Rubisco activity per mg soluble protein were usually much greater than the amounts of the known inhibitors (CA1P and ‘daytime inhibitor’) that can be recovered in acid extracts. Alternative explanations for the difference between maximal and total activities are discussed. PMID:12102509

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

PubMed

Heng, Sabrina; Stieglitz, Kimberly A; Eldo, Joby; Xia, Jiarong; Cardia, James P; Kantrowitz, Evan R

2006-08-22

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

PubMed

Mega, T; Nishijima, T; Ikenaka, T

1990-04-01

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.

Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sultatos, L.G.; Kaushik, R.

2008-08-01

The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, thesemore » changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.« less

A designed inhibitor of a CLC antiporter blocks function through a unique binding mode

PubMed Central

Howery, Andrew E.; Elvington, Shelley; Abraham, Sherwin J.; Choi, Kee-Hyun; Phillips, Sabrina; Ryan, Christopher M.; Sanford, R. Lea; Simpson-Dworschak, Sierra; Almqvist, Jonas; Tran, Kevin; Chew, Thomas A.; Zachariae, Ulrich; Andersen, Olaf S.; Whitelegge, Julian; Matulef, Kimberly; Du Bois, Justin; Maduke, Merritt C.

2012-01-01

SUMMARY The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC “Chloride Channel” family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of the first specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4′-octanamidostilbene-2,2′-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid-dependence of inhibition suggest potential mechanisms of action. The discovery of this compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders. PMID:23177200

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Broad-spectrum non-nucleoside inhibitors for caliciviruses.

PubMed

Netzler, Natalie E; Enosi Tuipulotu, Daniel; Eltahla, Auda A; Lun, Jennifer H; Ferla, Salvatore; Brancale, Andrea; Urakova, Nadya; Frese, Michael; Strive, Tanja; Mackenzie, Jason M; White, Peter A

2017-10-01

Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC 50 4.3-16.6 μM), TMC-647055 (IC 50 range: 18.8-45.4 μM) and Beclabuvir (IC 50 range: 23.8->100 μM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC 50 range: 0.1-2.3 μM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket, Site-B. We also revealed three broad-spectrum HCV NNIs that could be used as antiviral scaffolds for further development against caliciviruses and other viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Pyrrole-indolinone SU11652 targets the nucleoside diphosphate kinase from Leishmania parasites.

PubMed

Vieira, Plínio Salmazo; Souza, Tatiana de Arruda Campos Brasil; Honorato, Rodrigo Vargas; Zanphorlin, Letícia Maria; Severiano, Kelven Ulisses; Rocco, Silvana Aparecida; de Oliveira, Arthur Henrique Cavalcante; Cordeiro, Artur Torres; Oliveira, Paulo Sérgio Lopes; de Giuseppe, Priscila Oliveira; Murakami, Mário Tyago

2017-07-01

Nucleoside diphosphate kinases (NDKs) are key enzymes in the purine-salvage pathway of trypanosomatids and have been associated with the maintenance of host-cell integrity for the benefit of the parasite, being potential targets for rational drug discovery and design. The NDK from Leishmania major (LmNDK) and mutants were expressed and purified to homogeneity. Thermal shift assays were employed to identify potential inhibitors for LmNDK. Calorimetric experiments, site-directed mutagenesis and molecular docking analysis were performed to validate the interaction and to evaluate the structural basis of ligand recognition. Furthermore, the anti-leishmanial activity of the newly identified and validated compound was tested in vitro against different Leishmania species. The molecule SU11652, a Sunitinib analog, was identified as a potential inhibitor for LmNDK and structural studies indicated that this molecule binds to the active site of LmNDK in a similar conformation to nucleotides, mimicking natural substrates. Isothermal titration calorimetry experiments combined with site-directed mutagenesis revealed that the residues H50 and H117, considered essential for catalysis, play an important role in ligand binding. In vitro cell studies showed that SU11652 had similar efficacy to Amphotericin b against some Leishmania species. Together, our results indicate the pyrrole-indolinone SU11652 as a promising scaffold for the rational design of new drugs targeting the enzyme NDK from Leishmania parasites. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Misaghi, S.; Galardy, P.J.; Meester, W.J.

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 {angstrom} resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structuremore » confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.« less

Prediction of luciferase inhibitors by the high-performance MIEC-GBDT approach based on interaction energetic patterns.

PubMed

Chen, Fu; Sun, Huiyong; Liu, Hui; Li, Dan; Li, Youyong; Hou, Tingjun

2017-04-12

High-throughput screening (HTS) is widely applied in many fields ranging from drug discovery to clinical diagnostics and toxicity assessment. Firefly luciferase is commonly used as a reporter to monitor the effect of chemical compounds on the activity of a specific target or pathway in HTS. However, the false positive rate of luciferase-based HTS is relatively high because many artifacts or promiscuous compounds that have direct interaction with the luciferase reporter enzyme are usually identified as active compounds (hits). Therefore, it is necessary to develop a rapid screening method to identify these compounds that can inhibit the luciferase activity directly. In this study, a virtual screening (VS) classification model called MIEC-GBDT (MIEC: Molecular Interaction Energy Components; GBDT: Gradient Boosting Decision Tree) was developed to distinguish luciferase inhibitors from non-inhibitors. The MIECs calculated by Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition were used to energetically characterize the binding pattern of each small molecule at the active site of luciferase, and then the GBDT algorithm was employed to construct the classifiers based on MIECs. The predictions to the test set show that the optimized MIEC-GBDT model outperformed molecular docking and MM/GBSA rescoring. The best MIEC-GBDT model based on the MIECs with the energy terms of ΔG ele , ΔG vdW , ΔG GB , and ΔG SA achieves the prediction accuracies of 87.2% and 90.3% for the inhibitors and non-inhibitors in the test sets, respectively. Moreover, the energetic analysis of the vital residues suggests that the energetic contributions of the vital residues to the binding of inhibitors are quite different from those to the binding of non-inhibitors. These results suggest that the MIEC-GBDT model is reliable and can be used as a powerful tool to identify potential interference compounds in luciferase-based HTS experiments.

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

PubMed

Xiao, Sheng; Yosef, Nir; Yang, Jianfei; Wang, Yonghui; Zhou, Ling; Zhu, Chen; Wu, Chuan; Baloglu, Erkan; Schmidt, Darby; Ramesh, Radha; Lobera, Mercedes; Sundrud, Mark S; Tsai, Pei-Yun; Xiang, Zhijun; Wang, Jinsong; Xu, Yan; Lin, Xichen; Kretschmer, Karsten; Rahl, Peter B; Young, Richard A; Zhong, Zhong; Hafler, David A; Regev, Aviv; Ghosh, Shomir; Marson, Alexander; Kuchroo, Vijay K

2014-04-17

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination ofmore » an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.« less

Role of glutamate-104 in generating a transition state analogue inhibitor at the active site of cytidine deaminase.

PubMed

Carlow, D C; Short, S A; Wolfenden, R

1996-01-23

The 19F-NMR resonance of 5-[19F]fluoropyrimidin-2-one ribonucleoside moves upfield when it is bound by wild-type cytidine deaminase from Escherichia coli, in agreement with UV and X-ray spectroscopic indications that this inhibitor is bound as the rate 3,4-hydrated species 5-fluoro-3,4-dihydrouridine, a transition state analogue inhibitor resembling an intermediate in direct water attack on 5-fluorocytidine. Comparison of pKa values of model compounds indicates that the equilibrium constant for 3,4-hydration of this inhibitor in free solution is 3.5 x 10(-4) M, so that the corrected dissociation constant of 5-fluoro-3,4-dihydrouridine from the wild-type enzyme is 3.9 x 10(-11) M. Very different behavior is observed for a mutant enzyme in which alanine replaces Glu-104 at the active site, and kcat has been reduced by a factor of 10(8). 5-[19F]Fluoropyrimidin-2-one ribonucleoside is strongly fluorescent, making it possible to observe that the mutant enzyme binds this inhibitor even more tightly (Kd = 4.4 x 10(-8) M) than does the native enzyme (Kd = 1.1 x 10(-7) M). 19F-NMR indicates, however, that the E104A mutant enzyme binds the inhibitor without modification, in a form that resembles the substrate in the ground state. These results are consistent with a major role for Glu-104, not only in stabilizing the ES++ complex in the transition state, but also in destabilizing the ES complex in the ground state.

The interaction of the Eco R1 restriction enzyme E.coli with nucleotides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollis, Donald F.

1979-11-01

The Eco R1 restriction enzyme can be shown to be inhibited by nucleotides which correspond to any part of its known site of phosphodiesterase activity. A series of di-, tetra-, and hexa-nucleotide fragments were synthesized and their effect on the activity of the enzyme upon superhelical Co1 E1 DNA studied. The inhibition caused by the individual mononucleotides were also studied. In general all the nucleotide fragments showed some form of interaction with the enzyme system. Tetranucleotides were stronger inhibitors than dinucleotides, which in turn were stronger inhibitors than the mononucleotides. Within each category of inhibitors, those containing the phosphodiester bondmore » which is acted upon by the enzyme were the strongest inhibitors. Only those fragments which were consistent with the enzymes site of activity showed competitive inhibition kinetics. Nucleotides which do not fit within the site of phosphodiesterase activity show non-competitive inhibition kinetics.« less

Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

PubMed

Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

2012-01-20

The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

Drosophila serpin 4 functions as a neuroserpin-like inhibitor of subtilisin-like proprotein convertases.

PubMed

Osterwalder, Thomas; Kuhnen, Angela; Leiserson, William M; Kim, You-Seung; Keshishian, Haig

2004-06-16

The proteolytic processing of neuropeptide precursors is believed to be regulated by serine proteinase inhibitors, or serpins. Here we describe the molecular cloning and functional expression of a novel member of the serpin family, Serine protease inhibitor 4 (Spn4), that we propose is involved in the regulation of peptide maturation in Drosophila. The Spn4 gene encodes at least two different serpin proteins, generated by alternate splicing of the last coding exon. The closest vertebrate homolog to Spn4 is neuroserpin. Like neuroserpin, one of the Spn4 proteins (Spn4.1) features a unique C-terminal extension, reminiscent of an endoplasmic reticulum (ER) retention signal; however, Spn4.1 and neuroserpin have divergent reactive site loops, with Spn4.1 showing a generic recognition site for furin/SPC1, the founding member of the intracellularly active family of subtilisin-like proprotein convertases (SPCs). In vitro, Spn4.1 forms SDS-stable complexes with the SPC furin and directly inhibits it. When Spn4.1 is overexpressed in specific peptidergic cells of Drosophila larvae, the animals exhibit a phenotype consistent with disrupted neuropeptide processing. This observation, together with the unique combination of an ER-retention signal, a target sequence for SPCs in the reactive site loop, and the in vitro inhibitory activity against furin, strongly suggests that Spn4.1 is an intracellular regulator of SPCs.

Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

PubMed

Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

2016-09-12

CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.

Anaplastic lymphoma kinase inhibitors in brain metastases from ALK+ non-small cell lung cancer: hitting the target even in the CNS.

PubMed

Klempner, Samuel J; Ou, Sai-Hong Ignatius

2015-06-01

The paradigm shift occurring in non-small cell lung cancer (NSCLC) is encapsulated by the management of patients harboring oncogenic anaplastic lymphoma kinase (ALK) rearrangements. The unprecedented improvements in patient outcomes resulting from ALK-directed therapy have led to the appreciation of patterns of disease progression. Early studies have suggested that some tyrosine kinase inhibitors (TKIs), including ALK TKIs, inefficiently penetrated the blood brain barrier. With the increasing appreciation of the CNS as a sanctuary site in ALK TKI-treated patients, there is increasing focus and importance on the prevention and control of CNS metastases in ALK-rearranged NSCLC. The spectrum of CNS activity is variable among the currently available ALK TKI therapies and further studies are ongoing. In the following review we discuss the ability of current and future ALK inhibitors (ALK-i) to control and prevent CNS progression in patients with ALK-rearranged NSCLC. The potential implications for TKI sequencing and important future research directions are discussed.

The herpesvirus proteases as targets for antiviral chemotherapy.

PubMed

Waxman, L; Darke, P L

2000-01-01

Viruses of the family Herpesviridae are responsible for a diverse set of human diseases. The available treatments are largely ineffective, with the exception of a few drugs for treatment of herpes simplex virus (HSV) infections. For several members of this DNA virus family, advances have been made recently in the biochemistry and structural biology of the essential viral protease, revealing common features that may be possible to exploit in the development of a new class of anti-herpesvirus agents. The herpesvirus proteases have been identified as belonging to a unique class of serine protease, with a Ser-His-His catalytic triad. A new, single domain protein fold has been determined by X-ray crystallography for the proteases of at least three different herpesviruses. Also unique for serine proteases, dimerization has been shown to be required for activity of the cytomegalovirus and HSV proteases. The dimerization requirement seriously impacts methods needed for productive, functional analysis and inhibitor discovery. The conserved functional and catalytic properties of the herpesvirus proteases lead to common considerations for this group of proteases in the early phases of inhibitor discovery. In general, classical serine protease inhibitors that react with active site residues do not readily inactivate the herpesvirus proteases. There has been progress however, with activated carbonyls that exploit the selective nucleophilicity of the active site serine. In addition, screening of chemical libraries has yielded novel structures as starting points for drug development. Recent crystal structures of the herpesvirus proteases now allow more direct interpretation of ligand structure-activity relationships. This review first describes basic functional aspects of herpesvirus protease biology and enzymology. Then we discuss inhibitors identified to date and the prospects for their future development.

Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

PubMed

Gay, Sean C; Shah, Manish B; Talakad, Jyothi C; Maekawa, Keiko; Roberts, Arthur G; Wilderman, P Ross; Sun, Ling; Yang, Jane Y; Huelga, Stephanie C; Hong, Wen-Xu; Zhang, Qinghai; Stout, C David; Halpert, James R

2010-04-01

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

PubMed

Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

2016-12-01

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be applied in both health care and agricultural industries, and could lead to new methods for breeding fungus-resistant transgenic crops and antifungal transgenic silkworm strains. Copyright © 2016 Elsevier Inc. All rights reserved.

Design of potent and selective human cathepsin K inhibitors that span the active site

PubMed Central

Thompson, Scott K.; Halbert, Stacie M.; Bossard, Mary J.; Tomaszek, Thaddeus A.; Levy, Mark A.; Zhao, Baoguang; Smith, Ward W.; Abdel-Meguid, Sherin S.; Janson, Cheryl A.; D’Alessio, Karla J.; McQueney, Michael S.; Amegadzie, Bernard Y.; Hanning, Charles R.; DesJarlais, Renee L.; Briand, Jacques; Sarkar, Susanta K.; Huddleston, Michael J.; Ijames, Carl F.; Carr, Steven A.; Garnes, Keith T.; Shu, Art; Heys, J. Richard; Bradbeer, Jeremy; Zembryki, Denise; Lee-Rykaczewski, Liz; James, Ian E.; Lark, Michael W.; Drake, Fred H.; Gowen, Maxine; Gleason, John G.; Veber, Daniel F.

1997-01-01

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention. PMID:9405598

Inhibitors for human glutaminyl cyclase by structure based design and bioisosteric replacement.

PubMed

Buchholz, Mirko; Hamann, Antje; Aust, Susanne; Brandt, Wolfgang; Böhme, Livia; Hoffmann, Torsten; Schilling, Stephan; Demuth, Hans-Ulrich; Heiser, Ulrich

2009-11-26

The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs

PubMed Central

Scott, Fiona L; Denault, Jean-Bernard; Riedl, Stefan J; Shin, Hwain; Renatus, Martin; Salvesen, Guy S

2005-01-01

The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP. PMID:15650747

Allosteric Inhibition of the Neuropeptidase Neurolysin*

PubMed Central

Hines, Christina S.; Ray, Kallol; Schmidt, Jack J.; Xiong, Fei; Feenstra, Rolf W.; Pras-Raves, Mia; de Moes, Jan Peter; Lange, Jos H. M.; Melikishvili, Manana; Fried, Michael G.; Mortenson, Paul; Charlton, Michael; Patel, Yogendra; Courtney, Stephen M.; Kruse, Chris G.; Rodgers, David W.

2014-01-01

Neuropeptidases specialize in the hydrolysis of the small bioactive peptides that play a variety of signaling roles in the nervous and endocrine systems. One neuropeptidase, neurolysin, helps control the levels of the dopaminergic circuit modulator neurotensin and is a member of a fold group that includes the antihypertensive target angiotensin converting enzyme. We report the discovery of a potent inhibitor that, unexpectedly, binds away from the enzyme catalytic site. The location of the bound inhibitor suggests it disrupts activity by preventing a hinge-like motion associated with substrate binding and catalysis. In support of this model, the inhibition kinetics are mixed, with both noncompetitive and competitive components, and fluorescence polarization shows directly that the inhibitor reverses a substrate-associated conformational change. This new type of inhibition may have widespread utility in targeting neuropeptidases. PMID:25378390

Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, Aβ aggregation, and β-secretase.

PubMed

Sun, Qi; Peng, Da-Yong; Yang, Sheng-Gang; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu

2014-09-01

Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer's disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine-coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (Ki=16.7nM) against human AChE and about 2-fold lower potency (Ki=16.1nM) against BChE than tacrine (Ki=35.7nM for AChE, Ki=8.7nM for BChE), respectively. In addition, some of the tacrine-coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine-coumarin hybrid is an interesting multifunctional lead for the AD drug discovery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates.

PubMed

Askin, Samuel P; Morin, Isabelle; Schaeffer, Patrick M

2011-08-15

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus-GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus-GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader. Copyright © 2011 Elsevier Inc. All rights reserved.

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

PubMed

Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung; Swanstrom, Ronald; Zeldovich, Konstantin B; Kurt-Yilmaz, Nese; Schiffer, Celia A

2017-11-14

HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

Miglustat (NB-DNJ) works as a chaperone for mutated acid beta-glucosidase in cells transfected with several Gaucher disease mutations.

PubMed

Alfonso, Pilar; Pampín, Sandra; Estrada, Jorge; Rodríguez-Rey, José Carlos; Giraldo, Pilar; Sancho, Javier; Pocoví, Miguel

2005-01-01

Gaucher disease (GD) is a disorder of glycosphinglipid metabolism caused by deficiency of lysosomal acid beta-glucosidase (GC), resulting in progressive deposition of glucosylceramide in macrophages. The glucose analogue, N-butyl-deoxynojirimycin (NB-DNJ, Miglustat), is an inhibitor of the ceramide-specific glucosyltransferase (CSG) which catalyzes the first step of glycosphingolipids biosynthesis and is currently approved for the oral treatment of type 1 GD. Using site-directed mutagenesis, we constructed plasmids containing wild-type and several mutations in glucocerebrosidase (GBA) gene. The plasmids were transfected into COS-7 cells and stable transfected cell lines were obtained by geneticin (G418) selection. Cells were cultured during 6 days with medium with or without 10 microM NB-DNJ. The addition of NB-DNJ to COS-7 cell medium leads to 1.3-, 2.1-, 2.3-, 3.6-, and 9.9-fold increase in the activity of S364R, wild-type, N370S, V15M, and M123T GC, respectively. However, no significant changes were observed in the activity of the L444P, L336P, and S465del mutated proteins, but a small decrease in the rare P266L variant was observed. These results suggest that NB-DNJ, in addition to the inhibitory effect on CSG, also works as a "chemical chaperone", increasing the activity of acid beta-glucosidase of wild-type and several GC mutated proteins, including the most frequent N370S mutation. The specific location of the Miglustat binding site in GC is unknown. Potential binding sites in the enzyme have been searched for using computational molecular docking. The searching strategy identified three potential GC binding sites for Miglustat, one being the substrate-binding site of the enzyme, which was the best-ranked site by AutoDock program. Therefore, it is possible that Miglustat exerts its chaperoning activity on acid beta-glucosidase by acting as an inhibitor bound at the active site. This increase on the activity of the acid beta-glucosidase would imply that Miglustat is not only a substrate reducer but also an inhibitor of the GC degradation, with very promising clinical implications for the treatment of GD patients.

Role of different types of potassium channels in the antidepressant-like effect of agmatine in the mouse forced swimming test.

PubMed

Budni, Josiane; Gadotti, Vinícius M; Kaster, Manuella P; Santos, Adair R S; Rodrigues, Ana Lúcia S

2007-12-01

The administration of agmatine elicits an antidepressant-like effect in the mouse forced swimming test by a mechanism dependent on the inhibition of the NMDA receptors and the L-arginine-nitric oxide (NO) pathway. Since it has been reported that the NO can activate different types of potassium (K(+)) channels in several tissues, the present study investigates the possibility of synergistic interactions between different types of K(+) channel inhibitors and agmatine in the forced swimming test. Treatment of mice by i.c.v. route with subeffective doses of tetraethylammonium (a non specific inhibitor of K(+) channels, 25 pg/site), glibenclamide (an ATP-sensitive K(+) channels inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K(+) channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K(+) channel inhibitor, 10 pg/site), augmented the effect of agmatine (0.001 mg/kg, i.p.) in the forced swimming test. Furthermore, the administration of agmatine and the K(+) channel inhibitors, alone or in combination, did not affect locomotion in the open-field test. Moreover, the reduction in the immobility time elicited by an active dose of agmatine (10 mg/kg, i.p.) in the forced swimming test was prevented by the pre-treatment of mice with the K(+) channel openers cromakalim (10 microg/site, i.c.v.) and minoxidil (10 microg/site, i.c.v.), without affecting locomotion. Together these data raise the possibility that the antidepressant-like effect of agmatine in the forced swimming test is related to its modulatory effects on neuronal excitability, via inhibition of K(+) channels.

The role of small molecule Kit protein-tyrosine kinase inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-04-25

The Kit proto-oncogene was found as a consequence of the discovery of the feline v-kit sarcoma oncogene. Stem cell factor (SCF) is the Kit ligand and it mediates Kit dimerization and activation. The Kit receptor contains an extracellular segment that is made up of five immunoglobulin-like domains (D1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of 77 amino acid residues, and a carboxyterminal tail. Activating somatic mutations in Kit have been documented in various neoplasms including gastrointestinal stromal tumors (GIST), mast cell overexpression (systemic mastocytosis), core-binding factor acute myeloid leukemias (AML), melanomas, and seminomas. In the case of gastrointestinal stromal tumors, most activating mutations occur in the juxtamembrane segment and these mutants are initially sensitive to imatinib. As with many targeted anticancer drugs, resistance to Kit antagonists occurs in about two years and is the result of secondary KIT mutations. An activation segment exon 17 D816V mutation is one of the more common resistance mutations in Kit and this mutant is resistant to imatinib and sorafenib. Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). Based upon the X-ray crystallographic structures, imatinib, sunitinib, and ponatinib are Type II Kit inhibitors. We used the Schrödinger induced fit docking protocol to model the interaction of midostaurin with Kit and the result indicates that it binds to the DFG-D in conformation of the receptor and is thus classified as type I inhibitor. This medication inhibits the notoriously resistant Kit D816V mutant and is approved for the treatment of systemic mastocytosis and is effective against tumors bearing the D816V activation/resistance mutation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Substituted Phosphonic Analogues of Phenylglycine as Inhibitors of Phenylalanine Ammonia Lyase from Potatoes.

PubMed

Wanat, Weronika; Talma, Michał; Hurek, Józef; Pawełczak, Małgorzata; Kafarski, Paweł

2018-06-08

A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato L-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of their binding performed using molecular modeling have shown that they might be bound either in active site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby the to the entrance channel for substrate into active site. Copyright © 2018. Published by Elsevier B.V.

Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

PubMed

van de Locht, A; Lamba, D; Bauer, M; Huber, R; Friedrich, T; Kröger, B; Höffken, W; Bode, W

1995-11-01

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937

Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster.

PubMed

Tatsumi, Tadashi; Awahara, Chiyuki; Naka, Hiromi; Aimoto, Saburo; Konno, Hiroyuki; Nosaka, Kazuto; Akaji, Kenichi

2010-04-01

Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor. Copyright 2010 Elsevier Ltd. All rights reserved.

Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

PubMed

Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

2015-10-15

Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

Understanding the molecular mechanism of aryl acylamidase activity of acetylcholinesterase - An in silico study.

PubMed

Chinnadurai, Raj Kumar; Saravanaraman, Ponne; Boopathy, Rathanam

2015-08-15

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Gaucher disease types 1, 2, and 3: differential mutations of the acid beta-glucosidase active site identified with conduritol B epoxide derivatives and sphingosine.

PubMed Central

Grabowski, G A; Dinur, T; Osiecki, K M; Kruse, J R; Legler, G; Gatt, S

1985-01-01

To elucidate the genetic heterogeneity in Gaucher disease, the residual beta-glucosidase in cultured fibroblasts from affected patients with each of the major phenotypes was investigated in vitro and/or in viable cells by inhibitor studies using the covalent catalytic site inhibitors, conduritol B epoxide or its bromo derivative, and the reversible cationic inhibitor, sphingosine. These studies delineated three distinct groups (designated A, B, and C) of residual activities with characteristic responses to these inhibitors. Group A residual enzymes had normal I50 values (i.e., the concentration of inhibitor that results in 50% inhibition) for the inhibitors and normal or nearly normal t1/2 values for conduritol B epoxide. All neuronopathic (types 2 and 3) and most non-Jewish nonneuronopathic (type 1) patients had group A residual activities and, thus, could not be distinguished by these inhibitor studies. Group B residual enzymes had about four- to fivefold increased I50 values for the inhibitors and similarly increased t1/2 values for conduritol B epoxide. All Ashkenazi Jewish type 1 and only two non-Jewish type 1 patients had group B residual activities. The differences in I50 values between groups A and B also were confirmed by determining the uninhibited enzyme activity after culturing the cells in the presence of bromo-conduritol B epoxide. Group C residual activity had intermediate I50 values for the inhibitors and represented a single Afrikaner type 1 patient: this patient was a genetic compound for the group A (type 2) and group B (type 1) mutations. These inhibition studies indicated that: Gaucher disease type 1 is biochemically heterogeneous, neuronopathic and non-Jewish nonneuronopathic phenotypes cannot be reliably distinguished by these inhibitor studies, and the Ashkenazi Jewish form of Gaucher disease type 1 results from a unique mutation in a specific active site domain of acid beta-glucosidase that leads to a defective enzyme with a decreased Vmax. PMID:4003396

A structure-based virtual screening approach toward the discovery of histone deacetylase inhibitors: identification of promising zinc-chelating groups.

PubMed

Park, Hwangseo; Kim, Sukyoung; Kim, Yong Eun; Lim, Soo-Jeong

2010-04-06

The inhibitors of histone deacetylases (HDACs) have drawn a great deal of attention due to their promising potential as small-molecule therapeutics for the treatment of cancer. By means of virtual screening with docking simulations under consideration of the effects of ligand solvation, we were able to identify six novel HDAC inhibitors with IC(50) values ranging from 1 to 100 muM. These newly identified inhibitors are structurally diverse and have various chelating groups for the active site zinc ion, including N-[1,3,4]thiadiazol-2-yl sulfonamide, N-thiazol-2-yl sulfonamide, and hydroxamic acid moieties. The former two groups are included in many drugs in current clinical use and have not yet been reported as HDAC inhibitors. Therefore, they can be considered as new inhibitor scaffolds for the development of anticancer drugs by structure-activity relationship studies to improve the inhibitory activities against HDACs. Interactions with the HDAC1 active site residues responsible for stabilizing these new inhibitors are addressed in detail.

A novel Death Defying Domain in Met entraps the active site of Caspase-3 and blocks apoptosis in hepatocytes

PubMed Central

Ma, Jihong; Zou, Chunbin; Guo, Lida; Seneviratne, Danushka S.; Tan, Xinping; Kwon, Yong-Kook; An, Jiyan; Bowser, Robert; DeFrances, Marie C.; Zarnegar, Reza

2013-01-01

Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF) is known to function as a potent anti-apoptotic mediator in normal and neoplastic cells. Herein we report that intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of Caspase-3 cleavage sites, which bait, trap and disable the active site of Caspase-3, thereby blocking the execution of apoptosis. We call this Caspase-3 cleavage motif the ‘Death Defying Domain’ (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by Caspase-3, the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of Caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of Caspase-3 activity. By gain and loss-of-function studies using restoration of DDD expression in DDD deficient hepatocytic cells, we found that both Caspase-3 sites in DDD are necessary for inhibition of Caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met’s enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of Caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. Conclusion Our findings show that Met can directly inhibit Caspase-3 via a novel mechanism and promote hepato-cyte survival. Results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play. PMID:24122846

Crystal Structures of Trypanosoma brucei Sterol 14[alpha]-Demethylase and Implications for Selective Treatment of Human Infections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lepesheva, Galina I.; Park, Hee-Won; Hargrove, Tatiana Y.

2010-01-25

Sterol 14{alpha}-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 {angstrom} resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that ofmore » the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.« less

Inhibition of phospholipase A1, lipase and galactolipase activities of pancreatic lipase-related protein 2 by methyl arachidonyl fluorophosphonate (MAFP).

PubMed

Amara, Sawsan; Delorme, Vincent; Record, Michel; Carrière, Frédéric

2012-11-01

Methyl arachidonyl fluorophosphonate (MAFP) is a known inhibitor of cytosolic phospholipase A2 and some other serine enzymes. MAFP was found here to be an irreversible inhibitor of human pancreatic lipase-related protein 2 (HPLRP2), an enzyme displaying lipase, phospholipase A1 and galactolipase activities. In the presence of MAFP, mass spectrometry analysis of HPLRP2 revealed a mass increase of 351Da, suggesting a covalent binding of MAFP to the active site serine residue. When HPLRP2 was pre-incubated with MAFP before measuring residual activity, a direct inhibition of HPLRP2 occurred, confirming that HPLRP2 has an active site freely accessible to solvent and differs from most lipases in solution. HPLRP2 activities on tributyrin (TC4), phosphatidylcholine (PC) and monogalactosyl dioctanoylglycerol (C8-MGDG) were equally inhibited under these conditions. Bile salts were not required to trigger the inhibition, but they significantly increased the rate of HPLRP2 inhibition, probably because of MAFP micellar solubilization. Since HPLRP2 is active on various substrates that self-organize differently in the presence of water, HPLRP2 inhibition by MAFP was tested in the presence of these substrates after adding MAFP in the course of the lipolysis reaction. In this case, the rates of inhibition of lipase, phospholipase A1 and galactolipase activities were not equivalent (triglycerides>PC>MGDG), suggesting different enzyme/inhibitor partitioning between the aqueous phase and lipid aggregates. The inhibition by MAFP of a well identified phospholipase A1 (HPLRP2), present in pancreatic juice and also in human monocytes, indicates that MAFP cannot be used for discriminating phospholipase A2 from A1 activities at the cellular level. Copyright © 2012 Elsevier B.V. All rights reserved.

Sepiapterin Reductase Mediates Chemical Redox Cycling in Lung Epithelial Cells*

PubMed Central

Yang, Shaojun; Jan, Yi-Hua; Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

2013-01-01

In the lung, chemical redox cycling generates highly toxic reactive oxygen species that can cause alveolar inflammation and damage to the epithelium, as well as fibrosis. In this study, we identified a cytosolic NADPH-dependent redox cycling activity in mouse lung epithelial cells as sepiapterin reductase (SPR), an enzyme important for the biosynthesis of tetrahydrobiopterin. Human SPR was cloned and characterized. In addition to reducing sepiapterin, SPR mediated chemical redox cycling of bipyridinium herbicides and various quinones; this activity was greatest for 1,2-naphthoquinone followed by 9,10-phenanthrenequinone, 1,4-naphthoquinone, menadione, and 2,3-dimethyl-1,4-naphthoquinone. Whereas redox cycling chemicals inhibited sepiapterin reduction, sepiapterin had no effect on redox cycling. Additionally, inhibitors such as dicoumarol, N-acetylserotonin, and indomethacin blocked sepiapterin reduction, with no effect on redox cycling. Non-redox cycling quinones, including benzoquinone and phenylquinone, were competitive inhibitors of sepiapterin reduction but noncompetitive redox cycling inhibitors. Site-directed mutagenesis of the SPR C-terminal substrate-binding site (D257H) completely inhibited sepiapterin reduction but had minimal effects on redox cycling. These data indicate that SPR-mediated reduction of sepiapterin and redox cycling occur by distinct mechanisms. The identification of SPR as a key enzyme mediating chemical redox cycling suggests that it may be important in generating cytotoxic reactive oxygen species in the lung. This activity, together with inhibition of sepiapterin reduction by redox-active chemicals and consequent deficiencies in tetrahydrobiopterin, may contribute to tissue injury. PMID:23640889

Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

NASA Astrophysics Data System (ADS)

Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

Discovery and antiplatelet activity of a selective PI3Kβ inhibitor (MIPS-9922).

PubMed

Zheng, Zhaohua; Pinson, Jo-Anne; Mountford, Simon J; Orive, Stephanie; Schoenwaelder, Simone M; Shackleford, David; Powell, Andrew; Nelson, Erin M; Hamilton, Justin R; Jackson, Shaun P; Jennings, Ian G; Thompson, Philip E

2016-10-21

A series of amino-substituted triazines were developed and examined for PI3Kβ inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kβ selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kβ selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbβ3 activation and αIIbβ3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K.

PubMed

Law, Simon; Panwar, Preety; Li, Jody; Aguda, Adeleke H; Jamroz, Andrew; Guido, Rafael V C; Brömme, Dieter

2017-01-01

Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC50 < 20 μM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 μM. Two compounds exhibited IC50 values below 5 μM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects.

A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K

PubMed Central

Law, Simon; Panwar, Preety; Li, Jody; Aguda, Adeleke H.; Jamroz, Andrew; Guido, Rafael V. C.

2017-01-01

Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC50 < 20 μM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 μM. Two compounds exhibited IC50 values below 5 μM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC50-value of approximately 300 nM and no cell toxicity effects. PMID:29088253

A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A.

PubMed

Harrell, William A; Vieira, Rebecca C; Ensel, Susan M; Montgomery, Vicki; Guernieri, Rebecca; Eccard, Vanessa S; Campbell, Yvette; Roxas-Duncan, Virginia; Cardellina, John H; Webb, Robert P; Smith, Leonard A

2017-02-01

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC 50 values ⩽1μM and 11 effective at ⩽2μM in an ex vivo assay. Published by Elsevier Ltd.

Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Xiaoyi; Gujjar, Ramesh; El Mazouni, Farah

Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes andmore » to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.« less

Inhibition of the archaeal beta-class (Cab) and gamma-class (Cam) carbonic anhydrases.

PubMed

Zimmerman, Sabrina A; Ferry, James G; Supuran, Claudiu T

2007-01-01

Five independently evolved classes (alpha-, beta-, gamma-, delta-, zeta-) of carbonic anhydrases facilitate the reversible hydration of carbon dioxide to bicarbonate of which the alpha-class is the most extensively studied. Detailed inhibition studies of the alpha-class with the two main classes of inhibitors, sulfonamides and metal-complexing anions, revealed many inhibitors that are used as therapeutic agents to prevent and treat many diseases. Recent inhibitor studies of the archaeal beta-class (Cab) and the gamma-class (Cam) carbonic anhydrases show differences in inhibition response to sulfonamides and metal-complexing anions, when compared to the alpha-class carbonic anhydrases. In addition, inhibition between Cab and Cam differ. These inhibition patterns are consistent with the idea that although, alpha-, beta-, and gamma-class carbonic anhydrases participate in the same two-step isomechanism, diverse active site architecture among these classes predicts variations on the catalytic mechanism. These inhibitor studies of the archaeal beta- and gamma-class carbonic anhydrases give insight to new applications of current day carbonic anhydrase inhibitors, as well as direct research to develop new compounds that may be specific inhibitors of prokaryotic carbonic anhydrases.

Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides

NASA Astrophysics Data System (ADS)

Chen, Shiyu; Gopalakrishnan, Ranganath; Schaer, Tifany; Marger, Fabrice; Hovius, Ruud; Bertrand, Daniel; Pojer, Florence; Heinis, Christian

2014-11-01

The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with different biological activities. Here, we describe the development of a dithiol amino acid (Dtaa) that can form two disulfide bridges at a single amino acid site. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor that contain disulfide constraints enhanced their inhibitory activities 40- and 7.6-fold, respectively. X-ray crystallographic and NMR structure analysis show that the peptide ligands containing Dtaas have retained their native tertiary structures. We furthermore show that replacement of two cysteines by Dtaas can avoid the formation of disulfide bond isomers. With these properties, Dtaas are likely to have broad application in the rational design or directed evolution of peptides and proteins with high activity and stability.

Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

PubMed

Duprez, Wilko; Bachu, Prabhakar; Stoermer, Martin J; Tay, Stephanie; McMahon, Róisín M; Fairlie, David P; Martin, Jennifer L

2015-01-01

Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

PubMed Central

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.

The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolicmore » port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.« less

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

PubMed

Hylsová, Michaela; Carbain, Benoit; Fanfrlík, Jindřich; Musilová, Lenka; Haldar, Susanta; Köprülüoğlu, Cemal; Ajani, Haresh; Brahmkshatriya, Pathik S; Jorda, Radek; Kryštof, Vladimír; Hobza, Pavel; Echalier, Aude; Paruch, Kamil; Lepšík, Martin

2017-01-27

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R 2  = 0.49). However, the addition of the active-site waters resulted in significant improvement (R 2  = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Cholinesterases: structure of the active site and mechanism of the effect of cholinergic receptor blockers on the rate of interaction with ligands

NASA Astrophysics Data System (ADS)

Antokhin, A. M.; Gainullina, E. T.; Taranchenko, V. F.; Ryzhikov, S. B.; Yavaeva, D. K.

2010-10-01

Modern views on the structure of cholinesterase active sites and the mechanism of their interaction with organophosphorus inhibitors are considered. The attention is focused on the mechanism of the effect of cholinergic receptor blockers, acetylcholine antagonists, on the rate of interaction of acetylcholine esterase with organophosphorus inhibitors.

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

PubMed

Frobert, Y; Créminon, C; Cousin, X; Rémy, M H; Chatel, J M; Bon, S; Bon, C; Grassi, J

1997-05-23

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 > 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh,A.; Sridhar, P.; Leshchenko, S.

