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Sample records for diverse kunitz inhibitors

  1. A Family of Diverse Kunitz Inhibitors from Echinococcus granulosus Potentially Involved in Host-Parasite Cross-Talk

    PubMed Central

    Margenat, Mariana; Durán, Rosario; González-Sapienza, Gualberto; Graña, Martín; Parkinson, John; Maizels, Rick M.; Salinas, Gustavo; Alvarez, Beatriz; Fernández, Cecilia

    2009-01-01

    The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H+-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 – EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (KI*) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection. PMID:19759914

  2. Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels.

    PubMed

    Fló, Martín; Margenat, Mariana; Pellizza, Leonardo; Graña, Martín; Durán, Rosario; Báez, Adriana; Salceda, Emilio; Soto, Enrique; Alvarez, Beatriz; Fernández, Cecilia

    2017-02-01

    We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold.

  3. Functional diversity of secreted cestode Kunitz proteins: Inhibition of serine peptidases and blockade of cation channels

    PubMed Central

    Fló, Martín; Margenat, Mariana; Pellizza, Leonardo; Durán, Rosario; Salceda, Emilio; Alvarez, Beatriz

    2017-01-01

    We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (Kv); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold. PMID:28192542

  4. Knock-down of transcript abundance of a family of Kunitz proteinase inhibitor genes in white clover (Trifolium repens) reveals a redundancy and diversity of gene function.

    PubMed

    Islam, Afsana; Leung, Susanna; Burgess, Elisabeth P J; Laing, William A; Richardson, Kim A; Hofmann, Rainer W; Dijkwel, Paul P; McManus, Michael T

    2015-12-01

    The transcriptional regulation of four phylogenetically distinct members of a family of Kunitz proteinase inhibitor (KPI) genes isolated from white clover (Trifolium repens; designated Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5) has been investigated to determine their wider functional role. The four genes displayed differential transcription during seed germination, and in different tissues of the mature plant, and transcription was also ontogenetically regulated. Heterologous over-expression of Tr-KPI1, Tr-KPI2, Tr-KPI4 and Tr-KPI5 in Nicotiana tabacum retarded larval growth of the herbivore Spodoptera litura, and an increase in the transcription of the pathogenesis-related genes PR1 and PR4 was observed in the Tr-KPI1 and Tr-KPI4 over-expressing lines. RNA interference (RNAi) knock-down lines in white clover displayed significantly altered vegetative growth phenotypes with inhibition of shoot growth and a stimulation of root growth, while knock-down of Tr-KPI1, Tr-KPI2 and Tr-KPI5 transcript abundance also retarded larval growth of S. litura. Examination of these RNAi lines revealed constitutive stress-associated phenotypes as well as altered transcription of cellular signalling genes. These results reveal a functional redundancy across members of the KPI gene family. Further, the regulation of transcription of at least one member of the family, Tr-KPI2, may occupy a central role in the maintenance of a cellular homeostasis.

  5. Kunitz-type protease inhibitors group B from Solanum palustre.

    PubMed

    Speransky, Anna S; Cimaglia, Fabio; Krinitsina, Anastasya A; Poltronieri, Palmiro; Fasano, Pasqua; Bogacheva, Anna M; Valueva, Tatiana A; Halterman, Dennis; Shevelev, Alexei B; Santino, Angelo

    2007-11-01

    Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.

  6. Precursor of kunitz trypsin inhibitor in soybean seeds

    SciTech Connect

    McGrain, A.; Chen, J.; Tan-Wilson, A. )

    1990-05-01

    Kunitz soybean trypsin inhibitor (KSTI) appears to be synthesized in precursor form which is converted by proteolytic digestion to the mature form of KSTI. Two forms of anti-cross-reacting material are evident when Western blots of extracts of developing seeds are analyzed. The precursor form increases to maximum levels as seed lengths increase to 11 mm. As the seed matures to 13 mm and turns yellow, precursor levels decrease while mature KSTI levels increase. The conversion of precursor to mature form could be demonstrated in vitro in seed extracts. The conversion could also be demonstrated in excised seeds pulse-labeled with ({sup 14}C)-leucine as loss of radioactivity from the precursor and appearance in the mature KSTI form.

  7. A Spider-Derived Kunitz-Type Serine Protease Inhibitor That Acts as a Plasmin Inhibitor and an Elastase Inhibitor

    PubMed Central

    Wan, Hu; Lee, Kwang Sik; Kim, Bo Yeon; Zou, Feng Ming; Yoon, Hyung Joo; Je, Yeon Ho; Li, Jianhong; Jin, Byung Rae

    2013-01-01

    Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor. PMID:23308198

  8. Unexpected Activity of a Novel Kunitz-type Inhibitor

    PubMed Central

    Smith, David; Tikhonova, Irina G.; Jewhurst, Heather L.; Drysdale, Orla C.; Dvořák, Jan; Robinson, Mark W.; Cwiklinski, Krystyna; Dalton, John P.

    2016-01-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host. PMID:27422822

  9. A four-domain Kunitz-type proteinase inhibitor from Solen grandis is implicated in immune response.

    PubMed

    Wei, Xiumei; Yang, Jialong; Yang, Jianmin; Liu, Xiangquan; Liu, Meijun; Yang, Dinglong; Xu, Jie; Hu, Xiaoke

    2012-12-01

    Serine proteinase inhibitor (SPI) serves as a negative regulator in immune signal pathway by restraining the activities of serine proteinase (SP) and plays an essential role in the innate immunity. In the present study, a Kunitz-type SPI was identified from the mollusk razor clam Solen grandis (designated as SgKunitz). The full-length cDNA of SgKunitz was of 1284 bp, containing an open reading frame (ORF) of 768 bp. The ORF encoded four Kunitz domains, and their amino acids were well conserved when compared with those in other Kunitz-type SPIs, especially the six cysteines involved in forming of three disulfide bridges in each domain. In addition, the tertiary structure of all the four domains adopted a typical model of Kunitz-type SPI family, indicating SgKunitz was a new member of Kunitz-type SPI superfamily. The mRNA transcripts of SgKunitz were detected in all tested tissues of razor clam, including muscle, mantle, gonad, gill, hepatopancreas and hemocytes, and with the highest expression level in gill. When the razor clams were stimulated by LPS, PGN or β-1, 3-glucan, the expression level of SgKunitz mRNA in hemocytes was significantly up-regulated (P < 0.01), suggesting SgKunitz might involved in the processes of inhibiting the activity of SPs during the immune responses triggered by various pathogens. Furthermore, the recombinant protein of SgKunitz could effectively inhibit the activities of SP trypsin and chymotrypsin in vitro. The present results suggested SgKunitz could serve as an inhibitor of SP involving in the immune response of S. grandis, and provided helpful evidences to understand the regulation mechanism of immune signal pathway in mollusk.

  10. Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

    SciTech Connect

    Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela . E-mail: olivaml.bioq@epm.br

    2007-09-07

    Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 {sup o}C. The refined structure shows the conservative {beta}-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.

  11. Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor and Kunitz trypsin concentrations on the stabil...

  12. Structural and functional properties of kunitz proteinase inhibitors from leguminosae: a mini review.

    PubMed

    Oliva, Maria Luiza Vilela; Ferreira, Rodrigo da Silva; Ferreira, Joana Gasperazzo; de Paula, Cláudia Alessandra Andrade; Salas, Carlos E; Sampaio, Misako Uemura

    2011-08-01

    Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.

  13. Joannsin, a novel Kunitz-type FXa inhibitor from the venom of Prospirobolus joannsi.

    PubMed

    Luan, Ning; Zhou, Chunling; Li, Pengpeng; Ombati, Rose; Yan, Xiuwen; Mo, Guoxiang; Rong, Mingqiang; Lai, Ren; Duan, Zilei; Zheng, Ruiqiang

    2017-03-09

    The repugnatorial glands of millipedes release various defensive chemical secretions. Although varieties of such defensive secretions have been studied, none of them is protein or peptide. Herein, a novel factor Xa (FXa) inhibitor named joannsin was identified and characterised from repugnatorial glands of Prospirobolus joannsi. Joannsin is composed of 72 amino acid residues including six cysteines, which form three intra-molecular disulfide bridges. It is a member of Kunitz-type protease inhibitor family, members of which are also found in the secretory glands of other arthropods. Recombinant joannsin exhibited remarkable inhibitory activity against trypsin and FXa with a Ki of 182.7 ± 14.6 and 29.5 ± 4.7 nM, respectively. Joannsin showed strong anti-thrombosis functions in vitro and in vivo. Joannsin is the first peptide component in millipede repugnatorial glands to be identified and is a potential candidate and/or template for the development of anti-thrombotic agents. These results also indicated that there is Kunitz-type protease inhibitor toxin in millipede repugnatorial glands as in other arthropods secretory glands.

  14. Kunitz-type trypsin inhibitor with high stability from Spinacia oleracea L. seeds.

    PubMed

    Kang, Zhuang; Jiang, Jia-hong; Wang, Dong; Liu, Ke; Du, Lin-fang

    2009-01-01

    The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography. This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation of stable SOTI-trypsin complex (K(i) = 2.3x10(-6) M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors.