2006-01-01

Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps.more » A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.« less

Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolutionmore » X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.« less

Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein*

PubMed Central

Sawano, Yoriko; Hatano, Ken-ichi; Miyakawa, Takuya; Tanokura, Masaru

2008-01-01

Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed φ dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition. PMID:18948264

Potent selective nonpeptidic inhibitors of human lung tryptase

PubMed Central

Burgess, Laurence E.; Newhouse, Bradley J.; Ibrahim, Prabha; Rizzi, James; Kashem, Mohammed A.; Hartman, Ann; Brandhuber, Barbara J.; Wright, Clifford D.; Thomson, David S.; Vigers, Guy P. A.; Koch, Kevin

1999-01-01

Human lung tryptase, a homotetrameric serine protease unique to mast cell secretory granules, has been implicated in the pathogenesis of asthma. A hypothesis that tethered symmetrical inhibitors might bridge two adjacent active sites was explored via a rationally designed series of bisbenzamidines. These compounds demonstrated a remarkable distanced-defined structure–activity relationship against human tryptase with one series possessing subnanomolar potencies. Additional evidence supporting the concept of active-site bridging is also presented. PMID:10411878

Antidepressant-like effect of folic acid: Involvement of NMDA receptors and L-arginine-nitric oxide-cyclic guanosine monophosphate pathway.

PubMed

Brocardo, Patrícia de Souza; Budni, Josiane; Lobato, Kelly Ribas; Kaster, Manuella Pinto; Rodrigues, Ana Lúcia S

2008-11-19

Antidepressant-like activity of folic acid in forced swimming test and in the tail suspension test was demonstrated previously by our group. In this study we investigated the involvement of N-methyl-d-aspartate (NMDA) receptors and l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate pathway in its antidepressant-like effect in the forced swimming test in mice. The antidepressant-like effect of folic acid (10 nmol/site, i.c.v.) was prevented by the pretreatment of mice with NMDA (0.1 pmol/site, i.c.v.), l-arginine (750 mg/kg, i.p., substrate for nitric oxide synthase), S-nitroso-N-acetyl-penicillamine (SNAP, 25 microg/site, i.c.v, a NO donor) or sildenafil (5 mg/kg, i.p., phosphodiesterase 5 inhibitor). The administration of 7-nitroindazole (25 and 50 mg/kg, i.p., a specific neuronal nitric oxide synthase (nNOS) inhibitor) or methylene blue (20 mg/kg, i.p., direct inhibitor of both nitric oxide synthase and soluble guanylate cyclase) in combination with a sub-effective dose of folic acid (1 nmol/site, i.c.v.) reduced the immobility time in the FST as compared with either drug alone. Together the results suggest that the antidepressant-like effect of folic acid in the forced swimming test is dependent on an inhibition of either NMDA receptors or NO and cGMP synthesis.

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

PubMed Central

Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.

2011-01-01

Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377

Inhibition of thrombin by functionalized C60 nanoparticles revealed via in vitro assays and in silico studies.

PubMed

Liu, Yanyan; Fu, Jianjie; Pan, Wenxiao; Xue, Qiao; Liu, Xian; Zhang, Aiqian

2018-01-01

The studies on the human toxicity of nanoparticles (NPs) are far behind the rapid development of engineered functionalized NPs. Fullerene has been widely used as drug carrier skeleton due to its reported low risk. However, different from other kinds of NPs, fullerene-based NPs (C 60 NPs) have been found to have an anticoagulation effect, although the potential target is still unknown. In the study, both experimental and computational methods were adopted to gain mechanistic insight into the modulation of thrombin activity by nine kinds of C 60 NPs with diverse surface chemistry properties. In vitro enzyme activity assays showed that all tested surface-modified C 60 NPs exhibited thrombin inhibition ability. Kinetic studies coupled with competitive testing using 3 known inhibitors indicated that six of the C 60 NPs, of greater hydrophobicity and hydrogen bond (HB) donor acidity or acceptor basicity, acted as competitive inhibitors of thrombin by directly interacting with the active site of thrombin. A simple quantitative nanostructure-activity relationship model relating the surface substituent properties to the inhibition potential was then established for the six competitive inhibitors. Molecular docking analysis revealed that the intermolecular HB interactions were important for the specific binding of C 60 NPs to the active site canyon, while the additional stability provided by the surface groups through van der Waals interaction also play a key role in the thrombin binding affinity of the NPs. Our results suggest that thrombin is a possible target of the surface-functionalized C 60 NPs relevant to their anticoagulation effect. Copyright © 2017. Published by Elsevier B.V.

DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

PubMed

Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

2010-06-18

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

PubMed

Law, Ruby H P; Wu, Guojie; Leung, Eleanor W W; Hidaka, Koushi; Quek, Adam J; Caradoc-Davies, Tom T; Jeevarajah, Devadharshini; Conroy, Paul J; Kirby, Nigel M; Norton, Raymond S; Tsuda, Yuko; Whisstock, James C

2017-05-09

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO ( trans -4-aminomethylcyclohexanecarbonyl-l-tyrosine- n -octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.

X-ray crystal structure of plasmin with tranexamic acid–derived active site inhibitors

PubMed Central

Wu, Guojie; Leung, Eleanor W. W.; Hidaka, Koushi; Quek, Adam J.; Caradoc-Davies, Tom T.; Jeevarajah, Devadharshini; Kirby, Nigel M.; Norton, Raymond S.; Tsuda, Yuko; Whisstock, James C.

2017-01-01

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders. PMID:29296720

Discovery of a Novel Series of Inhibitors of Lymphoid Tyrosine Phosphatase with Activity in Human T Cells†

PubMed Central

Stanford, Stephanie M.; Krishnamurthy, Divya; Falk, Matthew D.; Messina, Rossella; Debnath, Bikash; Li, Sheng; Liu, Tong; Kazemi, Roza; Dahl, Russell; He, Yantao; Yu, Xiao; Chan, Andrew C.; Zhang, Zhong-Yin; Barrios, Amy M.; Woods, Virgil L.; Neamati, Nouri; Bottini, Nunzio

2011-01-01

The lymphoid tyrosine phosphatase LYP, encoded by the PTPN22 gene, is a critical regulator of signaling in T cells and recently emerged as a candidate target for therapy of autoimmune diseases. Here, by library screening, we identified a series of noncompetitive inhibitors of LYP that showed activity in primary T cells. Kinetic analysis confirmed that binding of the compounds to the phosphatase is nonmutually exclusive with respect to a known bidentate competitive inhibitor. The mechanism of action of the lead inhibitor compound 4e was studied by a combination of hydrogen/deuterium-exchange mass spectrometry and molecular modeling. The results suggest that the inhibitor interacts critically with a hydrophobic patch located outside the active site of the phosphatase. Targeting of secondary allosteric sites is viewed as a promising yet unexplored approach to develop pharmacological inhibitors of protein tyrosine phosphatases. Our novel scaffold could be a starting point to attempt development of “nonactive site” anti-LYP pharmacological agents. PMID:21341673

Molecular design and structure--activity relationships leading to the potent, selective, and orally active thrombin active site inhibitor BMS-189664.

PubMed

Das, Jagabandhu; Kimball, S David; Hall, Steven E; Han, Wen Ching; Iwanowicz, Edwin; Lin, James; Moquin, Robert V; Reid, Joyce A; Sack, John S; Malley, Mary F; Chang, Chiehying Y; Chong, Saeho; Wang-Iverson, David B; Roberts, Daniel G M; Seiler, Steven M; Schumacher, William A; Ogletree, Martin L

2002-01-07

A series of structurally novel small molecule inhibitors of human alpha-thrombin was prepared to elucidate their structure-activity relationships (SARs), selectivity and activity in vivo. BMS-189664 (3) is identified as a potent, selective, and orally active reversible inhibitor of human alpha-thrombin which is efficacious in vivo in a mouse lethality model, and at inhibiting both arterial and venous thrombosis in cynomolgus monkey models.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

PubMed

Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

2016-03-28

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

PubMed Central

Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

2016-01-01

6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity

PubMed Central

TRIPATHI, ASHUTOSH; DURRANT, DAVID; LEE, RAY M.; BARUCHELLO, RICCARDO; ROMAGNOLI, ROMEO; SIMONI, DANIELE; KELLOGG, GLEN E.

2009-01-01

The crucial role of the microtubule in the cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the αβ-tubulin colchicine site. Computational docking along with HINT score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 μM) such that calculated and measured free energies of binding correlate with an r2 of 0.89 (standard error ± 0.85 kcal mol−1). This correlation suggests that the activity of unknown compounds may be predicted. PMID:19912057

Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.

PubMed

Adler, Marc; Bryant, Judi; Buckman, Brad; Islam, Imadul; Larsen, Brent; Finster, Silke; Kent, Lorraine; May, Karen; Mohan, Raju; Yuan, Shendong; Whitlow, Marc

2005-07-05

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.

Human γ-Glutamyl Transpeptidase 1: STRUCTURES OF THE FREE ENZYME, INHIBITOR-BOUND TETRAHEDRAL TRANSITION STATES, AND GLUTAMATE-BOUND ENZYME REVEAL NOVEL MOVEMENT WITHIN THE ACTIVE SITE DURING CATALYSIS.

PubMed

Terzyan, Simon S; Burgett, Anthony W G; Heroux, Annie; Smith, Clyde A; Mooers, Blaine H M; Hanigan, Marie H

2015-07-10

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Multi-phasic bi-directional chemotactic responses of the growth cone

PubMed Central

Naoki, Honda; Nishiyama, Makoto; Togashi, Kazunobu; Igarashi, Yasunobu; Hong, Kyonsoo; Ishii, Shin

2016-01-01

The nerve growth cone is bi-directionally attracted and repelled by the same cue molecules depending on the situations, while other non-neural chemotactic cells usually show uni-directional attraction or repulsion toward their specific cue molecules. However, how the growth cone differs from other non-neural cells remains unclear. Toward this question, we developed a theory for describing chemotactic response based on a mathematical model of intracellular signaling of activator and inhibitor. Our theory was first able to clarify the conditions of attraction and repulsion, which are determined by balance between activator and inhibitor, and the conditions of uni- and bi-directional responses, which are determined by dose-response profiles of activator and inhibitor to the guidance cue. With biologically realistic sigmoidal dose-responses, our model predicted tri-phasic turning response depending on intracellular Ca2+ level, which was then experimentally confirmed by growth cone turning assays and Ca2+ imaging. Furthermore, we took a reverse-engineering analysis to identify balanced regulation between CaMKII (activator) and PP1 (inhibitor) and then the model performance was validated by reproducing turning assays with inhibitions of CaMKII and PP1. Thus, our study implies that the balance between activator and inhibitor underlies the multi-phasic bi-directional turning response of the growth cone. PMID:27808115

Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sanches, Mario; Duffy, Nicole M.; Talukdar, Manisha

2014-10-24

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy–aldehyde moieties, termed hydroxy–aryl–aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892.more » Structure–activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.« less

Structural investigation of protein kinase C inhibitors

NASA Technical Reports Server (NTRS)

Barak, D.; Shibata, M.; Rein, R.

1991-01-01

The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

AKT Kinase Activity Is Required for Lithium to Modulate Mood-Related Behaviors in Mice

PubMed Central

Pan, Jen Q; Lewis, Michael C; Ketterman, Josh K; Clore, Elizabeth L; Riley, Misha; Richards, Keenan R; Berry-Scott, Erin; Liu, Xiulin; Wagner, Florence F; Holson, Edward B; Neve, Rachael L; Biechele, Travis L; Moon, Randall T; Scolnick, Edward M; Petryshen, Tracey L; Haggarty, Stephen J

2011-01-01

Bipolar disorder (BP) is a debilitating psychiatric disorder, affecting ∼2% of the worldwide population, for which the etiological basis, pathogenesis, and neurocircuitry remain poorly understood. Individuals with BP suffer from recurrent episodes of mania and depression, which are commonly treated with the mood stabilizer lithium. However, nearly half of BP patients do not respond adequately to lithium therapy and the clinically relevant mechanisms of lithium for mood stabilization remain elusive. Here, we modeled lithium responsiveness using cellular assays of glycogen synthase kinase 3 (GSK-3) signaling and mood-related behavioral assays in inbred strains of mice that differ in their response to lithium. We found that activating AKT through phosphosrylation of a key regulatory site (Thr308) was associated with lithium response—activation of signaling pathways downstream of GSK-3 in cells and attenuation of mood-related behaviors in mice—and this response was attenuated by selective and direct inhibition of AKT kinase activity. Conversely, the expression of constitutively active AKT1 in both the cellular and behavioral assays conferred lithium sensitivity. In contrast, selective and direct GSK-3 inhibition by the ATP-competitive inhibitor CHIR99021 bypassed the requirement for AKT activation and modulated behavior in both lithium-responsive and non-responsive mouse strains. These results distinguish the mechanism of action of lithium from direct GSK-3 inhibition both in vivo and in vitro, and highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment. PMID:21389981

Interaction of Triton X-100 with acyl pocket of butyrylcholinesterase: effect on esterase activity and inhibitor sensitivity of the enzyme.

PubMed

Jaganathan, L; Boopathy, R

1998-06-01

The effect of non-ionic detergents like Triton X-100, Lubrol PX, Brij 35 and Tween 80 on the esterase activity and inhibitor sensitivity of human serum butyrylcholinesterase (BuChE) were studied. The results showed that though BuChE is not a detergent dependent enzyme, the esterase activity and inhibitor sensitivity of it can be modulated by the presence of detergents. All the detergents caused a marginal activation of the esterase activity. The presence of Lubrol PX, Brij 35 or Tween 80 did not affect the 50% molar inhibition concentration (IC50) of the inhibitors tested. But in the presence of Triton X-100 the IC50 values were increased for neostigmine, eserine and tetraisopropylpyrophosphoramide (acylation site interacting inhibitors), whereas for inhibitors like ethopropazine, imipramine and procainamide (choline binding pocket specific inhibitors) the IC50 values were unaltered. In addition, in the presence of Triton X-100 the bimolecular reaction constant for phosphorylation reaction (ki) of BuChE for the acyl pocket specific tetraisopropylpyrophosphoramide was reduced. Triton X-100 partially protected BuChE against this tetraisopropylpyrophosphoramide inactivation. These results indicate that Triton X-100 by interacting with the acyl pocket hydrophobic region is able to activate the esterase activity of BuChE. Further it reduces the capacity of the enzyme to react with inhibitors that inactivate it by interacting with the serine residue of the acylation site.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

PubMed

Bello, Angelica M; Poduch, Ewa; Fujihashi, Masahiro; Amani, Merhnaz; Li, Yan; Crandall, Ian; Hui, Raymond; Lee, Ping I; Kain, Kevin C; Pai, Emil F; Kotra, Lakshmi P

2007-03-08

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

SAR and characterization of non-substrate isoindoline urea inhibitors of nicotinamide phosphoribosyltransferase (NAMPT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curtin, Michael L.; Heyman, H. Robin; Clark, Richard F.

Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.

Prostate Cancer Cell–Stromal Cell Cross-Talk via FGFR1 Mediates Antitumor Activity of Dovitinib in Bone Metastases

PubMed Central

Wan, Xinhai; Corn, Paul G.; Yang, Jun; Palanisamy, Nallasivam; Starbuck, Michael W.; Efstathiou, Eleni; Li-Ning Tapia, Elsa M.; Zurita, Amado J.; Aparicio, Ana; Ravoori, Murali K.; Vazquez, Elba S.; Robinson, Dan R.; Wu, Yi-Mi; Cao, Xuhong; Iyer, Matthew K.; McKeehan, Wallace; Kundra, Vikas; Wang, Fen; Troncoso, Patricia; Chinnaiyan, Arul M.; Logothetis, Christopher J.; Navone, Nora M.

2015-01-01

Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell–bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors. PMID:25186177

Prostate cancer cell-stromal cell crosstalk via FGFR1 mediates antitumor activity of dovitinib in bone metastases.

PubMed

Wan, Xinhai; Corn, Paul G; Yang, Jun; Palanisamy, Nallasivam; Starbuck, Michael W; Efstathiou, Eleni; Li Ning Tapia, Elsa M; Tapia, Elsa M Li-Ning; Zurita, Amado J; Aparicio, Ana; Ravoori, Murali K; Vazquez, Elba S; Robinson, Dan R; Wu, Yi-Mi; Cao, Xuhong; Iyer, Matthew K; McKeehan, Wallace; Kundra, Vikas; Wang, Fen; Troncoso, Patricia; Chinnaiyan, Arul M; Logothetis, Christopher J; Navone, Nora M

2014-09-03

Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell-bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in serum prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors. Copyright © 2014, American Association for the Advancement of Science.

Inhibition of delta-secretase improves cognitive functions in mouse models of Alzheimer's disease

PubMed Central

Zhang, Zhentao; Obianyo, Obiamaka; Dall, Elfriede; Du, Yuhong; Fu, Haian; Liu, Xia; Kang, Seong Su; Song, Mingke; Yu, Shan-Ping; Cabrele, Chiara; Schubert, Mario; Li, Xiaoguang; Wang, Jian-Zhi; Brandstetter, Hans; Ye, Keqiang

2017-01-01

δ-secretase, also known as asparagine endopeptidase (AEP) or legumain, is a lysosomal cysteine protease that cleaves both amyloid precursor protein (APP) and tau, mediating the amyloid-β and tau pathology in Alzheimer's disease (AD). Here we report the therapeutic effect of an orally bioactive and brain permeable δ-secretase inhibitor in mouse models of AD. We performed a high-throughput screen and identified a non-toxic and selective δ-secretase inhibitor, termed compound 11, that specifically blocks δ-secretase but not other related cysteine proteases. Co-crystal structure analysis revealed a dual active site-directed and allosteric inhibition mode of this compound class. Chronic treatment of tau P301S and 5XFAD transgenic mice with this inhibitor reduces tau and APP cleavage, ameliorates synapse loss and augments long-term potentiation, resulting in protection of memory. Therefore, these findings demonstrate that this δ-secretase inhibitor may be an effective clinical therapeutic agent towards AD. PMID:28345579

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group.

PubMed

Lobera, Mercedes; Madauss, Kevin P; Pohlhaus, Denise T; Wright, Quentin G; Trocha, Mark; Schmidt, Darby R; Baloglu, Erkan; Trump, Ryan P; Head, Martha S; Hofmann, Glenn A; Murray-Thompson, Monique; Schwartz, Benjamin; Chakravorty, Subhas; Wu, Zining; Mander, Palwinder K; Kruidenier, Laurens; Reid, Robert A; Burkhart, William; Turunen, Brandon J; Rong, James X; Wagner, Craig; Moyer, Mary B; Wells, Carrow; Hong, Xuan; Moore, John T; Williams, Jon D; Soler, Dulce; Ghosh, Shomir; Nolan, Michael A

2013-05-01

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Dual-tail approach to discovery of novel carbonic anhydrase IX inhibitors by simultaneously matching the hydrophobic and hydrophilic halves of the active site.

PubMed

Hou, Zhuang; Lin, Bin; Bao, Yu; Yan, Hai-Ning; Zhang, Miao; Chang, Xiao-Wei; Zhang, Xin-Xin; Wang, Zi-Jie; Wei, Gao-Fei; Cheng, Mao-Sheng; Liu, Yang; Guo, Chun

2017-05-26

Dual-tail approach was employed to design novel Carbonic Anhydrase (CA) IX inhibitors by simultaneously matching the hydrophobic and hydrophilic halves of the active site, which also contains a zinc ion as part of the catalytic center. The classic sulfanilamide moiety was used as the zinc binding group. An amino glucosamine fragment was chosen as the hydrophilic part and a cinnamamide fragment as the hydrophobic part in order to draw favorable interactions with the corresponding halves of the active site. In comparison with sulfanilamide which is largely devoid of the hydrophilic and hydrophobic interactions with the two halves of the active site, the compounds so designed and synthesized in this study showed 1000-fold improvement in binding affinity. Most of the compounds inhibited the CA effectively with IC 50 values in the range of 7-152 nM. Compound 14e (IC 50 : 7 nM) was more effective than the reference drug acetazolamide (IC 50 : 30 nM). The results proved that the dual-tail approach to simultaneously matching the hydrophobic and hydrophilic halves of the active site by linking hydrophobic and hydrophilic fragments was useful for designing novel CA inhibitors. The effectiveness of those compounds was elucidated by both the experimental data and molecular docking simulations. This work laid a solid foundation for further development of novel CA IX inhibitors for cancer treatment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

PubMed

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Promiscuity and selectivity of small-molecule inhibitors across TAM receptor tyrosine kinases in pediatric leukemia.

PubMed

Liu, Mao-Hua; Chen, Shi-Bing; Yu, Juan; Liu, Cheng-Jun; Zhang, Xiao-Jing

2017-08-01

The TAM receptor tyrosine kinase family member Mer has been recognized as an attractive therapeutic target for pediatric leukemia. Beside Mer the family contains other two kinases, namely, Tyro3 and Axl, which are highly homologues with Mer and thus most existing small-molecule inhibitors show moderate or high promiscuity across the three kinases. Here, the structural basis and energetic property of selective binding of small-molecule inhibitors to the three kinases were investigated at molecular level. It is found that the selectivity is primarily determined by the size, shape and configuration of kinase's ATP-binding site; the Mer and Axl possess a small, closed active pocket as compared to the bulky, open pocket of Tyro3. The location and conformation of active-site residues of Mer and Axl are highly consistent, suggesting that small-molecule inhibitors generally have a low Mer-over-Axl selectivity and a high Mer-over-Tyro3 selectivity. We demonstrated that the difference in ATP binding potency to the three kinases is also responsible for inhibitor selectivity. We also found that the long-range interactions and allosteric effect arising from rest of the kinase's active site can indirectly influence inhibitor binding and selectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Phosphatidylinositol 3-Kinase (PI3K) δ blockade increases genomic instability in B cells

PubMed Central

Compagno, Mara; Wang, Qi; Pighi, Chiara; Cheong, Taek-Chin; Meng, Fei-Long; Poggio, Teresa; Yeap, Leng-Siew; Karaca, Elif; Blasco, Rafael B.; Langellotto, Fernanda; Ambrogio, Chiara; Voena, Claudia; Wiestner, Adrian; Kasar, Siddha N.; Brown, Jennifer R.; Sun, Jing; Wu, Catherine J.; Gostissa, Monica; Alt, Frederick W.; Chiarle, Roberto

2017-01-01

Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme that targets immunoglobulin (Ig) genes to initiate class switch recombination (CSR) and somatic hypermutation (SHM)1. Through off-target activity, however, AID has a much broader impact on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in lymphoma development and progression2. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation3. The phosphatidylinositol 3-kinase (PI3K) δ pathway plays a key role in AID regulation by suppressing its expression in B cells4. Novel drugs for leukemia or lymphoma therapy such as idelalisib, duvelisib or ibrutinib block PI3Kδ activity directly or indirectly5–8, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation (SHM) and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both these effects were completely abrogated in AID deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumors in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IgH and AID off-target sites in human chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased SHM in AID off-targets. In summary, we show that PI3Kδ or BTK inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism, an effect that should be carefully considered as such inhibitors are administered for years to patients. PMID:28199309

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme.

PubMed

Ueno, E; Sakai, H; Kato, Y; Yamamoto, K

1989-06-01

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.

Inhibition of AcpA phosphatase activity with ascorbate attenuates Francisella tularensis intramacrophage survival.

PubMed

McRae, Steven; Pagliai, Fernando A; Mohapatra, Nrusingh P; Gener, Alejandro; Mahmou, Asma Sayed Abdelgeliel; Gunn, John S; Lorca, Graciela L; Gonzalez, Claudio F

2010-02-19

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (K(i) = 380 +/- 160 microM) and 2-phosphoascorbate (K(i) = 3.2 +/- 0.85 microM) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia.

Inhibition of AcpA Phosphatase Activity with Ascorbate Attenuates Francisella tularensis Intramacrophage Survival

PubMed Central

McRae, Steven; Pagliai, Fernando A.; Mohapatra, Nrusingh P.; Gener, Alejandro; Abdelgeliel Mahmou, Asma Sayed; Gunn, John S.; Lorca, Graciela L.; Gonzalez, Claudio F.

2010-01-01

Acid phosphatase activity in the highly infectious intracellular pathogen Francisella tularensis is directly related with the ability of these bacteria to survive inside host cells. Pharmacological inactivation of acid phosphatases could potentially help in the treatment of tularemia or even be utilized to neutralize the infection. In the present work, we report inhibitory compounds for three of the four major acid phosphatases produced by F. tularensis SCHU4: AcpA, AcpB, and AcpC. The inhibitors were identified using a catalytic screen from a library of chemicals approved for use in humans. The best results were obtained against AcpA. The two compounds identified, ascorbate (Ki = 380 ± 160 μm) and 2-phosphoascorbate (Ki = 3.2 ± 0.85 μm) inhibit AcpA in a noncompetitive, nonreversible fashion. A potential ascorbylation site in the proximity of the catalytic pocket of AcpA was identified using site-directed mutagenesis. The effects of the inhibitors identified in vitro were evaluated using bioassays determining the ability of F. tularensis to survive inside infected cells. The presence of ascorbate or 2-phosphoascorbate impaired the intramacrophage survival of F. tularensis in an AcpA-dependent manner as it was probed using knockout strains. The evidence presented herein indicated that ascorbate could be a good alternative to be used clinically to improve treatments against tularemia. PMID:20028980

Design and Development of Microsomal Prostaglandin E2 Synthase-1 Inhibitors: Challenges and Future Directions.

PubMed

Koeberle, Andreas; Laufer, Stefan A; Werz, Oliver

2016-07-14

Microsomal prostaglandin E2 synthase (mPGES)-1 is responsible for the massive prostaglandin E2 (PGE2) formation during inflammation. Increasing evidence reveals mPGES-1 inhibitors as a safe alternative to nonsteroidal anti-inflammatory drugs. The first selective mPGES-1 inhibitors recently entered clinical trials. Major challenges for drug development have been the high plasma protein binding of lead structures, interspecies discrepancies, nuisance inhibition, sophisticated enzyme assays, and limited structural information about the mPGES-1 inhibitor binding site. Since most of these drawbacks could be solved during the past few years, we are standing at the threshold of a new era of mPGES-1-targeting anti-inflammatory drugs. This perspective introduces mPGES-1 as a key player within the network of eicosanoid biosynthesis and summarizes our current understanding of its structure and mechanism. Moreover, we present high-throughput and in silico screening techniques and discuss the structure-activity relationship and pharmacological potential of major mPGES-1 inhibitor classes in light of recent insights from pharmacophore models and cocrystallization studies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Byung Hak; Lee, Jun-Young; Seo, Jee Hee

Nuclear factor (NF)-{kappa}B regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-{kappa}B inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-{kappa}B activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of I{kappa}B kinase {beta} (IKK{beta}), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-{kappa}B activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKK{beta} and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKK{beta} activity was completely abolished by substitution of Cys-179 residue ofmore » IKK{beta} to Ala residue, indicating direct targeting site of ATM. ATM could inhibit I{kappa}B{alpha} phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-{kappa}B activation. Further, we demonstrate that ATM down-regulates NF-{kappa}B-dependent TNF-{alpha} expression. Taken together, this study provides a pharmacological potential of ATM in NF-{kappa}B-dependent inflammatory disorders.« less

Small-molecule inhibitors of APE1 DNA repair function: an overview.

PubMed

Al-Safi, Rasha I; Odde, Srinivas; Shabaik, Yumna; Neamati, Nouri

2012-01-01

APE1 is a multifaceted protein that orchestrates multiple activities in the cell, one of which is the preservation of genomic integrity; a vital process that takes place in the context of the base excision repair (BER) pathway. Studies have implicated APE1 in rendering cancerous cells less vulnerable to the effects of DNA-damaging agents that are commonly used for the treatment of cancer. Furthermore, suppression of APE1 expression in cancer cell lines is accompanied by the potentiation of the activity of cytotoxic agents. As a result, major efforts have been directed towards the identification of small-molecule inhibitors of this DNA-repair enzyme. Herein, we review all patented small-molecule APE1 inhibitors reported prior to 2011. Unfortunately, the potency and selectivity of many of the reported inhibitors were not disclosed by the original authors, and at present it is unclear if APE1 is a bona fide target for many of the purported inhibitors. Moreover, cellular activity and toxicity of many inhibitors remain to be established. Since this is the first comprehensive review of small molecule APE1 inhibitors, we present all compounds reported to inhibit APE1 activity with an IC50 value ≤ 25 μM. Efforts towards a careful validation and optimization of these compounds are warranted. Furthermore, we explore potential allosteric drug-binding sites on the protein as an alternative approach for modulating the activity of this multifunctional protein. In addition, we give an overview of APE2, as well as other APE1 homologues in some disease-causing pathogens. Finally, given the universal importance of DNA repair, as well as the considerable conservation of repair proteins across all living organisms, we propose targeting the AP endonuclease activity of pathogens by the compounds discussed in this review, thereby expanding their therapeutic potential and application.

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

PubMed

Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

2012-04-01

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX

DOE PAGES

Pinard, Melissa A.; Aggarwal, Mayank; Mahon, Brian P.; ...

2015-09-23

Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO 2to HCO 3 $-$, thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-basedmore » drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, anR cryst of 18.0% and anR free of 21.2%. Finally, the binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.« less

Inactivation of muscle adenylate kinase by site-specific destruction of tyrosine 95 using potassium ferrate.

PubMed

Crivellone, M D; Hermodson, M; Axelrod, B

1985-03-10

Potassium ferrate, an analog of orthophosphate and a potent oxidizing agent, was found to irreversibly inactivate porcine muscle adenylate kinase. Inhibition was prevented by competitive inhibitors or substrates, indicating that the action of ferrate was site-specific. Inactivation was accompanied by the loss of Cys-25 and Tyr-95. P1,P5-di(adenosine 5')-pentaphosphate (10(-7) M), a powerful competitive inhibitor, gave 50% protection to the enzyme from ferrate inactivation. No loss of tyrosine or cysteine residues was observed under conditions of total protection. The degree of inactivation was proportional to the amount of Tyr-95 destroyed. However, Cys-25 was totally oxidized when only 55% inactivation had occurred. Partially inactivated enzyme exhibited a Km for ATP and AMP similar to that of the untreated enzyme. It appears that Cys-25 may be proximate to a phosphate-binding site but is not directly involved in the catalytic reaction. The results suggest that Tyr-95 is located in the vicinity of a phosphate-binding region of adenylate kinase and is essential for enzyme activity.

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

PubMed Central

Matts, Robert L.; Dixit, Anshuman; Peterson, Laura B.; Sun, Liang; Voruganti, Sudhakar; Kalyanaraman, Palgunan; Hartson, Steve D.; Verkhivker, Gennady M.; Blagg, Brian S. J.

2011-01-01

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein. PMID:21548602

Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

PubMed

Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

Chalcone-based small-molecule inhibitors attenuate malignant phenotype via targeting deubiquitinating enzymes

PubMed Central

Issaenko, Olga A.; Amerik, Alexander Yu

2012-01-01

The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27Kip1 and p16Ink4A. These changes are associated with arrest in S-G2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors. PMID:22510564

Differential 3-bromopyruvate inhibition of cytosolic and mitochondrial human serine hydroxymethyltransferase isoforms, key enzymes in cancer metabolic reprogramming.

PubMed

Paiardini, Alessandro; Tramonti, Angela; Schirch, Doug; Guiducci, Giulia; di Salvo, Martino Luigi; Fiascarelli, Alessio; Giorgi, Alessandra; Maras, Bruno; Cutruzzolà, Francesca; Contestabile, Roberto

2016-11-01

The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP. Copyright © 2016 Elsevier B.V. All rights reserved.

Potential anti-cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 inhibitory activities of cornuside and gallotannins from Cornus officinalis fruits.

PubMed

Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Tanaka, Takashi; Jung, Hyun Ah; Choi, Jae Sue

2017-07-01

Cholinesterase (ChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors are promising agents for the treatment of Alzheimer's disease (AD). In the present study, we examined the inhibitory activity of seven compounds isolated from the fruits of Cornus officinalis, cornuside, polymeric proanthocyanidins, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, tellimagrandin I, tellimagrandin II, and isoterchebin, against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1. All of the compounds displayed concentration-dependent in vitro inhibitory activity toward the ChEs and BACE1. Among them, tellimagrandin II exhibited the best inhibitory activity toward ChEs, whereas the best BACE1 inhibitor was 1,2,3,6-tetra-O-galloyl-β-D-glucose. Isoterchebin and polymeric proanthocyanidins were also significant ChE inhibitors. The kinetic and docking studies demonstrated that all compounds interacted with both the catalytic active sites and the peripheral anionic sites of the ChEs and BACE1. Tellimagrandin II, isoterchebin, and the polymeric proanthocyanidins exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. In conclusion, we identified significant ChE and BACE1 inhibitors from Corni Fructus that could have value as new multi-targeted compounds for anti-AD agents.

Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.

PubMed

Young, Luke; May, Benjamin; Pendlebury-Watt, Alice; Shearman, Julia; Elliott, Catherine; Albury, Mary S; Shiba, Tomoo; Inaoka, Daniel Ken; Harada, Shigeharu; Kita, Kiyoshi; Moore, Anthony L

2014-07-01

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two-leucine residues. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Copyright © 2014 Elsevier B.V. All rights reserved.

Activation by Phoneutria nigriventer (armed spider) venom of tissue kallikrein-kininogen-kinin system in rabbit skin in vivo.

PubMed

Marangoni, R A; Antunes, E; Brain, S D; de Nucci, G

1993-06-01

1. The purpose of the present study was to investigate the mechanisms by which venom from Phoneutria nigriventer spider induces increases in vascular permeability in rabbit skin. 2. Local oedema formation, in response to intradermally-injected agents, was measured in male New Zealand white rabbits as the local accumulation of i.v. injected 125I-labelled human serum albumin into skin sites. 3. Phoneutria nigriventer venom (10-30 micrograms/site) increased vascular permeability, which was inhibited by trasylol (10 micrograms/site) and the bradykinin B2 receptor antagonists D-Arg,[Hyp3,Thi5,8,D-Phe7]-BK (3 nmol/site) and Hoe 140 (0.3 nmol/site). In addition, the oedema induced by the venom was potentiated by the kinase II inhibitor, captopril (1 nmol/site). The lipoxygenase inhibitor, BWA4C (10 nmol/site) and the PAF antagonist, WEB 2086 (100 nmol/site) had no effect on the venom-induced increase in vascular permeability. 4. Incubation of rabbit plasma with Phoneutria nigriventer venom in vitro did not cause bradykinin formation. Further, the plasma kallikrein inhibitor, soybean trypsin inhibitor (10 micrograms/site), had no effect on the venom-induced increase in vascular permeability in rabbit skin. 5. These results indicate that the oedema produced by Phoneutria nigriventer venom is dependent on the activation of the tissue kallikrein-kinin system.

Activation by Phoneutria nigriventer (armed spider) venom of tissue kallikrein-kininogen-kinin system in rabbit skin in vivo.

PubMed Central

Marangoni, R. A.; Antunes, E.; Brain, S. D.; de Nucci, G.

1993-01-01

1. The purpose of the present study was to investigate the mechanisms by which venom from Phoneutria nigriventer spider induces increases in vascular permeability in rabbit skin. 2. Local oedema formation, in response to intradermally-injected agents, was measured in male New Zealand white rabbits as the local accumulation of i.v. injected 125I-labelled human serum albumin into skin sites. 3. Phoneutria nigriventer venom (10-30 micrograms/site) increased vascular permeability, which was inhibited by trasylol (10 micrograms/site) and the bradykinin B2 receptor antagonists D-Arg,[Hyp3,Thi5,8,D-Phe7]-BK (3 nmol/site) and Hoe 140 (0.3 nmol/site). In addition, the oedema induced by the venom was potentiated by the kinase II inhibitor, captopril (1 nmol/site). The lipoxygenase inhibitor, BWA4C (10 nmol/site) and the PAF antagonist, WEB 2086 (100 nmol/site) had no effect on the venom-induced increase in vascular permeability. 4. Incubation of rabbit plasma with Phoneutria nigriventer venom in vitro did not cause bradykinin formation. Further, the plasma kallikrein inhibitor, soybean trypsin inhibitor (10 micrograms/site), had no effect on the venom-induced increase in vascular permeability in rabbit skin. 5. These results indicate that the oedema produced by Phoneutria nigriventer venom is dependent on the activation of the tissue kallikrein-kinin system. PMID:8395291

SbnG, a Citrate Synthase in Staphylococcus aureus

PubMed Central

Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; Heinrichs, David E.; Murphy, Michael E. P.

2014-01-01

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. PMID:25336653

Discovery of Selective Inhibitors of Imidazoleglycerol-Phosphate Dehydratase from Mycobacterium tuberculosis by Virtual Screening

NASA Astrophysics Data System (ADS)

Podshivalov, D.; Mandzhieva, Yu. B.; Sidorov-Biryukov, D. D.; Timofeev, V. I.; Kuranova, I. P.

2018-01-01

Bacterial imidazoleglycerol-phosphate dehydratase from Mycobacterium tuberculosis (HisB- Mt) is a convenient target for the discovery of selective inhibitors as potential antituberculosis drugs. The virtual screening was performed to find compounds suitable for the design of selective inhibitors of HisB- Mt. The positions of four ligands, which were selected based on the docking scoring function and docked to the activesite region of the enzyme, were refined by molecular dynamics simulation. The nearest environment of the ligands was determined. These compounds selectively bind to functionally essential active-site residues, thus blocking access of substrates to the active site of the enzyme, and can be used as lead compounds for the design of selective inhibitors of HisB- M.

Discovery and Development of Kelch-like ECH-Associated Protein 1. Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction Inhibitors: Achievements, Challenges, and Future Directions.

PubMed

Jiang, Zheng-Yu; Lu, Meng-Chen; You, Qi-Dong

2016-12-22

The transcription factor Nrf2 is the primary regulator of the cellular defense system, and enhancing Nrf2 activity has potential usages in various diseases, especially chronic age-related and inflammatory diseases. Recently, directly targeting Keap1-Nrf2 protein-protein interaction (PPI) has been an emerging strategy to selectively and effectively activate Nrf2. This Perspective summarizes the progress in the discovery and development of Keap1-Nrf2 PPI inhibitors, including the Keap1-Nrf2 regulatory mechanisms, biochemical techniques for inhibitor identification, and approaches for identifying peptide and small-molecule inhibitors, as well as discusses privileged structures and future directions for further development of Keap1-Nrf2 PPI inhibitors.

Prediction of enzyme binding: human thrombin inhibition study by quantum chemical and artificial intelligence methods based on X-ray structures.

PubMed

Mlinsek, G; Novic, M; Hodoscek, M; Solmajer, T

2001-01-01

Thrombin is a serine protease which plays important roles in the human body, the key one being the control of thrombus formation. The inhibition of thrombin has become a target for new antithrombotics. The aim of our work was to (i) construct a model which would enable us to predict Ki values for the binding of an inhibitor into the active site of thrombin based on a database of known X-ray structures of inhibitor-enzyme complexes and (ii) to identify the structural and electrostatic characteristics of inhibitor molecules crucially important to their effective binding. To retain as much of the 3D structural information of the bound inhibitor as possible, we implemented the quantum mechanical/molecular mechanical (QM/MM) procedure for calculating the molecular electrostatic potential (MEP) at the van der Waals surfaces of atoms in the protein's active site. The inhibitor was treated quantum mechanically, while the rest of the complex was treated by classical means. The obtained MEP values served as inputs into the counter-propagation artificial neural network (CP-ANN), and a genetic algorithm was subsequently used to search for the combination of atoms that predominantly influences the binding. The constructed CP-ANN model yielded Ki values predictions with a correlation coefficient of 0.96, with Ki values extended over 7 orders of magnitude. Our approach also shows the relative importance of the various amino acid residues present in the active site of the enzyme for inhibitor binding. The list of residues selected by our automatic procedure is in good correlation with the current consensus regarding the importance of certain crucial residues in thrombin's active site.

Thiol Versus Hydroxamate as Zinc Binding Group In HDAC Inhibition: An Ab Initio QM/MM Molecular Dynamics Study

PubMed Central

Gong, Wenjing; Wu, Ruibo; Zhang, Yingkai

2015-01-01

Zinc-dependent histone deacetylases (HDACs) play a critical role in transcriptional repression and gene silencing, and are among the most attractive targets for the development of new therapeutics against cancer and various other diseases. Two HDAC inhibitors have been approved by FDA as anti-cancer drugs: one is SAHA whose hydroxamate is directly bound to zinc, the other is FK228 whose active form may use thiol as the zinc binding group. In spite of extensive studies, it remains to be ambiguous regarding how thiol and hydroxamate are bound to the zinc active site of HDACs. In this work, our computational approaches center on Born-Oppenheimer ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics with umbrella sampling, which allow for modeling of the zinc active site with reasonable accuracy while properly including dynamics and effects of protein environment. Meanwhile, an improved short-long effective function (SLEF2) to describe non-bonded interactions between zinc and other atoms has been employed in initial MM equilibrations. Our ab initio QM/MM MD simulations have confirmed that hydroxamate is neutral when it is bound to HDAC8, and found that thiol is deprotonated when directly bound to zinc in the HDAC active site. By comparing thiol and hydroxamate, our results elucidated the differences in their binding environment in the HDAC active sites, and emphasized the importance of the linker design to achieve more specific binding towards class IIa HDACs. PMID:26452222

Thiol versus hydroxamate as zinc binding group in HDAC inhibition: An ab initio QM/MM molecular dynamics study.

PubMed

Gong, Wenjing; Wu, Ruibo; Zhang, Yingkai

2015-11-15

Zinc-dependent histone deacetylases (HDACs) play a critical role in transcriptional repression and gene silencing, and are among the most attractive targets for the development of new therapeutics against cancer and various other diseases. Two HDAC inhibitors have been approved by FDA as anti-cancer drugs: one is SAHA whose hydroxamate is directly bound to zinc, the other is FK228 whose active form may use thiol as the zinc binding group. In spite of extensive studies, it remains to be ambiguous regarding how thiol and hydroxamate are bound to the zinc active site of HDACs. In this work, our computational approaches center on Born-Oppenheimer ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics with umbrella sampling, which allow for modeling of the zinc active site with reasonable accuracy while properly including dynamics and effects of protein environment. Meanwhile, an improved short-long effective function (SLEF2) to describe non-bonded interactions between zinc and other atoms has been employed in initial MM equilibrations. Our ab initio QM/MM MD simulations have confirmed that hydroxamate is neutral when it is bound to HDAC8, and found that thiol is deprotonated when directly bound to zinc in the HDAC active site. By comparing thiol and hydroxamate, our results elucidated the differences in their binding environment in the HDAC active sites, and emphasized the importance of the linker design to achieve more specific binding toward class IIa HDACs. © 2015 Wiley Periodicals, Inc.

Discovery of novel high potent and cellular active ADC type PTP1B inhibitors with selectivity over TC-PTP via modification interacting with C site.

PubMed

Du, Yongli; Zhang, Yanhui; Ling, Hao; Li, Qunyi; Shen, Jingkang

2018-01-20

PTP1B serving as a key negative regulator of insulin signaling is a novel target for type 2 diabetes and obesity. Modification at ring B of N-{4-[(3-Phenyl-ureido)-methyl]-phenyl}-methane-sulfonamide template to interact with residues Arg47 and Lys41 in the C site of PTP1B by molecular docking aided design resulted in the discovery of a series of novel high potent and selective inhibitors of PTP1B. The structure activity relationship interacting with the C site of PTP1B was well illustrated. Compounds 8 and 18 were shown to be the high potent and most promising PTP1B inhibitors with cellular activity and great selectivity over the highly homologous TCPTP and other PTPs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands for the Treatment of Alzheimer's Disease: Design, Synthesis, Biological Evaluation, and X-ray Crystallography.

PubMed

Zha, Xiaoming; Lamba, Doriano; Zhang, Lili; Lou, Yinghan; Xu, Changxu; Kang, Di; Chen, Li; Xu, Yungen; Zhang, Luyong; De Simone, Angela; Samez, Sarah; Pesaresi, Alessandro; Stojan, Jure; Lopez, Manuela G; Egea, Javier; Andrisano, Vincenza; Bartolini, Manuela

2016-01-14

Twenty-six new tacrine-benzofuran hybrids were designed, synthesized, and evaluated in vitro on key molecular targets for Alzheimer's disease. Most hybrids exhibited good inhibitory activities on cholinesterases and β-amyloid self-aggregation. Selected compounds displayed significant inhibition of human β-secretase-1 (hBACE-1). Among the 26 hybrids, 2e showed the most interesting profile as a subnanomolar selective inhibitor of human acetylcholinesterase (hAChE) (IC50 = 0.86 nM) and a good inhibitor of both β-amyloid aggregation (hAChE- and self-induced, 61.3% and 58.4%, respectively) and hBACE-1 activity (IC50 = 1.35 μM). Kinetic studies showed that 2e acted as a slow, tight-binding, mixed-type inhibitor, while X-ray crystallographic studies highlighted the ability of 2e to induce large-scale structural changes in the active-site gorge of Torpedo californica AChE (TcAChE), with significant implications for structure-based drug design. In vivo studies confirmed that 2e significantly ameliorates performances of scopolamine-treated ICR mice. Finally, 2e administration did not exhibit significant hepatotoxicity.

Polyphenol Compound as a Transcription Factor Inhibitor.

PubMed

Park, Seyeon

2015-10-30

A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

Designed inhibitors with hetero linkers for gastric proton pump H+,K+-ATPase: Steered molecular dynamics and metadynamics studies.

PubMed

Jana, Kalyanashis; Bandyopadhyay, Tusar; Ganguly, Bishwajit

2017-11-01

Acid suppressant SCH28080 and its derivatives reversibly reduce acid secretion activity of the H + ,K + -ATPase in a K + competitive manner. The results on homologation of the SCH28080 by varying the linker chain length suggested the improvement in efficacy. However, the pharmacokinetic studies reveal that the hydrophobic nature of the CH 2 linker units may not help it to function as a better acid suppressant. We have exploited the role of linker unit to enhance the efficacy of such reversible acid suppressant drug molecules using hetero linker, i.e., disulfide and peroxy linkers. The logarithm of partition coefficient defined for a drug molecule relates to the partition coefficient, which allows the optimum solubility characteristics to reach the active site. The logarithm of partition coefficient calculated for the designed inhibitors suggests that inhibitors would possibly reach the active site in sufficient concentration like in the case of SCH28080. The steered molecular dynamics studies have revealed that the Inhibitor-1 with disulfide linker unit is more stable at the active site due to greater noncovalent interactions compared to the SCH28080. Centre of mass distance analysis suggests that the Cysteine-813 amino acid residue selectively plays an important role in the inhibition of H + ,K + -ATPase for Inhibitor-1. Furthermore, the quantum chemical calculations with M11L/6-31+G(d,p) level of theory have been performed to account the noncovalent interactions responsible for the stabilization of inhibitor molecules in the active site gorge of the gastric proton pump at different time scale. The hydrogen bonding and hydrophobic interaction studies corroborate the center of mass distance analysis as well. Well-tempered metadynamics free energy surface and center of mass separation analysis for the Inhibitor-1 is in good agreement with the steered molecular dynamics results. The torsional angle of the linker units seems to be crucial for better efficacy of drug molecules. The torsional angle of linker units of SCH28080 (COCH 2 C) and of Inhibitor 1 (CSSC) prefers to lie within ∼60°-90° for a longer time during the simulations, whereas, the peroxy linker (COOC) of Inhibitor 2 prefers to adopt ∼120-160°. Therefore, it appears that the smaller torsion angle of linker units can achieve better interactions with the active site residues of H + ,K + -ATPase to inhibit the acid secretion activity. The reversible drug molecules with disulfide linker unit would be a promising candidate as proton pump antagonist to H + ,K + -ATPase. Copyright © 2017 Elsevier Inc. All rights reserved.

Direct covalent modification as a strategy to inhibit nuclear factor-kappa B.

PubMed

Pande, Vineet; Sousa, Sérgio F; Ramos, Maria João

2009-01-01

Nuclear Factor-KkappaB (NF-kappaB) is a transcription factor whose inappropriate activation may result in the development of a number of diseases including cancer, inflammation, neurodegeneration and AIDS. Recent studies on NF-kappaB mediated pathologies, made therapeutic interventions leading to its inhibition an emerging theme in pharmaceutical research. NF-kappaB resides in the cytoplasm and is activated by several time-dependent factors, leading to proteasome-dependent degradation of its inhibitory protein (IkappaB), resulting in free NF-kappaB (p50 and p65 subunits, involved in disease states), which binds to target DNA sites, further resulting in enhanced transcription of several disease associated proteins. The complex pathway of NF-kappaB, finally leading to its DNA binding, has attracted several approaches interfering with this pathway. One such approach is that of a direct covalent modification of NF-kappaB. In this article, we present a critical review on the pharmacological agents that have been studied as inhibitors of NF-kappaB by covalently modifying redox-regulated cysteine residues in its subunits, ultimately resulting in the inhibition of kappaB DNA recognition and binding. Beginning with a general overview of NF-kappaB pathway and several possibilities of chemical interventions, the significance of redox-regulation in NF-kappaB activation and DNA binding is presented. Further, protein S-thiolation, S-nitrosylation and irreversible covalent modification are described as regular biochemical events in the cell, having provided a guideline for the development of NF-kappaB inhibitors discussed further. Although just a handful of inhibitors, with most of them being alkylating agents have been studied in the present context, this approach presents potential for the development of a new class of NF-kappaB-inhibitors.

Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

PubMed

Bimmler, D; Graf, R; Scheele, G A; Frick, T W

1997-01-31

Apart from digestive enzymes, pancreatic juice contains several proteins that are not directly involved in digestion. One of these, lithostathine, has been reported to exhibit calcite crystal inhibitor activity in vitro. As pancreatic juice is supersaturated with respect to calcium carbonate, it was hypothesized that lithostathine stabilizes pancreatic juice. Lithostathine is cleaved by trace amounts of trypsin, resulting in a C-terminal polypeptide and an N-terminal undecapeptide, which has been identified as the active site of lithostathine regarding crystal inhibition. We produced rat lithostathine in a baculovirus expression system. In order to test its functional activity, the protein was purified using a nondenaturing multi-step procedure. In the low micromolar range, recombinant rat lithostathine in vitro exhibited calcite crystal inhibitor activity, confirming earlier reports. Limited tryptic proteolysis of recombinant lithostathine was performed, and the two cleavage products were separated; the C-terminal polypeptide was precipitated by centrifugation, and the N-terminal undecapeptide was purified by high performance liquid chromatography. Only the C-terminal peptide displayed measurable calcite crystal inhibitory activity. Furthermore, synthetic undecapeptides with identical sequence to the N-terminal undecapeptides of rat or human lithostathine were inactive. However, when tested in the same in vitro assays, other pancreatic or extra-pancreatic proteins show inhibitory activity in the same concentration range as lithostathine, and inorganic phosphate is active as well. Based on these findings it seems unlikely that lithostathine is a physiologically relevant calcite crystal inhibitor. The name "lithostathine" is therefore inappropriate, and the protein's key function remains to be elucidated.

Virtual lead identification of farnesyltransferase inhibitors based on ligand and structure-based pharmacophore techniques.

PubMed

Al-Balas, Qosay A; Amawi, Haneen A; Hassan, Mohammad A; Qandil, Amjad M; Almaaytah, Ammar M; Mhaidat, Nizar M

2013-05-27

Farnesyltransferase enzyme (FTase) is considered an essential enzyme in the Ras signaling pathway associated with cancer. Thus, designing inhibitors for this enzyme might lead to the discovery of compounds with effective anticancer activity. In an attempt to obtain effective FTase inhibitors, pharmacophore hypotheses were generated using structure-based and ligand-based approaches built in Discovery Studio v3.1. Knowing the presence of the zinc feature is essential for inhibitor's binding to the active site of FTase enzyme; further customization was applied to include this feature in the generated pharmacophore hypotheses. These pharmacophore hypotheses were thoroughly validated using various procedures such as ROC analysis and ligand pharmacophore mapping. The validated pharmacophore hypotheses were used to screen 3D databases to identify possible hits. Those which were both high ranked and showed sufficient ability to bind the zinc feature in active site, were further refined by applying drug-like criteria such as Lipiniski's "rule of five" and ADMET filters. Finally, the two candidate compounds (ZINC39323901 and ZINC01034774) were allowed to dock using CDOCKER and GOLD in the active site of FTase enzyme to optimize hit selection.

Structure-Based Design and Synthesis of Potent and Selective Matrix Metalloproteinase 13 Inhibitors.

PubMed

Choi, Jun Yong; Fuerst, Rita; Knapinska, Anna M; Taylor, Alexander B; Smith, Lyndsay; Cao, Xiaohang; Hart, P John; Fields, Gregg B; Roush, William R

2017-07-13

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn 2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn 2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn 2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.

Binding and Inhibition of Spermidine Synthase from Plasmodium falciparum and Implications for In Vitro Inhibitor Testing

PubMed Central

Sprenger, Janina; Carey, Jannette; Svensson, Bo; Wengel, Verena

2016-01-01

The aminopropyltransferase spermidine synthase (SpdS) is a promising drug target in cancer and in protozoan diseases including malaria. Plasmodium falciparum SpdS (PfSpdS) transfers the aminopropyl group of decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine or to spermidine to form spermidine or spermine, respectively. In an effort to understand why efficient inhibitors of PfSpdS have been elusive, the present study uses enzyme activity assays and isothermal titration calorimetry with verified or predicted inhibitors of PfSpdS to analyze the relationship between binding affinity as assessed by KD and inhibitory activity as assessed by IC50. The results show that some predicted inhibitors bind to the enzyme with high affinity but are poor inhibitors. Binding studies with PfSpdS substrates and products strongly support an ordered sequential mechanism in which the aminopropyl donor (dcAdoMet) site must be occupied before the aminopropyl acceptor (putrescine) site can be occupied. Analysis of the results also shows that the ordered sequential mechanism adequately accounts for the complex relationship between IC50 and KD and may explain the limited success of previous efforts at structure-based inhibitor design for PfSpdS. Based on PfSpdS active-site occupancy, we suggest a classification of ligands that can help to predict the KD−IC50 relations in future design of new inhibitors. The present findings may be relevant for other drug targets that follow an ordered sequential mechanism. PMID:27661085

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

Molecular dynamics simulation studies of novel β-lactamase inhibitor.

PubMed

Ul Haq, Farhan; Abro, Asma; Raza, Saad; Liedl, Klaus R; Azam, Syed Sikander

2017-06-01

New Delhi Metallo-β-Lactamase-1 (NDM-1) has drawn great attention due to its diverse antibiotic resistant activity. It can hydrolyze almost all clinically available β-lactam antibiotics. To inhibit the activity of NDM-1 a new strategy is proposed using computational methods. Molecular dynamics (MD) simulations are used to analyze the molecular interactions between selected inhibitor candidates and NDM-1 structure. The enzyme-ligand complex is subject to binding free energy calculations using MM(PB/GB)SA methods. The role of each residue of the active site contributing in ligand binding affinity is explored using energy decomposition analysis. Furthermore, a hydrogen bonding network between ligand and enzyme active site is observed and key residues are identified ensuring that the ligand stays inside the active site and maintains its movement towards the active site pocket. A production run of 150ns is carried out and results are analyzed using root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) to explain the stability of enzyme ligand complex. Important active site residue e.g. PHE70, VAL73, TRP93, HIS122, GLN123, ASP124, HIS189, LYS216, CYS208, LYS211, ALA215, HIS250, and SER251 were observed to be involved in ligand attachemet inside the active site pocket, hence depicting its inhibitor potential. Hydrogen bonds involved in structural stability are analyzed through radial distribution function (RDF) and contribution of important residues involved in ligand movement is explained using a novel analytical tool, axial frequency distribution (AFD) to observe the role of important hydrogen bonding partners between ligand atoms and active site residues. Copyright © 2017 Elsevier Inc. All rights reserved.

A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

PubMed

Pöhler, Robert; Krahn, Jan H; van den Boom, Johannes; Dobrynin, Grzegorz; Kaschani, Farnusch; Eggenweiler, Hans-Michael; Zenke, Frank T; Kaiser, Markus; Meyer, Hemmo

2018-02-05

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Zinc binding in HDAC inhibitors: a DFT study.

PubMed

Wang, Difei; Helquist, Paul; Wiest, Olaf

2007-07-06

Histone deacetylases (HDACs) are attractive targets for the treatment of cancers and a variety of other diseases. Most currently studied HDAC inhibitors contain hydroxamic acids, which are potentially problematic in the development of practical drugs. DFT calculations of the binding modes and free energies of binding for a variety of other functionalities in a model active site of HDAC are described. The protonation state of hydroxamic acids in the active site and the origin of the high affinity are discussed. These results emphasize the importance of a carefully chosen pKa for zinc binding and provide guidance for the design of novel, non-hydroxamic acid HDAC inhibitors.

A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa.

PubMed

Lu, Genmin; DeGuzman, Francis R; Hollenbach, Stanley J; Karbarz, Mark J; Abe, Keith; Lee, Gail; Luan, Peng; Hutchaleelaha, Athiwat; Inagaki, Mayuko; Conley, Pamela B; Phillips, David R; Sinha, Uma

2013-04-01

Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

Synthesis, QSAR, and Molecular Dynamics Simulation of Amidino-substituted Benzimidazoles as Dipeptidyl Peptidase III Inhibitors.

PubMed

Rastija, Vesna; Agić, Dejan; Tomiš, Sanja; Nikolič, Sonja; Hranjec, Marijana; Grace, Karminski-Zamola; Abramić, Marija

2015-01-01

A molecular modeling study is performed on series of benzimidazol-based inhibitors of human dipeptidyl peptidase III (DPP III). An eight novel compounds were synthesized in excellent yields using green chemistry approach. This study is aimed to elucidate the structural features of benzimidazole derivatives required for antagonism of human DPP III activity using Quantitative Structure-Activity Relationship (QSAR) analysis, and to understand the mechanism of one of the most potent inhibitor binding into the active site of this enzyme, by molecular dynamics (MD) simulations. The best model obtained includes S3K and RDF045m descriptors which have explained 89.4 % of inhibitory activity. Depicted moiety for strong inhibition activity matches to the structure of most potent compound. MD simulation has revealed importance of imidazolinyl and phenyl groups in the mechanism of binding into the active site of human DPP III.

Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration.

PubMed

Bianchi, Mariarita; De Lucchini, Stefania; Marin, Oriano; Turner, David L; Hanks, Steven K; Villa-Moruzzi, Emma

2005-10-15

In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3beta. Furthermore, GSK3beta was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3beta underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714-730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3beta and PP1.

Changes in signal transducer and activator of transcription 3 (STAT3) dynamics induced by complexation with pharmacological inhibitors of Src homology 2 (SH2) domain dimerization.

PubMed

Resetca, Diana; Haftchenary, Sina; Gunning, Patrick T; Wilson, Derek J

2014-11-21

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

DTIC Science & Technology

2014-10-01

during hypoxia were biologically meaningful, we investigated the activity of a reporter with multiple HIF binding sites ( HRE ) in front of a luciferase...inhibitors in a dose dependent fashion blocked the activity of the HRE to activate luciferase mRNA and protein production. This result demonstrates that

Second-generation CK2α inhibitors targeting the αD pocket† †Electronic supplementary information (ESI) available: All experimental details, crystallographic data collection and refinement statistics, details of chemical synthesis, additional figures and tables. See DOI: 10.1039/c7sc05122k

PubMed Central

Iegre, Jessica; Brear, Paul; De Fusco, Claudia; Yoshida, Masao; Mitchell, Sophie L.; Rossmann, Maxim; Carro, Laura; Sore, Hannah F.

2018-01-01

CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 (IC50 = 7 μM, GI50 = 10.0 ± 3.6 μM), has numerous advantages over the previously reported CAM4066, including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066, there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC50) and the ability to inhibit cell proliferation (GI50). PMID:29732088

Discovery and evaluation of inhibitors to the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1): Probing the active site-inhibitor interactions.

PubMed

Tomek, Petr; Palmer, Brian D; Flanagan, Jack U; Sun, Chuanwen; Raven, Emma L; Ching, Lai-Ming

2017-01-27

High expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) for a broad range of malignancies is associated with poor patient prognosis, and the enzyme is a validated target for cancer intervention. To identify novel IDO1 inhibitors suitable for drug development, 1597 compounds in the National Cancer Institute Diversity Set III library were tested for inhibitory activity against recombinant human IDO1. We retrieved 35 hits that inhibited IDO1 activity >50% at 20 μM. Five structural filters and the PubChem Bioassay database were used to guide the selection of five inhibitors with IC 50 between 3 and 12 μM for subsequent experimental evaluation. A pyrimidinone scaffold emerged as being the most promising. It showed excellent cell penetration, negligible cytotoxicity and passed four out of the five structural filters applied. To evaluate the importance of Ser167 and Cys129 residues in the IDO1 active site for inhibitor binding, the entire NCI library was subsequently screened against alanine-replacement mutant enzymes of these two residues. The results established that Ser167 but not Cys129 is important for inhibitory activity of a broad range of IDO1 inhibitors. Structure-activity-relationship studies proposed substituents interacting with Ser167 on four investigated IDO1 inhibitors. Three of these four Ser167 interactions associated with an increased IDO1 inhibition and were correctly predicted by molecular docking supporting Ser167 as an important mediator of potency for IDO1 inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles.

PubMed

Janciauskiene, S

2001-03-26

Serine proteinase inhibitors (Serpins) are irreversible suicide inhibitors of proteases that regulate diverse physiological processes such as coagulation, fibrinolysis, complement activation, angiogenesis, apoptosis, inflammation, neoplasia and viral pathogenesis. The molecular structure and physical properties of serpins permit these proteins to adopt a number of variant conformations under physiological conditions including the native inhibitory form and several inactive, non-inhibitory forms, such as complexes with protease or other ligands, cleaved, polymerised and oxidised. Alterations of a serpin which affect its structure and/or secretion and thus reduce its functional levels may result in pathology. Serpin dysfunction has been implicated in thrombosis, emphysema, liver cirrhosis, immune hypersensitivity and mental disorders. The loss of inhibitory activity of serpins necessarily results in an imbalance between proteases and their inhibitors, but it may also have other physiological effects through the generation of abnormal concentrations of modified, non-inhibitory forms of serpins. Although these forms of inhibitory serpins are detected in tissues and fluids recovered from inflammatory sites, the important questions of which conditions result in generation of different molecular forms of serpins, what biological function these forms have, and which of them are directly linked to pathologies and/or may be useful markers for characterisation of disease states, remain to be answered. Elucidation of the biological activities of non-inhibitory forms of serpins may provide useful insights into the pathogenesis of diseases and suggest new therapeutic strategies.

Substrate specificity determinants of human macrophage elastase (MMP-12) based on the 1.1 A crystal structure.

PubMed

Lang, R; Kocourek, A; Braun, M; Tschesche, H; Huber, R; Bode, W; Maskos, K

2001-09-28

The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP. Copyright 2001 Academic Press.

Role of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in psychological stress and depression.

PubMed

Tsai, Shih-Jen

2017-12-22

Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Furthermore, the neurotrophic hypothesis of depression postulates that compromised neurotrophin brain-derived neurotrophic factor (BDNF) function is directly involved in the pathophysiology of depression. In the brain, the proteolytic cleavage of proBDNF, a BDNF precursor, to mature BDNF through plasmin represents one mechanism that can change the direction of BDNF action. We also discuss the implications of tissue-type plasminogen activator and plasminogen activator inhibitor-1 alterations as biomarkers for major depressive disorder. Using drugs that increase tissue-type plasminogen activator or decrease plasminogen activator inhibitor-1 levels may open new avenues to develop conceptually novel therapeutic strategies for depression treatment.

A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

PubMed

De Fusco, Claudia; Brear, Paul; Iegre, Jessica; Georgiou, Kathy Hadje; Sore, Hannah F; Hyvönen, Marko; Spring, David R

2017-07-01

Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors. Copyright © 2017. Published by Elsevier Ltd.

Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease.

PubMed

Kóna, Juraj

2008-01-21

Two possible mechanisms of the irreversible inhibition of HIV-1 protease by epoxide inhibitors are investigated on an enzymatic model using ab initio (MP2) and density functional theory (DFT) methods (B3LYP, MPW1K and M05-2X). The calculations predict the inhibition as a general acid-catalyzed nucleophilic substitution reaction proceeding by a concerted SN2 mechanism with a reaction barrier of ca. 15-21 kcal mol(-1). The irreversible nature of the inhibition is characterized by a large negative reaction energy of ca. -17-(-24) kcal mol(-1). A mechanism with a direct proton transfer from an aspartic acid residue of the active site onto the epoxide ring has been shown to be preferred compared to one with the proton transfer from the acid catalyst facilitated by a bridging catalytic water molecule. Based on the geometry of the transition state, structural data important for the design of irreversible epoxide inhibitors of HIV-1 protease were defined. Here we also briefly discuss differences between the epoxide ring-opening reaction in HIV-1 protease and epoxide hydrolase, and the accuracy of the DFT method used.

Inhibition of carboxylesterase activity of THP1 monocytes/macrophages and recombinant human carboxylesterase 1 by oxysterols and fatty acids

PubMed Central

Crow, J. Allen; Herring, Katye L.; Xie, Shuqi; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2009-01-01

Summary Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC50=8.1 μM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (Kiapp=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (Kiapp=1.7 μM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo. PMID:19761868

Discovery of Highly Selective and Nanomolar Carbamate-Based Butyrylcholinesterase Inhibitors by Rational Investigation into Their Inhibition Mode.

PubMed

Sawatzky, Edgar; Wehle, Sarah; Kling, Beata; Wendrich, Jan; Bringmann, Gerhard; Sotriffer, Christoph A; Heilmann, Jörg; Decker, Michael

2016-03-10

Butyrylcholinesterase (BChE) is a promising target for the treatment of later stage cognitive decline in Alzheimer's disease. A set of pseudo-irreversible BChE inhibitors with high selectivity over hAChE was synthesized based on carbamates attached to tetrahydroquinazoline scaffolds with the 2-thiophenyl compound 2p as the most potent inhibitor of eqBChE (KC = 14.3 nM) and also of hBChE (KC = 19.7 nM). The inhibitors transfer the carbamate moiety onto the active site under release of the phenolic tetrahydroquinazoline scaffolds that themselves act as neuroprotectants. By combination of kinetic data with molecular docking studies, a plausible binding model was probed describing how the tetrahydroquinazoline scaffold guides the carbamate into a close position to the active site. The model explains the influence of the carrier scaffold onto the affinity of an inhibitor just before carbamate transfer. This strategy can be used to utilize the binding mode of other carbamate-based inhibitors.

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

PubMed Central

Garcin, Elsa D.; Arvai, Andrew S.; Rosenfeld, Robin J.; Kroeger, Matt D.; Crane, Brian R.; Andersson, Gunilla; Andrews, Glen; Hamley, Peter J.; Mallinder, Philip R.; Nicholls, David J.; St-Gallay, Stephen A.; Tinker, Alan C.; Gensmantel, Nigel P.; Mete, Antonio; Cheshire, David R.; Connolly, Stephen; Stuehr, Dennis J.; Åberg, Anders; Wallace, Alan V.; Tainer, John A.; Getzoff, Elizabeth D.

2008-01-01

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low levels, and a defensive cytotoxin at higher levels. The high active-site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock, and cancer. Our structural and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a novel specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents towards remote specificity pockets, accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active-site conservation. PMID:18849972

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cody, Vivian, E-mail: cody@hwi.buffalo.edu; University of Buffalo, Buffalo, NY 14260; Pace, Jim

The structures of mouse DHFR holo enzyme and a ternary complex with NADPH and a potent inhibitor are described. It has been shown that 2, 4-diamino-6-arylmethylpteridines and 2, 4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure–activity profile observed for a series of substituted dibenz[b, f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2, 4-diamino-6-(2′-hydroxydibenz[b,more » f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 Å resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2′-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59–64) by 0.6 Å compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.« less

Structural Analysis of ADP-Glucose Pyrophosphorylase From the Bacterium Agrobacterium Tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cupp-Vickery, J.R.; Igarashi, R.Y.; Perez, M.

2009-05-14

ADP-glucose pyrophosphorylase (ADPGlc PPase) catalyzes the conversion of glucose 1-phosphate and ATP to ADP-glucose and pyrophosphate. As a key step in glucan synthesis, the ADPGlc PPases are highly regulated by allosteric activators and inhibitors in accord with the carbon metabolism pathways of the organism. Crystals of Agrobacterium tumefaciens ADPGlc PPase were obtained using lithium sulfate as a precipitant. A complete anomalous selenomethionyl derivative X-ray diffraction data set was collected with unit cell dimensions a = 85.38 {angstrom}, b = 93.79 {angstrom}, and c = 140.29 {angstrom} ({alpha} = {beta} = {gamma} = 90{sup o}) and space group I{sub 222}. Themore » A. tumefaciens ADPGlc PPase model was refined to 2.1 {angstrom} with an R{sub factor} = 22% and R{sub free} = 26.6%. The model consists of two domains: an N-terminal {alpha}{beta}{alpha} sandwich and a C-terminal parallel {beta}-helix. ATP and glucose 1-phosphate were successfully modeled in the proposed active site, and site-directed mutagenesis of conserved glycines in this region (G20, G21, and G23) resulted in substantial loss of activity. The interface between the N- and the C-terminal domains harbors a strong sulfate-binding site, and kinetic studies revealed that sulfate is a competitive inhibitor for the allosteric activator fructose 6-phosphate. These results suggest that the interface between the N- and C-terminal domains binds the allosteric regulator, and fructose 6-phosphate was modeled into this region. The A. tumefaciens ADPGlc PPase/fructose 6-phosphate structural model along with sequence alignment analysis was used to design mutagenesis experiments to expand the activator specificity to include fructose 1,6-bisphosphate. The H379R and H379K enzymes were found to be activated by fructose 1,6-bisphosphate.« less

Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase.

PubMed

Malmquist, Nicholas A; Gujjar, Ramesh; Rathod, Pradipsinh K; Phillips, Margaret A

2008-02-26

Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is a flavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in CoQ binding and catalysis, we undertook site-directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (H185, F188, and F227) to Ala substantially decreased the affinity of pfDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the Kmapp for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kdox for CoQ. As with the mutants, inhibitors that bind this site block the CoQ-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for CoQ and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and CoQ or by stabilizing a conformation that excludes CoQ binding.

Indirubin core structure of glycogen synthase kinase-3 inhibitors as novel chemotype for intervention with 5-lipoxygenase.

PubMed

Pergola, Carlo; Gaboriaud-Kolar, Nicolas; Jestädt, Nadine; König, Stefanie; Kritsanida, Marina; Schaible, Anja M; Li, Haokun; Garscha, Ulrike; Weinigel, Christina; Barz, Dagmar; Albring, Kai F; Huber, Otmar; Skaltsounis, Alexios L; Werz, Oliver

2014-05-08

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 μM), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface*

PubMed Central

Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

2016-01-01

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. PMID:26912659

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

PubMed Central

Jaffe, Eileen K.; Shanmugam, Dhanasekaran; Gardberg, Anna; Dieterich, Shellie; Sankaran, Banumathi; Stewart, Lance J.; Myler, Peter J.; Roos, David S.

2011-01-01

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors. PMID:21383008

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

PubMed Central

Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

2014-01-01

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner. PMID:24256730

The discovery and the structural basis of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor.

PubMed

Park, Jeung Kuk; Kim, Sunmin; Han, Yu Jin; Kim, Seong Hwan; Kang, Nam Sook; Lee, Hyuk; Park, SangYoun

2016-06-01

p21-Activated kinases (PAKs) which belong to the family of ste20 serine/threonine protein kinases regulate cytoskeletal reorganization, cell motility, cell proliferation, and oncogenic transformation which are all related to the cellular functions during cancer induction and metastasis. The fact that PAK mutations are detected in multiple tumor tissues makes PAKs a novel therapeutic drug target. In this study, an imidazo[4,5-b]pyridine-based PAK4 inhibitor, KY-04045 (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine), was discovered using a virtual site-directed fragment-based drug design and was validated using an inhibition assay. Although PAK4 affinity to KY-04045 seems much weaker than that of the reported PAK4 inhibitors, the location of KY-04045 is clearly defined in the structure of PAK4 co-crystallized with KY-04045. The crystal structure illustrates that the pyrazole and imidazopyridine rings of KY-04045 are sufficient for mediating PAK4 hinge loop interaction. Hence, we believe that KY-04045 can be exploited as a basic building block in designing novel imidazo[4,5-b]pyridine-based PAK4 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reduced Aβ secretion by human neurons under conditions of strongly increased BACE activity.

PubMed

Scholz, Diana; Chernyshova, Yana; Ückert, Anna-Katharina; Leist, Marcel

2018-05-27

The initial step in the amyloidogenic cascade of amyloid precursor protein (APP) processing is catalyzed by beta-site APP-cleaving enzyme (BACE), and this protease has increased activities in affected areas of Alzheimer's disease brains. We hypothesized that altered APP processing, due to augmented BACE activity, would affect the actions of direct and indirect BACE inhibitors. We therefore compared postmitotic human neurons (LUHMES) with their BACE-overexpressing counterparts (BLUHMES). Although β-cleavage of APP was strongly increased in BLUHMES, they produced less full-length and truncated amyloid beta (Aβ) than LUHMES. Moreover, low concentrations of BACE inhibitors decreased cellular BACE activity as expected, but increased Aβ 1-40 levels. Several other approaches to modulate BACE activity led to a similar, apparently paradoxical, behavior. For instance, reduction of intracellular acidification by bepridil increased Aβ production in parallel with decreased BACE activity. In contrast to BLUHMES, the respective control cells (LUHMES or BLUHMES with catalytically inactive BACE) showed conventional pharmacological responses. Other non-canonical neurochemical responses (so-called 'rebound effects') are well-documented for the Aβ pathway, especially for γ-secretase: a partial block of its activity leads to an increased Aβ secretion by some cell types. We therefore compared LUHMES and BLUHMES regarding rebound effects of γ-secretase inhibitors and found an Aβ rise in LUHMES but not in BLUHMES. Thus, different cellular factors are responsible for the γ-secretase- vs. BACE-related Aβ rebound. We conclude that increased BACE activity, possibly accompanied by an altered cellular localization pattern, can dramatically influence Aβ generation in human neurons and affect pharmacological responses to secretase inhibitors. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Aller, Glenn S., E-mail: glenn.s.van.aller@gsk.com; Carson, Jeff D.; Tang, Wei

Research highlights: {yields} Epigallocatechin-3-gallate (EGCG) is an ATP-competitive inhibitor of PI3K and mTOR with Ki values around 300 nM. {yields} EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231and A549 cells. {yields} Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site. {yields} These results suggest another important molecular mechanism for the anticancer activities of EGCG. -- Abstract: The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The keymore » nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K{sub i} values of 380 and 320 nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.« less

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

PubMed Central

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; Akers-Rodriguez, Suzanne; Cunningham, Mark L.; Kwan, Bryan P.; Nelson, Kirk J.; Castellano, Amanda; Locke, Jeff B.; Brown-Driver, Vickie; Murphy, Timothy M.; Ong, Voon S.; Pillar, Chris M.; Shinabarger, Dean L.; Nix, Jay; Lightstone, Felice C.; Wong, Sergio E.; Nguyen, Toan B.; Shaw, Karen J.; Finn, John

2013-01-01

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models. PMID:24386374

Calmodulin is a phospholipase C-beta interacting protein.

PubMed

McCullar, Jennifer S; Larsen, Shana A; Millimaki, Ryan A; Filtz, Theresa M

2003-09-05

Phospholipase C-beta 3 (PLC beta 3) is an important effector enzyme in G protein-coupled signaling pathways. Activation of PLC beta 3 by G alpha and G beta gamma subunits has been fairly well characterized, but little is known about other protein interactions that may also regulate PLC beta 3 function. A yeast two-hybrid screen of a mouse brain cDNA library with the amino terminus of PLC beta 3 has yielded potential PLC beta 3 interacting proteins including calmodulin (CaM). Physical interaction between CaM and PLC beta 3 is supported by a positive secondary screen in yeast and the identification of a CaM binding site in the amino terminus of PLC beta 3. Co-precipitation of in vitro translated and transcribed amino- and carboxyl-terminal PLC beta 3 revealed CaM binding at a putative amino-terminal binding site. Direct physical interaction of PLC beta 3 and PLC beta 1 isoforms with CaM is supported by pull-down of both isoenzymes with CaM-Sepharose beads from 1321N1 cell lysates. CaM inhibitors reduced M1-muscarinic receptor stimulation of inositol phospholipid hydrolysis in 1321N1 astrocytoma cells consistent with a physiologic role for CaM in modulation of PLC beta activity. There was no effect of CaM kinase II inhibitors, KN-93 and KN-62, on M1-muscarinic receptor stimulation of inositol phosphate hydrolysis, consistent with a direct interaction between PLC beta isoforms and CaM.