  15. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    Salameh, M.A.; Soares, A.; Navaneetham, D.; Sinha, D.; Walsh, P. N.; Radisky, E. S.

    2010-11-19

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P1 and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin {center_dot} APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  16. Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds.

    PubMed

    Cruz, Ana C B; Massena, Fábio S; Migliolo, Ludovico; Macedo, Leonardo L P; Monteiro, Norberto K V; Oliveira, Adeliana S; Macedo, Francisco P; Uchoa, Adriana F; Grossi de Sá, Maria F; Vasconcelos, Ilka M; Murad, Andre M; Franco, Octavio L; Santos, Elizeu A

    2013-09-01

    The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.

  17. Determinants of Affinity and Proteolytic Stability in Interactions of Kunitz Family Protease Inhibitors with Mesotrypsin

    SciTech Connect

    M Salameh; A Soares; D Navaneetham; D Sinha; P Walsh; E Radisky

    2011-12-31

    An important functional property of protein protease inhibitors is their stability to proteolysis. Mesotrypsin is a human trypsin that has been implicated in the proteolytic inactivation of several protein protease inhibitors. We have found that bovine pancreatic trypsin inhibitor (BPTI), a Kunitz protease inhibitor, inhibits mesotrypsin very weakly and is slowly proteolyzed, whereas, despite close sequence and structural homology, the Kunitz protease inhibitor domain of the amyloid precursor protein (APPI) binds to mesotrypsin 100 times more tightly and is cleaved 300 times more rapidly. To define features responsible for these differences, we have assessed the binding and cleavage by mesotrypsin of APPI and BPTI reciprocally mutated at two nonidentical residues that make direct contact with the enzyme. We find that Arg at P{sub 1} (versus Lys) favors both tighter binding and more rapid cleavage, whereas Met (versus Arg) at P'{sub 2} favors tighter binding but has minimal effect on cleavage. Surprisingly, we find that the APPI scaffold greatly enhances proteolytic cleavage rates, independently of the binding loop. We draw thermodynamic additivity cycles analyzing the interdependence of P{sub 1} and P'{sub 2} substitutions and scaffold differences, finding multiple instances in which the contributions of these features are nonadditive. We also report the crystal structure of the mesotrypsin-APPI complex, in which we find that the binding loop of APPI displays evidence of increased mobility compared with BPTI. Our data suggest that the enhanced vulnerability of APPI to mesotrypsin cleavage may derive from sequence differences in the scaffold that propagate increased flexibility and mobility to the binding loop.

  18. A Kunitz proteinase inhibitor from corms of Xanthosoma blandum with bactericidal activity.

    PubMed

    Lima, Thaís B; Silva, Osmar N; Migliolo, Ludovico; Souza-Filho, Carlos R; Gonçalves, Eduardo G; Vasconcelos, Ilka M; Oliveira, José T A; Amaral, André C; Franco, Octávio L

    2011-05-27

    Bacterial infections directly affect the world's population, and this situation has been aggravated by indiscriminate use of antimicrobial agents, which can generate resistant microorganisms. In this report, an initial screening of proteins with antibacterial activity from corms of 15 species of the Xanthosoma genus was conducted. Since Xanthosoma blandum corms showed enhanced activity toward bacteria, a novel protein with bactericidal activity was isolated from this particular species. Edman degradation was used for protein N-termini determination; the primary structure showed similarities with Kunitz inhibitors, and this protein was named Xb-KTI. This protein was further challenged against serine proteinases from different sources, showing clear inhibitory activities. Otherwise, no hemolytic activity was observed for Xb-KTI. The results demonstrate the biotechnological potential of Xb-KTI, the first proteinase inhibitor with antimicrobial activity described in the Xanthosoma genus.

  19. Purification and characterization of a stable Kunitz trypsin inhibitor from Trigonella foenum-graecum (fenugreek) seeds.

    PubMed

    Oddepally, Rajender; Sriram, Gopi; Guruprasad, Lalitha

    2013-12-01

    Kunitz trypsin inhibitor was purified from the seeds of Trigonella foenum-graecum (TfgKTI) belonging to fabaceae family by ammonium sulphate precipitation, cation exchange, gel filtration and hydrophobic chromatography. Purity of the protein was analyzed by RP-HPLC and native-PAGE. SDS-PAGE analysis under reducing and non-reducing conditions showed that protein consists of a single polypeptide chain with molecular mass of approximately 20 kDa. Mass spectroscopy analysis revealed that the intact mass of purified inhibitor is 19,842.154 Da. One dimensional SDS gel was tryptically digested, resulting peptides were subjected to MALDI-TOF-MS analysis, and peptide mass fingerprinting (PMF) analysis of TfgKTI shows sequence similarity with Kunitz trypsin inhibitor in database search. Two dimensional electrophoresis identified presence of four isoinhibitors (pI values of 5.1, 5.4, 5.7 and 6.1). Kinetic studies showed that the protein is a competitive inhibitor and has high binding affinity with trypsin (Ki 3.01×10(-9)M) and chymotrypsin (Ki 0.52×10(-9)M). The TfgKTI retained the inhibitory activity over a broad range of pH (pH 3-10), temperature (37-100°C) and salt concentration (up to 3.5%). Far-UV circular dichroism measurements revealed that TfgKTI is predominantly composed of β-sheets (39%) and unordered structures (48%) with slight helical content (13%). TfgKTI retained over 90% trypsin inhibition upon storage at 4°C for over a period of six months.

  20. Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

    PubMed Central

    Vodkin, Lila O.

    1981-01-01

    The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA. Images PMID:16661996

  1. Identification of a new soybean Kunitz trypsin inhibitor mutation and its effect on Bowman-Birk protease inhibitor content in soybean seed

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean seeds possess anti-nutritional compounds which inactivate digestive proteases, principally corresponding to two families: Kunitz Trypsin Inhibitors (KTi) and Bowman-Birk Inhibitors (BBI). High levels of raw soybeans/soybean meal in feed mixtures can cause poor weight gain and pancreatic abno...

  2. Characterization and Pharmacological Properties of a Novel Multifunctional Kunitz Inhibitor from Erythrina velutina Seeds

    PubMed Central

    Machado, Richele J. A.; Monteiro, Norberto K. V.; Migliolo, Ludovico; Silva, Osmar N.; Pinto, Michele F. S.; Oliveira, Adeliana S.; Franco, Octávio L.; Kiyota, Sumika; Bemquerer, Marcelo P.; Uchoa, Adriana F.; Morais, Ana H. A.; Santos, Elizeu A.

    2013-01-01

    Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30–60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10−8 mol.L−1 and constant inhibition (Ki) of 1.0×10−8 mol.L−1, by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation. PMID

  3. Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

    PubMed

    Machado, Richele J A; Monteiro, Norberto K V; Migliolo, Ludovico; Silva, Osmar N; Pinto, Michele F S; Oliveira, Adeliana S; Franco, Octávio L; Kiyota, Sumika; Bemquerer, Marcelo P; Uchoa, Adriana F; Morais, Ana H A; Santos, Elizeu A

    2013-01-01

    Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.

  4. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds

    PubMed Central

    Patil, Dipak N.; Preeti; Chaudhry, Anshul; Sharma, Ashwani K.; Tomar, ­Shailly; Kumar, Pravindra

    2009-01-01

    A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS–PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 Å. Diffraction data were collected to a resolution of 2.7 Å. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%. PMID:19574654

  5. Inhibitory effects of a Kunitz-type inhibitor from Pithecellobium dumosum (Benth) seeds against insect-pests' digestive proteinases.

    PubMed

    Rufino, Fabiola P S; Pedroso, Vanessa M A; Araujo, Jonalson N; França, Anderson F J; Rabêlo, Luciana M A; Migliolo, Ludovico; Kiyota, Sumika; Santos, Elizeu A; Franco, Octavio L; Oliveira, Adeliana S

    2013-02-01

    Pithecellobium dumosum is a tree belonging to the Mimosoideae subfamily that presents various previously characterized Kunitz-type inhibitors. The present study provides a novel Kunitz-trypsin inhibitor isoform purified from P. dumosum seeds. Purification procedure was performed by TCA precipitation followed by a trypsin-Sepharose chromatography and a further reversed-phase HPLC. Purified inhibitor (PdKI-4) showed enhanced inhibitory activity against bovine trypsin and chymotrypsin. Furthermore, PdKI-4 showed remarkable inhibitory activity against serine proteases from the coleopterans Callosobruchus maculatus and Zabrotes subfasciatus, and the lepidopterans Alabama argillacea and Telchin licus. However, PdKI-4 was unable to inhibit porcine pancreatic elastase, pineapple bromelain and Carica papaya papain. SDS-PAGE showed that PdKI-4 consisted of a single polypeptide chain with molecular mass of 21 kDa. Kinetic studies demonstrated that PdKI-4 is probably a competitive inhibitor with a Ki value of 5.7 × 10(-10) M for bovine trypsin. PdKI-4 also showed higher stability over a wide range of temperature (37-100 °C) and pH (2-12). N-termini sequence was obtained by Edman degradation showing higher identity with other Mimosoideae subfamily Kunitz-type inhibitor members. In summary, data here reported indicate the biotechnological potential of PdKI-4 for development of products against insect-pests.