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

PubMed

Xu, Hong-Tao; Colby-Germinario, Susan P; Hassounah, Said; Quashie, Peter K; Han, Yingshan; Oliveira, Maureen; Stranix, Brent R; Wainberg, Mark A

2016-01-01

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 μM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 μM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal, R.; Schmidt, J; Stafford, R

2009-01-01

Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exositesmore » away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.« less

Quantum mechanics/molecular mechanics modeling of covalent addition between EGFR-cysteine 797 and N-(4-anilinoquinazolin-6-yl) acrylamide.

PubMed

Capoferri, Luigi; Lodola, Alessio; Rivara, Silvia; Mor, Marco

2015-03-23

Irreversible epidermal growth factor receptor (EGFR) inhibitors can circumvent resistance to first-generation ATP-competitive inhibitors in the treatment of nonsmall-cell lung cancer. They covalently bind a noncatalytic cysteine (Cys797) at the surface of EGFR active site by an acrylamide warhead. Herein, we used a hybrid quantum mechanics/molecular mechanics (QM/MM) potential in combination with umbrella sampling in the path-collective variable space to investigate the mechanism of alkylation of Cys797 by the prototypical covalent inhibitor N-(4-anilinoquinazolin-6-yl) acrylamide. Calculations show that Cys797 reacts with the acrylamide group of the inhibitor through a direct addition mechanism, with Asp800 acting as a general base/general acid in distinct steps of the reaction. The obtained reaction free energy is negative (ΔA = -12 kcal/mol) consistent with the spontaneous and irreversible alkylation of Cys797 by N-(4-anilinoquinazolin-6-yl) acrylamide. Our calculations identify desolvation of Cys797 thiolate anion as a key step of the alkylation process, indicating that changes in the intrinsic reactivity of the acrylamide would have only a minor impact on the inhibitor potency.

Inhibitors Selective for Mycobacterial Versus Human Proteasomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, G.; Li, D; Sorio de Carvalho, L

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-onemore » compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.« less

Structural Determinants of the 5′-Methylthioinosine Specificity of Plasmodium Purine Nucleoside Phosphorylase

PubMed Central

Donaldson, Teraya M.; Ting, Li-Min; Zhan, Chenyang; Shi, Wuxian; Zheng, Renjian; Almo, Steven C.; Kim, Kami

2014-01-01

Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5′-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5′-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5′-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5′-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket. PMID:24416224

Interdependence of Inhibitor Recognition in HIV-1 Protease

PubMed Central

2017-01-01

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1′ and S2′ subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1′ subsite highly influences other subsites: the extension of the hydrophobic P1′ moiety results in 1) reduced van der Waals contacts in the P2′ subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor. PMID:28358514

Interdependence of Inhibitor Recognition in HIV-1 Protease.

PubMed

Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-09

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

NASA Astrophysics Data System (ADS)

Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

2014-07-01

Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.

Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi

2008-04-02

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) againstmore » hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.« less

Mechanism of MenE Inhibition by Acyl-Adenylate Analogues and Discovery of Novel Antibacterial Agents

PubMed Central

Sharma, Indrajeet; Lavaud, Lubens J.; Ngo, Stephen C.; Shek, Roger; Rajashankar, Kanagalaghatta R.; French, Jarrod B.; Tan, Derek S.; Tonge, Peter J.

2015-01-01

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1) which has an IC50 value of ≤ 25 nM for the Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in S. aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ~1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure–activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto-acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively-charged keto-acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future. PMID:26394156

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

PubMed

Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

2015-10-27

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations.

PubMed

De Simone, Giuseppina; Langella, Emma; Esposito, Davide; Supuran, Claudiu T; Monti, Simona Maria; Winum, Jean-Yves; Alterio, Vincenzo

2017-12-01

Sulphamate and sulphamide derivatives have been largely investigated as carbonic anhydrase inhibitors (CAIs) by means of different experimental techniques. However, the structural determinants responsible for their different binding mode to the enzyme active site were not clearly defined so far. In this paper, we report the X-ray crystal structure of hCA II in complex with a sulphamate inhibitor incorporating a nitroimidazole moiety. The comparison with the structure of hCA II in complex with its sulphamide analogue revealed that the two inhibitors adopt a completely different binding mode within the hCA II active site. Starting from these results, we performed a theoretical study on sulphamate and sulphamide derivatives, demonstrating that electrostatic interactions with residues within the enzyme active site play a key role in determining their binding conformation. These findings open new perspectives in the design of effective CAIs using the sulphamate and sulphamide zinc binding groups as lead compounds.

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containingmore » the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less

Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

DOE PAGES

Song, Min-Suk; Kumar, Gyanendra; Shadrick, William R.; ...

2016-03-14

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. Furthermore, these mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containingmore » the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. When using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ratia, Kiira; Pegan, Scott; Takayama, Jun

We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC{sub 50} value of 20 {mu}M, which was improved to 600 nM via synthetic optimization.more » The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC{sub 50} of 15 {mu}M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.« less

Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

PubMed Central

Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

1994-01-01

Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

Computational insights into the interaction of small molecule inhibitors with HRI kinase domain.

PubMed

Palrecha, Sourabh; Lakade, Dushant; Kulkarni, Abhijeet; Pal, Jayanta K; Joshi, Manali

2018-05-07

The Heme-Regulated Inhibitor (HRI) kinase regulates globin synthesis in a heme-dependent manner in reticulocytes and erythroid cells in bone marrow. Inhibitors of HRI have been proposed to lead to an increased amount of haemoglobin, benefitting anaemia patients. A series of indeno[1,2-c]pyrazoles were discovered to be the first known in vitro inhibitors of HRI. However, the structural mechanism of inhibition is yet to be understood. The aim of this study was to unravel the binding mechanism of these inhibitors using molecular dynamic simulations and docking. The docking scores were observed to correlate well with experimentally determined pIC 50 values. The inhibitors were observed to bind in the ATP-binding site forming hydrogen bonds with the hinge region and van der Waals interactions with non-polar residues in the binding site. Further, quantitative structure-activity relationship (QSAR) studies were performed to correlate the structural features of the inhibitors with their biological activity. The developed QSAR models were found to be statistically significant in terms of internal and external predictabilities. The presence of chlorine atoms and the hydroxymethyl groups were found to correlate with higher activity. The identified binding modes and the descriptors can support future rational identification of more potent and selective small molecule inhibitors for this kinase which are of therapeutic importance in the context of various human pathological disorders.

Novel human D-amino acid oxidase inhibitors stabilize an active-site lid-open conformation

PubMed Central

Terry-Lorenzo, Ryan T.; Chun, Lawrence E.; Brown, Scott P.; Heffernan, Michele L. R.; Fang, Q. Kevin; Orsini, Michael A.; Pollegioni, Loredano; Hardy, Larry W.; Spear, Kerry L.; Large, Thomas H.

2014-01-01

The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors. PMID:25001371

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cody, V.; Pace, J.; Rosowsky, A.

It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2{prime}-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 A resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed nomore » electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2{prime}-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6 A compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.« less

[Adenylate cyclase from rabbit heart: substrate binding site].

PubMed

Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

1981-08-01

The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

[Insect cholinesterases and irreversible inhibitors. Statistical treatment of the data].

PubMed

Moralev, S N

2010-01-01

The data on sensitivity of cholinesterases (ChE) of different insects to reversible inhibitors, as well as the data on physico-chemical parameters of amino acids constituting their active centers, were treated by factor analysis and juxtaposed. It is shown that both these characteristics are related to taxonomical belonging of insects. It is revealed the "material substrate" of the factors determining inhibitor action specificity, which are specific sites in ChE active center.

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain*S⃞

PubMed Central

Bottomley, Matthew J.; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-01-01

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions. PMID:18614528

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

PubMed

Bottomley, Matthew J; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-09-26

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors.

PubMed

Shimamoto, Yasuhiro; Hattori, Yasunao; Kobayashi, Kazuya; Teruya, Kenta; Sanjoh, Akira; Nakagawa, Atsushi; Yamashita, Eiki; Akaji, Kenichi

2015-02-15

The design and evaluation of a novel decahydroisoquinolin scaffold as an inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro)) are described. Focusing on hydrophobic interactions at the S2 site, the decahydroisoquinolin scaffold was designed by connecting the P2 site cyclohexyl group of the substrate-based inhibitor to the main-chain at the α-nitrogen atom of the P2 position via a methylene linker. Starting from a cyclohexene enantiomer obtained by salt resolution, trans-decahydroisoquinolin derivatives were synthesized. All decahydroisoquinolin inhibitors synthesized showed moderate but clear inhibitory activities for SARS 3CL(pro), which confirmed the fused ring structure of the decahydroisoquinolin functions as a novel scaffold for SARS 3CL(pro) inhibitor. X-ray crystallographic analyses of the SARS 3CL(pro) in a complex with the decahydroisoquinolin inhibitor revealed the expected interactions at the S1 and S2 sites, as well as additional interactions at the N-substituent of the inhibitor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Drug Resistance Mechanism of L10F, L10F/N88S and L90M mutations in CRF01_AE HIV-1 protease: Molecular dynamics simulations and binding free energy calculations.

PubMed

Vasavi, C S; Tamizhselvi, Ramasamy; Munusami, Punnagai

2017-08-01

HIV-1 protease plays a crucial role in viral replication and maturation, which makes it one of the most attractive targets for anti-retroviral therapy. The majority of HIV infections in developing countries are due to non-B subtype. Subtype AE is spreading rapidly and infecting huge population worldwide. The mutations in the active site of subtype AE directly impair the interactions with the inhibitor. The non-active site mutations influence the binding of the inhibitor indirectly and their resistance mechanism is not well understood. It is important to design new effective inhibitors that combat drug resistance in subtype AE protease. In this work, we examined the effect of non active site mutations L10F, L10F/N88S and L90M with nelfinavir using molecular dynamics simulation and binding free energy calculations. The simulations suggested that the L10F and L10F/N88S mutants decrease the binding affinity of nelfinavir, whereas the L90M mutant increases the binding affinity. The formation of hydrogen bonds between nelfinavir and Asp30 is crucial for effective binding. The benzamide moiety of nelfinavir shows large positional deviation in L10F and L10F/N88S complexes and the L10F/N88S mutation changes the hydrogen bond between the side chain atoms of 30th residue and the 88th residue. Consequently the hydrogen bond interaction between Asp30 and nelfinavir are destroyed leading to drug resistance. Our present study shed light on the resistance mechanism of the strongly linked mutation L10F/N88S observed experimentally in AE subtype. Copyright © 2017 Elsevier Inc. All rights reserved.

Accelerated Disassembly of IgE:Receptor Complexes by a Disruptive Macromolecular Inhibitor

PubMed Central

Kim, Beomkyu; Eggel, Alexander; Tarchevskaya, Svetlana S.; Vogel, Monique; Prinz, Heino; Jardetzky, Theodore S.

2012-01-01

IgE antibodies bind the high affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE:FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE:FcεRI complexes that prime cells prior to allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6–8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_799–11, acts through a non-classical inhibition mechanism, not only blocking IgE:FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79:IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE:FcεRI complex, with Site 1 distant from the receptor and Site 2 exhibiting partial steric overlap. While the structure is suggestive of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modeling indicate that E2_79 acts through a facilitated dissociation mechanism at Site 2 alone. These results demonstrate that high affinity IgE:FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein:protein complexes may be more generally amenable to active disruption by macromolecular inhibitors. PMID:23103871

Design and synthesis of dual inhibitors of acetylcholinesterase and serotonin transporter targeting potential agents for Alzheimer's disease.

PubMed

Kogen, Hiroshi; Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio

2002-10-03

Highly efficient acetylcholinesterase (AChE) and serotonin transporter (SERT) dual inhibitors, (S)-4 and (R)-13 were designed and synthesized on the basis of the hypothetical model of AChE active site. Both compounds showed potent inhibitory activities against AChE and SERT. [structure: see text

Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

PubMed Central

Conner, Kip P.; Cruce, Alex A.; Krzyaniak, Matthew D.; Schimpf, Alina M.; Frank, Daniel J.; de Montellano, Paul Ortiz; Atkins, William M.; Bowman, Michael K.

2015-01-01

Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP–inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenyl-propyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of “classic” type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A 15N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution. PMID:25591012

Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms.

PubMed

Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

2015-01-01

Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ.

Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms

PubMed Central

Sehgal, Sheikh Arslan; Mannan, Shazia; Kanwal, Sumaira; Naveed, Ishrat; Mir, Asif

2015-01-01

Schizophrenia (SZ), a chronic mental and heritable disorder characterized by neurophysiological impairment and neuropsychological abnormalities, is strongly associated with D-amino acid oxidase activator (DAOA, G72). Research studies emphasized that overexpression of DAOA may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like SZ. In the present study, a hybrid approach of comparative modeling and molecular docking followed by inhibitor identification and structure modeling was employed. Screening was performed by two-dimensional similarity search against selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Here, we report an inhibitor compound which showed maximum binding affinity against four selected isoforms of DAOA. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 are critical residues for receptor–ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. We propose that selected inhibitor might be more potent on the basis of binding energy values. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful in designing novel therapeutic targets to cure SZ. PMID:26170631

SbnG, a citrate synthase in Staphylococcus aureus: A new fold on an old enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. In this paper, we present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic genemore » clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. Finally, a structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production.« less

SbnG, a citrate synthase in Staphylococcus aureus: A new fold on an old enzyme

DOE PAGES

Kobylarz, Marek J.; Grigg, Jason C.; Sheldon, Jessica R.; ...

2014-10-21

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. In this paper, we present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic genemore » clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. Finally, a structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production.« less

SbnG, a citrate synthase in Staphylococcus aureus: a new fold on an old enzyme.

PubMed

Kobylarz, Marek J; Grigg, Jason C; Sheldon, Jessica R; Heinrichs, David E; Murphy, Michael E P

2014-12-05

In response to iron deprivation, Staphylococcus aureus produces staphyloferrin B, a citrate-containing siderophore that delivers iron back to the cell. This bacterium also possesses a second citrate synthase, SbnG, that is necessary for supplying citrate to the staphyloferrin B biosynthetic pathway. We present the structure of SbnG bound to the inhibitor calcium and an active site variant in complex with oxaloacetate. The overall fold of SbnG is structurally distinct from TCA cycle citrate synthases yet similar to metal-dependent class II aldolases. Phylogenetic analyses revealed that SbnG forms a separate clade with homologs from other siderophore biosynthetic gene clusters and is representative of a metal-independent subgroup in the phosphoenolpyruvate/pyruvate domain superfamily. A structural superposition of the SbnG active site to TCA cycle citrate synthases and site-directed mutagenesis suggests a case for convergent evolution toward a conserved catalytic mechanism for citrate production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study.

PubMed

Miled, Nabil; Roussel, Alain; Bussetta, Cécile; Berti-Dupuis, Liliane; Rivière, Mireille; Buono, Gérard; Verger, Robert; Cambillau, Christian; Canaan, Stéphane

2003-10-14

The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.

Protein kinase C epsilon mediates the inhibition of angiotensin II on the slowly activating delayed-rectifier potassium current through channel phosphorylation.

PubMed

Gou, Xiangbo; Wang, Wenying; Zou, Sihao; Qi, Yajuan; Xu, Yanfang

2018-03-01

The slowly activating delayed rectifier K + current (I Ks ) is one of the main repolarizing currents in the human heart. Evidence has shown that angiotensin II (Ang II) regulates I Ks through the protein kinase C (PKC) pathway, but the related results are controversial. This study was designed to identify PKC isoenzymes involved in the regulation of I Ks by Ang II and the underlying molecular mechanism. The whole-cell patch-clamp technique was used to record I Ks in isolated guinea pig ventricular cardiomyocytes and in human embryonic kidney (HEK) 293 cells co-transfected with human KCNQ1/KCNE1 genes and Ang II type 1 receptor genes. Ang II inhibited I Ks in a concentration-dependent manner in native cardiomyocytes. A broad PKC inhibitor Gö6983 (not inhibiting PKCε) and a selective cPKC inhibitor Gö6976 did not affect the inhibitory action of Ang II. In contrast, the inhibition was significantly attenuated by PKCε-selective peptide inhibitor εV1-2. However, direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) increased the cloned human I Ks in HEK293 cells. Similarly, the cPKC peptide activator significantly enhanced the current. In contrast, the PKCε peptide activator inhibited the current. Further evidence showed that PKCε knockdown by siRNA antagonized the Ang II-induced inhibition on KCNQ1/KCNE1 current, whereas knockdown of cPKCs (PKCα and PKCβ) attenuated the potentiation of the current by PMA. Moreover, deletion of four putative phosphorylation sites in the C-terminus of KCNQ1 abolished the action of PMA. Mutation of two putative phosphorylation sites in the N-terminus of KCNQ1 and one site in KCNE1 (S102) blocked the inhibition of Ang II. Our results demonstrate that PKCε isoenzyme mediates the inhibitory action of Ang II on I Ks and by phosphorylating distinct sites in KCNQ1/KCNE1, cPKC and PKCε isoenzymes produce the contrary regulatory effects on the channel. These findings have provided new insight into the molecular mechanism underlying the modulation of the KCNQ1/KCNE1 channel. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

DOE PAGES

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; ...

2013-12-26

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

PubMed

Oyama, Takahiro; Yoshimori, Atsushi; Takahashi, Satoshi; Yamamoto, Tetsuya; Sato, Akira; Kamiya, Takanori; Abe, Hideaki; Abe, Takehiko; Tanuma, Sei-Ichi

2017-07-01

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibition of tyrosinase by 4H-chromene analogs: Synthesis, kinetic studies, and computational analysis.

PubMed

Brasil, Edikarlos M; Canavieira, Luciana M; Cardoso, Érica T C; Silva, Edilene O; Lameira, Jerônimo; Nascimento, José L M; Eifler-Lima, Vera L; Macchi, Barbarella M; Sriram, Dharmarajan; Bernhardt, Paul V; Silva, José Rogério Araújo; Williams, Craig M; Alves, Cláudio N

2017-11-01

Inhibition of mushroom tyrosinase was observed with synthetic dihydropyrano[3,2-b]chromenediones. Among them, DHPC04 displayed the most potent tyrosinase inhibitory activity with a K i value of 4 μm, comparable to the reference standard inhibitor kojic acid. A kinetic study suggested that these synthetic heterocyclic compounds behave as competitive inhibitors for the L-DOPA binding site of the enzyme. Furthermore, molecular modeling provided important insight into the mechanism of binding interactions with the tyrosinase copper active site. © 2017 John Wiley & Sons A/S.

Lipid Sulfates and Sulfonates Are Allosteric Competitive Inhibitors of the N-Terminal Phosphatase Activity of the Mammalian Soluble Epoxide Hydrolase†

PubMed Central

Tran, Katherine L.; Aronov, Pavel A.; Tanaka, Hiromasa; Newman, John W.; Hammock, Bruce D.; Morisseau, Christophe

2006-01-01

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with KI in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH. PMID:16142916

Thoughts on the current assessment of Polo-like kinase inhibitor drug discovery.

PubMed

Strebhardt, Klaus; Becker, Sven; Matthess, Yves

2015-01-01

The Polo-like kinase 1 (Plk1) plays a key role in regulating a broad spectrum of critical cell cycle events. Plk1 is a marker of cellular proliferation and has prognostic potential in different types of human tumors. In a series of preclinical studies, Plk1 has been validated as a cancer target. This prompted many pharmaceutical companies to develop small-molecule inhibitors targeting the classical ATP-binding site of Plk1 for anticancer drug development. Recently, FDA has granted a Breakthrough Therapy designation to the Plk inhibitor BI 6727 (volasertib), which provided a survival benefit for patients suffering from acute myeloid leukemia. Remarkably, a new generation of Plk1 inhibitors that target the second druggable domain of Plk1, the Polo-box domain, is currently being tested preclinically. Since various ATP-competitive compounds of Plk1 inhibit also the activities of Plk2 and Plk3, which act as tumor suppressors, the roles of closely related Plk-family members in cancer cells need to be considered carefully. In this article, the authors highlight recent insights into the biology of Plks in cancer cells and discuss the progress in the development of small-molecule Plk1 inhibitors. The authors believe that the greatest therapeutic benefit might come through leukemic cells that are in direct contact with the inhibitor in the blood stream. The identification of biomarkers and studies that document Plk activities in treated patients would also be beneficial to better understand the role of Plk inhibition in tumor development and anticancer therapy.

Crystal Structure Analyses of the Fosmidomycin-Target Enzyme from Plasmodium Falciparum

NASA Astrophysics Data System (ADS)

Umeda, Tomonobu; Kusakabe, Yoshio; Tanaka, Nobutada

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. Fosmidomycin has proved to be efficient in the treatment of P. falciparum malaria through the inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway of isoprenoid biosynthesis, which is absent in humans. Crystal structure analyses of P. falciparum DXR (PfDXR) revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for the induced-fit movement to accommodate the bound inhibitor in the active site, and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We believe that our study will serve as a useful guide to develop more potent PfDXR inhibitors.

The xylanase inhibitor TAXI-III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants.

PubMed

Moscetti, Ilaria; Faoro, Franco; Moro, Stefano; Sabbadin, Davide; Sella, Luca; Favaron, Francesco; D'Ovidio, Renato

2015-08-01

The xylanase inhibitor TAXI-III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI-III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre-incubation of xylanase FGSG_03624 with TAXI-III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non-transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI-III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI-III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI-III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI-III to avoid host cell death. © 2014 BSPP AND JOHN WILEY & SONS LTD.

SKLB060 Reversibly Binds to Colchicine Site of Tubulin and Possesses Efficacy in Multidrug-Resistant Cell Lines.

PubMed

Yan, Wei; Yang, Tao; Yang, Jianhong; Wang, Taijin; Yu, Yamei; Wang, Yuxi; Chen, Qiang; Bai, Peng; Li, Dan; Ye, Haoyu; Qiu, Qiang; Zhou, Yongzhao; Hu, Yiguo; Yang, Shengyong; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2018-05-22

Many tubulin inhibitors are in clinical use as anti-cancer drugs. In our previous study, a novel series of 4-substituted coumarins derivatives were identified as novel tubulin inhibitors. Here, we report the anti-cancer activity and underlying mechanism of one of the 4-substituted coumarins derivatives (SKLB060). The anti-cancer activity of SKLB060 was tested on 13 different cancer cell lines and four xenograft cancer models. Immunofluorescence staining, cell cycle analysis, and tubulin polymerization assay were employed to study the inhibition of tubulin. N, N '-Ethylenebis(iodoacetamide) assay was used to measure binding to the colchicine site. Wound-healing migration and tube formation assays were performed on human umbilical vascular endothelial cells to study anti-vascular activity (the ability to inhibit blood vessel growth). Mitotic block reversibility and structural biology assays were used to investigate the SKLB060-tubulin bound model. SKLB060 inhibited tubulin polymerization and subsequently induced G2/M cell cycle arrest and apoptosis in cancer cells. SKLB060 bound to the colchicine site of β-tubulin and showed antivascular activity in vitro. Moreover, SKLB060 induced reversible cell cycle arrest and reversible inhibition of tubulin polymerization. A mitotic block reversibility assay showed that the effects of SKLB060 have greater reversibility than those of colcemid (a reversible tubulin inhibitor), indicating that SKLB060 binds to tubulin in a totally reversible manner. The crystal structures of SKLB060-tubulin complexes confirmed that SKLB060 binds to the colchicine site, and the natural coumarin ring in SKLB060 enables reversible binding. These results reveal that SKLB060 is a powerful and reversible microtubule inhibitor that binds to the colchicine site and is effective in multidrug-resistant cell lines. © 2018 The Author(s). Published by S. Karger AG, Basel.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

PubMed

Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

2018-02-15

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Novel tetrahydrocarbazole benzyl pyridine hybrids as potent and selective butryl cholinesterase inhibitors with neuroprotective and β-secretase inhibition activities.

PubMed

Ghobadian, Roshanak; Mahdavi, Mohammad; Nadri, Hamid; Moradi, Alireza; Edraki, Najmeh; Akbarzadeh, Tahmineh; Sharifzadeh, Mohammad; Bukhari, Syed Nasir Abbas; Amini, Mohsen

2018-05-23

Butyrylcholinesterase (BuChE) inhibitors have become interesting target for treatment of Alzheimer's disease (AD). A series of dual binding site BuChE inhibitors were designed and synthesized based on 2,3,4,9-tetrahydro-1H-carbazole attached benzyl pyridine moieties. In-vitro assay revealed that all of the designed compounds were selective and potent BuChE inhibitors. The most potent BuChE inhibitor was compound 6i (IC 50  = 0.088 ± 0.0009 μM) with the mixed-type inhibition. Docking study revealed that 6i is a dual binding site BuChE inhibitor. Also, Pharmacokinetic properties for 6i were accurate to Lipinski's rule. In addition, compound 6i demonstrated neuroprotective and β-secretase (BACE1) inhibition activities. This compound could also inhibit AChE-induced and self-induced Aβ peptide aggregation at concentration of 100 μM and 10 μM respectively. Generally, the results are presented as new potent selective BuChE inhibitors with a therapeutic potential for the treatment of AD. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

PubMed Central

Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

2006-01-01

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

A New Covalent Inhibitor of Class C β-Lactamases Reveals Extended Active Site Specificity.

PubMed

Tilvawala, Ronak; Cammarata, Michael; Adediran, S A; Brodbelt, Jennifer S; Pratt, R F

2015-12-22

O-Aryloxycarbonyl hydroxamates have previously been shown to efficiently inactivate class C β-lactamases by cross-linking serine and lysine residues in the active site. A new analogue of these inhibitors, D-(R)-O-(phenoxycarbonyl)-N-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine, designed to inactivate certain low-molecular mass dd-peptidases, has now been synthesized. Although the new molecule was found to be only a poor inactivator of the latter enzymes, it proved, unexpectedly, to be a very effective inactivator (ki = 3.5 × 10(4) M(-1) s(-1)) of class C β-lactamases, more so than the original lead compound, O-phenoxycarbonyl-N-(benzyloxycarbonyl)hydroxylamine. Furthermore, the mechanism of inactivation is different. Mass spectrometry demonstrated that β-lactamase inactivation by the new molecule involved formation of an O-alkoxycarbonylhydroxamate with the nucleophilic active site serine residue. This acyl-enzyme did not cyclize to cross-link the active site as did that from the lead compound. Model building suggested that the rapid enzyme acylation by the new molecule may occur because of favorable interaction between the polar terminus of its side chain and elements of the Ω loop that abuts the active site, Arg 204 in particular. This interaction should be considered in the design of new covalent β-lactamase inhibitors. The initially formed acyl-enzyme partitions (ratio of ∼ 1) between hydrolysis, which regenerates the active enzyme, and formation of an inert second acyl-enzyme. Structural modeling suggests that the latter intermediate arises from conformational movement of the acyl group away from the reaction center, probably enforced by the inflexibility of the acyl group. The new molecule is thus a mechanism-based inhibitor in which an inert complex is formed by noncovalent rearrangement. Phosphyl analogues of the new molecule were efficient inactivators of neither dd-peptidases nor β-lactamases.

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

PubMed

Brzoska, Tomasz; Suzuki, Yuko; Sano, Hideto; Suzuki, Seiichirou; Tomczyk, Martyna; Tanaka, Hiroki; Urano, Tetsumei

2017-04-03

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

PL-100, a novel HIV-1 protease inhibitor displaying a high genetic barrier to resistance: an in vitro selection study.

PubMed

Dandache, Serge; Coburn, Craig A; Oliveira, Maureen; Allison, Timothy J; Holloway, M Katharine; Wu, Jinzi J; Stranix, Brent R; Panchal, Chandra; Wainberg, Mark A; Vacca, Joseph P

2008-12-01

The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.

Key binding and susceptibility of NS3/4A serine protease inhibitors against hepatitis C virus.

PubMed

Meeprasert, Arthitaya; Hannongbua, Supot; Rungrotmongkol, Thanyada

2014-04-28

Hepatitis C virus (HCV) causes an infectious disease that manifests itself as liver inflammation, cirrhosis, and can lead to the development of liver cancer. Its NS3/4A serine protease is a potent target for drug design and development since it is responsible for cleavage of the scissile peptide bonds in the polyprotein important for the HCV life cycle. Herein, the ligand-target interactions and the binding free energy of the four current NS3/4A inhibitors (boceprevir, telaprevir, danoprevir, and BI201335) were investigated by all-atom molecular dynamics simulations with three different initial atomic velocities. The per-residue free energy decomposition suggests that the key residues involved in inhibitor binding were residues 41-43, 57, 81, 136-139, 155-159, and 168 in the NS3 domain. The van der Waals interactions yielded the main driving force for inhibitor binding at the protease active site for the cleavage reaction. In addition, the highest number of hydrogen bonds was formed at the reactive P1 site of the four studied inhibitors. Although the hydrogen bond patterns of these inhibitors were different, their P3 site was most likely to be recognized by the A157 backbone. Both molecular mechanic (MM)/Poisson-Boltzmann surface area and MM/generalized Born surface area approaches predicted the relative binding affinities of the four inhibitors in a somewhat similar trend to their experimentally derived biological activities.

Identification of novel inhibitors of human Chk1 using pharmacophore-based virtual screening and their evaluation as potential anti-cancer agents

NASA Astrophysics Data System (ADS)

Kumar, Vikash; Khan, Saman; Gupta, Priyanka; Rastogi, Namrata; Mishra, Durga Prasad; Ahmed, Shakil; Siddiqi, Mohammad Imran

2014-12-01

Kinases are one of the major players in cancer development and progression. Serine threonine kinases such as human checkpoint kinase-1 (Chk1), Mek1 and cyclin-dependent kinases have been identified as promising targets for cancer treatment. Chk1 is an important kinase with vital role in cell cycle arrest and many potent inhibitors targeted to Chk1 have been reported and few are currently in clinical trials. Considering the emerging importance of Chk1 inhibitors in cancer treatment there is a need to widen the chemical space of Chk1 inhibitors. In this study, we are reporting an integrated in silico approach to identify novel competitive Chk1 inhibitors. A 4-features pharmacophore model was derived from a co-crystallized structure of known potent Chk1 inhibitor and subjected to screen Maybridge compound library. Hits obtained from the screening were docked into the Chk1 active site and filtered on the basis of docking score and the number of pharmacophoric features showing conserved interaction within the active site of Chk1. Further, five compounds from the top ranking hits were subjected to in vitro evaluation as Chk1 inhibitor. After the kinase assay, four compounds were found to be active against human Chk1 (IC50 range from 4.2 to 12.5 µM). Subsequent study using the cdc25-22 mutant yeast cells revealed that one of compound (SPB07479; IC50 = 4.24 µM) promoted the formation of multinucleated cells, therefore overriding the cell cycle checkpoint. Validation studies using normal and human cancer cell lines, indicated that SPB07479 significantly inhibited proliferation of cervical cancer cells as a single agent and chemosensitized glioma and pancreatic cancer cell lines to standard chemotherapy while sparing normal cells. Additionally SPB07479 did not show significant cytotoxicity in normal cells. In conclusion we report that SPB07479 appear promising for further development of Chk1 inhibitors. This study also highlights the role of conserved water molecules in the active site of Chk1 for the successful identification of novel inhibitors.

Inhibition of Mammalian 15-Lipoxygenase by Three Ebselen-like Drugs. A QM/MM and MM/PBSA Comparative Study.

PubMed

Cebrián-Prats, Anna; Rovira, Tiffani; Saura, Patricia; González-Lafont, Àngels; Lluch, José M

2017-12-28

Ebselen is a potent competitive inhibitor of the active form of rabbit 15-lipoxygenase, an enzyme involved in many inflammatory diseases. Light-induced Z-to-E isomerization of the ebselen-like 2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester (BODTCM) molecule was used to convert the weak (Z)-BOTDCM inhibitor into the (E)-isomer with much higher inhibitory capacity. In this study, the binding modes of ebselen, (E)-BOTDCM and (Z)-BOTDCM, have been analyzed to provide molecular insights on the inhibitory potency of ebselen and on the geometric-isomer specificity of (E)- and (Z)-BOTDCM inhibitors. The inhibitor-enzyme structures obtained from docking and molecular dynamics simulations as well as from QM/MM calculations show that the inhibitor molecules are not coordinated to the nonheme iron in the active site. Thermal motion allows ebselen and (E)-BOTDCM to visit a wide range of the configurational space competing with the polyunsaturated fatty acid for binding at the active site. Both molecules present similar MM/PBSA binding free energies. The energy penalty for the bigger geometric deformation undergone by (E)-BODTCM would explain its lower inhibitor potency. The (Z)-isomer is the weakest inhibitor because thermal motion moves it to a region very far from the first coordination sphere of Fe, where it could not compete with the fatty acid substrate.

Nanoscale liposomal formulation of a SYK P-site inhibitor against B-precursor leukemia

PubMed Central

Qazi, Sanjive; Cely, Ingrid; Sahin, Kazim; Shahidzadeh, Anoush; Ozercan, Ibrahim; Yin, Qian; Gaynon, Paul; Termuhlen, Amanda; Cheng, Jianjun

2013-01-01

We report preclinical proof of principle for effective treatment of B-precursor acute lymphoblastic leukemia (ALL) by targeting the spleen tyrosine kinase (SYK)–dependent antiapoptotic blast cell survival machinery with a unique nanoscale pharmaceutical composition. This nanoscale liposomal formulation (NLF) contains the pentapeptide mimic 1,4-Bis (9-O dihydroquinidinyl) phthalazine/hydroquinidine 1,4-phathalazinediyl diether (C61) as the first and only selective inhibitor of the substrate binding P-site of SYK. The C61 NLF exhibited a very favorable pharmacokinetic and safety profile in mice, induced apoptosis in primary B-precursor ALL blast cells taken directly from patients as well as in vivo clonogenic ALL xenograft cells, destroyed the in vivo clonogenic fraction of ALL blast cells, and, at nontoxic dose levels, exhibited potent in vivo antileukemic activity against patient-derived ALL cells in xenograft models of aggressive B-precursor ALL. Our findings establish SYK as an attractive molecular target for therapy of B-precursor ALL. Further development of the C61 NLF may provide the foundation for therapeutic innovation against therapy-refractory B-precursor ALL. PMID:23568490

Interaction between wheat alpha-amylase/trypsin bi-functional inhibitor and mammalian digestive enzymes: Kinetic, equilibrium and structural characterization of binding.

PubMed

Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Ali, Ishtiaq; Bonfili, Laura; Cecarini, Valentina; Eleuteri, Anna Maria; Angeletti, Mauro

2016-12-15

Alpha-amylase/trypsin bi-functional inhibitors (ATIs) are non-gluten protein components of wheat and other cereals that can hypersensitise the human gastrointestinal tract, eventually causing enteropathies in predisposed individuals. These inhibitory proteins can act both directly by targeting specific pro-inflammatory receptors, and indirectly by impairing the activity of digestive enzymes, the latter event causing the accumulation of undigested peptides with potential immunogenic properties. Herein, according to a concerted approach based on in vitro and in silico methods we characterized kinetics, equilibrium parameters and modes of binding of the complexes formed between wheat ATI and two representative mammalian digestive enzymes, namely trypsin and alpha-amylase. Interestingly, we demonstrated ATI to target both enzymes with independent binding sites and with moderately high affinity. Copyright © 2016 Elsevier Ltd. All rights reserved.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, J. Richard; Dunham, Steve; Mochalkin, Igor

2009-06-25

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidinesmore » target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.« less

Discovery of Allosteric and Selective Inhibitors of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

PubMed

Pang, Allan H; Garzan, Atefeh; Larsen, Martha J; McQuade, Thomas J; Garneau-Tsodikova, Sylvie; Tsodikov, Oleg V

2016-11-18

Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PP i ), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-s-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically Mycobacterium tuberculosis (Mtb) PPiase (MtPPiase). The best analogue (compound 16) with a K i of 11 μM for MtPPiase is a species-specific inhibitor. Crystal structures of MtPPiase in complex with the lead compound and one of its analogues (compound 6) demonstrate that the inhibitors bind in a nonconserved interface between monomers of the hexameric MtPPiase in a yet unprecedented pairwise manner, while the remote conserved active site of the enzyme is occupied by a bound PP i substrate. Consistent with the structural studies, the kinetic analysis of the most potent inhibitor has indicated that it functions uncompetitively, by binding to the enzyme-substrate complex. The inhibitors appear to allosterically lock the active site in a closed state causing its dysfunctionalization and blocking the hydrolysis. These inhibitors are the first examples of allosteric, species-selective inhibitors of PPiases, serving as a proof-of-principle that PPiases can be selectively targeted.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

PubMed

Halasi, Marianna; Wang, Ming; Chavan, Tanmay S; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L

2013-09-01

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5*

PubMed Central

Liu, Liqiong; Richard, Jessica; Kim, Sunyoung; Wojcik, Edward J.

2014-01-01

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease. PMID:24737313

Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

PubMed Central

Verespy III, Stephen; Mehta, Akul Y.; Afosah, Daniel; Al-Horani, Rami A.; Desai, Umesh R.

2016-01-01

Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

Carbonic anhydrase inhibitors. Comparison of chlorthalidone, indapamide, trichloromethiazide, and furosemide X-ray crystal structures in adducts with isozyme II, when several water molecules make the difference.

PubMed

Temperini, Claudia; Cecchi, Alessandro; Scozzafava, Andrea; Supuran, Claudiu T

2009-02-01

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K(I)s of 65-138 nM), whereas indapamide is a much weaker one (K(I) of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2-3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.

Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans.