  6. Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus

    PubMed Central

    Ranasinghe, Shiwanthi L.; Fischer, Katja; Zhang, Wenbao; Gobert, Geoffrey N.; McManus, Donald P.

    2015-01-01

    The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic. PMID:26645974

  7. Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues.

    PubMed

    Pereira, Keitty R B; Botelho-Júnior, Sylvio; Domingues, Dalvania P; Machado, Olga L T; Oliveira, Antônia E A; Fernandes, Kátia V S; Madureira, Herika C; Pereira, Telma N S; Jacinto, Tânia

    2011-11-01

    In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.

  8. Marker assisted accelerated introgression of null allele of kunitz trypsin inhibitor in soybean

    PubMed Central

    Kumar, Vineet; Rani, Anita; Rawal, Reena; Mourya, Vaishali

    2015-01-01

    Development of kunitz trypsin inhibitor (KTI)-free soybean is crucial for soy-food industry as the heat inactivation employed to inactivate the anti-nutritional factor in regular soybean incurs extra cost and affects protein solubility. In the presented work, a null allele of KTI from PI542044 was introgressed into cultivar ‘JS97-52’ (recurrent parent) through marker assisted backcrossing. Foreground selection in BC1F2, BC2F2 and BC3F2 was carried out using the null allele-specific marker in tandem with SSR marker Satt228, tightly linked with a trypsin inhibitor Ti locus. Background selection in null allele-carrying plants through 106 polymorphic SSR markers across the genome led to the identification of 9 KTI-free lines exhibiting 98.6% average recurrent parent genome content (RPGC) after three backcrosses, which otherwise had required 5–6 backcrosses through conventional method. Introgressed lines (ILs) were free from KTI and yielded at par with recurrent parent. Reduction of 68.8–83.5% in trypsin inhibitor content (TIC) in ILs compared to the recurrent parent (‘JS97-52’) was attributed to the elimination of KTI. PMID:26719748

  9. A genetically engineered human Kunitz protease inhibitor with increased kallikrein inhibition in an ovine model of cardiopulmonary bypass.

    PubMed

    Ohri, S K; Parratt, R; White, T; Becket, J; Brannan, J J; Hunt, B J; Taylor, K M

    2001-05-01

    A recombinant human serine protease inhibitor known as Kunitz protease inhibitor (KPI) wild type has functional similarities to the bovine Kunitz inhibitor, aprotinin, and had shown a potential to reduce bleeding in an ovine model of cardiopulmonary bypass (CPB). The aim of this study was to assess KPI-185, a modification of KPI-wild type that differs from KPI-wild type in two amino acid residues and which enhances anti-kallikrein activity in a further double-blind, randomized study in an ovine model of CPB, and to compare with our previous study of KPI-wild type and aprotinin in the same ovine model. Post-operative drain losses and subjective assessment of wound 'dryness' showed no significant differences between KPI-185 and KPI-wild type, despite the significant enhancement of kallikrein inhibition using KPI-185 seen in serial kallikrein inhibition assays. These preliminary findings support the hypothesis that kallikrein inhibition is not the major mechanism by which Kunitz inhibitors such as aprotinin reduce perioperative bleeding.

  10. AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

    PubMed

    Xu, Feng; Previti, Mary Lou; Nieman, Marvin T; Davis, Judianne; Schmaier, Alvin H; Van Nostrand, William E

    2009-04-29

    The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

  11. A Kunitz-type protease inhibitor regulates programmed cell death during flower development in Arabidopsis thaliana.

    PubMed

    Boex-Fontvieille, Edouard; Rustgi, Sachin; Reinbothe, Steffen; Reinbothe, Christiane

    2015-10-01

    Flower development and fertilization are tightly controlled in Arabidopsis thaliana. In order to permit the fertilization of a maximum amount of ovules as well as proper embryo and seed development, a subtle balance between pollen tube growth inside the transmitting tract and pollen tube exit from the septum is needed. Both processes depend on a type of programmed cell death that is still poorly understood. Here, it is shown that a Kunitz protease inhibitor related to water-soluble chlorophyll proteins of Brassicaceae (AtWSCP, encoded by At1g72290) is involved in controlling cell death during flower development in A. thaliana. Genetic, biochemical, and cell biology approaches revealed that WSCP physically interacts with RD21 (RESPONSIVE TO DESICCATION) and that this interaction in turn inhibits the activity of RD21 as a pro-death protein. The regulatory circuit identified depends on the restricted expression of WSCP in the transmitting tract and the septum epidermis. In a respective Atwscp knock-out mutant, flowers exhibited precocious cell death in the transmitting tract and unnatural death of septum epidermis cells. As a consequence, apical-basal pollen tube growth, fertilization of ovules, as well as embryo development and seed formation were perturbed. Together, the data identify a unique mechanism of cell death regulation that fine-tunes pollen tube growth.

  12. Promising pharmacological profile of a Kunitz-type inhibitor in murine renal cell carcinoma model

    PubMed Central

    de Souza, Jean Gabriel; Morais, Katia L.P.; Anglés-Cano, Eduardo; Boufleur, Pamela; de Mello, Evandro Sobroza; Maria, Durvanei Augusto; Origassa, Clarice Silvia Taemi; Zampolli, Hamilton de Campos; Câmara, Niels Olsen Saraiva; Berra, Carolina Maria; Bosch, Rosemary Viola; Chudzinski-Tavassi, Ana Marisa

    2016-01-01

    Renal cell carcinoma (RCC), also called kidney cancer or renal adenocarcinoma, is highly resistant to current treatments. It has been previously reported that a Kunitz-type inhibitor domain-containing protein, isolated from the salivary glands of the Amblyomma cajennense tick, triggers apoptosis in murine renal adenocarcinoma cells (Renca) by inhibiting the proteasome and endoplasmic reticulum stress. Of note, Amblyomin-X is the corresponding recombinant protein identified in the cDNA library from A. cajennense salivary glands. Herein, using orthotopic kidney tumors in mice, we demonstrate that Amblyomin-X is able to drastically reduce the incidence of lung metastases by inducing cell cycle arrest and apoptosis. The in vitro assays show that Amblyomin-X is capable of reducing the proliferation rate of Renca cells, promoting cell cycle arrest, and down-regulating the expression of crucial proteins (cyclin D1, Ki67 and Pgp) involved in the aggressiveness and resistance of RCC. Regarding non-tumor cells (NIH3T3), Amblyomin-X produced minor effects in the cyclin D1 levels. Interestingly, observing the image assays, the fluorescence-labelled Amblyomin-X was indeed detected in the tumor stroma whereas in healthy animals it was rapidly metabolized and excreted. Taken the findings together, Amblyomin-X can be considered as a potential anti-RCC drug candidate. PMID:27566592

  13. Identification of a new soybean kunitz trypsin inhibitor mutation and its effect on bowman-birk protease inhibitor content in soybean seed.

    PubMed

    Gillman, Jason D; Kim, Won-Seok; Krishnan, Hari B

    2015-02-11

    Soybean seed contains antinutritional compounds that inactivate digestive proteases, principally corresponding to two families: Kunitz trypsin inhibitors (KTi) and Bowman-Birk inhibitors (BBI). High levels of raw soybean/soybean meal in feed mixtures can cause poor weight gain and pancreatic abnormalities via inactivation of trypsin/chymotrypsin enzymes. Soybean protein meal is routinely heat-treated to inactivate inhibitors, a practice that is energy-intensive and costly and can degrade certain essential amino acids. In this work, we screened seed from 520 soybean accessions, using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots with anti-Kunitz trypsin inhibitor antibodies. A soybean germplasm accession was identified with a mutation affecting an isoform annotated as nonfunctional (KTi1), which was determined to be synergistic with a previously identified mutation (KTi3-). We observed significant proteome rebalancing in all KTi mutant lines, resulting in dramatically increased BBI protein levels.