PubMed

Díaz-Sánchez, Ángel Gabriel; Alvarez-Parrilla, Emilio; Martínez-Martínez, Alejandro; Aguirre-Reyes, Luis; Orozpe-Olvera, Jesica Aline; Ramos-Soto, Miguel Armando; Núñez-Gastélum, José Alberto; Alvarado-Tenorio, Bonifacio; de la Rosa, Laura Alejandra

2016-11-26

Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2'-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a "hinge" located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

Epirubicin, Identified Using a Novel Luciferase Reporter Assay for Foxp3 Inhibitors, Inhibits Regulatory T Cell Activity.

PubMed

Kashima, Hajime; Momose, Fumiyasu; Umehara, Hiroshi; Miyoshi, Nao; Ogo, Naohisa; Muraoka, Daisuke; Shiku, Hiroshi; Harada, Naozumi; Asai, Akira

2016-01-01

Forkhead box protein p3 (Foxp3) is crucial to the development and suppressor function of regulatory T cells (Tregs) that have a significant role in tumor-associated immune suppression. Development of small molecule inhibitors of Foxp3 function is therefore considered a promising strategy to enhance anti-tumor immunity. In this study, we developed a novel cell-based assay system in which the NF-κB luciferase reporter signal is suppressed by the co-expressed Foxp3 protein. Using this system, we screened our chemical library consisting of approximately 2,100 compounds and discovered that a cancer chemotherapeutic drug epirubicin restored the Foxp3-inhibited NF-κB activity in a concentration-dependent manner without influencing cell viability. Using immunoprecipitation assay in a Treg-like cell line Karpas-299, we found that epirubicin inhibited the interaction between Foxp3 and p65. In addition, epirubicin inhibited the suppressor function of murine Tregs and thereby improved effector T cell stimulation in vitro. Administration of low dose epirubicin into tumor-bearing mice modulated the function of immune cells at the tumor site and promoted their IFN-γ production without direct cytotoxicity. In summary, we identified the novel action of epirubicin as a Foxp3 inhibitor using a newly established luciferase-based cellular screen. Our work also demonstrated our screen system is useful in accelerating discovery of Foxp3 inhibitors.

Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface.

PubMed

Wojcik, John; Lamontanara, Allan Joaquim; Grabe, Grzegorz; Koide, Akiko; Akin, Louesa; Gerig, Barbara; Hantschel, Oliver; Koide, Shohei

2016-04-15

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar KD values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Tacrine-based dual binding site acetylcholinesterase inhibitors as potential disease-modifying anti-Alzheimer drug candidates.

PubMed

Camps, Pelayo; Formosa, Xavier; Galdeano, Carles; Gómez, Tània; Muñoz-Torrero, Diego; Ramírez, Lorena; Viayna, Elisabet; Gómez, Elena; Isambert, Nicolás; Lavilla, Rodolfo; Badia, Albert; Clos, M Victòria; Bartolini, Manuela; Mancini, Francesca; Andrisano, Vincenza; Bidon-Chanal, Axel; Huertas, Oscar; Dafni, Thomai; Luque, F Javier

2010-09-06

Two novel families of dual binding site acetylcholinesterase (AChE) inhibitors have been developed, consisting of a tacrine or 6-chlorotacrine unit as the active site interacting moiety, either the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone fragment of donepezil (or the indane derivative thereof) or a 5-phenylpyrano[3,2-c]quinoline system, reminiscent to the tryciclic core of propidium, as the peripheral site interacting unit, and a linker of suitable length as to allow the simultaneous binding at both sites. These hybrid compounds are all potent and selective inhibitors of human AChE, and more interestingly, are able to interfere in vitro both formation and aggregation of the beta-amyloid peptide, the latter effects endowing these compounds with the potential to modify Alzheimer's disease progression. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Spectroscopic characterization of the SH2- and active site-directed peptide sequences of a bivalent Src kinase inhibitor.

PubMed

Desamero, Ruel Z B; Kang, Jeonghee; Dol, Chrystel; Chinwong, Justina; Walters, Karim; Sivarajah, Thulashie; Profit, Adam A

2009-07-01

The spectral properties of the SH2 and active site-directed sequences of the bivalent Src kinase inhibitor Ac-EELL(F5)Phe-(GABA)3-pYEEIE-amide (1) have been determined. Ac-pYEEIE-amide (2) and AcEELL(F5)Phe-amide (3), as well as the amino acids phosphotyrosine (pTyr) and pentafluorophenylalanine (F5)Phe, have been characterized by electronic absorption, fluorescence, and vibrational spectroscopy. Specific and unique marker bands that originate from the phosphate group of pTyr and the fluorinated aromatic ring of (F5)Phe have been identified, with the latter showing some solvent dependence. Peptide 2 was found to have excitation and emission wavelengths emanating from pTyr at 268 and 295 nm, respectively, whereas peptide 3 displayed excitation and emission peaks attributable to (F5)Phe at 274 and 315 nm, respectively. Fourier transform infrared (FT-IR) analysis of the amino acid pTyr identified distinct marker bands at approximately 930, 1090, and 1330 cm(-1) that could be attributed to the phosphate group. These markers were also observed in the IR spectrum of peptide 2. Likewise, peptide 3 displayed a characteristic C-F stretching mode at 961 cm(-1) due to the presence of (F5)Phe, including two C-F reporting ring modes at 1509 and 1527 cm(-1). Identifying and monitoring spectroscopic changes in these marker bands may afford a means to observe the molecular interactions that occur when peptides 1-3 bind to the Src kinase.

Loss of Asparagine-Linked Glycosylation Sites in Variable Region 5 of Human Immunodeficiency Virus Type 1 Envelope Is Associated with Resistance to CD4 Antibody Ibalizumab ▿

PubMed Central

Toma, Jonathan; Weinheimer, Steven P.; Stawiski, Eric; Whitcomb, Jeannette M.; Lewis, Stanley T.; Petropoulos, Christos J.; Huang, Wei

2011-01-01

Ibalizumab (formerly TNX-355) is a first-in-class, monoclonal antibody inhibitor of CD4-mediated human immunodeficiency type 1 (HIV-1) entry. Multiple clinical trials with HIV-infected patients have demonstrated the antiviral activity, safety, and tolerability of ibalizumab treatment. A 9-week phase Ib study adding ibalizumab monotherapy to failing drug regimens led to transient reductions in HIV viral loads and the evolution of HIV-1 variants with reduced susceptibility to ibalizumab. This report characterizes these variants by comparing the phenotypic susceptibilities and envelope (env) sequences of (i) paired baseline and on-treatment virus populations, (ii) individual env clones from selected paired samples, and (iii) env clones containing site-directed mutations. Viruses with reduced susceptibility to ibalizumab were found to exhibit reduced susceptibility to the anti-CD4 antibody RPA-T4. Conversely, susceptibility to soluble CD4, which targets the HIV-1 gp120 envelope protein, was enhanced. No changes in susceptibility to the fusion inhibitor enfuvirtide or the CCR5 antagonist maraviroc were observed. Functionally, viruses with reduced ibalizumab susceptibility also displayed high levels of infectivity relative to those of paired baseline viruses. Individual env clones exhibiting reduced ibalizumab susceptibility contained multiple amino acid changes in different regions relative to the paired baseline clones. In particular, clones with reduced susceptibility to ibalizumab contained fewer potential asparagine-linked glycosylation sites (PNGSs) in variable region 5 (V5) than did paired ibalizumab-susceptible clones. The reduction in ibalizumab susceptibility due to the loss of V5 PNGSs was confirmed by site-directed mutagenesis. Taken together, these findings provide important insights into resistance to this new class of antiretroviral drug. PMID:21289125

Novobiocin: redesigning a DNA gyrase inhibitor for selective inhibition of hsp90.

PubMed

Burlison, Joseph A; Neckers, Len; Smith, Andrew B; Maxwell, Anthony; Blagg, Brian S J

2006-12-06

Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure-activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure-activity relationships for the coumermycin family of antibiotics.

Differential response of orthologous L,L-diaminopimelate aminotransferases (DapL) to enzyme inhibitory antibiotic lead compounds.

PubMed

McKinnie, Shaun M K; Rodriguez-Lopez, Eva M; Vederas, John C; Crowther, Jennifer M; Suzuki, Hironori; Dobson, Renwick C J; Leustek, Thomas; Triassi, Alexander J; Wheatley, Matthew S; Hudson, André O

2014-01-01

L,L-Diaminopimelate aminotransferase (DapL) is an enzyme required for the biosynthesis of meso-diaminopimelate (m-DAP) and L-lysine (Lys) in some bacteria and photosynthetic organisms. m-DAP and Lys are both involved in the synthesis of peptidoglycan (PG) and protein synthesis. DapL is found in specific eubacterial and archaeal lineages, in particular in several groups of pathogenic bacteria such as Leptospira interrogans (LiDapL), the soil/water bacterium Verrucomicrobium spinosum (VsDapL) and the alga Chlamydomonas reinhardtii (CrDapL). Here we present the first comprehensive inhibition study comparing the kinetic activity of DapL orthologs using previously active small molecule inhibitors formerly identified in a screen with the DapL of Arabidopsis thaliana (AtDapL), a flowering plant. Each inhibitor is derived from one of four classes with different central structural moieties: a hydrazide, a rhodanine, a barbiturate, or a thiobarbituate functionality. The results show that all five compounds tested were effective at inhibiting the DapL orthologs. LiDapL and AtDapL showed similar patterns of inhibition across the inhibitor series, whereas the VsDapL and CrDapL inhibition patterns were different from that of LiDapL and AtDapL. CrDapL was found to be insensitive to the hydrazide (IC₅₀ >200 μM). VsDapL was found to be the most sensitive to the barbiturate and thiobarbiturate containing inhibitors (IC₅₀ ∼5 μM). Taken together, the data shows that the homologs have differing sensitivities to the inhibitors with IC₅₀ values ranging from 4.7 to 250 μM. In an attempt to understand the basis for these differences the four enzymes were modeled based on the known structure of AtDapL. Overall, it was found that the enzyme active sites were conserved, although the second shell of residues close to the active site were not. We conclude from this that the altered binding patterns seen in the inhibition studies may be a consequence of the inhibitors forming additional interactions with residues proximal to the active site, or that the inhibitors may not act by binding to the active site. Compounds that are specific for DapL could be potential biocides (antibiotic, herbicide or algaecide) that are nontoxic to animals since animals do not contain the enzymes necessary for PG or Lys synthesis. This study provides important information to expand our current understanding of the structure/activity relationship of DapL and putative inhibitors that are potentially useful for the design and or discovery of novel biocides. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Active site similarity between human and Plasmodium falciparum phosphodiesterases: considerations for antimalarial drug design

NASA Astrophysics Data System (ADS)

Howard, Brittany L.; Thompson, Philip E.; Manallack, David T.

2011-08-01

The similarity between Plasmodium falciparum phosphodiesterase enzymes ( PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase

NASA Astrophysics Data System (ADS)

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-01

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2‧,3‧-O-(2,4,6-trinitrophenyl)adenosine 5‧-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase.

New insight into the binding modes of TNP-AMP to human liver fructose-1,6-bisphosphatase.

PubMed

Han, Xinya; Huang, Yunyuan; Zhang, Rui; Xiao, San; Zhu, Shuaihuan; Qin, Nian; Hong, Zongqin; Wei, Lin; Feng, Jiangtao; Ren, Yanliang; Feng, Lingling; Wan, Jian

2016-08-05

Human liver fructose-1,6-bisphosphatase (FBPase) contains two binding sites, a substrate fructose-1,6-bisphosphate (FBP) active site and an adenosine monophosphate (AMP) allosteric site. The FBP active site works by stabilizing the FBPase, and the allosteric site impairs the activity of FBPase through its binding of a nonsubstrate molecule. The fluorescent AMP analogue, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) has been used as a fluorescent probe as it is able to competitively inhibit AMP binding to the AMP allosteric site and, therefore, could be used for exploring the binding modes of inhibitors targeted on the allosteric site. In this study, we have re-examined the binding modes of TNP-AMP to FBPase. However, our present enzyme kinetic assays show that AMP and FBP both can reduce the fluorescence from the bound TNP-AMP through competition for FBPase, suggesting that TNP-AMP binds not only to the AMP allosteric site but also to the FBP active site. Mutagenesis assays of K274L (located in the FBP active site) show that the residue K274 is very important for TNP-AMP to bind to the active site of FBPase. The results further prove that TNP-AMP is able to bind individually to the both sites. Our present study provides a new insight into the binding mechanism of TNP-AMP to the FBPase. The TNP-AMP fluorescent probe can be used to exam the binding site of an inhibitor (the active site or the allosteric site) using FBPase saturated by AMP and FBP, respectively, or the K247L mutant FBPase. Copyright © 2016 Elsevier B.V. All rights reserved.

The molecular mechanism for interaction of ceruloplasmin and myeloperoxidase

NASA Astrophysics Data System (ADS)

Bakhautdin, Bakytzhan; Bakhautdin, Esen Göksöy

2016-04-01

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp-MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp-MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp's crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp-MPO binding and rescues MPO activity. Collectively, our results confirm Cp-MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change.

Current Status of New Anticoagulants in the Management of Venous Thromboembolism

PubMed Central

Montoya, Roberto C.; Gajra, Ajeet

2012-01-01

Venous Thromboembolism, manifested as deep venous thrombosis and pulmonary embolism, is a common problem associated with significant morbidity, mortality, and resource expenditure. Unfractionated heparin, low-molecular-weight heparin, and vitamin K antagonists are the most common treatment and prophylaxis, and have demonstrated their efficacy in a vast number of previous studies. Despite their broad use, these agents have important limitations that have led to the development of new drugs in a bid to overcome the disadvantages of the old ones without decreasing their therapeutic effect. These novel medications, some approved and others in different stages of development, include direct thrombin inhibitors like dabigatran etexilate, and direct activated factor X inhibitors like rivaroxaban. The current paper will review the characteristics, clinical trial results, and current and potential therapeutic uses of these new agents with a focus on the categories of direct thrombin inhibitors and activated factor X inhibitors. PMID:22496694

Targeting S-adenosylmethionine biosynthesis with a novel allosteric inhibitor of Mat2A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quinlan, Casey L.; Kaiser, Stephen E.; Bolaños, Ben

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzymemore » turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.« less

Virtual Lead Identification of Farnesyltransferase Inhibitors Based on Ligand and Structure-Based Pharmacophore Techniques

PubMed Central

Al-Balas, Qosay A.; Amawi, Haneen A.; Hassan, Mohammad A.; Qandil, Amjad M.; Almaaytah, Ammar M.; Mhaidat, Nizar M.

2013-01-01

Farnesyltransferase enzyme (FTase) is considered an essential enzyme in the Ras signaling pathway associated with cancer. Thus, designing inhibitors for this enzyme might lead to the discovery of compounds with effective anticancer activity. In an attempt to obtain effective FTase inhibitors, pharmacophore hypotheses were generated using structure-based and ligand-based approaches built in Discovery Studio v3.1. Knowing the presence of the zinc feature is essential for inhibitor’s binding to the active site of FTase enzyme; further customization was applied to include this feature in the generated pharmacophore hypotheses. These pharmacophore hypotheses were thoroughly validated using various procedures such as ROC analysis and ligand pharmacophore mapping. The validated pharmacophore hypotheses were used to screen 3D databases to identify possible hits. Those which were both high ranked and showed sufficient ability to bind the zinc feature in active site, were further refined by applying drug-like criteria such as Lipiniski’s “rule of five” and ADMET filters. Finally, the two candidate compounds (ZINC39323901 and ZINC01034774) were allowed to dock using CDOCKER and GOLD in the active site of FTase enzyme to optimize hit selection. PMID:24276257

Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*

PubMed Central

Mautner, Henry G.; Bartels, Eva

1970-01-01

p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholine-receptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or p-nitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptor-activator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue. PMID:5272331

Molecular dynamics simulations on the inhibition of cyclin-dependent kinases 2 and 5 in the presence of activators.

PubMed

Zhang, Bing; Tan, Vincent B C; Lim, Kian Meng; Tay, Tong Earn

2006-06-01

Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ting; Ooi, Amy; Lee, Hooi Chen

An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pHmore » 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.« less

A combinatorial feature selection approach to describe the QSAR of dual site inhibitors of acetylcholinesterase.

PubMed

Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz; Barkooie, Seyyed Mohsen Hosseini; Jahandideh, Samad; Rezaei, Mohammad Ali

2009-12-01

Regarding the great potential of dual binding site inhibitors of acetylcholinesterase as the future potent drugs of Alzheimer's disease, this study was devoted to extraction of the most effective structural features of these inhibitors from among a large number of quantitative descriptors. To do this, we adopted a unique approach in quantitative structure-activity relationships. An efficient feature selection method was emphasized in such an approach, using the confirmative results of different routine and novel feature selection methods. The proposed methods generated quite consistent results ensuring the effectiveness of the selected structural features.

Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases

PubMed Central

Patterson, H; Nibbs, R; McInnes, I; Siebert, S

2014-01-01

Protein kinases mediate protein phosphorylation, which is a fundamental component of cell signalling, with crucial roles in most signal transduction cascades: from controlling cell growth and proliferation to the initiation and regulation of immunological responses. Aberrant kinase activity is implicated in an increasing number of diseases, with more than 400 human diseases now linked either directly or indirectly to protein kinases. Protein kinases are therefore regarded as highly important drug targets, and are the subject of intensive research activity. The success of small molecule kinase inhibitors in the treatment of cancer, coupled with a greater understanding of inflammatory signalling cascades, has led to kinase inhibitors taking centre stage in the pursuit for new anti-inflammatory agents for the treatment of immune-mediated diseases. Herein we discuss the main classes of kinase inhibitors; namely Janus kinase (JAK), mitogen-activated protein kinase (MAPK) and spleen tyrosine kinase (Syk) inhibitors. We provide a mechanistic insight into how these inhibitors interfere with kinase signalling pathways and discuss the clinical successes and failures in the implementation of kinase-directed therapeutics in the context of inflammatory and autoimmune disorders. PMID:24313320

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

PubMed

Lassmann, G; Curtis, J; Liermann, B; Mason, R P; Eling, T E

1993-01-01

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction.

PubMed

Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S; Somlyo, Avril V; Eto, Masumi

2009-08-07

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC(50) = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of beta-escin-permeabilized ileum SM at constant pCa 6.3 (EC(50) = 2 microm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697-880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca(2+) sensitization of smooth muscle force.

The Molecular Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors

PubMed Central

Romano, Keith P.; Ali, Akbar; Aydin, Cihan; Soumana, Djade; Özen, Ayşegül; Deveau, Laura M.; Silver, Casey; Cao, Hong; Newton, Alicia; Petropoulos, Christos J.; Huang, Wei; Schiffer, Celia A.

2012-01-01

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus. PMID:22910833

Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

PubMed

Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

2011-12-01

In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

Structure based drug design: development of potent and selective factor IXa (FIXa) inhibitors.

PubMed

Wang, Shouming; Beck, Richard; Burd, Andrew; Blench, Toby; Marlin, Frederic; Ayele, Tenagne; Buxton, Stuart; Dagostin, Claudio; Malic, Maja; Joshi, Rina; Barry, John; Sajad, Mohammed; Cheung, Chiming; Shaikh, Shaheda; Chahwala, Suresh; Chander, Chaman; Baumgartner, Christine; Holthoff, Hans-Peter; Murray, Elizabeth; Blackney, Michael; Giddings, Amanda

2010-02-25

On the basis of our understanding on the binding interactions of the benzothiophene template within the FIXa active site by X-ray crystallography and molecular modeling studies, we developed our SAR strategy by targeting the 4-position of the template to access the S1 beta and S2-S4 sites. A number of highly selective and potent factor Xa (FXa) and FIXa inhibitors were identified by simple switch of functional groups with conformational changes toward the S2-S4 sites.

Molecular modeling studies of novel retro-binding tripeptide active-site inhibitors of thrombin.

PubMed

Lau, W F; Tabernero, L; Sack, J S; Iwanowicz, E J

1995-08-01

A novel series of retro-binding tripeptide thrombin active-site inhibitors was recently developed (Iwanowicz, E. I. et al. J. Med. Chem. 1994, 37, 2111(1)). It was hypothesized that the binding mode for these inhibitors is similar to that of the first three N-terminal residues of hirudin. This binding hypothesis was subsequently verified when the crystal structure of a member of this series, BMS-183,507 (N-[N-[N-[4-(Aminoiminomethyl)amino[-1-oxobutyl]-L- phenylalanyl]-L-allo-threonyl]-L-phenylalanine, methyl ester), was determined (Taberno, L.J. Mol. Biol. 1995, 246, 14). The methodology for developing the binding models of these inhibitors, the structure-activity relationships (SAR) and modeling studies that led to the elucidation of the proposed binding mode is described. The crystal structure of BMS-183,507/human alpha-thrombin is compared with the crystal structure of hirudin/human alpha-thrombin (Rydel, T.J. et al. Science 1990, 249,227; Rydel, T.J. et al. J. Mol Biol. 1991, 221, 583; Grutter, M.G. et al. EMBO J. 1990, 9, 2361) and with the computational binding model of BMS-183,507.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

PubMed

Szaszkó, Mária; Hajdú, István; Flachner, Beáta; Dobi, Krisztina; Magyar, Csaba; Simon, István; Lőrincz, Zsolt; Kapui, Zoltán; Pázmány, Tamás; Cseh, Sándor; Dormán, György

2017-02-01

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

Kinetics and molecular docking studies of loganin, morroniside and 7-O-galloyl-D-sedoheptulose derived from Corni fructus as cholinesterase and β-secretase 1 inhibitors.

PubMed

Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Min, Byung-Sun; Jung, Hyun Ah; Choi, Jae Sue

2016-06-01

We evaluated the major active components isolated from Corni Fructus: loganin, morroniside, and 7-O-galloyl-D-sedoheptulose as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) for use in Alzheimer's disease treatment. These compounds exhibited predominant cholinesterase (ChEs) inhibitory effects with IC50 values of 0.33, 3.95, and 10.50 ± 1.16 µM, respectively, for AChE, and 33.02, 37.78, and 87.94 ± 4.66 µM, respectively, for BChE. Kinetics studies revealed that loganin and 7-O-galloyl-D-sedoheptulose inhibited AChE with characteristics typical of mixed inhibitors, while morroniside was found to be a noncompetitive inhibitor against AChE and also exerted mixed BChE inhibitory activities. For BACE1, loganin showed noncompetitive type inhibitory effects, while morroniside and 7-O-galloyl-D-sedoheptulose were found to be mixed inhibitors. Furthermore, these compounds exhibited dose-dependent inhibitory activity with ONOO(-)-mediated protein tyrosine nitration. Molecular docking simulation of these compounds demonstrated negative binding energies for ChEs, and BACE1, indicating high affinity and tighter binding capacity for the active site of the enzyme. Loganin was the most potent inhibitor against both ChEs and BACE1. The data suggest that these compounds together can act as a triple inhibitor of AChE, BChE, and BACE1, providing a preventive and therapeutic strategy for Alzheimer's disease treatment.

Targeted delivery of antigen processing inhibitors to antigen presenting cells via mannose receptors.

PubMed

Raiber, Eun-Ang; Tulone, Calogero; Zhang, Yanjing; Martinez-Pomares, Luisa; Steed, Emily; Sponaas, Anna M; Langhorne, Jean; Noursadeghi, Mahdad; Chain, Benjamin M; Tabor, Alethea B

2010-05-21

Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope-specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells. This does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Preliminary results suggest that access to nonlymphoid tissues is very limited in the steady state but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.

The discovery of novel tartrate-based TNF-[alpha] converting enzyme (TACE) inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosner, Kristin E.; Guo, Zhuyan; Orth, Peter

2010-09-17

A novel series of TNF-{alpha} convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of L-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP's. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.

LEDGINs, non-catalytic site inhibitors of HIV-1 integrase: a patent review (2006 - 2014).

PubMed

Demeulemeester, Jonas; Chaltin, Patrick; Marchand, Arnaud; De Maeyer, Marc; Debyser, Zeger; Christ, Frauke

2014-06-01

Integration of the viral genome into the host cell chromatin is a central step in the replication cycle of the HIV. Blocking the viral integrase (IN) enzyme therefore provides an attractive therapeutic strategy, as evidenced by the recent clinical approval of three IN strand transfer inhibitors. Viral resistance and cross-resistance among these inhibitors, however, warrant the search for compounds targeting HIV integration through alternative mechanisms of action. The most potent class of allosteric IN inhibitors was independently identified at the University of Leuven, Belgium, and at Boehringer Ingelheim, Canada. These compounds, coined LEDGINs (after the lens epithelium-derived growth factor/p75 cofactor binding pocket on IN) or non-catalytic site IN inhibitors (NCINIs) by the respective groups, have shown remarkable antiviral activity. This review provides a brief introduction to the compound class and discusses the recent patent literature (2006 to the present). LEDGINs are still early in development. Trials with clinical candidate BI-224436 were put on hold despite promising results. Literature, however, reveals that almost all major pharmaceutical companies active in the treatment of HIV/AIDS have taken a significant interest in this class. As a result, several of these inhibitors may soon enter clinical trials.

cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

PubMed

Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

2009-09-01

The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

Direct imaging of the disruption of hepatitis C virus replication complexes by inhibitors of lipid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lyn, Rodney K.; Department of Chemistry, University of Ottawa, 10 Marie-Curie, Ottawa, K1N 6N5; Kennedy, David C.

2009-11-10

Here we have simultaneously characterized the influence of inhibitors of peroxisome proliferator-activated receptor alpha (PPARalpha) and the mevalonate pathway on hepatocyte lipid metabolism and the subcellular localization of hepatitis C virus (HCV) RNA using two-photon fluorescence (TPF) and coherent anti-Stokes Raman scattering (CARS) microscopy. Using this approach, we demonstrate that modulators of PPARalpha signaling rapidly cause the dispersion of HCV RNA from replication sites and simultaneously induce lipid storage and increases in lipid droplet size. We demonstrate that reductions in the levels of cholesterol resulting from inhibition of the mevalonate pathway upregulates triglyceride levels. We also show that the ratemore » of dispersion of HCV RNA is very rapid when using a PPARalpha antagonist. This occurs with a faster rate to that of direct inhibition of 3-hydroxy-3-methyglutaryl CoA reductase (HMG-CoA reductase) using lovastatin in living cells, demonstrating the potential therapeutic value of modulating host cell pathways as part of a strategy to eliminate chronic HCV infection.« less

Activation of AMPK reduces the co-transporter activity of NKCC1.

PubMed

Fraser, Scott A; Davies, Matthew; Katerelos, Marina; Gleich, Kurt; Choy, Suet-Wan; Steel, Rohan; Galic, Sandra; Mount, Peter F; Kemp, Bruce E; Power, David A

2014-01-01

The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.

Metallochaperone UreG serves as a new target for design of urease inhibitor: A novel strategy for development of antimicrobials.

PubMed

Yang, Xinming; Koohi-Moghadam, Mohamad; Wang, Runming; Chang, Yuen-Yan; Woo, Patrick C Y; Wang, Junwen; Li, Hongyan; Sun, Hongzhe

2018-01-01

Urease as a potential target of antimicrobial drugs has received considerable attention given its versatile roles in microbial infection. Development of effective urease inhibitors, however, is a significant challenge due to the deeply buried active site and highly specific substrate of a bacterial urease. Conventionally, urease inhibitors are designed by either targeting the active site or mimicking substrate of urease, which is not efficient. Up to now, only one effective inhibitor-acetohydroxamic acid (AHA)-is clinically available, but it has adverse side effects. Herein, we demonstrate that a clinically used drug, colloidal bismuth subcitrate, utilizes an unusual way to inhibit urease activity, i.e., disruption of urease maturation process via functional perturbation of a metallochaperone, UreG. Similar phenomena were also observed in various pathogenic bacteria, suggesting that UreG may serve as a general target for design of new types of urease inhibitors. Using Helicobacter pylori UreG as a showcase, by virtual screening combined with experimental validation, we show that two compounds targeting UreG also efficiently inhibited urease activity with inhibitory concentration (IC)50 values of micromolar level, resulting in attenuated virulence of the pathogen. We further demonstrate the efficacy of the compounds in a mammalian cell infection model. This study opens up a new opportunity for the design of more effective urease inhibitors and clearly indicates that metallochaperones involved in the maturation of important microbial metalloenzymes serve as new targets for devising a new type of antimicrobial drugs.

Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

PubMed

Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

2015-02-13

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

Direct interaction between caffeic acid phenethyl ester and human neutrophil elastase inhibits the growth and migration of PANC-1 cells.

PubMed

Duan, Jianhui; Xiaokaiti, Yilixiati; Fan, Shengjun; Pan, Yan; Li, Xin; Li, Xuejun

2017-05-01

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors of the digestive system, but the mechanisms of its development and progression are unclear. Inflammation is thought to be fundamental to pancreatic cancer development and caffeic acid phenethyl ester (CAPE) is an active component of honey bee resin or propolis with anti-inflammatory and anticancer activities. We investigated the inhibitory effects of CAPE on cell growth and migration induced by human neutrophil elastase (HNE) and report that HNE induced cancer cell migration at low doses and growth at higher doses. In contrast, lower CAPE doses inhibited migration and higher doses of CAPE inhibited the growth induced by HNE. HNE activity was significantly inhibited by CAPE (7.5-120 µM). Using quantitative real-time PCR and western blotting, we observed that CAPE (18-60 µM) did not affect transcription and translation of α1-antitrypsin (α1-AT), an endogenous HNE inhibitor. However, in an in silico drug target docking model, we found that CAPE directly bound to the binding pocket of HNE (25.66 kcal/mol) according to CDOCKER, and the residue of the catalytic site stabilized the interaction between CAPE and HNE as evidenced by molecular dynamic simulation. Response unit (RU) values of surface plasmon resonance (SPR) significantly increased with incremental CAPE doses (7.5-120 µM), indicating that CAPE could directly bind to HNE in a concentration-dependent manner. Thus, CAPE is an effective inhibitor of HNE via direct interaction whereby it inhibits the migration and growth of PANC-1 cells in a dose-dependent manner.

A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11).

PubMed

Tiraboschi, G; Jullian, N; Thery, V; Antonczak, S; Fournie-Zaluski, M C; Roques, B P

1999-02-01

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

The Ndc80 complex targets Bod1 to human mitotic kinetochores

PubMed Central

2017-01-01

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores. PMID:29142109

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity.

PubMed

Nancolas, Bethany; Sessions, Richard B; Halestrap, Andrew P

2015-02-15

The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523-530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7-10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278.

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity

PubMed Central

Nancolas, Bethany; Sessions, Richard B.; Halestrap, Andrew P.

2014-01-01

The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523–530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7–10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278. PMID:25437897

Protease inhibitors and indoleamines selectively inhibit cholinesterases in the histopathologic structures of Alzheimer disease.

PubMed Central

Wright, C I; Guela, C; Mesulam, M M

1993-01-01

Neurofibrillary tangles and amyloid plaques express acetylcholinesterase and butyrylcholinesterase activity in Alzheimer disease. We previously reported that traditional acetylcholinesterase inhibitors such as BW284C51, tacrine, and physostigmine were more potent inhibitors of the acetylcholinesterase in normal axons and cell bodies than of the acetylcholinesterase in plaques and tangles. We now report that the reverse pattern is seen with indoleamines (such as serotonin and its precursor 5-hydroxytryptophan), carboxypeptidase inhibitor, and the nonspecific protease inhibitor bacitracin. These substances are more potent inhibitors of the cholinesterases in plaques and tangles than of those in normal axons and cell bodies. These results show that the enzymatic properties of plaque and tangle-associated cholinesterases diverge from those of normal axons and cell bodies. The selective susceptibility to bacitracin and carboxypeptidase inhibitor indicates that the catalytic sites of plaque and tangle-bound cholinesterases are more closely associated with peptidase or protease-like properties than the catalytic sites of cholinesterases in normal axons and cell bodies. This shift in enzymatic affinity may lead to the abnormal protein processing that is thought to play a major role in the pathogenesis of Alzheimer disease. The availability of pharmacological and dietary means for altering brain indoleamines raises therapeutic possibilities for inhibiting the abnormal cholinesterase activity associated with Alzheimer disease. Images PMID:8421706

DOE Office of Scientific and Technical Information (OSTI.GOV)

Totoritis, Rachel; Duraiswami, Chaya; Taylor, Amy N.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes inmore » time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.« less

Structural insights into binding of small molecule inhibitors to Enhancer of Zeste Homolog 2

NASA Astrophysics Data System (ADS)

Kalinić, Marko; Zloh, Mire; Erić, Slavica

2014-11-01

Enhancer of Zeste Homolog 2 (EZH2) is a SET domain protein lysine methyltransferase (PKMT) which has recently emerged as a chemically tractable and therapeutically promising epigenetic target, evidenced by the discovery and characterization of potent and highly selective EZH2 inhibitors. However, no experimental structures of the inhibitors co-crystallized to EZH2 have been resolved, and the structural basis for their activity and selectivity remains unknown. Considering the need to minimize cross-reactivity between prospective PKMT inhibitors, much can be learned from understanding the molecular basis for selective inhibition of EZH2. Thus, to elucidate the binding of small-molecule inhibitors to EZH2, we have developed a model of its fully-formed cofactor binding site and used it to carry out molecular dynamics simulations of protein-ligand complexes, followed by molecular mechanics/generalized born surface area calculations. The obtained results are in good agreement with biochemical inhibition data and reflect the structure-activity relationships of known ligands. Our findings suggest that the variable and flexible post-SET domain plays an important role in inhibitor binding, allowing possibly distinct binding modes of inhibitors with only small variations in their structure. Insights from this study present a good basis for design of novel and optimization of existing compounds targeting the cofactor binding site of EZH2.

Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottea, J.A.; Payne, G.T.; Soderlund, D.M.

1990-08-01

Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealedmore » that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.« less

Binding Properties of a Peptide Derived from β-Lactamase Inhibitory Protein

PubMed Central

Rudgers, Gary W.; Huang, Wanzhi; Palzkill, Timothy

2001-01-01

To overcome the antibiotic resistance mechanism mediated by β-lactamases, small-molecule β-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in β-lactamases no longer sensitive to β-lactamase inhibitors. On the basis of the structure of β-lactamase inhibitor protein (BLIP), novel peptide inhibitors of β-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 β-lactamase (Ki = 0.3 nM). The cocrystal structure of TEM-1 β-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 β-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 β-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 β-lactamase, the peptide also inhibits a class A β-lactamase and a class C β-lactamase that are not inhibited by BLIP. The crystal structures of class A and C β-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of β-lactamases and PBPs. PMID:11709298

The role of small molecule platelet-derived growth factor receptor (PDGFR) inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-03-01

Platelet-derived growth factor (PDGF) was discovered as a serum-derived component necessary for the growth of smooth muscle cells, fibroblasts, and glial cells. The PDGF family is a product of four gene products and consists of five dimeric isoforms: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and the PDGF-AB heterodimer. This growth factor family plays an essential role in embryonic development and in wound healing in the adult. These growth factors mediate their effects by binding to and activating their receptor protein-tyrosine kinases, which are encoded by two genes: PDGFRA and PDGFRB. The functional receptors consist of the PDGFRα/α and PDGFRβ/β homodimers and the PDGFRα/β heterodimer. Although PDGF signaling is most closely associated with mesenchymal cells, PDGFs and PDGF receptors are widely expressed in the mammalian central nervous system. The PDGF receptors contain an extracellular domain that is made up of five immunoglobulin-like domains (Ig-d1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of about 100 amino acid residues, and a carboxyterminal tail. Although uncommon, activating mutations in the genes for PDGF or PDGF receptors have been documented in various neoplasms including dermatofibrosarcoma protuberans (DFSP) and gastrointestinal stromal tumors (GIST). In most neoplastic diseases, PDGF expression and action appear to involve the tumor stroma. Moreover, this family is pro-angiogenic. More than ten PDGFRα/β multikinase antagonists have been approved by the FDA for the treatment of several neoplastic disorders and interstitial pulmonary fibrosis (www.brimr.org/PKI/PKIs.htm). Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). We used the Schrödinger induced-fit docking protocol to model the interaction of several antagonists with PDGFRα including imatinib, sorafenib, and sunitinib. The results indicate that these antagonists are able to bind to the DFG-D out conformation of the receptor and are thus classified as type II inhibitors. Owing to the multiplicity of less active protein kinase conformations when compared with the canonical more active conformation, it was hypothesized that type II drugs would be less promiscuous than type I drugs which bind to the typical active conformation. Although type II inhibitors may be more selective, most - if not all - inhibit more than one target protein kinase and the differences are a matter of degree only. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural insights into activation and inhibition of histo-aspartic protease (HAP) from Plasmodium falciparum

PubMed Central

Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander

2012-01-01

Histo-aspartic protease (HAP) from Plasmodium falciparum offers a promising target for the development of novel antimalarial drugs. HAP exhibits high sequence similarity to pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced by histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 Å resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C- terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter “U”, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to the fragment of helix 2 comprising residues 34p–38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A′ of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP. PMID:21928835

Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi

2012-09-17

Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215more » and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.« less

Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

PubMed

Zhu, Shun; Travis, Sue M; Elcock, Adrian H

2013-07-09

A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant. Imposition of position restraints in corresponding simulations that used the Amber ff99SB-ILDN force field had little effect on their ability to match experiment. Overall, the study shows that both force fields can work well for predicting the effects of active-site mutations on small molecule binding affinities and demonstrates how a direct combination of experiment and computation can be a powerful strategy for developing an understanding of protein-inhibitor interactions.

Thioredoxin-1 Negatively Modulates ADAM17 Activity Through Direct Binding and Indirect Reductive Activity.