  14. Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme*

    PubMed Central

    García-Fernández, Rossana; Perbandt, Markus; Rehders, Dirk; Ziegelmüller, Patrick; Piganeau, Nicolas; Hahn, Ulrich; Betzel, Christian; Chávez, María de los Ángeles; Redecke, Lars

    2015-01-01

    Elastase-like enzymes are involved in important diseases such as acute pancreatitis, chronic inflammatory lung diseases, and cancer. Structural insights into their interaction with specific inhibitors will contribute to the development of novel anti-elastase compounds that resist rapid oxidation and proteolysis. Proteinaceous Kunitz-type inhibitors homologous to the bovine pancreatic trypsin inhibitor (BPTI) provide a suitable scaffold, but the structural aspects of their interaction with elastase-like enzymes have not been elucidated. Here, we increased the selectivity of ShPI-1, a versatile serine protease inhibitor from the sea anemone Stichodactyla helianthus with high biomedical and biotechnological potential, toward elastase-like enzymes by substitution of the P1 residue (Lys13) with leucine. The variant (rShPI-1/K13L) exhibits a novel anti-porcine pancreatic elastase (PPE) activity together with a significantly improved inhibition of human neuthrophil elastase and chymotrypsin. The crystal structure of the PPE·rShPI-1/K13L complex determined at 2.0 Å resolution provided the first details of the canonical interaction between a BPTI-Kunitz-type domain and elastase-like enzymes. In addition to the essential impact of the variant P1 residue for complex stability, the interface is improved by increased contributions of the primary and secondary binding loop as compared with similar trypsin and chymotrypsin complexes. A comparison of the interaction network with elastase complexes of canonical inhibitors from the chelonian in family supports a key role of the P3 site in ShPI-1 in directing its selectivity against pancreatic and neutrophil elastases. Our results provide the structural basis for site-specific mutagenesis to further improve the binding affinity and/or direct the selectivity of BPTI-Kunitz-type inhibitors toward elastase-like enzymes. PMID:25878249

  15. Isolation and partial sequence of a Kunitz-type elastase specific inhibitor from marama bean (Tylosema esculentum).

    PubMed

    Nadaraja, Deepa; Weintraub, Susan T; Hakala, Kevin W; Sherman, Nicholas E; Starcher, Barry

    2010-06-01

    An isolation procedure utilizing ammonium sulfate fractionation and affinity chromatography was used to purify an elastase inhibitor present in large amounts in marama beans (Tylosema esculentum). The protein appeared to be heterogeneous due to carbohydrate differences, demonstrating two bands on SDS gels with molecular weights of 17.8 kDa and 20 kDa. Partial sequence, derived from mass spectrometry, indicated that the protein is a Kunitz-type inhibitor distinct from other known plant serine protease inhibitors. The marama bean inhibitor is specific for elastase, with very low K(i) for both pancreatic and neutrophil elastase. The quantity of elastase inhibitor present in marama beans is many times greater than in soybean or any other bean or nut source reported to date. This raises the question of why a bean found in an arid corner of the Kalahari Desert would be so rich in a very potent elastase inhibitor.

  16. Allelic differentiation of Kunitz trypsin inhibitor in wild soybean (Glycine soja).

    PubMed

    Wang, K J; Takahata, Y; Kono, Y; Kaizuma, N

    2008-08-01

    Soybean Kunitz trypsin inhibitor (SKTI) has several polymorphic types, which are controlled by co-dominant multiple alleles at a single locus. Of these types, Tia and Tib are predominant types, and there are nine differences in amino acids between Tia and Tib. Recently, an intermediate transitional type (Tibi5) between them was detected. However, other transitional types have not been detected despite surveys of many cultivated and wild soybeans. One of the reasons why other transitional variants have not been found is inferred to be due to the difficulty of the detection of SKTI protein variants by polyacrylamide gel electrophoresis (PAGE). To detect novel variants of SKTI, nucleotide sequence analysis in addition to PAGE was carried out. Four new variants were found from many Japanese wild soybeans. Of these variants, three (designated as Tiaa1, Tiaa2, Tiab1) were detected through gene sequence analysis on wild soybeans having the same electrophoretic mobility as Tia, and one (Tig) was detected through PAGE. The Tig variant showed a slightly lower electrophoretic mobility than Tic. The nucleotide sequences of Tig were identical to those of Tib except for one T-->C transitional mutation at position +340. The sequences of Tiaa1 and Tiaa2 genes were identical to those of Tia with the exception of a G-->A mutation at position +376 and a T-->C mutation at +404, respectively. The sequence of Tiab1 differed from Tia by three nucleotides: C-->A at position +331, T-->C at +459 and A-->G at +484. Of the three nucleotide changes, two were common to Tiab1, Tibi5 and Tib, suggesting that Tiab1 is an intermediate transitional type between Tia and Tib. Our results suggest that Tib type has been differentiated through a series of mutations from Tia before the domestication of cultivated soybean.

  17. Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

    SciTech Connect

    Millers, Emma-Karin I.; Masci, Paul P.; Lavin, Martin F.; Jersey, John de; Guddat, Luke W.

    2006-07-01

    Crystals of a canonical inhibitor of plasmin from Australian Brown snake venom has been obtained. In complex with trypsin these diffract to 2.0 Å resolution, while the free inhibitor diffracts to 1.63 Å. Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1–bovine trypsin complex diffracted to 2.0 Å resolution, while crystals of free rTxln-1 diffracted to 1.63 Å resolution.

  18. Effect of the enzymatic inhibitor of Kunitz on the gastric lesions from reserpine, from phenylbutazone, from pyloric ligation and by restraint in the rat

    NASA Technical Reports Server (NTRS)

    Guerrin, F.; Demaille, A.; Merveille, P.; Bel, C.

    1980-01-01

    The protective effects of certain polypeptides on gastric ulcerations caused from reserpine and phenylbutazone in the rate were studied. It was found that the Kunitz enzymatic inhibitor exerts a protective action in regard to gastric lesions. However, the inhibitor did not change the development of Shay ulcers and stress ulcers from restraint.

  19. Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor.

    PubMed

    Petersen, L C; Bjørn, S E; Olsen, O H; Nordfang, O; Norris, F; Norris, K

    1996-01-15

    Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI-(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.

  20. Identification of a Kunitz-type proteinase inhibitor from Pithecellobium dumosum seeds with insecticidal properties and double activity.

    PubMed

    Oliveira, A S; Migliolo, L; Aquino, R O; Ribeiro, J K C; Macedo, L L P; Andrade, L B S; Bemquerer, M P; Santos, E A; Kiyota, S; Sales, M P

    2007-09-05

    A trypsin inhibitor, PdKI, was purified from Pithecellobium dumosum seeds by TCA precipitation, trypsin-sepharose chromatography, and reversed-phase-HPLC. PdKI was purified 217.6-fold and recovered 4.7%. SDS-PAGE showed that PdKI is a single polypeptide chain of 18.9 kDa and 19.7 kDa by MALDI-TOF. The inhibition on trypsin was stable in the pH range 2-10 and at a temperature of 50 degrees C. The Ki values were 3.56 x 10(-8)and 7.61 x 10(-7) M with competitive and noncompetitive inhibition mechanisms for trypsin and papain, respectively. The N-terminal sequence identified with members of Kunitz-type inhibitors from the Mimosoideae and Caesalpinoideae subfamilies. PdKI was effective against digestive proteinase from Zabrotes subfasciatus, Ceratitis capitata, Plodia interpunctella, Alabama argillaceae, and Callosobruchus maculatus, with 69, 66, 44, 38, and 29% inhibition, respectively. Results support that PdKI is a member of the Kunitz inhibitor family and its insecticidal properties indicate a potent insect antifeedant.

  1. Amino acid sequence and disulfide bridges of affinity purified Kunitz-type chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC).

    PubMed

    Kortt, A A; Burns, J E; Strike, P M

    1990-11-01

    The primary sequence of the affinity purified chymotrypsin inhibitor, WBCI, isolated from the albumin fraction of Psophocarpus tetragonolobus (L.) DC cv. UPS-122 seed was determined. The inhibitor consisted of a single polypeptide chain of 183 amino acids (Mr 20285) and the four half-cystine residues in the molecule formed two intramolecular disulfide bridges equivalent to those in other Kunitz-type seed inhibitors. The sequence of this chymotrypsin inhibitor was identical to that of chymotrypsin inhibitor-3 from cultivar UPS-31 and it showed about 50% sequence similarity to the winged bean acidic (WBTI-2, pI 5.1) and basic (WBTI-1, pI 8.9) trypsin inhibitors. Sequence similarities to other Kunitz-type seed inhibitors are discussed.

  2. Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family

    PubMed Central

    Shamsi, Tooba Naz; Parveen, Romana; Amir, Mohd.; Baig, Mohd. Affan; Qureshi, M. Irfan; Ali, Sher; Fatima, Sadaf

    2016-01-01

    Purpose This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. Methods Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. Results ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2–12 showing a decline in the activity around pH 4–5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10–80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (μM/min) and Km value of 0.12 μM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% β -sheets at native pH. Conclusions To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids

  3. Differential Expression of Kunitz and Bowman-Birk Soybean Proteinase Inhibitors in Plant and Callus Tissue 1

    PubMed Central

    Tan-Wilson, Anna L.; Hartl, Philippe M.; Delfel, Norman E.; Wilson, Karl A.

    1985-01-01

    Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred. Images Fig. 1 Fig. 4 PMID:16664236

  4. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    PubMed

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.

  5. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

    PubMed

    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  6. Crystallization and preliminary X-ray analysis of a novel Kunitz-type kallikrein inhibitor from Bauhinia bauhinioides

    SciTech Connect

    Navarro, Marcos Vicente de A. S.; Vierira, Débora F.; Nagem, Ronaldo A. P.; Araújo, Ana Paula U. de; Oliva, Maria Luiza V.; Garratt, Richard C.

    2005-10-01

    Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges. A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V{sub M} = 2.5 Å{sup 3} Da{sup −1}). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.

  7. Structure of the recombinant BPTI/Kunitz-type inhibitor rShPI-1A from the marine invertebrate Stichodactyla helianthus

    PubMed Central

    García-Fernández, Rossana; Pons, Tirso; Meyer, Arne; Perbandt, Markus; González-González, Yamile; Gil, Dayrom; de los Angeles Chávez, María; Betzel, Christian; Redecke, Lars

    2012-01-01

    The BPTI/Kunitz-type inhibitor family includes several extremely potent serine protease inhibitors. To date, the inhibitory mechanisms have only been studied for mammalian inhibitors. Here, the first crystal structure of a BPTI/Kunitz-type inhibitor from a marine invertebrate (rShPI-1A) is reported to 2.5 Å resolution. Crystallization of recombinant rShPI-1A required the salt-induced dissociation of a trypsin complex that was previously formed to avoid intrinsic inhibitor aggregates in solution. The rShPI-1A structure is similar to the NMR structure of the molecule purified from the natural source, but allowed the assignment of disulfide-bridge chiralities and the detection of an internal stabilizing water network. A structural comparison with other BPTI/Kunitz-type canonical inhibitors revealed unusual ϕ angles at positions 17 and 30 to be a particular characteristic of the family. A significant clustering of ϕ and ψ angle values in the glycine-rich remote fragment near the secondary binding loop was additionally identified, but its impact on the specificity of rShPI-1A and similar molecules requires further study. PMID:23143234

  8. Isolation and characterization of a Kunitz-type trypsin inhibitor with antiproliferative activity from Gymnocladus chinensis (Yunnan bean) seeds.

    PubMed

    Zhu, M J; Zhang, G Q; Wang, H X; Ng, T B

    2011-04-01

    A 20-kDa Kunitz-type trypsin inhibitor was isolated from Gymnocladus chinensis (Yunnan bean) seeds. The isolation procedure involved ion exchange chromatography on diethylaminoethyl cellulose (DEAE-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on sulfopropyl sepharose (SP-sepharose), and gel filtration by FPLC on Superdex 75. The trypsin inhibitor was adsorbed on DEAE-cellulose, unadsorbed on Affi-gel blue gel, and adsorbed on SP-Sepharose. It dose-dependently inhibited trypsin with an IC(50) value of 0.4 μM. Dithiothreitol reduced its trypsin inhibitory activity, suggesting that an intact disulfide bond is indispensable to the activity. It suppressed [methyl-(3)H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC(50) value of 4.7 and 9.4 μM, respectively. There was no effect on human immunodeficiency virus(4)-1 reverse transcriptase activity and fungal growth when the trypsin inhibitor was tested up to 100 μM.

  9. Selective Inhibition of Prostasin in Human Enterocytes by the Integral Membrane Kunitz-Type Serine Protease Inhibitor HAI-2

    PubMed Central

    Shiao, Frank; Liu, Li-Ching O.; Huang, Nanxi; Lai, Ying-Jung J.; Barndt, Robert J.; Tseng, Chun-Che; Wang, Jehng-Kang; Jia, Bailing; Johnson, Michael D.

    2017-01-01

    Mutations of hepatocyte growth factor activator inhibitor (HAI)-2 in humans cause sodium loss in the gastrointestinal (GI) tract in patients with syndromic congenital sodium diarrhea (SCSD). Aberrant regulation of HAI-2 target protease(s) was proposed as the cause of the disease. Here functional linkage of HAI-2 with two membrane-associated serine proteases, matriptase and prostasin was analyzed in Caco-2 cells and the human GI tract. Immunodepletion-immunoblot analysis showed that significant proportion of HAI-2 is in complex with activated prostasin but not matriptase. Unexpectedly, prostasin is expressed predominantly in activated forms and was also detected in complex with HAI-1, a Kunitz inhibitor highly related to HAI-2. Immunohistochemistry showed a similar tissue distribution of prostasin and HAI-2 immunoreactivity with the most intense labeling near the brush borders of villus epithelial cells. In contrast, matriptase was detected primarily at the lateral plasma membrane, where HAI-1 was also detected. The tissue distribution profiles of immunoreactivity against these proteins, when paired with the species detected suggests that prostasin is under tight control by both HAI-1 and HAI-2 and matriptase by HAI-1 in human enterocytes. Furthermore, HAI-1 is a general inhibitor of prostasin in a variety of epithelial cells. In contrast, HAI-2 was not found to be a significant inhibitor for prostasin in mammary epithelial cells or keratinocytes. The high levels of constitutive prostasin zymogen activation and the selective prostasin inhibition by HAI-2 in enterocytes suggest that dysregulated prostasin proteolysis may be particularly important in the GI tract when HAI-2 function is lost and/or dysregulated. PMID:28125689

  10. Functional characterization and novel rickettsiostatic effects of a Kunitz-type serine protease inhibitor from the tick Dermacentor variabilis.

    PubMed

    Ceraul, Shane M; Dreher-Lesnick, Sheila M; Mulenga, Albert; Rahman, M Sayeedur; Azad, Abdu F

    2008-11-01

    Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPI's anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPI's trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the tick's defense response to rickettsiae.

  11. Structural and functional characterization of complex formation between two Kunitz-type serine protease inhibitors from Russell's Viper venom.

    PubMed

    Mukherjee, Ashis K; Dutta, Sumita; Kalita, Bhargab; Jha, Deepak K; Deb, Pritam; Mackessy, Stephen P

    2016-01-01

    Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

  12. Antimicrobial Activity of ILTI, a Kunitz-Type Trypsin Inhibitor from Inga laurina (SW.) Willd.

    PubMed

    Macedo, Maria Lígia R; Ribeiro, Suzanna F F; Taveira, Gabriel B; Gomes, Valdirene M; de Barros, Karina M C A; Maria-Neto, Simone

    2016-05-01

    Over the last few years, a growing number of proteinase inhibitors have been isolated from plants and particularly from seeds and have shown antimicrobial activity. A 20,000 Da serine peptidase inhibitor, named ILTI, was isolated from Inga laurina seeds and showed potent inhibitory enzymatic activity against trypsin. The aim of this study was to determine the effects of ILTI on the growth of pathogenic and non-pathogenic microorganisms. We observed that ILTI strongly inhibited in particular the growth of Candida tropicalis and Candida buinensis, inducing cellular agglomeration. However, it was ineffective against human pathogenic bacteria. We also investigated the potential of ILTI to permeabilize the plasma membrane of yeast cells. C. tropicalis and C. buinensis were incubated for 24 h with the ILTI at different concentrations, which showed that this inhibitor induced changes in the membranes of yeast cells, leading to their permeabilization. Interestingly, ILTI induced the production of reactive oxygen species (ROS) in C. tropicalis and C. buinensis cells. Finally, ILTI was coupled with fluorescein isothiocyanate, and subsequent treatment of C. tropicalis and C. buinensis with DAPI revealed the presence of the labeled protein in the intracellular spaces. In conclusion, our results indicated the ability of peptidase inhibitors to induce microbial inhibition; therefore, they might offer templates for the design of new antifungal agents.

  13. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    PubMed

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

  14. Pharmacological properties and pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-II) and its protein complex (Rusvikunin complex) purified from Daboia russelii russelii venom.

    PubMed

    Mukherjee, Ashis K; Mackessy, Stephen P

    2014-10-01

    A 7.1 kDa basic peptide (Rusvikunin-II) was purified from a previously described protein complex (Rusvikunin complex, consists of Rusvikunin and Rusvikunin-II) of Daboia russelii russelii venom. The N-terminal sequence of Rusvikunin-II was found to be blocked, but peptide mass fingerprinting analysis indicated its identity as Kunitz-type basic protease inhibitor 2, previously reported from Russell's Viper venom. A tryptic peptide sequence of Rusvikunin-II containing the N-terminal sequence HDRPTFCNLFPESGR demonstrated significant sequence homology to venom basic protease inhibitors, Kunitz-type protease inhibitors and trypsin inhibitors. The secondary structure of Rusvikunin-II was dominated by β-sheets (60.4%), followed by random coil (38.2%), whereas α-helix (1.4%) contributes the least to its secondary structure. Both Rusvikunin-II and the Rusvikunin complex demonstrated dose-dependent anticoagulant activity; however, the anticoagulant potency of latter was found to be higher. Both inhibited the amidolytic activity of trypsin > plasmin > FXa, fibrinogen clotting activity of thrombin, and, to a lesser extent, the prothrombin activation property of FXa; however, the inhibitory effect of the Rusvikunin complex was more pronounced. Neither Rusvikunin-II nor Rusvikunin complex inhibited the amidolytic activity of chymotrypsin and thrombin. Rusvikunin-II at 10 μg/ml was not cytotoxic to Colo-205, MCF-7 or 3T3 cancer cells; conversely, Rusvikunin complex showed ∼30% reduction of MCF-7 cells under identical experimental conditions. Rusvikunin-II (5.0 mg/kg body weight, i.p. injection) was not lethal to mice or House Geckos; nevertheless, it showed in vivo anticoagulant action in mice. However, the Rusvikunin complex (at 5.0 mg/kg) was toxic to NSA mice, but not to House Geckos, suggesting it has prey-specific toxicity. Rusvikunin complex-treated mice exhibited dyspnea and hind-limb paresis prior to death. The present study indicates that the Kunitz

  15. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    PubMed

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.

  16. A conserved tryptophan (W91) at the barrel-lid junction modulates the packing and stability of Kunitz (STI) family of inhibitors.