PubMed

Granato, Daniela C; E Costa, Rute A P; Kawahara, Rebeca; Yokoo, Sami; Aragão, Annelize Z; Domingues, Romênia R; Pauletti, Bianca A; Honorato, Rodrigo V; Fattori, Juliana; Figueira, Ana Carolina M; Oliveira, Paulo S L; Consonni, Silvio R; Fernandes, Denise; Laurindo, Francisco; Hansen, Hinrich P; Paes Leme, Adriana F

2018-02-27

A disintegrin and metalloprotease 17 (ADAM17) modulates signaling events by releasing surface protein ectodomains such as TNFa and the EGFR-ligands. We have previously characterized cytoplasmic thioredoxin-1 (Trx-1) as a partner of ADAM17 cytoplasmic domain. Still, the mechanism of ADAM17 regulation by Trx-1 is unknown, and it has become of paramount importance to assess the degree of influence that Trx-1 has on metalloproteinase ADAM17. Combining discovery and targeted proteomic approaches, we uncovered that Trx-1 negatively regulates ADAM17 by direct and indirect effect. We performed cell-based assays with synthetic peptides and site-directed mutagenesis, and we demonstrated that the interaction interface of Trx-1 and ADAM17 is important for the negative regulation of ADAM17 activity. However, both Trx-1 K72A and catalytic site mutant Trx-1 C32/35S rescued ADAM17 activity, although the interaction with Trx-1 C32/35S was unaffected, suggesting an indirect effect of Trx-1. We confirmed that the Trx-1 C32/35S mutant showed diminished reductive capacity, explaining this indirect effect on increasing ADAM17 activity through oxidant levels. Interestingly, Trx-1 K72A mutant showed similar oxidant levels to Trx-1 C32/35S , even though its catalytic site was preserved. We further demonstrated that the general reactive oxygen species inhibitor, Nacetylcysteine (NAC), maintained the regulation of ADAM17 dependent of Trx-1 reductase activity levels; whereas the electron transport chain modulator, rotenone, abolished Trx-1 effect on ADAM17 activity. We show for the first time that the mechanism of ADAM17 regulation, Trx-1 dependent, can be by direct interaction and indirect effect, bringing new insights into the cross-talk between isomerases and mammalian metalloproteinases. This unexpected Trx-1 K72A behavior was due to more dimer formation and, consequently, the reduction of its Trx-1 reductase activity, evaluated through dimer verification, by gel filtration and mass spectrometry analysis. Antioxid. Redox Signal. 00, 000-000.

Human HDAC7 Harbors a Class IIa Histone Deacetylase-specific Zinc Binding Motif and Cryptic Deacetylase Activity*S⃞

PubMed Central

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P.; Lewis, Timothy A.; Maglathin, Rebecca L.; McLean, Thomas H.; Bochkarev, Alexey; Plotnikov, Alexander N.; Vedadi, Masoud; Arrowsmith, Cheryl H.

2008-01-01

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators. PMID:18285338

Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity.

PubMed

Schuetz, Anja; Min, Jinrong; Allali-Hassani, Abdellah; Schapira, Matthieu; Shuen, Michael; Loppnau, Peter; Mazitschek, Ralph; Kwiatkowski, Nick P; Lewis, Timothy A; Maglathin, Rebecca L; McLean, Thomas H; Bochkarev, Alexey; Plotnikov, Alexander N; Vedadi, Masoud; Arrowsmith, Cheryl H

2008-04-25

Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.

Novel dual small-molecule HIV inhibitors: scaffolds and discovery strategies.

PubMed

Song, Anran; Yu, Haiqing; Wang, Changyuan; Zhu, Xingqi; Liu, Kexin; Ma, Xiaodong

2015-01-01

Searching for safe and effective treatments for HIV infection is still a great challenge worldwide in spite of the 27 marketed anti-HIV drugs and the powerful highly active antiretroviral therapy (HAART). As a promising prospect for generation of new HIV therapy drugs, multiple ligands (MDLs) were greatly focused on recently due to their lower toxicity, simplified dosing and patient adherence than single-target drugs. Till now, by disrupting two active sites or steps of HIV replications, a number of HIV dual inhibitors, such as CD4-gssucap120 inhibitors, CXCR4-gp20 inhibitors, RT-CXCR4 inhibitors, RT-protease inhibitors, RT-integrase inhibitors, and RTassociated functions inhibitors have been identified. Generally, these dual inhibitors were discovered mainly through screening approaches and design strategies. Of these compounds, the molecules bearing small skeletons exhibited strong anti-HIV activity and aroused great attention recently. Reviewing the progress of the dual small-molecule HIV inhibitors from the point of view of their scaffolds and discovery strategies will provide valuable information for producing more effective anti-HIV drugs. In this regard, novel dual small-molecule HIV inhibitors were illustrated, and their discovery paradigms as the major contents were also summarized in this manuscript.

Lung Adenocarcinoma with Anaplastic Lymphoma Kinase (ALK) Rearrangement Presenting as Carcinoma of Unknown Primary Site: Recognition and Treatment Implications.

PubMed

Hainsworth, John D; Anthony Greco, F

Molecular cancer classifier assays are being used with increasing frequency to predict tissue of origin and direct site-specific therapy for patients with carcinoma of unknown primary site (CUP). We postulated some CUP patients predicted to have non-small-cell lung cancer (NSCLC) by molecular cancer classifier assay may have anaplastic lymphoma kinase (ALK) rearranged tumors, and benefit from treatment with ALK inhibitors. We retrospectively reviewed CUP patients who had the 92-gene molecular cancer classifier assay (CancerTYPE ID; bioTheranostics, Inc.) performed on tumor biopsies to identify patients predicted to have NSCLC. Beginning in 2011, we have tested these patients for ALK rearrangements and epidermal growth factor receptor (EGFR) activating mutations, based on the proven therapeutic value of these targets in NSCLC. We identified CUP patients with predicted NSCLC who were subsequently found to have ALK rearrangements. NSCLC was predicted by the molecular cancer classifier assay in 37 of 310 CUP patients. Twenty-one of these patients were tested for ALK rearrangements, and four had an EML4-ALK fusion gene detected. The diagnosis of lung cancer was strongly suggested in only one patient prior to molecular testing. One patient received ALK inhibitor treatment and has had prolonged benefit. We report on patients with lung adenocarcinoma and ALK rearrangements originally diagnosed as CUP who were identified using a molecular cancer classifier assay. Although ALK inhibitors treatment experience is limited, this newly identifiable group of lung cancer patients should be considered for therapy according to guidelines for stage IV ALK-positive NSCLC.

Lung Adenocarcinoma with Anaplastic Lymphoma Kinase (ALK) Rearrangement Presenting as Carcinoma of Unknown Primary Site: Recognition and Treatment Implications.

PubMed

Hainsworth, John D; Anthony Greco, F

2016-03-01

Molecular cancer classifier assays are being used with increasing frequency to predict tissue of origin and direct site-specific therapy for patients with carcinoma of unknown primary site (CUP). We postulated some CUP patients predicted to have non-small-cell lung cancer (NSCLC) by molecular cancer classifier assay may have anaplastic lymphoma kinase (ALK) rearranged tumors, and benefit from treatment with ALK inhibitors. We retrospectively reviewed CUP patients who had the 92-gene molecular cancer classifier assay (CancerTYPE ID; bioTheranostics, Inc.) performed on tumor biopsies to identify patients predicted to have NSCLC. Beginning in 2011, we have tested these patients for ALK rearrangements and epidermal growth factor receptor (EGFR) activating mutations, based on the proven therapeutic value of these targets in NSCLC. We identified CUP patients with predicted NSCLC who were subsequently found to have ALK rearrangements. NSCLC was predicted by the molecular cancer classifier assay in 37 of 310 CUP patients. Twenty-one of these patients were tested for ALK rearrangements, and four had an EML4-ALK fusion gene detected. The diagnosis of lung cancer was strongly suggested in only one patient prior to molecular testing. One patient received ALK inhibitor treatment and has had prolonged benefit. We report on patients with lung adenocarcinoma and ALK rearrangements originally diagnosed as CUP who were identified using a molecular cancer classifier assay. Although ALK inhibitors treatment experience is limited, this newly identifiable group of lung cancer patients should be considered for therapy according to guidelines for stage IV ALK-positive NSCLC.

Inhibition of d-xylose isomerase by polyols: atomic details by joint X-ray/neutron crystallography

PubMed Central

Kovalevsky, Andrey; Hanson, B. Leif; Mason, Sax A.; Forsyth, V. Trevor; Fisher, Zoe; Mustyakimov, Marat; Blakeley, Matthew P.; Keen, David A.; Langan, Paul

2012-01-01

d-Xylose isomerase (XI) converts the aldo-sugars xylose and glucose to their keto analogs xylulose and fructose, but is strongly inhibited by the polyols xylitol and sorbitol, especially at acidic pH. In order to understand the atomic details of polyol binding to the XI active site, a 2.0 Å resolution room-temperature joint X-ray/neutron structure of XI in complex with Ni2+ cofactors and sorbitol inhibitor at pH 5.9 and a room-temperature X-ray structure of XI containing Mg2+ ions and xylitol at the physiological pH of 7.7 were obtained. The protonation of oxygen O5 of the inhibitor, which was found to be deprotonated and negatively charged in previous structures of XI complexed with linear glucose and xylulose, was directly observed. The Ni2+ ions occupying the catalytic metal site (M2) were found at two locations, while Mg2+ in M2 is very mobile and has a high B factor. Under acidic conditions sorbitol gains a water-mediated interaction that connects its O1 hydroxyl to Asp257. This contact is not found in structures at basic pH. The new interaction that is formed may improve the binding of the inhibitor, providing an explanation for the increased affinity of the polyols for XI at low pH. PMID:22948921

The Crystal Structure Analysis of Group B Streptococcus Sortase C1: A Model for the ;Lid; Movement upon Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu

2012-02-07

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible 'lid' anchored in the active site. However, the mechanistic details of the 'lid' displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its 'lid' mutant and proposed a role of the 'lid' as a protectormore » of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.« less

Molecular modeling-driven approach for identification of Janus kinase 1 inhibitors through 3D-QSAR, docking and molecular dynamics simulations.

PubMed

Itteboina, Ramesh; Ballu, Srilata; Sivan, Sree Kanth; Manga, Vijjulatha

2017-10-01

Janus kinase 1 (JAK 1) belongs to the JAK family of intracellular nonreceptor tyrosine kinase. JAK-signal transducer and activator of transcription (JAK-STAT) pathway mediate signaling by cytokines, which control survival, proliferation and differentiation of a variety of cells. Three-dimensional quantitative structure activity relationship (3 D-QSAR), molecular docking and molecular dynamics (MD) methods was carried out on a dataset of Janus kinase 1(JAK 1) inhibitors. Ligands were constructed and docked into the active site of protein using GLIDE 5.6. Best docked poses were selected after analysis for further 3 D-QSAR analysis using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methodology. Employing 60 molecules in the training set, 3 D-QSAR models were generate that showed good statistical reliability, which is clearly observed in terms of r 2 ncv and q 2 loo values. The predictive ability of these models was determined using a test set of 25 molecules that gave acceptable predictive correlation (r 2 Pred ) values. The key amino acid residues were identified by means of molecular docking, and the stability and rationality of the derived molecular conformations were also validated by MD simulation. The good consonance between the docking results and CoMFA/CoMSIA contour maps provides helpful clues about the reasonable modification of molecules in order to design more efficient JAK 1 inhibitors. The developed models are expected to provide some directives for further synthesis of highly effective JAK 1 inhibitors.

Modeling and Proposed Molecular Mechanism of Hydroxyurea Through Docking and Molecular Dynamic Simulation to Curtail the Action of Ribonucleotide Reductase.

PubMed

Iman, Maryam; Khansefid, Zeynab; Davood, Asghar

2016-01-01

Ribonucleotide Reductase (RNR) is an important anticancer chemotherapy target. It has main key role in DNA synthesis and cell growth. Therefore several RNR inhibitors, such as hydroxyurea, have entered the clinical trials. Based on our proposed mechanism, radical site of RNR protein reacts with hydroxyurea in which hydroxyurea is converted into its oxidized form compound III, and whereby the tyrosyl radical is converted into a normal tyrosine residue. In this study, docking and molecular dynamics simulations were used for proposed molecular mechanism of hydroxyurea in RNR inhibition as anticancer agent. The binding affinity of hydroxyurea and compound III to RNR was studied by docking method. The docking study was performed for the crystal structure of human RNR with the radical scavenger Hydroxyurea and its oxidized form to inhibit the human RNR. hydroxyurea and compound III bind at the active site with Tyr-176, which are essential for free radical formation. This helps to understand the functional aspects and also aids in the development of novel inhibitors for the human RNR2. To confirm the binding mode of inhibitors, the molecular dynamics (MD) simulations were performed using GROMACS 4.5.5, based upon the docked conformation of inhibitors. Both of the studied compounds stayed in the active site. The results of MD simulations confirmed the binding mode of ligands, accuracy of docking and the reliability of active conformations which were obtained by AutoDock. MD studies confirm our proposed mechanism in which compound III reacts with the active site residues specially Tyr-176, and inhibits the radical generation and subsequently inhibits the RNR enzyme.

Different inhibitory potency of febuxostat towards mammalian and bacterial xanthine oxidoreductases: insight from molecular dynamics

PubMed Central

Kikuchi, Hiroto; Fujisaki, Hiroshi; Furuta, Tadaomi; Okamoto, Ken; Leimkühler, Silke; Nishino, Takeshi

2012-01-01

Febuxostat, a drug recently approved in the US, European Union and Japan for treatment of gout, inhibits xanthine oxidoreductase (XOR)-mediated generation of uric acid during purine catabolism. It inhibits bovine milk XOR with a Ki in the picomolar-order, but we found that it is a much weaker inhibitor of Rhodobacter capsulatus XOR, even though the substrate-binding pockets of mammalian and bacterial XOR are well-conserved as regards to catalytically important residues and three-dimensional structure, and both permit the inhibitor to be accommodated in the active site, as indicated by computational docking studies. To clarify the reason for the difference of inhibitory potency towards the two XORs, we performed molecular dynamics simulations. The results indicate that differences in mobility of hydrophobic residues that do not directly interact with the substrate account for the difference in inhibitory potency. PMID:22448318

Structure-activity relationships of benzimidazole-based glutaminyl cyclase inhibitors featuring a heteroaryl scaffold.

PubMed

Ramsbeck, Daniel; Buchholz, Mirko; Koch, Birgit; Böhme, Livia; Hoffmann, Torsten; Demuth, Hans-Ulrich; Heiser, Ulrich

2013-09-12

Glutaminyl cyclase (hQC) has emerged as a new potential target for the treatment of Alzheimer's disease (AD). The inhibition of hQC prevents of the formation of the Aβ3(pE)-40,42 species which were shown to be of elevated neurotoxicity and are likely to act as a seeding core, leading to an accelerated formation of Aβ-oligomers and fibrils. This work presents a new class of inhibitors of hQC, resulting from a pharmacophore-based screen. Hit molecules were identified, containing benzimidazole as the metal binding group connected to 1,3,4-oxadiazole as the central scaffold. The subsequent optimization resulted in benzimidazolyl-1,3,4-thiadiazoles and -1,2,3-triazoles with an inhibitory potency in the nanomolar range. Further investigation into the potential binding mode of the new compound classes combined molecular docking and site directed mutagenesis studies.

Molecular interaction of 2-mercaptobenzimidazole with catalase reveals a potentially toxic mechanism of the inhibitor.

PubMed

Teng, Yue; Zou, Luyi; Huang, Ming; Zong, Wansong

2014-12-01

2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment possesses a potential risk to human health. In this work, the toxic interaction of MBI with the important antioxidant enzyme catalase (CAT) was investigated using spectroscopic and molecular docking methods under physiological conditions. MBI can spontaneously bind with CAT with one binding site through hydrogen bonds and van der Waals forces to form MBI-CAT complex. The molecular docking study revealed that MBI bound into the CAT interface of chains B and C, which led to some conformational and microenvironmental changes of CAT and further resulted in the inhibition of CAT activity. This present study provides direct evidence at a molecular level to show that exposure to MBI could induce changes in the structure and function of the enzyme CAT. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

PubMed

Tang, Jing; Vernekar, Sanjeev Kumar V; Chen, Yue-Lei; Miller, Lena; Huber, Andrew D; Myshakina, Nataliya; Sarafianos, Stefan G; Parniak, Michael A; Wang, Zhengqiang

2017-06-16

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8-9 as exemplified with compounds 8c and 9c. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Wei; NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032; Li, Juan

The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC{sub 50} values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC{sub 50} values of 49.1 μM (for ZLA) and 55.3more » μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD. -- Highlights: ► Two novel bis-(−)-nor-meptazinol derivatives are designed and synthesized. ► ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency. ► They are potential leads for disease-modifying treatment of Alzheimer's disease.« less

Molecular mechanism of respiratory syncytial virus fusion inhibitors

PubMed Central

Battles, Michael B; Langedijk, Johannes P; Furmanova-Hollenstein, Polina; Chaiwatpongsakorn, Supranee; Costello, Heather M; Kwanten, Leen; Vranckx, Luc; Vink, Paul; Jaensch, Steffen; Jonckers, Tim H M; Koul, Anil; Arnoult, Eric; Peeples, Mark E; Roymans, Dirk; McLellan, Jason S

2016-01-01

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors. PMID:26641933

A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

PubMed

Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

1996-09-01

The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

Fluoride-Mediated Capture of a Noncovalent Bound State of a Reversible Covalent Enzyme Inhibitor: X-ray Crystallographic Analysis of an Exceptionally Potent [alpha]-Ketoheterocycle Inhibitor of Fatty Acid Amide Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine

2011-11-02

Two cocrystal X-ray structures of the exceptionally potent {alpha}-ketoheterocycle inhibitor 1 (K{sub i} = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitormore » within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same 'in action' state with the three catalytic residues Ser241-Ser217-Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of {alpha}-ketoheterocycle-based inhibitors bound to FAAH recently disclosed.« less

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2.

PubMed

Wang, Hong; Brautigan, David L

2002-12-20

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.

Inhibiting prolyl isomerase activity by hybrid organic-inorganic molecules containing rhodium(II) fragments.

PubMed

Coughlin, Jane M; Kundu, Rituparna; Cooper, Julian C; Ball, Zachary T

2014-11-15

A small molecule containing a rhodium(II) tetracarboxylate fragment is shown to be a potent inhibitor of the prolyl isomerase FKBP12. The use of small molecules conjugates of rhodium(II) is presented as a general strategy for developing new protein inhibitors based on distinct structural and sequence features of the enzyme active site. Copyright © 2014 Elsevier Ltd. All rights reserved.

Non-coding nucleotides and amino acids near the active site regulate peptide deformylase expression and inhibitor susceptibility in Chlamydia trachomatis

PubMed Central

Bao, Xiaofeng; Pachikara, Niseema D.; Oey, Christopher B.; Balakrishnan, Amit; Westblade, Lars F.; Tan, Ming; Chase, Theodore; Nickels, Bryce E.

2011-01-01

Chlamydia trachomatis, an obligate intracellular bacterium, is a highly prevalent human pathogen. Hydroxamic-acid-based matrix metalloprotease inhibitors can effectively inhibit the pathogen both in vitro and in vivo, and have exhibited therapeutic potential. Here, we provide genome sequencing data indicating that peptide deformylase (PDF) is the sole target of the inhibitors in this organism. We further report molecular mechanisms that control chlamydial PDF (cPDF) expression and inhibition efficiency. In particular, we identify the σ66-dependent promoter that controls cPDF gene expression and demonstrate that point mutations in this promoter lead to resistance by increasing cPDF transcription. Furthermore, we show that substitution of two amino acids near the active site of the enzyme alters enzyme kinetics and protein stability. PMID:21719536

2-(Hetero(aryl)methylene)hydrazine-1-carbothioamides as potent urease inhibitors.

PubMed

Saeed, Aamer; Imran, Aqeel; Channar, Pervaiz A; Shahid, Mohammad; Mahmood, Wajahat; Iqbal, Jamshed

2015-02-01

A small series of 2-(hetero(aryl)methylene) hydrazine-1-carbothioamides including two aryl derivatives was synthesized and tested for their inhibitory activity against urease. Compound (E)-2-(Furan-2-ylmethylene) hydrazine-1-carbothioamide (3f), having a furan ring, was the most potent inhibitor of urease with an IC50 value of 0.58 μM. Molecular modeling was carried out through docking the designed compounds into the urease binding site to predict whether these derivatives have analogous binding mode to the urease inhibitors. The study revealed that all of the tested compounds bind with both metal atoms at the active site of the enzyme. The aromatic ring of the compounds forms ionic interactions with the residues, Ala(440), Asp(494), Ala(636), and Met(637). © 2014 John Wiley & Sons A/S.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

PubMed

Manoharan, Prabu; Ghoshal, Nanda

2018-05-01

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Upregulation of RhoB via c-Jun N-terminal kinase signaling induces apoptosis of the human gastric carcinoma NUGC-3 cells treated with NSC12618.

PubMed

Kim, Bo-Kyung; Kim, Hwan Mook; Chung, Kyung-Sook; Kim, Dong-Myung; Park, Song-Kyu; Song, Alexander; Won, Kyoung-Jae; Lee, Kiho; Oh, Yu-Kyoung; Lee, Kyeong; Song, Kyung-Bin; Simon, Julian A; Han, Gyoonhee; Won, Misun

2011-03-01

RhoB expression is reduced in most invasive tumors, with loss of RhoB expression correlating significantly with tumor stage. Here, we demonstrate that upregulation of RhoB by the potent anticancer agent NSC126188 induces apoptosis of NUGC-3 human gastric carcinoma cells. The crucial role of RhoB in NSC126188-induced apoptosis is indicated by the rescue of NUGC-3 cells from apoptosis by knockdown of RhoB. In the presence of NSC126188, c-Jun N-terminal kinase (JNK) signaling was activated, and the JNK inhibitor SP600125 reduced RhoB expression and suppressed the apoptosis of NUGC-3 cells. Knockdowns of mitogen-activated protein kinase kinase (MKK) 4/7, JNK1/2 and c-Jun downregulated RhoB expression and rescued cells from apoptotic death in the presence of NSC126188. The JNK inhibitor SP600125 suppressed transcriptional activation of RhoB in the presence of NSC126188, as indicated by a reporter assay that used luciferase under the RhoB promoter. The ability of NSC126188 to increase luciferase activity through both the p300-binding site and the inverted CCAAT sequence (iCCAAT box) suggests that JNK signaling to upregulate RhoB expression is mediated through both the p300-binding site and the iCCAAT box. However, the JNK inhibitor SP600125 did not inhibit the upregulation of RhoB by farnesyltransferase inhibitor (FTI)-277. The p300-binding site did not affect activation of the RhoB promoter by FTI-277 in NUGC-3 cells, suggesting that the transcriptional activation of RhoB by NSC126188 occurs by a different mechanism than that reported for FTIs. Our data indicate that NSC126188 increases RhoB expression via JNK-mediated signaling through a p300-binding site and iCCAAT box resulting in apoptosis of NUGC-3 cells.

Mutagenesis of threonine to serine in the active site of Mycobacterium tuberculosis fructose-1,6-bisphosphatase (Class II) retains partial enzyme activity.

PubMed

Bondoc, Jasper Marc G; Wolf, Nina M; Ndichuck, Michael; Abad-Zapatero, Celerino; Movahedzadeh, Farahnaz

2017-09-01

The glpX gene encodes for the Class II fructose-1,6-bisphosphatase enzyme in Mycobacterium tuberculosis ( Mt ), an essential enzyme for pathogenesis. We have performed site directed mutagenesis to introduce two mutations at residue Thr84, T84A and T84S, to explore the binding affinity of the substrate and the catalytic mechanism. The T84A mutant fully abolishes enzyme activity while retaining substrate binding affinity. In contrast, the T84S mutant retains some activity having a 10 times reduction in V max and exhibited similar sensitivity to lithium when compared to the wildtype. Homology modeling using the Escherichia coli enzyme structure suggests that the replacement of the critical nucleophile OH - in the Thr84 residue of the wildtype of Mt FBPase by Ser84 results in subtle alterations of the position and orientation that reduce the catalytic efficiency. This mutant could be used to trap reaction intermediates, through crystallographic methods, facilitating the design of potent inhibitors via structure-based drug design.

Discovery of Tyk2 inhibitors via the virtual site-directed fragment-based drug design.

PubMed

Jang, Woo Dae; Kim, Jun-Tae; Son, Hoon Young; Park, Seung Yeon; Cho, Young Sik; Koo, Tae-sung; Lee, Hyuk; Kang, Nam Sook

2015-09-15

In this study, we synthesized compound 12 with potent Tyk2 inhibitory activity from FBDD study and carried out a cell-based assay for Tyk2/STAT3 signaling activation upon IFNα5 stimulation. Compound 12 completely suppressed the IFNα5-mediated Tyk2/STAT3 signaling pathway as well as the basal levels of pSTAT3. Stimulation with IFNα/β leads to the tyrosine phosphorylation of the JAK1 and Tyk2 receptor-associated kinases with subsequent STATs activation, transmitting signals from the cell surface receptor to the nucleus. In conclusion, the potency of compound 12 to interrupt the signal transmission of Tyk2/STAT3 appeared to be equivalent or superior to that of the reference compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

New Perspectives in the Renin-Angiotensin-Aldosterone System (RAAS) I: Endogenous Angiotensin Converting Enzyme (ACE) Inhibition

PubMed Central

Fagyas, Miklós; Úri, Katalin; Siket, Ivetta M.; Daragó, Andrea; Boczán, Judit; Bányai, Emese; Édes, István; Papp, Zoltán; Tóth, Attila

2014-01-01

Angiotensin-converting enzyme (ACE) inhibitors represent the fifth most often prescribed drugs. ACE inhibitors decrease 5-year mortality by approximately one-fifth in cardiovascular patients. Surprisingly, there are reports dating back to 1979 suggesting the existence of endogenous ACE inhibitors, which endogenous inhibitory effects are much less characterized than that for the clinically administered ACE inhibitors. Here we aimed to investigate this endogenous ACE inhibition in human sera. It was hypothesized that ACE activity is masked by an endogenous inhibitor, which dissociates from the ACE when its concentration decreases upon dilution. ACE activity was measured by FAPGG hydrolysis first. The specific (dilution corrected) enzyme activities significantly increased by dilution of human serum samples (23.2±0.7 U/L at 4-fold dilution, 51.4±0.3 U/L at 32-fold dilution, n = 3, p = 0.001), suggesting the presence of an endogenous inhibitor. In accordance, specific enzyme activities did not changed by dilution when purified renal ACE was used, where no endogenous inhibitor was present (655±145 U/L, 605±42 U/L, n = 3, p = 0.715, respectively). FAPGG conversion strongly correlated with angiotensin I conversion suggesting that this feature is not related to the artificial substrate. Serum samples were ultra-filtered to separate ACE (MW: 180 kDa) and the hypothesized inhibitor. Filtering through 50 kDa filters was without effect, while filtering through 100 kDa filters eliminated the inhibiting factor (ACE activity after <100 kDa filtering: 56.4±2.4 U/L, n = 4, control: 26.4±0.7 U/L, n = 4, p<0.001). Lineweaver-Burk plot indicated non-competitive inhibition of ACE by this endogenous factor. The endogenous inhibitor had higher potency on the C-terminal active site than N-terminal active site of ACE. Finally, this endogenous ACE inhibition was also present in mouse, donkey, goat, bovine sera besides men (increasing of specific ACE activity from 4-fold to 32-fold dilution: 2.8-fold, 1.7-fold, 1.5-fold, 1.8-fold, 2.6-fold, respectively). We report here the existence of an evolutionary conserved mechanism suppressing circulating ACE activity, in vivo, similarly to ACE inhibitory drugs. PMID:24691160

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

PubMed Central

Ding, Kejia; Wang, Annie; Boerneke, Mark A.; Dibrov, Sergey M.; Hermann, Thomas

2014-01-01

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex. PMID:24856063

Resistance to AHAS inhibitor herbicides: current understanding.

PubMed

Yu, Qin; Powles, Stephen B

2014-09-01

Acetohydroxyacid synthase (AHAS) inhibitor herbicides currently comprise the largest site-of-action group (with 54 active ingredients across five chemical groups) and have been widely used in world agriculture since they were first introduced in 1982. Resistance evolution in weeds to AHAS inhibitors has been rapid and identified in populations of many weed species. Often, evolved resistance is associated with point mutations in the target AHAS gene; however non-target-site enhanced herbicide metabolism occurs as well. Many AHAS gene resistance mutations can occur and be rapidly enriched owing to a high initial resistance gene frequency, simple and dominant genetic inheritance and lack of major fitness cost of the resistance alleles. Major advances in the elucidation of the crystal structure of the AHAS (Arabidopsis thaliana) catalytic subunit in complex with various AHAS inhibitor herbicides have greatly improved current understanding of the detailed molecular interactions between AHAS, cofactors and herbicides. Compared with target-site resistance, non-target-site resistance to AHAS inhibitor herbicides is less studied and hence less understood. In a few well-studied cases, non-target-site resistance is due to enhanced rates of herbicide metabolism (metabolic resistance), mimicking that occurring in tolerant crop species and often involving cytochrome P450 monooxygenases. However, the specific herbicide-metabolising, resistance-endowing genes are yet to be identified in resistant weed species. The current state of mechanistic understanding of AHAS inhibitor herbicide resistance is reviewed, and outstanding research issues are outlined. © 2013 Society of Chemical Industry.

Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1.

PubMed

Stamogiannos, Athanasios; Maben, Zachary; Papakyriakou, Athanasios; Mpakali, Anastasia; Kokkala, Paraskevi; Georgiadis, Dimitris; Stern, Lawrence J; Stratikos, Efstratios

2017-03-14

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.

Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations.

PubMed

Fang, Lei; Zhang, Huai; Cui, Wei; Ji, Mingjun

2008-10-01

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.

Role of asparagine 152 in catalysis of beta-lactam hydrolysis by Escherichia coli AmpC beta-lactamase studied by site-directed mutagenesis.

PubMed

Dubus, A; Normark, S; Kania, M; Page, M G

1995-06-13

The role of asparagine 152 in the catalytic mechanism of Escherichia coli AmpC beta-lactamase has been investigated by site-directed mutagenesis. The residue has been replaced by aspartic acid, glutamic acid, histidine, and leucine. All the substitutions had similar effects on the activity toward substrates and inhibitors. The rate of substrate hydrolysis decreased by factors of 500-5000. The rates of both acylation (2-50-fold decrease) and deacylation (50-500-fold decrease) were affected, indicating a role for Asn152 in both processes. The wild-type AmpC beta-lactamase appears to exist as an equilibrium mixture of two forms, identified by their different kinetic properties. The Asn152 mutations affected the activity of the slow-reacting form much more than that of the fast-reacting form, but they did not appear to affect the interconversion of these two kinetic forms. Comparison of these observations with results obtained with mutation of the equivalent residues in other classes of penicillin-sensitive enzyme indicates that there are quite profound differences between the catalytic mechanisms of these enzymes despite a high degree of conservation of amino acids in the active center, and of the overall three-dimensional structure.

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

PubMed

Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

2003-07-01

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

Identification of new allosteric sites and modulators of AChE through computational and experimental tools.

PubMed

Roca, Carlos; Requena, Carlos; Sebastián-Pérez, Víctor; Malhotra, Sony; Radoux, Chris; Pérez, Concepción; Martinez, Ana; Antonio Páez, Juan; Blundell, Tom L; Campillo, Nuria E

2018-12-01

Allosteric sites on proteins are targeted for designing more selective inhibitors of enzyme activity and to discover new functions. Acetylcholinesterase (AChE), which is most widely known for the hydrolysis of the neurotransmitter acetylcholine, has a peripheral allosteric subsite responsible for amyloidosis in Alzheimer's disease through interaction with amyloid β-peptide. However, AChE plays other non-hydrolytic functions. Here, we identify and characterise using computational tools two new allosteric sites in AChE, which have allowed us to identify allosteric inhibitors by virtual screening guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. These new compounds may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.

Synthesis and discovery of highly functionalized mono- and bis-spiro-pyrrolidines as potent cholinesterase enzyme inhibitors.

PubMed

Kia, Yalda; Osman, Hasnah; Suresh Kumar, Raju; Basiri, Alireza; Murugaiyah, Vikneswaran

2014-04-01

Novel mono and bis spiropyrrolidine derivatives were synthesized via an efficient ionic liquid mediated, 1,3-dipolar cycloaddition methodology and evaluated in vitro for their AChE and BChE inhibitory activities in search for potent cholinesterase enzyme inhibitors. Most of the synthesized compounds displayed remarkable AChE inhibitory activities with IC50 values ranging from 1.68 to 21.85 μM, wherein compounds 8d and 8j were found to be most active inhibitors against AChE and BChE with IC50 values of 1.68 and 2.75 μM, respectively. Molecular modeling simulation on Torpedo californica AChE and human BChE receptors, showed good correlation between IC50 values and binding interaction template of the most active inhibitors docked into the active site of their relevant enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rhodium(II) proximity-labeling identifies a novel target site on STAT3 for inhibitors with potent anti-leukemia activity

PubMed Central

Minus, Matthew B.; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M.; Long, Xin; Krueger, Michael; Stevens, Alexandra; Kolosov, Mikhail I.; Sison, Edward Allen R.; Ball, Zachary T.

2015-01-01

Nearly 40% of children with acute myeloid leukemia (AML) suffer relapse due to chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). In this paper, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. PMID:26480340

The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.

PubMed

Jevtuševskaja, Jekaterina; Krõlov, Katrin; Tulp, Indrek; Langel, Ülo

2017-04-01

The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases. These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.

Analogs of methyllycaconitine as novel noncompetitive inhibitors of nicotinic receptors: pharmacological characterization, computational modeling, and pharmacophore development.

PubMed

McKay, Dennis B; Chang, Cheng; González-Cestari, Tatiana F; McKay, Susan B; El-Hajj, Raed A; Bryant, Darrell L; Zhu, Michael X; Swaan, Peter W; Arason, Kristjan M; Pulipaka, Aravinda B; Orac, Crina M; Bergmeier, Stephen C

2007-05-01

As a novel approach to drug discovery involving neuronal nicotinic acetylcholine receptors (nAChRs), our laboratory targeted nonagonist binding sites (i.e., noncompetitive binding sites, negative allosteric binding sites) located on nAChRs. Cultured bovine adrenal cells were used as neuronal models to investigate interactions of 67 analogs of methyllycaconitine (MLA) on native alpha3beta4* nAChRs. The availability of large numbers of structurally related molecules presents a unique opportunity for the development of pharmacophore models for noncompetitive binding sites. Our MLA analogs inhibited nicotine-mediated functional activation of both native and recombinant alpha3beta4* nAChRs with a wide range of IC(50) values (0.9-115 microM). These analogs had little or no inhibitory effects on agonist binding to native or recombinant nAChRs, supporting noncompetitive inhibitory activity. Based on these data, two highly predictive 3D quantitative structure-activity relationship (comparative molecular field analysis and comparative molecular similarity index analysis) models were generated. These computational models were successfully validated and provided insights into the molecular interactions of MLA analogs with nAChRs. In addition, a pharmacophore model was constructed to analyze and visualize the binding requirements to the analog binding site. The pharmacophore model was subsequently applied to search structurally diverse molecular databases to prospectively identify novel inhibitors. The rapid identification of eight molecules from database mining and our successful demonstration of in vitro inhibitory activity support the utility of these computational models as novel tools for the efficient retrieval of inhibitors. These results demonstrate the effectiveness of computational modeling and pharmacophore development, which may lead to the identification of new therapeutic drugs that target novel sites on nAChRs.

Design, synthesis and biological activity of novel donepezil derivatives bearing N-benzyl pyridinium moiety as potent and dual binding site acetylcholinesterase inhibitors.

PubMed

Lan, Jin-Shuai; Zhang, Tong; Liu, Yun; Yang, Jing; Xie, Sai-Sai; Liu, Jing; Miao, Ze-Yang; Ding, Yue

2017-06-16

A series of new donepezil derivatives were designed synthesized and evaluated as multifunctional cholinesterase inhibitors against Alzheimer's disease (AD). In vitro studies showed that most of them exhibited significant potency to inhibit acetylcholinesterase and self-induced β-amyloid (Aβ) aggregation, and moderate antioxidant activity. Especially, compound 5b presented the greatest ability to inhibit cholinesterase (IC 50 , 1.9 nM for eeAChE and 0.8 nM for hAChE), good inhibition of Aβ aggregation (53.7% at 20 μM) and good antioxidant activity (0.54 trolox equivalents). Kinetic and molecular modeling studies indicated that compound 5b was a mixed-type inhibitor, binding simultaneously to the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. In addition, compound 5b could reduce PC12 cells death induced by oxidative stress and Aβ (1-42). Moreover, in vivo experiments showed that compound 5b was nontoxic and tolerated at doses up to 2000 mg/kg. These results suggested that compound 5b might be an excellent multifunctional agent for AD treatment. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.

Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

ENDOTHELIN-STIMULATED HUMAN B-TYPE NATRIURETIC PEPTIDE GENE EXPRESSION IS MEDIATED BY YY1 IN ASSOCIATION WITH HDAC2

PubMed Central

Glenn, Denis J.; Wang, Feng; Chen, Songcang; Nishimoto, Minobu; Gardner, David G.

2009-01-01

Increased B-type natriuretic peptide (BNP) gene expression is regarded as one of the hallmarks of cardiac myocyte hypertrophy. Here we demonstrate that both basal and endothelin-1 (ET-1) -dependent stimulation of human (h) BNP gene transcription requires the presence of an intact Yin Yang 1 (YY1) binding site positioned at -62 bp relative to the transcription start site. Mutation of this site reduced both basal and stimulated hBNP promoter activity. This site was shown to bind YY1 both in vitro and within the context of the intact cell. The latter interaction increased following ET-1 treatment. Exposure to ET-1 also resulted in increased nuclear localization of YY1 and a reduction in acetylation of the YY1 protein. Overexpression of wild type YY1 increased both basal and endothelin-stimulated hBNP promoter activity, while a carboxy terminal deletion mutant of YY1 was devoid of activity. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) resulted in decreased hBNP reporter activity. YY1 was shown to associate with histone deacetylase 2 (HDAC2), and HDAC2 was shown to associate directly with the hBNP promoter in the intact cell. Collectively these findings demonstrate that YY1 plays an important role in regulating the transcriptional activity of the hBNP gene promoter. These data suggest a model in which YY1 activates hBNP transcription through interaction with HDAC2. PMID:19139378

Structures of Human Golgi-resident Glutaminyl Cyclase and Its Complexes with Inhibitors Reveal a Large Loop Movement upon Inhibitor Binding*

PubMed Central

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H.-J.

2011-01-01

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05–1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC. PMID:21288892

Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

PubMed

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

2011-04-08

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

Cheminformatics-aided discovery of small-molecule Protein-Protein Interaction (PPI) dual inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL).