    PubMed

    Majumder, Sudip; Khamrui, Susmita; Banerjee, Ramanuj; Bhowmik, Pallab; Sen, Udayaditya

    2015-01-01

    β-trefoil fold, consisting of a six stranded β-barrel capped at one end by a lid comprising of another six β-strands, is one of the most important folds among proteins. Important classes of proteins like Interleukins (ILs), Fibroblast Growth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Their core is packed by hydrophobic residues contributed by the 6 stranded β-barrel and three β-hairpins that make essential contacts with each other and keep the protein in 'topologically minimal frustrated state'. A complete database analysis of the core residues of the β-trefoil fold proteins presented here identified a conserved tryptophan (W91) residue in the Kunitz (STI) family of inhibitors that projects from the lid and interacts with the bottom layer residues of the barrel. This kind of interactions is unique in Kunitz (STI) family because no other families of β-trefoil fold have such a shear sized residue at the barrel lid junction; suggesting its possible importance in packing and stability. We took WCI as a representative of this family and prepared four cavity creating mutants W91F-WCI, W91M-WCI, W91I-WCI & W91A-WCI. CD experiments show that the secondary structure of the mutants remains indistinguishable with the wild type. Crystal structures of the mutants W91F-WCI, W91M-WCI & W91A-WCI also show the same feature. However, slight readjustments of the side chains around the site of mutation have been observed so as to minimize the cavity created due to mutation. Comparative stability of these mutants, estimated using heat denaturation CD spectroscopy, indicates that stability of the mutants inversely correlates with the size of the cavity inside the core. Interestingly, although we mutated at the core, mutants show varying susceptibility against tryptic digestion that grossly follow their instability determined by CD. Our findings suggest that the W91 residue plays an important role in determining the stability and packing of the

  17. Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin

    PubMed Central

    Tomar, Sakshi; Patil, Dipak N.; Datta, Manali; Tapas, Satya; Preeti; Chaudhary, Anshul; Sharma, Ashwani K.; Tomar, Shailly; Kumar, Pravindra

    2009-01-01

    The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS–PAGE. The crystals diffracted to 2.0 Å resolution and belonged to the tetragonal space group P41, with unit-cell parameters a = b = 57.1, c = 120.1 Å. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor–trypsin complex per asymmetric unit, with a solvent content of 45%. PMID:19923745

  18. In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus

    PubMed Central

    Assumpção, Teresa C.; Ma, Dongying; Mizurini, Daniella M.; Kini, R. Manjunatha; Ribeiro, José M. C.; Kotsyfakis, Michail; Monteiro, Robson Q.; Francischetti, Ivo M. B.

    2016-01-01

    Background Hematophagous mosquitos and ticks avoid host hemostatic system through expression of enzyme inhibitors targeting proteolytic reactions of the coagulation and complement cascades. While most inhibitors characterized to date were found in the salivary glands, relatively few others have been identified in the midgut. Among those, Boophilin is a 2-Kunitz multifunctional inhibitor targeting thrombin, elastase, and kallikrein. However, the kinetics of Boophilin interaction with these enzymes, how it modulates platelet function, and whether it inhibits thrombosis in vivo have not been determined. Methodology/Principal Findings Boophilin was expressed in HEK293 cells and purified to homogeneity. Using amidolytic assays and surface plasmon resonance experiments, we have demonstrated that Boophilin behaves as a classical, non-competitive inhibitor of thrombin with respect to small chromogenic substrates by a mechanism dependent on both exosite-1 and catalytic site. Inhibition is accompanied by blockade of platelet aggregation, fibrin formation, and clot-bound thrombin in vitro. Notably, we also identified Boophilin as a non-competitive inhibitor of FXIa, preventing FIX activation. In addition, Boophilin inhibits kallikrein activity and the reciprocal activation, indicating that it targets the contact pathway. Furthermore, Boophilin abrogates cathepsin G- and plasmin-induced platelet aggregation and partially affects elastase-mediated cleavage of Tissue Factor Pathway Inhibitor (TFPI). Finally, Boophilin inhibits carotid artery occlusion in vivo triggered by FeCl3, and promotes bleeding according to the mice tail transection method. Conclusion/Significance Through inhibition of several enzymes involved in proteolytic cascades and cell activation, Boophilin plays a major role in keeping the midgut microenvironment at low hemostatic and inflammatory tonus. This response allows ticks to successfully digest a blood meal which is critical for metabolism and egg

  19. Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products

    PubMed Central

    2011-01-01

    Background We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function. Results We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas. Conclusions The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in

  20. The kunitz protease inhibitor domain of protease nexin-2 inhibits factor XIa and murine carotid artery and middle cerebral artery thrombosis

    PubMed Central

    Wu, Wenman; Li, Hongbo; Navaneetham, Duraiswamy; Reichenbach, Zachary W.; Tuma, Ronald F.

    2012-01-01

    Coagulation factor XI (FXI) plays an important part in both venous and arterial thrombosis, rendering FXIa a potential target for the development of antithrombotic therapy. The kunitz protease inhibitor (KPI) domain of protease nexin-2 (PN2) is a potent, highly specific inhibitor of FXIa, suggesting its possible role in the inhibition of FXI-dependent thrombosis in vivo. Therefore, we examined the effect of PN2KPI on thrombosis in the murine carotid artery and the middle cerebral artery. Intravenous administration of PN2KPI prolonged the clotting time of both human and murine plasma, and PN2KPI inhibited FXIa activity in both human and murine plasma in vitro. The intravenous administration of PN2KPI into WT mice dramatically decreased the progress of FeCl3-induced thrombus formation in the carotid artery. After a similar initial rate of thrombus formation with and without PN2KPI treatment, the propagation of thrombus formation after 10 minutes and the amount of thrombus formed were significantly decreased in mice treated with PN2KPI injection compared with untreated mice. In the middle cerebral artery occlusion model, the volume and fraction of ischemic brain tissue were significantly decreased in PN2KPI-treated compared with untreated mice. Thus, inhibition of FXIa by PN2KPI is a promising approach to antithrombotic therapy. PMID:22674803

  1. The Kunitz-protease inhibitor domain in amyloid precursor protein reduces cellular mitochondrial enzymes expression and function.

    PubMed

    Chua, Li-Min; Lim, Mei-Li; Wong, Boon-Seng

    2013-08-09

    Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) and this can be contributed by aberrant metabolic enzyme function. But, the mechanism causing this enzymatic impairment is unclear. Amyloid precursor protein (APP) is known to be alternatively spliced to produce three major isoforms in the brain (APP695, APP751, APP770). Both APP770 and APP751 contain the Kunitz Protease Inhibitory (KPI) domain, but the former also contain an extra OX-2 domain. APP695 on the other hand, lacks both domains. In AD, up-regulation of the KPI-containing APP isoforms has been reported. But the functional contribution of this elevation is unclear. In the present study, we have expressed and compared the effect of the non-KPI containing APP695 and the KPI-containing APP751 on mitochondrial function. We found that the KPI-containing APP751 significantly decreased the expression of three major mitochondrial metabolic enzymes; citrate synthase, succinate dehydrogenase and cytochrome c oxidase (COX IV). This reduction lowers the NAD(+)/NADH ratio, COX IV activity and mitochondrial membrane potential. Overall, this study demonstrated that up-regulation of the KPI-containing APP isoforms is likely to contribute to the impairment of metabolic enzymes and mitochondrial function in AD.

  2. Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor.

    PubMed

    Van Nostrand, W E; Schmaier, A H; Siegel, R S; Wagner, S L; Raschke, W C

    1995-09-29

    The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.

  3. Transient removal of proflavine inhibition of bovine beta-trypsin by the bovine basic pancreatic trypsin inhibitor (Kunitz). A case for "chronosteric effects".

    PubMed

    Antonini, E; Ascenzi, P; Bolognesi, M; Menegatti, E; Guarneri, M

    1983-04-25

    The formation of the bovine beta-trypsin-bovine basic pancreatic trypsin inhibitor (Kunitz) (BPTI) complex was monitored, making use of three different signals: proflavine displacement, optical density changes in the ultraviolet region, and the loss of the catalytic activity. The rates of the reactions indicated by the three different signals were similar at neutral pH, but diverged at low pH. At pH 3.50, proflavine displacement precedes the optical density changes in the ultraviolet and the loss of enzyme activity by several orders of magnitude in time (Antonini, E., Ascenzi, P., Menegatti, E., and Guarneri, M. (1983) Biopolymers 22, 363-375). These data indicated that the bovine beta-trypsin-BPTI complex formation is a multistage process and led to the prediction that, at pH 3.50, BPTI addition to the bovine beta-trypsin-proflavine complex would remove proflavine inhibition and the enzyme would recover transiently its catalytic activity before being irreversibly inhibited by completion of BPTI binding. The kinetic evidences, by completion of BPTI binding. The kinetic evidences, here shown, verified this prediction, indicating that during the bovine beta-trypsin-BPTI complex formation one transient intermediate occurs, which is not able to bind proflavine but may bind and hydrolyze the substrate. Thus, the observed peculiar catalytic behavior is in line with the proposed reaction mechanism for the bovine beta-trypsin-BPTI complex formation, which postulates a sequence of distinct polar and apolar interactions at the contact area.

  4. Sequential NMR resonance assignment and structure determination of the Kunitz-type inhibitor domain of the Alzheimer's beta-amyloid precursor protein.

    PubMed

    Heald, S L; Tilton, R F; Hammond, L J; Lee, A; Bayney, R M; Kamarck, M E; Ramabhadran, T V; Dreyer, R N; Davis, G; Unterbeck, A

    1991-10-29

    Certain precursor proteins (APP751 and APP770) of the amyloid beta-protein (AP) present in Alzheimer's disease contain a Kunitz-type serine protease inhibitor domain (APPI). In this study, the domain is obtained as a functional inhibitor through both recombinant (APPIr) and synthetic (APPIs) methodologies, and the solution structure of APPI is determined by 1H 2D NMR techniques. Complete sequence-specific resonance assignments (except for P13 and G37 NH) for both APPIr and APPIs are achieved using standard procedures. Ambiguities arising from degeneracies in the NMR resonances are resolved by varying sample conditions. Qualitative interpretation of short- and long-range NOEs reveals secondary structural features similar to those extensively documented by NMR for bovine pancreatic trypsin inhibitor (BPTI). A more rigorous interpretation of the NOESY spectra yields NOE-derived interresidue distance restraints which are used in conjunction with dynamic simulated annealing to generate a family of APPI structures. Within this family, the beta-sheet and helical regions are in good agreement with the crystal structure of BPTI, whereas portions of the protease-binding loops deviate from those in BPTI. These deviations are consistent with those recently described in the crystal structure of APPI (Hynes et al., 1990). Also supported in the NMR study is the hydrophobic patch in the protease-binding domain created by side chain-side chain NOE contacts between M17 and F34. In addition, the NMR spectra indicate that the rotation of the W21 ring in APPI is hindered, unlike Y21 in BPTI, showing a greater than 90% preference for one orientation in the hydrophobic groove.

  5. Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

    PubMed Central

    Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

    2008-01-01

    After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. PMID:18689443

  6. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

    PubMed

    Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

    1999-01-29

    Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

  7. A Kunitz-type inhibitor of coleopteran proteases, isolated from Adenanthera pavonina L. seeds and its effect on Callosobruchus maculatus.

    PubMed

    Macedo, Maria Lígia Rodrigues; de Sá, Claudia Mara; Freire, Maria Das Graças Machado; Parra, José Roberto Postali

    2004-05-05

    The cowpea weevil Callosobruchus maculatus is one of the major pests of Vigna unguiculata cowpea. Digestion in the cowpea weevil is facilitated by high levels of cysteine and aspartic acid proteinases. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (ApTI) isolated from Adenanthera pavonina seeds showed activity against papain. The inhibition of papain by ApTI was of the noncompetitive type, with a K(i) of 1 microM. ApTI was highly effective against digestive proteinases from C. maculatus, Acanthoscelides obtectus (bean weevil), and Zabrotes subfasciatus (Mexican bean weevil) and was moderately active against midgut proteinases from the boll weevil Anthonomus grandis and the mealworm Tenebrio molitor. In C. maculates fed an artificial diet containing 0.25% and 0.5% ApTI (w/w), the latter concentration caused 50% mortality and reduced larval weight gain by approximately 40%. The action of ApTI on C. maculatus larvae may involve the inhibition of ApTI-sensitive cysteine proteinases and binding to chitin components of the peritrophic membrane (or equivalent structures) in the weevil midgut.

  8. Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor.

    PubMed

    Fernández, Erlinda; Toledo, Jorge R; Mansur, Manuel; Sánchez, Oliberto; Gil, Dayrom F; González-González, Yamile; Lamazares, Emilio; Fernández, Yaiza; Parra, Francisco; Farnós, Omar

    2015-05-01

    The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116 mg L(-1) of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation.

  9. Polar Desolvation and Position 226 of Pancreatic and Neutrophil Elastases Are Crucial to their Affinity for the Kunitz-Type Inhibitors ShPI-1 and ShPI-1/K13L

    PubMed Central

    Hernández González, Jorge Enrique; García-Fernández, Rossana; Valiente, Pedro Alberto

    2015-01-01

    The Kunitz-type protease inhibitor ShPI-1 inhibits human neutrophil elastase (HNE, Ki = 2.35·10−8 M) but does not interact with the porcine pancreatic elastase (PPE); whereas its P1 site variant, ShPI-1/K13L, inhibits both HNE and PPE (Ki = 1.3·10−9 M, and Ki = 1.2·10−8 M, respectively). By employing a combination of molecular modeling tools, e.g., structural alignment, molecular dynamics simulations and Molecular Mechanics Generalized-Born/Poisson-Boltzmann Surface Area free energy calculations, we showed that D226 of HNE plays a critical role in the interaction of this enzyme with ShPI-1 through the formation of a strong salt bridge and hydrogen bonds with K13 at the inhibitor’s P1 site, which compensate the unfavorable polar-desolvation penalty of the latter residue. Conversely, T226 of PPE is unable to establish strong interactions with K13, thereby precluding the insertion of K13 side-chain into the S1 subsite of this enzyme. An alternative conformation of K13 site-chain placed at the entrance of the S1 subsite of PPE, similar to that observed in the crystal structure of ShPI-1 in complex with chymotrypsin (PDB: 3T62), is also unfavorable due to the lack of stabilizing pair-wise interactions. In addition, our results suggest that the higher affinity of ShPI-1/K13L for both elastases mainly arises from the lower polar-desolvation penalty of L13 compared to that of K13, and not from stronger pair-wise interactions of the former residue with those of each enzyme. These results provide insights into the PPE and HNE inhibition and may contribute to the design of more potent and/or specific inhibitors toward one of these proteases. PMID:26372354

  10. Strong and widespread action of site-specific positive selection in the snake venom Kunitz/BPTI protein family

    PubMed Central

    Župunski, Vera; Kordiš, Dušan

    2016-01-01

    S1 family of serine peptidases is the largest family of peptidases. They are specifically inhibited by the Kunitz/BPTI inhibitors. Kunitz domain is characterized by the compact 3D structure with the most important inhibitory loops for the inhibition of S1 peptidases. In the present study we analysed the action of site-specific positive selection and its impact on the structurally and functionally important parts of the snake venom Kunitz/BPTI family of proteins. By using numerous models we demonstrated the presence of large numbers of site-specific positively selected sites that can reach between 30–50% of the Kunitz domain. The mapping of the positively selected sites on the 3D model of Kunitz/BPTI inhibitors has shown that these sites are located in the inhibitory loops 1 and 2, but also in the Kunitz scaffold. Amino acid replacements have been found exclusively on the surface, and the vast majority of replacements are causing the change of the charge. The consequence of these replacements is the change in the electrostatic potential on the surface of the Kunitz/BPTI proteins that may play an important role in the precise targeting of these inhibitors into the active site of S1 family of serine peptidases. PMID:27841308

  11. APP with Kunitz type protease inhibitor domain (KPI) correlates with neuritic plaque density but not with cortical synaptophysin immunoreactivity in Alzheimer's disease and non-demented aged subjects: a multifactorial analysis.

    PubMed

    Zhan, S S; Sandbrink, R; Beyreuther, K; Schmitt, H P

    1995-01-01

    The formation of beta A4 amyloid protein in neuritic plaques in Alzheimer's disease (AD) and advanced age is a complex process that involves a number of both cellular and molecular mechanisms, the interrelations of which are not yet completely understood. We have examined quantitatively, in AD and aged controls an extended spectrum of amyloid plaque-related cellular and molecular factors and the cortical synaptophysin immunoreactivity (synaptic density) in order to check for interrelations between them by multifactorial analysis. In 3 cases of senile dementia of the Alzheimer type (SDAT) aged 72, 80 and 82 years, and 9 controls aged 43-88 (mean age 65) years, the cortical synaptophysin immunoreactivity was assessed, together with the numbers of neurons, astrocytes and microglial cells, senile plaques, of tangle-bearing neurons, and the amount of beta A4 amyloid precursor protein (APP) with and without the Kunitz type serine protease inhibitor (KPI) domain. The main results were: APP including the KPI domain (KPI-APP) correlated with the number of neuritic plaques, regardless of whether they occurred in SDAT or non-demented controls. There was no significant difference in the amount of KPI-APP between SDAT and controls. Conversely, APP695 (without KPI) was significantly reduced in SDAT. KPI-APP did not correlate with the synaptophysin immunoreactivity (RGVA), while APP695 showed a significant correlation with the latter in all evaluations. It also correlated with the neuron counts, which was not true for KPI-APP. These results support previous findings indicating that KPI-APP is an important local factor for amyloid deposition in the neuritic plaques, both in AD and in non-demented aged people. On the contrary, KPI-APP does not seem to be significantly involved in the mechanisms of synaptic change outside of the plaques.

  12. Purification of a Kunitz-type inhibitor from Acacia polyphyllaDC seeds: characterization and insecticidal properties against Anagasta kuehniella Zeller (Lepidoptera: Pyralidae).

    PubMed

    Machado, Suzy Wider; de Oliveira, Caio Fernando Ramalho; Bezerra, Cezar da Silva; Freire, Maria das Graças Machado; Regina Kill, Marta; Machado, Olga Lima Tavares; Marangoni, Sergio; Macedo, Maria Ligia Rodrigues

    2013-03-13

    Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serine-protease inhibitor was isolated from Acacia polyphylla seeds (AcKI).Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ∼20 kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ∼50% with the lower concentration of AcKI used in the study (0.5%). Although additionalassays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.