PubMed

Melagraki, Georgia; Ntougkos, Evangelos; Rinotas, Vagelis; Papaneophytou, Christos; Leonis, Georgios; Mavromoustakos, Thomas; Kontopidis, George; Douni, Eleni; Afantitis, Antreas; Kollias, George

2017-04-01

We present an in silico drug discovery pipeline developed and applied for the identification and virtual screening of small-molecule Protein-Protein Interaction (PPI) compounds that act as dual inhibitors of TNF and RANKL through the trimerization interface. The cheminformatics part of the pipeline was developed by combining structure-based with ligand-based modeling using the largest available set of known TNF inhibitors in the literature (2481 small molecules). To facilitate virtual screening, the consensus predictive model was made freely available at: http://enalos.insilicotox.com/TNFPubChem/. We thus generated a priority list of nine small molecules as candidates for direct TNF function inhibition. In vitro evaluation of these compounds led to the selection of two small molecules that act as potent direct inhibitors of TNF function, with IC50 values comparable to those of a previously-described direct inhibitor (SPD304), but with significantly reduced toxicity. These molecules were also identified as RANKL inhibitors and validated in vitro with respect to this second functionality. Direct binding of the two compounds was confirmed both for TNF and RANKL, as well as their ability to inhibit the biologically-active trimer forms. Molecular dynamics calculations were also carried out for the two small molecules in each protein to offer additional insight into the interactions that govern TNF and RANKL complex formation. To our knowledge, these compounds, namely T8 and T23, constitute the second and third published examples of dual small-molecule direct function inhibitors of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.

Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

PubMed

Neis, Vivian Binder; Moretti, Morgana; Bettio, Luis Eduardo B; Ribeiro, Camille M; Rosa, Priscila Batista; Gonçalves, Filipe Marques; Lopes, Mark William; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

2016-06-01

The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5μg/site, i.c.v.), BDNF antibody (1μg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1μg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3β inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, 0.01μg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3β, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

Inhibiting the HIV Integration Process: Past, Present, and the Future

PubMed Central

2013-01-01

HIV integrase (IN) catalyzes the insertion into the genome of the infected human cell of viral DNA produced by the retrotranscription process. The discovery of raltegravir validated the existence of the IN, which is a new target in the field of anti-HIV drug research. The mechanism of catalysis of IN is depicted, and the characteristics of the inhibitors of the catalytic site of this viral enzyme are reported. The role played by the resistance is elucidated, as well as the possibility of bypassing this problem. New approaches to block the integration process are depicted as future perspectives, such as development of allosteric IN inhibitors, dual inhibitors targeting both IN and other enzymes, inhibitors of enzymes that activate IN, activators of IN activity, as well as a gene therapy approach. PMID:24025027

Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crichlow, G.; Lubetsky, J; Leng, L

Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic datamore » indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.« less

Impact of protists on a hydrocarbon-degrading bacterial community from deep-sea Gulf of Mexico sediments: A microcosm study

NASA Astrophysics Data System (ADS)

Beaudoin, David J.; Carmichael, Catherine A.; Nelson, Robert K.; Reddy, Christopher M.; Teske, Andreas P.; Edgcomb, Virginia P.

2016-07-01

In spite of significant advancements towards understanding the dynamics of petroleum hydrocarbon degrading microbial consortia, the impacts (direct or indirect via grazing activities) of bacterivorous protists remain largely unknown. Microcosm experiments were used to examine whether protistan grazing affects the petroleum hydrocarbon degradation capacity of a deep-sea sediment microbial community from an active Gulf of Mexico cold seep. Differences in n-alkane content between native sediment microcosms and those treated with inhibitors of eukaryotes were assessed by comprehensive two-dimensional gas chromatography following 30-90 day incubations and analysis of shifts in microbial community composition using small subunit ribosomal RNA gene clone libraries. More biodegradation was observed in microcosms supplemented with eukaryotic inhibitors. SSU rRNA gene clone libraries from oil-amended treatments revealed an increase in the number of proteobacterial clones (particularly γ-proteobacteria) after spiking sediments with diesel oil. Bacterial community composition shifted, and degradation rates increased, in treatments where protists were inhibited, suggesting protists affect the hydrocarbon degrading capacity of microbial communities in sediments collected at this Gulf of Mexico site.

Identification and Characterization of BMS-955176, a Second-Generation HIV-1 Maturation Inhibitor with Improved Potency, Antiviral Spectrum, and Gag Polymorphic Coverage.

PubMed

Nowicka-Sans, Beata; Protack, Tricia; Lin, Zeyu; Li, Zhufang; Zhang, Sharon; Sun, Yongnian; Samanta, Himadri; Terry, Brian; Liu, Zheng; Chen, Yan; Sin, Ny; Sit, Sing-Yuen; Swidorski, Jacob J; Chen, Jie; Venables, Brian L; Healy, Matthew; Meanwell, Nicholas A; Cockett, Mark; Hanumegowda, Umesh; Regueiro-Ren, Alicia; Krystal, Mark; Dicker, Ira B

2016-07-01

BMS-955176 is a second-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor (MI). A first-generation MI, bevirimat, showed clinical efficacy in early-phase studies, but ∼50% of subjects had viruses with reduced susceptibility associated with naturally occurring polymorphisms in Gag near the site of MI action. MI potency was optimized using a panel of engineered reporter viruses containing site-directed polymorphic changes in Gag that reduce susceptibility to bevirimat (including V362I, V370A/M/Δ, and T371A/Δ), leading incrementally to the identification of BMS-955176. BMS-955176 exhibits potent activity (50% effective concentration [EC50], 3.9 ± 3.4 nM [mean ± standard deviation]) toward a library (n = 87) of gag/pr recombinant viruses representing 96.5% of subtype B polymorphic Gag diversity near the CA/SP1 cleavage site. BMS-955176 exhibited a median EC50 of 21 nM toward a library of subtype B clinical isolates assayed in peripheral blood mononuclear cells (PBMCs). Potent activity was maintained against a panel of reverse transcriptase, protease, and integrase inhibitor-resistant viruses, with EC50s similar to those for the wild-type virus. A 5.4-fold reduction in EC50 occurred in the presence of 40% human serum plus 27 mg/ml of human serum albumin (HSA), which corresponded well to an in vitro measurement of 86% human serum binding. Time-of-addition and pseudotype reporter virus studies confirm a mechanism of action for the compound that occurs late in the virus replication cycle. BMS-955176 inhibits HIV-1 protease cleavage at the CA/SP1 junction within Gag in virus-like particles (VLPs) and in HIV-1-infected cells, and it binds reversibly and with high affinity to assembled Gag in purified HIV-1 VLPs. Finally, in vitro combination studies showed no antagonistic interactions with representative antiretrovirals (ARVs) of other mechanistic classes. In conclusion, BMS-955176 is a second-generation MI with potent in vitro anti-HIV-1 activity and a greatly improved preclinical profile compared to that of bevirimat. Copyright © 2016 Nowicka-Sans et al.

High-throughput Screening Identification of Poliovirus RNA-dependent RNA Polymerase Inhibitors

PubMed Central

Campagnola, Grace; Gong, Peng; Peersen, Olve B.

2011-01-01

Viral RNA-dependent RNA polymerase (RdRP) enzymes are essential for the replication of positive-strand RNA viruses and established targets for the development of selective antiviral therapeutics. In this work we have carried out a high-throughput screen of 154,267 compounds to identify poliovirus polymerase inhibitors using a fluorescence based RNA elongation assay. Screening and subsequent validation experiments using kinetic methods and RNA product analysis resulted in the identification of seven inhibitors that affect the RNA binding, initiation, or elongation activity of the polymerase. X-ray crystallography data show clear density for five of the compounds in the active site of the poliovirus polymerase elongation complex. The inhibitors occupy the NTP binding site by stacking on the priming nucleotide and interacting with the templating base, yet competition studies show fairly weak IC50 values in the low μM range. A comparison with nucleotide bound structures suggests that weak binding is likely due to the lack of a triphosphate group on the inhibitors. Consequently, the inhibitors are primarily effective at blocking polymerase initiation and do not effectively compete with NTP binding during processive elongation. These findings are discussed in the context of the polymerase elongation complex structure and allosteric control of the viral RdRP catalytic cycle. PMID:21722674

Important Aspects of Post-Prandial Antidiabetic Drug, Acarbose.

PubMed

Singla, Rajeev Kumar; Singh, Radha; Dubey, Ashok Kumar

2016-01-01

Acarbose, a well known and efficacious α-amylase and α-glucosidase inhibitor, is a postprandial acting antidiabetic drug. DNS-based α-amylase inhibitory assays showed that use of acarbose at concentrations above 125 µg/ml resulted in release of reducing sugar in the reaction, an unexpected observation. Objective of the present study was to design experimental strategies to address this unusual finding. Acarbose was found to be susceptible to thermo-lysis. Further, besides being an inhibitor, it could also be hydrolyzed by porcine pancreatic α-amylase, but had weaker affinity for α - amylase compared to starch. GRIP docking was done for the mechanistic analysis of the active site in the enzyme for substrate, inhibitor and, inhibitor's metabolite (K2). Interaction between acarbose and α-amylase involved significant hydrogen binding compared to that of starch, producing a stronger enzyme-inhibitor complex. Further, docking analysis led us to predict the site on α-amylase where the inhibitor (acarbose) bound more tightly, which possibly affected the binding and hydrolysis of starch exerting its effective anti-diabetic function.

Computational Insight into Protein Tyrosine Phosphatase 1B Inhibition: A Case Study of the Combined Ligand- and Structure-Based Approach.

PubMed

Zhang, Xiangyu; Jiang, Hailun; Li, Wei; Wang, Jian; Cheng, Maosheng

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) is an attractive target for treating cancer, obesity, and type 2 diabetes. In our work, the way of combined ligand- and structure-based approach was applied to analyze the characteristics of PTP1B enzyme and its interaction with competitive inhibitors. Firstly, the pharmacophore model of PTP1B inhibitors was built based on the common feature of sixteen compounds. It was found that the pharmacophore model consisted of five chemical features: one aromatic ring (R) region, two hydrophobic (H) groups, and two hydrogen bond acceptors (A). To further elucidate the binding modes of these inhibitors with PTP1B active sites, four docking programs (AutoDock 4.0, AutoDock Vina 1.0, standard precision (SP) Glide 9.7, and extra precision (XP) Glide 9.7) were used. The characteristics of the active sites were then described by the conformations of the docking results. In conclusion, a combination of various pharmacophore features and the integration information of structure activity relationship (SAR) can be used to design novel potent PTP1B inhibitors.

Discovery and Optimization of Allosteric Inhibitors of Mutant Isocitrate Dehydrogenase 1 (R132H IDH1) Displaying Activity in Human Acute Myeloid Leukemia Cells.

PubMed

Jones, Stuart; Ahmet, Jonathan; Ayton, Kelly; Ball, Matthew; Cockerill, Mark; Fairweather, Emma; Hamilton, Nicola; Harper, Paul; Hitchin, James; Jordan, Allan; Levy, Colin; Lopez, Ruth; McKenzie, Eddie; Packer, Martin; Plant, Darren; Simpson, Iain; Simpson, Peter; Sinclair, Ian; Somervaille, Tim C P; Small, Helen; Spencer, Gary J; Thomson, Graeme; Tonge, Michael; Waddell, Ian; Walsh, Jarrod; Waszkowycz, Bohdan; Wigglesworth, Mark; Wiseman, Daniel H; Ogilvie, Donald

2016-12-22

A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.

Three camelid VHH domains in complex with porcine pancreatic alpha-amylase. Inhibition and versatility of binding topology.

PubMed

Desmyter, Aline; Spinelli, Silvia; Payan, Francoise; Lauwereys, Marc; Wyns, Lode; Muyldermans, Serge; Cambillau, Christian

2002-06-28

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.

Assessment of the Target Engagement and D-Serine Biomarker Profiles of the D-Amino Acid Oxidase Inhibitors Sodium Benzoate and PGM030756.

PubMed

Howley, Eimear; Bestwick, Michael; Fradley, Rosa; Harrison, Helen; Leveridge, Mathew; Okada, Kengo; Fieldhouse, Charlotte; Farnaby, Will; Canning, Hannah; Sykes, Andy P; Merchant, Kevin; Hazel, Katherine; Kerr, Catrina; Kinsella, Natasha; Walsh, Louise; Livermore, David G; Hoffman, Isaac; Ellery, Jonathan; Mitchell, Phillip; Patel, Toshal; Carlton, Mark; Barnes, Matt; Miller, David J

2017-11-01

Irregular N-methyl-D-aspartate receptor (NMDAR) function is one of the main hypotheses employed to facilitate understanding of the underlying disease state of schizophrenia. Although direct agonism of the NMDAR has not yielded promising therapeutics, advances have been made by modulating the NMDAR co-agonist site which is activated by glycine and D-serine. One approach to activate the co-agonist site is to increase synaptic D-serine levels through inhibition of D-amino acid oxidase (DAO), the major catabolic clearance pathway for this and other D-amino acids. A number of DAO inhibitors have been developed but most have not entered clinical trials. One exception to this is sodium benzoate which has demonstrated efficacy in small trials of schizophrenia and Alzheimer's disease. Herein we provide data on the effect of sodium benzoate and an optimised Takeda compound, PGM030756 on ex vivo DAO enzyme occupancy and cerebellar D-serine levels in mice. Both compounds achieve high levels of enzyme occupancy; although lower doses of PGM030756 (1, 3 and 10 mg/kg) were required to achieve this compared to sodium benzoate (300, 1000 mg/kg). Cerebellar D-serine levels were increased by both agents with a delay of approximately 6 h after dosing before the peak effect was achieved. Our data and methods may be useful in understanding the effects of sodium benzoate that have been seen in clinical trials of schizophrenia and Alzheimer's disease and to support the potential clinical assessment of other DAO inhibitors, such as PGM030756, which demonstrate good enzyme occupancy and D-serine increases following administration of low oral doses.

Angiotensin-I-converting enzyme and its relatives

PubMed Central

Riordan, James F

2003-01-01

Angiotensin-I-converting enzyme (ACE) is a monomeric, membrane-bound, zinc- and chloride-dependent peptidyl dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a carboxy-terminal dipeptide. ACE has long been known to be a key part of the renin angiotensin system that regulates blood pressure, and ACE inhibitors are important for the treatment of hypertension. There are two forms of the enzyme in humans, the ubiquitous somatic ACE and the sperm-specific germinal ACE, both encoded by the same gene through transcription from alternative promoters. Somatic ACE has two tandem active sites with distinct catalytic properties, whereas germinal ACE, the function of which is largely unknown, has just a single active site. Recently, an ACE homolog, ACE2, has been identified in humans that differs from ACE in being a carboxypeptidase that preferentially removes carboxy-terminal hydrophobic or basic amino acids; it appears to be important in cardiac function. ACE homologs (also known as members of the M2 gluzincin family) have been found in a wide variety of species, even in those that neither have a cardiovascular system nor synthesize angiotensin. X-ray structures of a truncated, deglycosylated form of germinal ACE and a related enzyme from Drosophila have been reported, and these show that the active site is deep within a central cavity. Structure-based drug design targeting the individual active sites of somatic ACE may lead to a new generation of ACE inhibitors, with fewer side-effects than currently available inhibitors. PMID:12914653

Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

NASA Astrophysics Data System (ADS)

Pautus, Stéphane; Alami, Mouad; Adam, Fréderic; Bernadat, Guillaume; Lawrence, Daniel A.; de Carvalho, Allan; Ferry, Gilles; Rupin, Alain; Hamze, Abdallah; Champy, Pierre; Bonneau, Natacha; Gloanec, Philippe; Peglion, Jean-Louis; Brion, Jean-Daniel; Bianchini, Elsa P.; Borgel, Delphine

2016-11-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

Screening of benzamidine-based thrombin inhibitors via a linear interaction energy in continuum electrostatics model

NASA Astrophysics Data System (ADS)

Nicolotti, Orazio; Giangreco, Ilenia; Miscioscia, Teresa Fabiola; Convertino, Marino; Leonetti, Francesco; Pisani, Leonardo; Carotti, Angelo

2010-02-01

A series of 27 benzamidine inhibitors covering a wide range of biological activity and chemical diversity was analysed to derive a Linear Interaction Energy in Continuum Electrostatics (LIECE) model for analysing the thrombin inhibitory activity. The main interactions occurring at the thrombin binding site and the preferred binding conformations of inhibitors were explicitly biased by including into the LIECE model 10 compounds extracted from X-ray solved thrombin-inhibitor complexes available from the Protein Data Bank (PDB). Supported by a robust statistics ( r 2 = 0.698; q 2 = 0.662), the LIECE model was successful in predicting the inhibitory activity for about 76% of compounds ( r ext 2 ≥ 0.600) from a larger external test set encompassing 88 known thrombin inhibitors and, more importantly, in retrieving, at high sensitivity and with better performance than docking and shape-based methods, active compounds from a thrombin combinatorial library of 10240 mimetic chemical products. The herein proposed LIECE model has the potential for successfully driving the design of novel thrombin inhibitors with benzamidine and/or benzamidine-like chemical structure.

Novel tacrine/acridine anticholinesterase inhibitors with piperazine and thiourea linkers.

PubMed

Hamulakova, Slavka; Imrich, Jan; Janovec, Ladislav; Kristian, Pavol; Danihel, Ivan; Holas, Ondrej; Pohanka, Miroslav; Böhm, Stanislav; Kozurkova, Maria; Kuca, Kamil

2014-09-01

A new series of substituted tacrine/acridine and tacrine/tacrine dimers with aliphatic or alkylene-thiourea linkers was synthesized and the potential of these compounds as novel human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE) inhibitors with nanomolar inhibition activity was evaluated. The most potent AChE inhibitor was found to be homodimeric tacrine derivative 14a, which demonstrated an IC50 value of 2 nM; this value indicates an activity rate which is 250-times higher than that of tacrine 1 and 7500-times higher than 7-MEOTA 15, the compounds which were used as standards in the study. IC50 values of derivatives 1, 9, 10, 14b and 15 were compared with the dissociation constants of the enzyme-inhibitor complex, Ki1, and the enzyme-substrate-inhibitor complex, Ki2, for. A dual binding site is presumed for the synthesized compounds which possess two tacrines or tacrine and acridine as terminal moieties show evidence of dual site binding. DFT calculations of theoretical desolvation free energies, ΔΔGtheor, and docking studies elucidate these suggestions in more detail. Copyright © 2014 Elsevier B.V. All rights reserved.

Crystal Structure of Ll-Diaminopimelate Aminotransferase From 'Arabidopsis Thaliana': a Recently-Discovered Enzyme in the Biosynthesis of L-Lysine By Plants And 'Chlamydia'

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, N.; Cherney, M.M.; van Belkum, M.J.

2007-07-13

The essential biosynthetic pathway to l-Lysine in bacteria and plants is an attractive target for the development of new antibiotics or herbicides because it is absent in humans, who must acquire this amino acid in their diet. Plants use a shortcut of a bacterial pathway to l-Lysine in which the pyridoxal-5-phosphate (PLP)-dependent enzyme ll-diaminopimelate aminotransferase (LL-DAP-AT) transforms l-tetrahydrodipicolinic acid (L-THDP) directly to LL-DAP. In addition, LL-DAP-AT was recently found in Chlamydia sp., suggesting that inhibitors of this enzyme may also be effective against such organisms. In order to understand the mechanism of this enzyme and to assist in the designmore » of inhibitors, the three-dimensional crystal structure of LL-DAP-AT was determined at 1.95 Angstroms resolution. The cDNA sequence of LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT) was optimized for expression in bacteria and cloned in Escherichia coli without its leader sequence but with a C-terminal hexahistidine affinity tag to aid protein purification. The structure of AtDAP-AT was determined using the multiple-wavelength anomalous dispersion (MAD) method with a seleno-methionine derivative. AtDAP-AT is active as a homodimer with each subunit having PLP in the active site. It belongs to the family of type I fold PLP-dependent enzymes. Comparison of the active site residues of AtDAP-AT and aspartate aminotransferases revealed that the PLP binding residues in AtDAP-AT are well conserved in both enzymes. However, Glu97* and Asn309* in the active site of AtDAP-AT are not found at similar positions in aspartate aminotransferases, suggesting that specific substrate recognition may require these residues from the other monomer. A malate-bound structure of AtDAP-AT allowed LL-DAP and L-glutamate to be modeled into the active site. These initial three-dimensional structures of LL-DAP-AT provide insight into its substrate specificity and catalytic mechanism.« less

The N Domain of Human Angiotensin-I-converting Enzyme

PubMed Central

Anthony, Colin S.; Corradi, Hazel R.; Schwager, Sylva L. U.; Redelinghuys, Pierre; Georgiadis, Dimitris; Dive, Vincent; Acharya, K. Ravi; Sturrock, Edward D.

2010-01-01

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding. PMID:20826823

Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan

2010-06-14

In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to designmore » novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.« less

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.

PubMed

Dömötör, Orsolya; Pelivan, Karla; Borics, Attila; Keppler, Bernhard K; Kowol, Christian R; Enyedy, Éva A

2018-05-30

Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding. Copyright © 2018 Elsevier B.V. All rights reserved.

Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

PubMed Central

Termathe, Martin; Grütter, Christian; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, Daniel

2012-01-01

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. PMID:22768308

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability.

PubMed

Andersson, Vincent; Bergström, Fredrik; Brånalt, Jonas; Grönberg, Gunnar; Gustafsson, David; Karlsson, Staffan; Polla, Magnus; Bergman, Joakim; Kihlberg, Jan

2016-07-28

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not.

Focused library with a core structure extracted from natural products and modified: application to phosphatase inhibitors and several biochemical findings.

PubMed

Hirai, Go; Sodeoka, Mikiko

2015-05-19

Synthesis of a focused library is an important strategy to create novel modulators of specific classes of proteins. Compounds in a focused library are composed of a common core structure and different diversity structures. In this Account, we describe our design and synthesis of libraries focused on selective inhibitors of protein phosphatases (PPases). We considered that core structures having structural and electronic features similar to those of PPase substrates, phosphate esters, would be a reasonable choice. Therefore, we extracted core structures from natural products already identified as PPase inhibitors. Since many PPases share similar active-site structures, such phosphate-mimicking core structures should interact with many enzymes in the same family, and therefore the choice of diversity structures is pivotal both to increase the binding affinity and to achieve specificity for individual enzymes. Here we present case studies of application of focused libraries to obtain PPase inhibitors, covering the overall process from selection of core structures to identification and evaluation of candidates in the focused libraries. To synthesize a library focused on protein serine-threonine phosphatases (PPs), we chose norcantharidin as a core structure, because norcantharidin dicarboxylate shows a broad inhibition profile toward several PPs. From the resulting focused library, we identified a highly selective PP2B inhibitor, NCA-01. On the other hand, to find inhibitors of dual-specificity protein phosphatases (DSPs), we chose 3-acyltetronic acid extracted from natural product RK-682 as a core structure, because its structure resembles the transition state in the dephosphorylation reaction of DSPs. However, a highly selective inhibitor was not found in the resulting focused library. Furthermore, an inherent drawback of compounds having the highly acidic 3-acyltetronic acid as a core structure is very weak potency in cellulo, probably due to poor cell membrane permeability. Therefore, we next modified the core structure from acidic to neutral by transformation to the enamine derivative and constructed a second-generation focused library (RE derivatives). The resulting compounds showed dramatically improved cell membrane permeability and inhibitory selectivity and included VHR (vaccinia VH1-related)-selective RE12 and CDC25A/B (cell division cycle 25A/B)-selective RE44. These inhibitors act on target enzymes in cellulo and do not generate reactive oxygen species, which is a potential problem with quinoid-type inhibitors of CDC25s. The cellular activity of RE12 was further improved by replacement of the side chain to afford RE176, which showed more potent antiproliferative activity than RE12 against HeLa cells. The dramatic change of inhibitory selectivity obtained by core structure modification from 3-acyltetronic acid to its enamine derivative was associated with a change in the mode of action. Namely, RE derivatives were found to be noncompetitive inhibitors with respect to a small-molecular substrate of CDC25A/B, whereas RK-682 was a competitive inhibitor of VHR. We identified the binding site of RE derivatives on the CDC25A as a pocket adjacent to the active site; this appears to be a promising target site for development of further novel inhibitors of CDC25s.

The naphthoquinone diospyrin is an inhibitor of DNA gyrase with a novel mechanism of action.

PubMed

Karkare, Shantanu; Chung, Terence T H; Collin, Frederic; Mitchenall, Lesley A; McKay, Adam R; Greive, Sandra J; Meyer, Jacobus J M; Lall, Namrita; Maxwell, Anthony

2013-02-15

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action*

PubMed Central

Karkare, Shantanu; Chung, Terence T. H.; Collin, Frederic; Mitchenall, Lesley A.; McKay, Adam R.; Greive, Sandra J.; Meyer, Jacobus J. M.; Lall, Namrita; Maxwell, Anthony

2013-01-01

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents. PMID:23275348

F99 is critical for dimerization and activation of South African HIV-1 subtype C protease.

PubMed

Naicker, Previn; Seele, Palesa; Dirr, Heini W; Sayed, Yasien

2013-10-01

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed 'lock-and-key' motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.

Synthesis of ST7612AA1, a Novel Oral HDAC Inhibitor, via Radical 
Thioacetic Acid Addition.

PubMed

Battistuzzi, Gianfranco; Giannini, Giuseppe

2016-12-01

In the expanding field of anticancer drugs, HDAC inhibitors are playing an increasingly important role. To date, four/five HDAC inhibitors have been approved by FDA. All these compounds fit the widely accepted HDAC inhibitors pharmacophore model characterized by a cap group, a linker chain and a zinc binding group (ZBG), able to bind the Zn 2+ ion in a pocket of the HDAC active site. Romidepsin, a natural compound, is the only thiol derivative. We have selected a new class of synthetic HDAC inhibitors, the thio-ω(lactam-carboxamide) derivatives, with ST7612AA1 as drug candidate, pan-inhibitor active in the range of single- to two-digit nanomolar concentrations. Preliminary results of a synthetic optimization attempt towards a fast scale-up process are here proposed. In the four steps of synthesis, from unsaturated amino acid intermediate to the final product, we explored different synthetic conditions in order to have a transferable process for a scale-up synthetic laboratory. In the first step, isobutyl chloroformate was used and, after a simple work up with 1M HCl, 2 (96% yield) was obtained as a white solid, which was used directly in the next step. For thioacetic acid addition to the double bond of intermediate 2 , two different routes were possible, with addition reaction in the first (D') or last step (D). Reactions of 2 to give 5 or of 4 to give ST7612AA1 were both performed in dioxane. Reactions were fast and did not need the usually advised radical quenching with cyclohexene. The corresponding products were obtained in good yields (step D', 89%; step D, 81%) after a flash chromatography. , a thiol derivative prodrug of ST7464AA1 , is the first of a new generation of HDAC inhibitors, very potent, orally administered, and well tolerated. Here, we have identified a synthetic route, competitive, versatile and easily transferable to industrial processes.

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

PubMed

Mori, Shigeo; Matsuzaki, Koichi; Yoshida, Katsunori; Furukawa, Fukiko; Tahashi, Yoshiya; Yamagata, Hideo; Sekimoto, Go; Seki, Toshihito; Matsui, Hirofumi; Nishizawa, Mikio; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2004-09-23

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

PubMed Central

Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

2015-01-01

Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter. PMID:26379245

A new TAO kinase inhibitor reduces tau phosphorylation at sites associated with neurodegeneration in human tauopathies.

PubMed

Giacomini, Caterina; Koo, Chuay-Yeng; Yankova, Natalia; Tavares, Ignatius A; Wray, Selina; Noble, Wendy; Hanger, Diane P; Morris, Jonathan D H

2018-05-07

In Alzheimer's disease (AD) and related tauopathies, the microtubule-associated protein tau is highly phosphorylated and aggregates to form neurofibrillary tangles that are characteristic of these neurodegenerative diseases. Our previous work has demonstrated that the thousand-and-one amino acid kinases (TAOKs) 1 and 2 phosphorylate tau on more than 40 residues in vitro. Here we show that TAOKs are phosphorylated and active in AD brain sections displaying mild (Braak stage II), intermediate (Braak stage IV) and advanced (Braak stage VI) tau pathology and that active TAOKs co-localise with both pre-tangle and tangle structures. TAOK activity is also enriched in pathological tau containing sarkosyl-insoluble extracts prepared from AD brain. Two new phosphorylated tau residues (T123 and T427) were identified in AD brain, which appear to be targeted specifically by TAOKs. A new small molecule TAOK inhibitor (Compound 43) reduced tau phosphorylation on T123 and T427 and also on additional pathological sites (S262/S356 and S202/T205/S208) in vitro and in cell models. The TAOK inhibitor also decreased tau phosphorylation in differentiated primary cortical neurons without affecting markers of synapse and neuron health. Notably, TAOK activity also co-localised with tangles in post-mortem frontotemporal lobar degeneration (FTLD) brain tissue. Furthermore, the TAOK inhibitor decreased tau phosphorylation in induced pluripotent stem cell derived neurons from FTLD patients, as well as cortical neurons from a transgenic mouse model of tauopathy (Tau35 mice). Our results demonstrate that abnormal TAOK activity is present at pre-tangles and tangles in tauopathies and that TAOK inhibition effectively decreases tau phosphorylation on pathological sites. Thus, TAOKs may represent a novel target to reduce or prevent tau-associated neurodegeneration in tauopathies.

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy

PubMed Central

Faisal, Amir; Mak, Grace W Y; Gurden, Mark D; Xavier, Cristina P R; Anderhub, Simon J; Innocenti, Paolo; Westwood, Isaac M; Naud, Sébastien; Hayes, Angela; Box, Gary; Valenti, Melanie R; De Haven Brandon, Alexis K; O'Fee, Lisa; Schmitt, Jessica; Woodward, Hannah L; Burke, Rosemary; vanMontfort, Rob L M; Blagg, Julian; Raynaud, Florence I; Eccles, Suzanne A; Hoelder, Swen; Linardopoulos, Spiros

2017-01-01

Background: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. Methods: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. Results: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. Conclusions: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850. PMID:28334731

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

PubMed

Faisal, Amir; Mak, Grace W Y; Gurden, Mark D; Xavier, Cristina P R; Anderhub, Simon J; Innocenti, Paolo; Westwood, Isaac M; Naud, Sébastien; Hayes, Angela; Box, Gary; Valenti, Melanie R; De Haven Brandon, Alexis K; O'Fee, Lisa; Schmitt, Jessica; Woodward, Hannah L; Burke, Rosemary; vanMontfort, Rob L M; Blagg, Julian; Raynaud, Florence I; Eccles, Suzanne A; Hoelder, Swen; Linardopoulos, Spiros

2017-04-25

The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850.

Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bodea, Smaranda; Funk, Michael A.; Balskus, Emily P.

We report that deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structuralmore » data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. Lastly, this work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production.« less

Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy

PubMed Central

2012-01-01

Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that prohibit PR enzymatic activities through non-competitive binding to PR, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chances of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible surrogate system to study the effects of HIV-1 protease on cellular functions and to explore its utility as a HTS system to search for new PIs to battle HIV-1 resistant strains. PMID:22971934

PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor.

PubMed

Devy, Laetitia; Rabbani, Shafaat A; Stochl, Mark; Ruskowski, Mary; Mackie, Ian; Naa, Laurent; Toews, Mark; van Gool, Reinoud; Chen, Jie; Ley, Art; Ladner, Robert C; Dransfield, Daniel T; Henderikx, Paula

2007-11-01

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

Isolation of a small molecule inhibitor of DNA base excision repair

PubMed Central

Madhusudan, Srinivasan; Smart, Fiona; Shrimpton, Paul; Parsons, Jason L.; Gardiner, Laurence; Houlbrook, Sue; Talbot, Denis C.; Hammonds, Timothy; Freemont, Paul A.; Sternberg, Michael J. E.; Dianov, Grigory L.; Hickson, Ian D.

2005-01-01

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3′-phosphodiesterase and 3′-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy. PMID:16113242

QSAR and molecular graphics analysis of N2-phenylguanines as inhibitors of herpes simplex virus thymidine kinases.

PubMed

Gaudio, A C; Richards, W G; Takahata, Y

2000-02-01

A quantitative structure-activity relationship study of N2-(substituted)-phenylguanines (PHG) as inhibitors of herpes simplex virus thymidine kinase (HSV TK) was performed. The activity of a set of PHG derivatives were analyzed against the thymidine kinase of herpes simplex virus types 1 (HSV1 TK) and 2 (HSV2 TK). Classic and calculated physicochemical parameters were included in the analysis. The results showed that there is an important difference in the activity of the meta substituted PHG derivatives against HSV1 TK and HSV2 TK. The activity of the meta derivatives against HSV2 TK is influenced by a steric effect, which is not observed against HSV1 TK. The superposition of the three-dimensional structures of the active sites of HSV1 TK (crystal structure) and HSV2 TK (homology model) revealed that the amino acid Ile97 is located near the meta position in the HSV1 TK active site, whereas the amino acid Leu97 is located near the meta position in the HSV2 TK active site. This single difference in the active sites of both enzymes can explain the source of the steric effect and serves as an indication that our previously proposed binding mode for the PHG derivatives is plausible. However, another observed mutation in the active site region, Ala168 by Ser168, suggests that an alternative binding mode, similar to that of ganciclovir, could be possible.

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives.

PubMed

Goins, Christopher M; Dajnowicz, Steven; Thanna, Sandeep; Sucheck, Steven J; Parks, Jerry M; Ronning, Donald R

2017-05-12

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis (Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitor orientation that were unobserved in previous Ag85C ebselen structures. The k inact /K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein-inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein-inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Furthermore, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.

Ensemble-based virtual screening reveals dual-inhibitors for the p53-MDM2/MDMX interactions.

PubMed

Barakat, Khaled; Mane, Jonathan; Friesen, Douglas; Tuszynski, Jack

2010-02-26

The p53 protein, a guardian of the genome, is inactivated by mutations or deletions in approximately half of human tumors. While in the rest of human tumors, p53 is expressed in wild-type form, yet it is inhibited by over-expression of its cellular regulators MDM2 and MDMX proteins. Although the p53-binding sites within the MDMX and MDM2 proteins are closely related, known MDM2 small-molecule inhibitors have been shown experimentally not to bind to its homolog, MDMX. As a result, the activity of these inhibitors including Nutlin3 is compromised in tumor cells over-expressing MDMX, preventing these compounds from fully activating the p53 protein. Here, we applied the relaxed complex scheme (RCS) to allow for the full receptor flexibility in screening for dual-inhibitors that can mutually antagonize the two p53-regulator proteins. First, we filtered the NCI diversity set, DrugBank compounds and a derivative library for MDM2-inhibitors against 28 dominant MDM2-conformations. Then, we screened the MDM2 top hits against the binding site of p53 within the MDMX target. Results described herein identify a set of compounds that have been computationally predicted to ultimately activate the p53 pathway in tumor cells retaining the wild-type protein. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

α-Keto phenylamides as P1'-extended proteasome inhibitors.

PubMed

Voss, Constantin; Scholz, Christoph; Knorr, Sabine; Beck, Philipp; Stein, Martin L; Zall, Andrea; Kuckelkorn, Ulrike; Kloetzel, Peter-Michael; Groll, Michael; Hamacher, Kay; Schmidt, Boris

2014-11-01

The major challenge for proteasome inhibitor design lies in achieving high selectivity for, and activity against, the target, which requires specific interactions with the active site. Novel ligands aim to overcome off-target-related side effects such as peripheral neuropathy, which is frequently observed in cancer patients treated with the FDA-approved proteasome inhibitors bortezomib (1) or carfilzomib (2). A systematic comparison of electrophilic headgroups recently identified the class of α-keto amides as promising for next generation drug development. On the basis of crystallographic knowledge, we were able to develop a structure-activity relationship (SAR)-based approach for rational ligand design using an electronic parameter (Hammett's σ) and in silico molecular modeling. This resulted in the tripeptidic α-keto phenylamide BSc4999 [(S)-3-(benzyloxycarbonyl-(S)-leucyl-(S)-leucylamino)-5-methyl-2-oxo-N-(2,4-dimethylphenyl)hexanamide, 6 a], a highly potent (IC50 = 38 nM), cell-permeable, and slowly reversible covalent inhibitor which targets both the primed and non-primed sites of the proteasome's substrate binding channel as a special criterion for selectivity. The improved inhibition potency and selectivity of this new α-keto phenylamide makes it a promising candidate for targeting a wider range of tumor subtypes than commercially available proteasome inhibitors and presents a new candidate for future studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mixture toxicity of three photosystem II inhibitors (atrazine, isoproturon, and diuron) toward photosynthesis of freshwater phytoplankton studied in outdoor mesocosms.

PubMed

Knauert, Stefanie; Escher, Beate; Singer, Heinz; Hollender, Juliane; Knauer, Katja

2008-09-01

Mixture toxicity of three herbicides with the same mode of action was studied in a long-term outdoor mesocosm study. Photosynthetic activity of phytoplankton as the direct target site of the herbicides was chosen as physiological response parameter. The three photosystem II (PSII) inhibitors atrazine, isoproturon, and diuron were applied as 30% hazardous concentrations (HC30), which we derived from species sensitivity distributions calculated on the basis of EC50 growth inhibition data. The respective herbicide mixture comprised 1/3 of the HC30 of each herbicide. Short-term laboratory experiments revealed that the HC30 values corresponded to EC40 values when regarding photosynthetic activity as the response parameter. In the outdoor mesocosm experiment, effects of atrazine, isoproturon, diuron and their mixture on the photosynthetic activity of phytoplankton were investigated during a five-week period with constant exposure and a subsequent five-month postexposure period when the herbicides dissipated. The results demonstrated that mixture effects determined at the beginning of constant exposure can be described by concentration addition since the mixture elicited a phytotoxic effect comparable to the single herbicides. Declining effects on photosynthetic activity during the experiment might be explained by both a decrease in water herbicide concentrations and by the induction of community tolerance.

Prevention of abdominal aortic aneurysm progression by targeted inhibition of matrix metalloproteinase activity with batimastat-loaded nanoparticles.

PubMed

Nosoudi, Nasim; Nahar-Gohad, Pranjal; Sinha, Aditi; Chowdhury, Aniqa; Gerard, Patrick; Carsten, Christopher G; Gray, Bruce H; Vyavahare, Naren R

2015-11-06

Matrix metalloproteinases (MMPs)-mediated extracellular matrix destruction is the major cause of development and progression of abdominal aortic aneurysms. Systemic treatments of MMP inhibitors have shown effectiveness in animal models, but it did not translate to clinical success either because of low doses used or systemic side effects of MMP inhibitors. We propose a targeted nanoparticle (NP)-based delivery of MMP inhibitor at low doses to the abdominal aortic aneurysms site. Such therapy will be an attractive option for preventing expansion of aneurysms in patients without systemic side effects. Our previous study showed that poly(d,l-lactide) NPs conjugated with an antielastin antibody could be targeted to the site of an aneurysm in a rat model of abdominal aortic aneurysms. In the study reported here, we tested whether such targeted NPs could deliver the MMP inhibitor batimastat (BB-94) to the site of an aneurysm and prevent aneurysmal growth. Poly(d,l-lactide) NPs were loaded with BB-94 and conjugated with an elastin antibody. Intravenous injections of elastin antibody-conjugated BB-94-loaded NPs targeted the site of aneurysms and delivered BB-94 in a calcium chloride injury-induced abdominal aortic aneurysms in rats. Such targeted delivery inhibited MMP activity, elastin degradation, calcification, and aneurysmal development in the aorta (269% expansion in control versus 40% elastin antibody-conjugated BB-94-loaded NPs) at a low dose of BB-94. The systemic administration of BB-94 alone at the same dose was ineffective in producing MMP inhibition. Targeted delivery of MMP inhibitors using NPs may be an attractive strategy to inhibit aneurysmal progression. © 2015 American Heart Association, Inc.

Fragment-based discovery of novel pentacyclic triterpenoid derivatives as cholesteryl ester transfer protein inhibitors.

PubMed

Chang, Yongzhi; Zhou, Shuxi; Li, Enqin; Zhao, Wenfeng; Ji, Yanpeng; Wen, Xiaoan; Sun, Hongbin; Yuan, Haoliang

2017-01-27

Cholesteryl Ester Transfer Protein (CETP) is an important therapeutic target for the treatment of atherosclerotic cardiovascular disease. Our molecular modeling study revealed that pentacyclic triterpenoid compounds could mimic the protein-ligand interactions of the endogenous ligand cholesteryl ester (CE) by occupying its binding site. Alignment of the docking conformations of oleanolic acid (OA), ursolic acid (UA) and the crystal conformations of known CETP inhibitor Torcetrapib in the active site proposed the applicability of fragment-based drug design (FBDD) approaches in this study. Accordingly, a series of pentacyclic triterpenoid derivatives have been designed and synthesized as novel CETP inhibitors. The most potent compound 12e (IC 50 :0.28 μM) validated our strategy for molecular design. Molecular dynamics simulations illustrated that the more stable hydrogen bond interaction of the UA derivative 12e with Ser191 and stronger hydrophobic interactions with Val198, Phe463 than those of OA derivative 12b mainly led to their significantly different CETP inhibitory activity. These novel potent CETP inhibitors based on ursane-type scaffold should deserve further investigation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Rhodium(II) Proximity-Labeling Identifies a Novel Target Site on STAT3 for Inhibitors with Potent Anti-Leukemia Activity.

PubMed

Minus, Matthew B; Liu, Wei; Vohidov, Farrukh; Kasembeli, Moses M; Long, Xin; Krueger, Michael J; Stevens, Alexandra; Kolosov, Mikhail I; Tweardy, David J; Sison, Edward Allan R; Redell, Michele S; Ball, Zachary T

2015-10-26

Nearly 40 % of children with acute myeloid leukemia (AML) suffer relapse arising from chemoresistance, often involving upregulation of the oncoprotein STAT3 (signal transducer and activator of transcription 3). Herein, rhodium(II)-catalyzed, proximity-driven modification identifies the STAT3 coiled-coil domain (CCD) as a novel ligand-binding site, and we describe a new naphthalene sulfonamide inhibitor that targets the CCD, blocks STAT3 function, and halts its disease-promoting effects in vitro, in tumor growth models, and in a leukemia mouse model, validating this new therapeutic target for resistant AML. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis, Activity and Structural Analysis of Novel α-Hydroxytropolone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Chung, Suhman; Himmel, Daniel M.; Jiang, Jian-Kang; Wojtak, Krzysztof; Bauman, Joseph D.; Rausch, Jason W.; Wilson, Jennifer A.; Beutler, John A.; Thomas, Craig J.; Arnold, Eddy; Le Grice, Stuart F.J.

2011-01-01

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one) potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at non-cytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogs can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use. PMID:21568335

Synthesis and biological evaluation of guanylhydrazone coactivator binding inhibitors for the estrogen receptor.

PubMed

LaFrate, Andrew L; Gunther, Jillian R; Carlson, Kathryn E; Katzenellenbogen, John A

2008-12-01

Most patients with hormone-responsive breast cancer eventually develop resistance to traditional antiestrogens such as tamoxifen, and this has become a major obstacle in their treatment. We prepared and characterized the activity of a series of 16 guanylhydrazone small molecules that are designed to block estrogen receptor (ER) activity through a non-traditional mechanism, by directly interfering with coactivator binding to agonist-liganded ER. The inhibitory activity of these compounds was determined in cell-based transcription assays using ER-responsive reporter gene and mammalian two-hybrid assays. Several of the compounds gave IC(50) values in the low micromolar range. Two secondary assays were used to confirm that these compounds were acting through the proposed non-traditional mode of estrogen inhibitory action and not as conventional antagonists at the ligand binding site.

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Rho-associated kinase inhibitors: a novel glaucoma therapy.

PubMed

Inoue, Toshihiro; Tanihara, Hidenobu

2013-11-01

The rho-associated kinase (ROCK) signaling pathway is activated via secreted bioactive molecules or via integrin activation after extracellular matrix binding. These lead to polymerization of actin stress fibers and formation of focal adhesions. Accumulating evidence suggests that actin cytoskeleton-modulating signals are involved in aqueous outflow regulation. Aqueous humor contains various biologically active factors, some of which are elevated in glaucomatous eyes. These factors affect aqueous outflow, in part, through ROCK signaling modulation. Various drugs acting on the cytoskeleton have also been shown to increase aqueous outflow by acting directly on outflow tissue. In vivo animal studies have shown that the trabecular meshwork (TM) actin cytoskeleton in glaucomatous eyes is more disorganized and more randomly oriented than in non-glaucomatous control eyes. In a previous study, we introduced ROCK inhibitors as a potential glaucoma therapy by showing that a selective ROCK inhibitor significantly lowered rabbit IOP. Rho-associated kinase inhibitors directly affect the TM and Schlemm's canal (SC), differing from the target sight of other glaucoma drugs. The TM is affected earlier and more strongly than ciliary muscle cells by ROCK inhibitors, largely because of pharmacological affinity differences stemming from regulatory mechanisms. Additionally, ROCK inhibitors disrupt tight junctions, result in F-actin depolymerization, and modulate intracellular calcium level, effectively increasing SC-cell monolayer permeability. Perfusion of an enucleated eye with a ROCK inhibitor resulted in wider empty spaces in the juxtacanalicular (JCT) area and more giant vacuoles in the endothelial cells of SC, while the endothelial lining of SC was intact. Interestingly, ROCK inhibitors also increase retinal blood flow by relaxing vascular smooth muscle cells, directly protecting neurons against various stresses, while promoting wound healing. These additional effects may help slow progressing visual field loss in glaucoma patients, making ROCK inhibitors an even more desirable anti-glaucoma agent. All evidence indicates that aqueous humor outflow is affected by cytoskeleton physiology and this information may provide valuable insight into understanding glaucoma pathology and treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Bioinorganic Approach to Fragment-Based Drug Discovery Targeting Metalloenzymes.

PubMed

Cohen, Seth M

2017-08-15

Metal-dependent enzymes (i.e., metalloenzymes) make up a large fraction of all enzymes and are critically important in a wide range of biological processes, including DNA modification, protein homeostasis, antibiotic resistance, and many others. Consequently, metalloenzymes represent a vast and largely untapped space for drug development. The discovery of effective therapeutics that target metalloenzymes lies squarely at the interface of bioinorganic and medicinal chemistry and requires expertise, methods, and strategies from both fields to mount an effective campaign. In this Account, our research program that brings together the principles and methods of bioinorganic and medicinal chemistry are described, in an effort to bridge the gap between these fields and address an important class of medicinal targets. Fragment-based drug discovery (FBDD) is an important drug discovery approach that is particularly well suited for metalloenzyme inhibitor development. FBDD uses relatively small but diverse chemical structures that allow for the assembly of privileged molecular collections that focus on a specific feature of the target enzyme. For metalloenzyme inhibition, the specific feature is rather obvious, namely, a metal-dependent active site. Surprisingly, prior to our work, the exploration of diverse molecular fragments for binding the metal active sites of metalloenzymes was largely unexplored. By assembling a modest library of metal-binding pharmacophores (MBPs), we have been able to find lead hits for many metalloenzymes and, from these hits, develop inhibitors that act via novel mechanisms of action. A specific case study on the use of this strategy to identify a first-in-class inhibitor of zinc-dependent Rpn11 (a component of the proteasome) is highlighted. The application of FBDD for the development of metalloenzyme inhibitors has raised several other compelling questions, such as how the metalloenzyme active site influences the coordination chemistry of bound fragments, how one can identify the best fragments for a given metalloenzyme, and many others. Among the most significant, and concerning, questions for metalloenzyme inhibition are those that reside around issues of specificity and whether metalloenzyme inhibitors can be as selective and specific as other small molecule inhibitors (i.e., compounds that inhibit enzymes that do not utilize a metal at their active site). This also leads to the question of whether metalloenzyme inhibitors might interfere more broadly with the metallome. Efforts to address these and related questions are discussed, with the expectation that our findings will illuminate some of these topics, alleviate some of these concerns, and encourage greater interest in this important, undervalued class of drug targets.

Inhibitors of MAO-A and MAO-B in Psychiatry and Neurology

PubMed Central

Finberg, John P. M.; Rabey, Jose M.

2016-01-01

Inhibitors of MAO-A and MAO-B are in clinical use for the treatment of psychiatric and neurological disorders respectively. Elucidation of the molecular structure of the active sites of the enzymes has enabled a precise determination of the way in which substrates and inhibitor molecules are metabolized, or inhibit metabolism of substrates, respectively. Despite the knowledge of the strong antidepressant efficacy of irreversible MAO inhibitors, their clinical use has been limited by their side effect of potentiation of the cardiovascular effects of dietary amines (“cheese effect”). A number of reversible MAO-A inhibitors which are devoid of cheese effect have been described in the literature, but only one, moclobemide, is currently in clinical use. The irreversible inhibitors of MAO-B, selegiline and rasagiline, are used clinically in treatment of Parkinson's disease, and a recently introduced reversible MAO-B inhibitor, safinamide, has also been found efficacious. Modification of the pharmacokinetic characteristics of selegiline by transdermal administration has led to the development of a new drug form for treatment of depression. The clinical potential of MAO inhibitors together with detailed knowledge of the enzyme's binding site structure should lead to future developments with these drugs. PMID:27803666

Inhibitors of MAO-A and MAO-B in Psychiatry and Neurology.

PubMed

Finberg, John P M; Rabey, Jose M

2016-01-01

Inhibitors of MAO-A and MAO-B are in clinical use for the treatment of psychiatric and neurological disorders respectively. Elucidation of the molecular structure of the active sites of the enzymes has enabled a precise determination of the way in which substrates and inhibitor molecules are metabolized, or inhibit metabolism of substrates, respectively. Despite the knowledge of the strong antidepressant efficacy of irreversible MAO inhibitors, their clinical use has been limited by their side effect of potentiation of the cardiovascular effects of dietary amines ("cheese effect"). A number of reversible MAO-A inhibitors which are devoid of cheese effect have been described in the literature, but only one, moclobemide, is currently in clinical use. The irreversible inhibitors of MAO-B, selegiline and rasagiline, are used clinically in treatment of Parkinson's disease, and a recently introduced reversible MAO-B inhibitor, safinamide, has also been found efficacious. Modification of the pharmacokinetic characteristics of selegiline by transdermal administration has led to the development of a new drug form for treatment of depression. The clinical potential of MAO inhibitors together with detailed knowledge of the enzyme's binding site structure should lead to future developments with these drugs.

Structural dynamics and quantum mechanical aspects of shikonin derivatives as CREBBP bromodomain inhibitors.

PubMed

Mitra, Sarmistha; Dash, Raju

2018-05-04

The Proteins involved in the chemical modification of lysine residues in histone, is currently being excessively focused as the therapeutic target for the treatment of cell related diseases like cancer. Among these proteins, the epigenetic reader, CREB-binding protein (CREBBP) bromodomain is one of the most prominent targets for effective anticancer drug design, which is responsible for the reorganization of acetylated histone lysine residues. Therefore, this study employed an integrative approach of structure based drug design, in combination with Molecular Dynamics (MD) and QM/MM study to identify as well as to describe the binding mechanism of two shikonin derivatives, acetylshikonin and propionylshikonin as inhibitors of CREBBP bromodomain. Here induced fit docking strategy was employed to explore the important intrinsic interactions of ligands with CREBBP bromodomain, consistently molecular dynamics simulation with two different methods and binding energy calculations by MM-GBSA and MM-PBSA were adopted to determine the stability of intermolecular interactions between protein and ligands. The results showed that both these derivatives made direct contacts with the important conserved residues of the active site, where propionylshikonin demonstrated stronger binding and stability than acetylshikonin, according to molecular dynamics simulation and binding free energy calculations. Further, QM/MM energy calculation was employed to study the chemical reactivity of the propionylshikonin and also to describe the mechanism of non bonded interactions between the propionylshikonin and CREBBP bromodomain. Though this study demands in vitro and in vivo experiments to evaluate the efficiency of the compound, these insights would assist to design more potent CREBBP bromodomain inhibitor, guiding the site of modification of propionylshikonin moiety for designing selective inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

Effects of phosphoramidon and pepstatin A on the secretion of endothelin-1 and big endothelin-1 by human umbilical vein endothelial cells: measurement by two-site enzyme-linked immunosorbent assays.

PubMed

Plumpton, C; Kalinka, S; Martin, R C; Horton, J K; Davenport, A P

1994-08-01

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and non-isotopic measurement of endothelin-1 and big endothelin-1. The sensitivities of detection were 0.5 and 0.3 fmol/well, with ED50 values of 13 and 12 fmol/well for the endothelin-1 and big endothelin-1 assays, respectively. Each assay is highly selective for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only recognizes the 21-amino acid biologically active peptide. 2. The two assays were used to measure the effects of two classes of protease inhibitor on the basal release of enothelin-1 and big endothelin-1 from cultured first-passage human umbilical vein endothelial cells. 3. The secretion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P < 0.05), with a concomitant increase in the secreted levels of big endothelin-1 (P < 0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significant decrease in the secretion of endothelin (P < 0.05). However, unlike phosphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affected the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s).(ABSTRACT TRUNCATED AT 250 WORDS)

Insights into structure and activity of natural compound inhibitors of pneumolysin

PubMed Central

Li, Hongen; Zhao, Xiaoran; Deng, Xuming; Wang, Jianfeng; Song, Meng; Niu, Xiaodi; Peng, Liping

2017-01-01

Pneumolysin is the one of the major virulence factor of the bacterium Streptococcus pneumoniae. In previous report, it is shown that β-sitosterol, a natural compound without antimicrobial activity, is a potent antagonist of pneumolysin. Here, two new pneumolysin natural compound inhibitors, with differential activity, were discovered via haemolysis assay. To explore the key factor of the conformation for the inhibition activity, the interactions between five natural compound inhibitors with differential activity and pneumolysin were reported using molecular modelling, the potential of mean force profiles. Interestingly, it is found that incorporation of the single bond (C22-C23-C24-C25) to replace the double bond (hydrocarbon sidechain) improved the anti-haemolytic activity. In view of the molecular modelling, binding of the five inhibitors to the conserved loop region (Val372, Leu460, and Tyr461) of the cholesterol binding sites led to stable complex systems, which was consistent with the result of β-sitosterol. Owing to the single bond (C22-C23-C24-C25), campesterol and brassicasterol could form strong interactions with Val372 and show higher anti-haemolytic activity, which indicated that the single bond (C22-C23-C24-C25) in inhibitors was required for the anti-haemolytic activity. Overall, the current molecular modelling work provides a starting point for the development of rational design and higher activity pneumolysin inhibitors. PMID:28165051

Mitoketoscins: Novel mitochondrial inhibitors for targeting ketone metabolism in cancer stem cells (CSCs)

PubMed Central

Ozsvari, Bela; Sotgia, Federica; Simmons, Katie; Trowbridge, Rachel; Foster, Richard; Lisanti, Michael P.

2017-01-01

Previous studies have now well-established that epithelial cancer cells can utilize ketone bodies (3-hydroxybutyrate and aceto-acetate) as mitochondrial fuels, to actively promote tumor growth and metastatic dissemination. The two critical metabolic enzymes implicated in this process are OXCT1 and ACAT1, which are both mitochondrial proteins. Importantly, over-expression of OXCT1 or ACAT1 in human breast cancer cells is sufficient to genetically drive tumorigenesis and/or lung metastasis, validating that they indeed behave as metabolic “tumor promoters”. Here, we decided to target these two enzymes, which give cancer cells the ability to recycle ketone bodies into Acetyl-CoA and, therefore, to produce increased ATP. Briefly, we used computational chemistry (in silico drug design) to select a sub-set of potentially promising compounds that spatially fit within the active site of these enzymes, based on their known 3D crystal structures. These libraries of compounds were then phenotypically screened for their effects on total cellular ATP levels. Positive hits were further validated by metabolic flux analysis. Our results indicated that four of these compounds effectively inhibited mitochondrial oxygen consumption. Two of these compounds also induced a reactive glycolytic phenotype in cancer cells. Most importantly, using the mammosphere assay, we showed that these compounds can be used to functionally inhibit cancer stem cell (CSC) activity and propagation. Finally, our molecular modeling studies directly show how these novel compounds are predicted to bind to the active catalytic sites of OXCT1 and ACAT1, within their Coenzyme A binding site. As such, we speculate that these mitochondrial inhibitors are partially mimicking the structure of Coenzyme A. Thus, we conclude that OXCT1 and ACAT1 are important new therapeutic targets for further drug development and optimization. We propose that this new class of drugs should be termed “mitoketoscins”, to reflect that they were designed to target ketone re-utilization and mitochondrial function. PMID:29108233

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications.

PubMed

Robertson, Brooklyn A; Johnson, William H; Lo, Herng-Hsiang; Whitman, Christian P

2008-08-19

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.

Structure-activity relationship for enantiomers of potent inhibitors of B. anthracis dihydrofolate reductase

PubMed Central

Bourne, Christina R.; Wakeham, Nancy; Nammalwar, Baskar; Tseitin, Vladimir; Bourne, Philip C.; Barrow, Esther W.; Mylvaganam, Shankari; Ramnarayan, Kal; Bunce, Richard A.; Berlin, K. Darrell; Barrow, William W.

2012-01-01

Background Bacterial resistance to antibiotic therapies is increasing and new treatment options are badly needed. There is an overlap between these resistant bacteria and organisms classified as likely bioterror weapons. For example, Bacillus anthracis is innately resistant to the anti-folate trimethoprim due to sequence changes found in the dihydrofolate reductase enzyme. Development of new inhibitors provides an opportunity to enhance the current arsenal of anti-folate antibiotics while also expanding the coverage of the anti-folate class. Methods We have characterized inhibitors of Bacillus anthracis dihydrofolate reductase by measuring the Ki and MIC values and calculating the energetics of binding. This series contains a core diaminopyrimidine ring, a central dimethoxybenzyl ring, and a dihydrophthalazine moiety. We have altered the chemical groups extended from a chiral center on the dihydropyridazine ring of the phthalazine moiety. The interactions for the most potent compounds were visualized by X-ray structure determination. Results We find that the potency of individual enantiomers is divergent with clear preference for the S-enantiomer, while maintaining a high conservation of contacts within the binding site. The preference for enantiomers seems to be predicated largely by differential interactions with protein residues Leu29, Gln30 and Arg53. Conclusions These studies have clarified the activity of modifications and of individual enantiomers, and highlighted the role of the less-active R-enantiomer in effectively diluting the more active S-enantiomer in racemic solutions. This directly contributes to the development of new antimicrobials, combating trimethoprim resistance, and treatment options for potential bioterrorism agents. PMID:22999981

N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide Deformylase Inhibitors with Antibacterial Activity

PubMed Central

Hackbarth, Corinne J.; Chen, Dawn Z.; Lewis, Jason G.; Clark, Kirk; Mangold, James B.; Cramer, Jeffrey A.; Margolis, Peter S.; Wang, Wen; Koehn, Jim; Wu, Charlotte; Lopez, S.; Withers III, George; Gu, Helen; Dunn, Elina; Kulathila, R.; Pan, Shi-Hao; Porter, Wilma L.; Jacobs, Jeff; Trias, Joaquim; Patel, Dinesh V.; Weidmann, Beat; White, Richard J.; Yuan, Zhengyu

2002-01-01

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P1′ site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P1′ site. Compounds with MICs of ≤4 μg/ml against gram-positive and gram-negative pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae, have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC50s) for Escherichia coli Ni-PDF were ≤0.1 μM, demonstrating the specificity of the inhibitors. In addition, these compounds were very selective for PDF, with IC50s of consistently >200 μM for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 Å. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents. PMID:12183225

Cholinesterases inhibition and molecular modeling studies of piperidyl-thienyl and 2-pyrazoline derivatives of chalcones.

PubMed

Shah, Muhammad Shakil; Khan, Shafi Ullah; Ejaz, Syeda Abida; Afridi, Saifullah; Rizvi, Syed Umar Farooq; Najam-Ul-Haq, Muhammad; Iqbal, Jamshed

2017-01-22

Super-activation of cholinesterases (acetylcholinesterase and butyrylcholinesterase) are linked to various neurological problems most precisely Alzheimer's disease (AD), which leads to senile dementia. Therefore, cholinesterases (AChE & BChE) inhibition are considered as a promising strategy for the treatment of Alzheimer's disease. FDA approved drugs for the treatment of AD, belong to a group of cholinesterase inhibitors. However, none of them is able to combat or completely abrogate the disease progression. Herein, we report a series of newly synthesized chalcone derivatives with anti-AD potential. For this purpose, a series of piperidyl-thienyl and 2-pyrazoline derivatives of chalcones were tested for their cholinesterases (AChE & BChE) inhibitory activity. All compounds were found as selective inhibitor of AChE. In piperidyl chalcones derivatives compound 1e having IC 50 of 0.16 ± 0.008 μM and 2m in 2-pyrazoline chalcones with IC 50 of 0.13 ± 0.006 μM, were found to be the most potent inhibitors of AChE, exhibiting ≈142 and ≈ 173-fold greater inhibitory potential compared to the reference inhibitor i.e., Neostigmine (IC 50  ± SEM = 22.2 ± 3.2 μM). Molecular docking studies of most potent inhibitors were carried out to investigate the binding interactions inside the active site. Molecular docking study revealed that potent compounds and co-crystalized ligand had same binding orientation within the active site of target enzyme. Most of these compounds are selective inhibitors of AChE with a potential use against progressive neurodegenerative disorder and age related problems. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Battles, Michael B.; Langedijk, Johannes P.; Furmanova-Hollenstein, Polina

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. In this paper, we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitorsmore » or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Finally and collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.« less

Probes of the catalytic site of cysteine dioxygenase.

PubMed

Chai, Sergio C; Bruyere, John R; Maroney, Michael J

2006-06-09

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.

Novel biphenyl ester derivatives as tyrosinase inhibitors: Synthesis, crystallographic, spectral analysis and molecular docking studies.

PubMed

Kwong, Huey Chong; Chidan Kumar, C S; Mah, Siau Hui; Chia, Tze Shyang; Quah, Ching Kheng; Loh, Zi Han; Chandraju, Siddegowda; Lim, Gin Keat

2017-01-01

Biphenyl-based compounds are clinically important for the treatments of hypertension and inflammatory, while many more are under development for pharmaceutical uses. In the present study, a series of 2-([1,1'-biphenyl]-4-yl)-2-oxoethyl benzoates, 2(a-q), and 2-([1,1'-biphenyl]-4-yl)-2-oxoethyl pyridinecarboxylate, 2(r-s) were synthesized by reacting 1-([1,1'-biphenyl]-4-yl)-2-bromoethan-1-one with various carboxylic acids using potassium carbonate in dimethylformamide at ambient temperature. Single-crystal X-ray diffraction studies revealed a more closely packed crystal structure can be produced by introduction of biphenyl moiety. Five of the compounds among the reported series exhibited significant anti-tyrosinase activities, in which 2p, 2r and 2s displayed good inhibitions which are comparable to standard inhibitor kojic acid at concentrations of 100 and 250 μg/mL. The inhibitory effects of these active compounds were further confirmed by computational molecular docking studies and the results revealed the primary binding site is active-site entrance instead of inner copper binding site which acted as the secondary binding site.

Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein.

PubMed

Tsai, Ting-Yueh; Yeh, Teng-Kuang; Chen, Xin; Hsu, Tsu; Jao, Yu-Chen; Huang, Chih-Hsiang; Song, Jen-Shin; Huang, Yu-Chen; Chien, Chia-Hui; Chiu, Jing-Huai; Yen, Shih-Chieh; Tang, Hung-Kuan; Chao, Yu-Sheng; Jiaang, Weir-Torn

2010-09-23

Fibroblast activation protein (FAP) belongs to the prolyl peptidase family. FAP inhibition is expected to become a new antitumor target. Most known FAP inhibitors often resemble the dipeptide cleavage products, with a boroproline at the P1 site; however, these inhibitors also inhibit DPP-IV, DPP-II, DPP8, and DPP9. Potent and selective FAP inhibitor is needed in evaluating that FAP as a therapeutic target. Therefore, it is important to develop selective FAP inhibitors for the use of target validation. To achieve this, optimization of the nonselective DPP-IV inhibitor 8 led to the discovery of a new class of substituted 4-carboxymethylpyroglutamic acid diamides as FAP inhibitors. SAR studies resulted in a number of FAP inhibitors having IC(50) of <100 nM with excellent selectivity over DPP-IV, DPP-II, DPP8, and DPP9 (IC(50) > 100 μM). Compounds 18a, 18b, and 19 are the only known potent and selective FAP inhibitors, which prompts us to further study the physiological role of FAP.

Longitudinal analysis of metalloproteinases, tissue inhibitors of metalloproteinases and clinical parameters in gingival crevicular fluid from periodontitis-affected patients.

PubMed

Pozo, P; Valenzuela, M A; Melej, C; Zaldívar, M; Puente, J; Martínez, B; Gamonal, J

2005-06-01

The aim of this work was to improve the assessment of the periodontal disease status through measurements of extracellular matrix metalloproteinases (MMPs) and their tissular inhibitors (TIMPs) in the gingival crevicular fluid from patients diagnosed with chronic periodontitis. Gingival crevicular fluid samples from patients (n = 13) were taken from 60 sites initially, and from 51 and 41 sites, respectively, 3 and 6 months after scaling and root planing. Gingival crevicular fluid samples were also taken from healthy subjects (n = 11, 24 sites). The presence of MMP-9 and MMP-8 was assessed by zymography and immunowestern blotting, respectively. The actual MMP activity (gelatinase and collagenase) was measured using the fluorogenic substrate assay. TIMP-1 and -2 levels were measured by immunodot blot. The fluorogenic substrate assay determinations showed higher MMP activity in sites with probing depth > or = 4 mm, with significant reduction post-treatment. Gelatinase activity followed by zymography consisted mainly of MMP-9. A different pattern of MMP-8 in control and patient sites was found. Controls only showed species of a partially active form (69 kDa), whereas patient sites showed a high frequency of the active form (56 kDa), and in some cases the latent form (85 kDa) was also observed. The active form reduced its frequency in sites with probing depth > or = 4 mm. TIMP-1 and -2 levels in patients were significantly lower than in controls, and after treatment the recovery of TIMP-1 level similar to control was observed. Significant correlations between the severity of the periodontal disease and the actual MMP activity, the active form of MMP-8 and the low level of both TIMP-1 and TIMP-2 were found.

Kinetic analysis of inhibition of glucoamylase and active site mutants via chemoselective oxime immobilization of acarbose on SPR chip surfaces.

PubMed

Sauer, Jørgen; Abou Hachem, Maher; Svensson, Birte; Jensen, Knud J; Thygesen, Mikkel B

2013-06-28

We here report a quantitative study on the binding kinetics of inhibition of the enzyme glucoamylase and how individual active site amino acid mutations influence kinetics. To address this challenge, we have developed a fast and efficient method for anchoring native acarbose to gold chip surfaces for surface plasmon resonance studies employing wild type glucoamylase and active site mutants, Y175F, E180Q, and R54L, as analytes. The key method was the chemoselective and protecting group-free oxime functionalization of the pseudo-tetrasaccharide-based inhibitor acarbose. By using this technique we have shown that at pH 7.0 the association and dissociation rate constants for the acarbose-glucoamylase interaction are 10(4)M(-1)s(-1) and 10(3)s(-1), respectively, and that the conformational change to a tight enzyme-inhibitor complex affects the dissociation rate constant by a factor of 10(2)s(-1). Additionally, the acarbose-presenting SPR surfaces could be used as a glucoamylase sensor that allowed rapid, label-free affinity screening of small carbohydrate-based inhibitors in solution, which is otherwise difficult with immobilized enzymes or other proteins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inhibition of protein tyrosine phosphatase 1B as a potential treatment of diabetes and obesity.

PubMed

Pei, Zhonghua; Liu, Gang; Lubben, Thomas H; Szczepankiewicz, Bruce G

2004-01-01

Diabetes is a prevalent disease which effects over 150 million people worldwide and there is a great medical need for new therapeutic agents to treat it. Inhibition of protein tyrosine phosphatase 1B (PTP1B) has emerged as a highly validated, attractive target for treatment of not only diabetes but also obesity. Discovery of small-molecule inhibitors has been pursued extensively in both academia and industry and a number of very potent and selective inhibitors have been identified. With X-ray crystallography, the binding interactions of several classes of inhibitors have been elucidated. This has resulted in significant progress in understanding important interactions between inhibitors and specific residues of PTP1B, which could help the design of future inhibitors. However, since the active site of PTP1B that most of these inhibitors bind to is highly hydrophilic, it remains a challenge to identify inhibitors with both excellent in vitro potency and drug-like physiochemical properties which would lead to good in vivo activities.

Targeting the disordered C-terminus of PTP1B with an allosteric inhibitor

PubMed Central

Krishnan, Navasona; Koveal, Dorothy; Miller, Daniel H.; Xue, Bin; Akshinthala, Sai Dipikaa; Kragelj, Jaka; Jensen, Malene Ringkjøbing; Gauss, Carla-Maria; Page, Rebecca; Blackledge, Martin; Muthuswamy, Senthil K.; Peti, Wolfgang; Tonks, Nicholas K.

2014-01-01

PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules. PMID:24845231

Synthesis and in vitro evaluation of new derivatives of 2-substituted-6-fluorobenzo[d]thiazoles as cholinesterase inhibitors.

PubMed

Imramovský, Aleš; Pejchal, Vladimír; Štěpánková, Šárka; Vorčáková, Katarína; Jampílek, Josef; Vančo, Ján; Šimůnek, Petr; Královec, Karel; Brůčková, Lenka; Mandíková, Jana; Trejtnar, František

2013-04-01

A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthesised and characterised by IR, (1)H, (13)C and (19)F NMR spectroscopy and HRMS. Purity was checked by elemental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure-activity relationships reflecting the dependence of cholinesterase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinesterase was determined using molecular docking of the most active compound. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of new 2,5-diketopiperazine derivatives as simultaneous effective inhibitors of αβ-tubulin and BCRP proteins: Molecular docking, Structure-Activity Relationships and virtual consensus docking studies

NASA Astrophysics Data System (ADS)

Fani, Najmeh; Sattarinezhad, Elham; Bordbar, Abdol-Khalegh

2017-06-01

In the first part of this paper, docking method was employed in order to study the binding mechanism of breast cancer resistance protein (BCRP) with a group of previously synthesized TPS-A derivatives which known as potent inhibitors of this protein to get insight into drug binding site of BCRP and to explore structure-activity relationship of these compounds. Molecular docking results showed that most of these compounds bind in the binding site of BCRP at the interface between the membrane and outer environment. In the second part, a group of designed TPS-A derivatives which showed good binding energies in the binding site of αβ-tubulin in the previous study were chosen to study their binding energies in the binding site of BCRP to investigate their simultaneous inhibitory effect on both αβ-tubulin and BCRP. The results showed that all of these compounds bind to the binding site of BCRP with relatively suitable binding energies and therefore could be potential inhibitors of both αβ-tubulin and BCRP proteins. Finally, virtual consensus docking method was utilized with the aim of design of new 2,5-diketopiperazine derivatives with significant inhibitory effect on both αβ-tubulin and BCRP proteins. For this purpose binding energies of a library of 2,5-diketopiperazine derivatives in the binding sites of αβ-tubulin and BCRP was investigated by using AutoDock and AutoDock vina tools. Molecular docking results revealed that a group of 36 compounds among them exhibit strong anti-tubulin and anti-BCRP activity.

Mechanism-based inactivation of dopamine beta-hydroxylase by p-cresol and related alkylphenols

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodhart, P.J.; DeWolf, W.E. Jr.; Kruse, L.I.

1987-05-05

The mechanism-based inhibition of dopamine beta-hydroxylase by p-cresol (4-methylphenol) and other simple structural analogues of dopamine, which lack a basic side-chain nitrogen, is reported. p-Cresol binds DBH by a mechanism that is kinetically indistinguishable from normal dopamine substrate binding. Under conditions (pH 6.6) of random oxygen and phenethylamine substrate addition p-cresol adds randomly, whereas at pH 4.5 or in the presence of fumarate activator addition of p-cresol precedes oxygen binding as is observed with phenethylamine substrate. p-Cresol is shown to be a rapid (kinact = 2.0 min-1, pH 5.0) mechanism-based inactivator of DBH. This inactivation exhibits pseudo-first-order kinetics, is irreversible,more » is prevented by tyramine substrate or competitive inhibitor, and is dependent upon oxygen and ascorbic acid cosubstrates. Inhibition occurs with partial covalent incorporation of p-cresol into DBH. A plot of -log kinact vs. pH shows maximal inactivation occurs at pH 5.0 with dependence upon enzymatic groups with apparent pK values of 4.51 +/- 0.06 and 5.12 +/- 0.06. p-Cresol and related alkylphenols, unlike other mechanism-based inhibitors of DBH, lack a latent electrophile. These inhibitors are postulated to covalently modify DBH by a direct insertion of an aberrant substrate-derived benzylic radical into an active site residue.« less

Extreme Entropy-Enthalpy Compensation in a Drug Resistant Variant of HIV-1 Protease

PubMed Central

King, Nancy M.; Prabu-Jeyabalan, Moses; Bandaranayake, Rajintha M.; Nalam, Madhavi N. L.; Nalivaika, Ellen A.; Özen, Ayşegül; Haliloglu, Türkan; Yılmaz, Neşe Kurt; Schiffer, Celia A.

2012-01-01

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5–15 kcal/mol, while losing only 1–3 kcal/mol in total binding free energy for any of six FDA approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wildtype protease and another drug resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design. PMID:22712830

Homology modeling and virtual screening of inhibitors against TEM- and SHV-type-resistant mutants: A multilayer filtering approach.

PubMed

Baig, Mohammad H; Balaramnavar, Vishal M; Wadhwa, Gulshan; Khan, Asad U

2015-01-01

TEM and SHV are class-A-type β-lactamases commonly found in Escherichia coli and Klebsiella pneumoniae. Previous studies reported S130G and K234R mutations in SHVs to be 41- and 10-fold more resistant toward clavulanic acid than SHV-1, respectively, whereas TEM S130G and R244S also showed the same level of resistance. These selected mutants confer higher level of resistance against clavulanic acid. They also show little susceptibility against other commercially available β-lactamase inhibitors. In this study, we have used docking-based virtual screening approach in order to screen potential inhibitors against some of the major resistant mutants of SHV and TEM types β-lactamase. Two different inhibitor-resistant mutants from SHV and TEM were selected. Moreover, we have retained the active site water molecules within each enzyme. Active site water molecules were placed within modeled structure of the mutant whose structure was unavailable with protein databank. The novelty of this work lies in the use of multilayer virtual screening approach for the prediction of best and accurate results. We are reporting five inhibitors on the basis of their efficacy against all the selected resistant mutants. These inhibitors were selected on the basis of their binding efficacies and pharmacophore features. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Structurally distinct toxicity inhibitors bind at common loci on β-amyloid fibril

PubMed Central

Keshet, Ben; Gray, Jeffrey J; Good, Theresa A

2010-01-01

The accumulation of aggregated β-Amyloid (Aβ) in the brain is a hallmark of Alzheimer's disease and is thought to play a role in the neurotoxicity associated with the disease. The mechanism by which Aβ aggregates induce toxicity is uncertain. Nonetheless, several small molecules have been found to interact with Aβ fibrils and to prevent their toxicity. In this paper we studied the binding of these known toxicity inhibitors to Aβ fibrils, as a means to explore surfaces or loci on Aβ aggregates that may be significant in the mechanism of action of these inhibitors. We believe knowledge of these binding loci will provide insight into surfaces on the Aβ fibrils important in Aβ biological activity. The program DOCK was used to computationally dock the inhibitors to an Aβ fibril. The inhibitors docked at two shared binding loci, near Lys28 and at the C-termini near Asn27 and Val39. The docking predictions were experimentally verified using lysine specific chemical modifications and Aβ fibrils mutated at Asn27. We found that both Congo red and Myricetin, despite being structurally different, bound at the same two sites. Additionally, our data suggests that three additional Aβ toxicity inhibitors may also bind in one of the sites. Identification of these common binding loci provides targets on the Aβ fibril surface that can be tested in the future for their role in Aβ biological activity. PMID:20882638