  13. Discovery of a Distinct Superfamily of Kunitz-Type Toxin (KTT) from Tarantulas

    PubMed Central

    Diao, Jian-Bo; Jiang, Li-Ping; Tang, Xing; Liang, Song-Ping

    2008-01-01

    Background Kuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone. The main ancestral function of Kunitz-type proteins was the inhibition of a diverse array of serine proteases, while toxic activities (such as ion-channel blocking) were developed under a variety of Darwinian selection pressures. How new functions were grafted onto an old protein scaffold and what effect Darwinian selection pressures had on KTT evolution remains a puzzle. Principal Findings Here we report the presence of a new superfamily of KTTs in spiders (Tarantulas: Ornithoctonus huwena and Ornithoctonus hainana), which share low sequence similarity to known KTTs and is clustered in a distinct clade in the phylogenetic tree of KTT evolution. The representative molecule of spider KTTs, HWTX-XI, purified from the venom of O. huwena, is a bi-functional protein which is a very potent trypsin inhibitor (about 30-fold more strong than BPTI) as well as a weak Kv1.1 potassium channel blocker. Structural analysis of HWTX-XI in 3-D by NMR together with comparative function analysis of 18 expressed mutants of this toxin revealed two separate sites, corresponding to these two activities, located on the two ends of the cone-shape molecule of HWTX-XI. Comparison of non-synonymous/synonymous mutation ratios (ω) for each site in spider and snake KTTs, as well as PBTI like body Kunitz proteins revealed high Darwinian selection pressure on the binding sites for Kv channels and serine proteases in snake, while only on the proteases in spider and none detected in body proteins, suggesting different rates and patterns of evolution among them. The results also revealed a series of key events in the history of spider KTT evolution, including the formation of a novel KTT family (named sub-Kuntiz-type toxins) derived from the ancestral native KTTs with the loss of the second disulfide bridge accompanied by several dramatic sequence modifications. Conclusions

  14. Action of plant proteinase inhibitors on enzymes of physiopathological importance.

    PubMed

    Oliva, Maria Luiza V; Sampaio, Misako U

    2009-09-01

    Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

  15. Proteases of Stored Product Insects and Their Inhibition by Specific Protease Inhibitors from Soybeans and Wheat Grain

    DTIC Science & Technology

    1988-10-16

    Tenebria molitor MIDGUT PROTEASES; LOCUST CAECAL PROTEASES; BOWMAN-BIRK TRYPSIN-CHMOTRYPSIN INHIBITOR (SOYBEANS) CHICKPEAS TRYPSIN-CHYMOTRYPSIN...and Kunitz (STI) from soybeans, CI from chickpeas , chicken ovomucoid and turkey ovomucoid. It was Jnactivated by phenylemthvsulfonyl fluoride (PMSF...soybeans and Cl from chickpeas , by chicken ovomucoid and turkey overmucoid, as well as by the Kunitz (STI) soybean trypsin inhibitor that hardly

  16. The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

    PubMed

    Beckmann, Anna-Madeleine; Glebov, Konstantin; Walter, Jochen; Merkel, Olaf; Mangold, Martin; Schmidt, Frederike; Becker-Pauly, Christoph; Gütschow, Michael; Stirnberg, Marit

    2016-08-01

    Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ.

  17. Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail

    PubMed Central

    Dy, Catherine Y.; Buczek, Pawel; Imperial, Julita S.; Bulaj, Grzegorz; Horvath, Martin P.

    2006-01-01

    Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50–200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine–cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as α-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and α-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 310–β–β–α Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in α-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed the cost of losing a disulfide bond by augmenting and optimizing weaker yet nonetheless effective non-covalent interactions. PMID:16929098

  18. Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail.

    PubMed

    Dy, Catherine Y; Buczek, Pawel; Imperial, Julita S; Bulaj, Grzegorz; Horvath, Martin P

    2006-09-01

    Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50-200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine-cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as alpha-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and alpha-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 3(10)-beta-beta-alpha Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in alpha-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed the cost of losing a disulfide bond by augmenting and optimizing weaker yet nonetheless effective non-covalent interactions.

  19. Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail

    SciTech Connect

    Dy, Catherine Y.; Buczek, Pawel; Imperial, Julita S.; Bulaj, Grzegorz; Horvath, Martin P.

    2006-09-01

    Most Kunitz proteins like BPTI and α-dendrotoxin are stabilized by three disulfide bonds. The crystal structure shows how subtle repacking of non-covalent interactions may compensate for disulfide bond loss in a naturally occurring two-disulfide variant, conkunitzin-S1, the first discovered member of a new conotoxin family. Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50–200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine–cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as α-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and α-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 3{sub 10}–β–β–α Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in α-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed

  20. Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

    PubMed

    Kato, Nobutaka; Comer, Eamon; Sakata-Kato, Tomoyo; Sharma, Arvind; Sharma, Manmohan; Maetani, Micah; Bastien, Jessica; Brancucci, Nicolas M; Bittker, Joshua A; Corey, Victoria; Clarke, David; Derbyshire, Emily R; Dornan, Gillian L; Duffy, Sandra; Eckley, Sean; Itoe, Maurice A; Koolen, Karin M J; Lewis, Timothy A; Lui, Ping S; Lukens, Amanda K; Lund, Emily; March, Sandra; Meibalan, Elamaran; Meier, Bennett C; McPhail, Jacob A; Mitasev, Branko; Moss, Eli L; Sayes, Morgane; Van Gessel, Yvonne; Wawer, Mathias J; Yoshinaga, Takashi; Zeeman, Anne-Marie; Avery, Vicky M; Bhatia, Sangeeta N; Burke, John E; Catteruccia, Flaminia; Clardy, Jon C; Clemons, Paul A; Dechering, Koen J; Duvall, Jeremy R; Foley, Michael A; Gusovsky, Fabian; Kocken, Clemens H M; Marti, Matthias; Morningstar, Marshall L; Munoz, Benito; Neafsey, Daniel E; Sharma, Amit; Winzeler, Elizabeth A; Wirth, Dyann F; Scherer, Christina A; Schreiber, Stuart L

    2016-10-20

    Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

  1. Dynein Function and Protein Clearance Changes in Tumor Cells Induced by a Kunitz-Type Molecule, Amblyomin-X

    PubMed Central

    Pacheco, Mario T. F.; Berra, Carolina M.; Morais, Kátia L. P.; Sciani, Juliana M.; Branco, Vania G.; Bosch, Rosemary V.; Chudzinski-Tavassi, Ana M.

    2014-01-01

    Amblyomin-X is a Kunitz-type recombinant protein identified from the transcriptome of the salivary glands of the tick Amblyomma cajennense and has anti-coagulant and antitumoral activity. The supposed primary target of this molecule is the proteasome system. Herein, we elucidated intracellular events that are triggered by Amblyomin-X treatment in an attempt to provide new insight into how this serine protease inhibitor, acting on the proteasome, could be comparable with known proteasome inhibitors. The collective results showed aggresome formation after proteasome inhibition that appeared to occur via the non-exclusive ubiquitin pathway. Additionally, Amblyomin-X increased the expression of various chains of the molecular motor dynein in tumor cells, modulated specific ubiquitin linkage signaling and inhibited autophagy activation by modulating mTOR, LC3 and AMBRA1 with probable dynein involvement. Interestingly, one possible role for dynein in the mechanism of action of Amblyomin-X was in the apoptotic response and its crosstalk with autophagy, which involved the factor Bim; however, we observed no changes in the apoptotic response related to dynein in the experiments performed. The characteristics shared among Amblyomin-X and known proteasome inhibitors included NF-κB blockage and nascent polypeptide-dependent aggresome formation. Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules. In investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects. These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition. PMID:25479096

  2. Distinct folding pathways of two homologous disulfide proteins: bovine pancreatic trypsin inhibitor and tick anticoagulant peptide.

    PubMed

    Chang, Jui-Yoa

    2011-01-01

    The folding pathways of disulfide proteins vary substantially (Arolas et al., Trends Biochem Sci 31: 292-301, 2006). The diversity is mainly manifested by (a) the extent of heterogeneity of folding intermediates, (b) the extent of presence of native-like intermediates, and (c) the variation of folding kinetics. Even among structurally similar proteins, the difference can be enormous. This is demonstrated in this concise review with two structurally homologous kunitz-type protease inhibitors, bovine pancreatic trypsin inhibitor and tick anticoagulant peptide, as well as a group of cystine knot proteins. The diversity of their folding mechanisms is illustrated with two different folding techniques: (a) the conventional method of disulfide oxidation (oxidative folding), and (b) the novel method of disulfide scrambling (Chang, J Biol Chem 277: 120-126, 2002). This review also highlights the convergence of folding models concluded form the conventional conformational folding and those obtained by oxidative folding.

  3. The Kunitz-Type Protein ShPI-1 Inhibits Serine Proteases and Voltage-Gated Potassium Channels

    PubMed Central

    García-Fernández, Rossana; Peigneur, Steve; Pons, Tirso; Alvarez, Carlos; González, Lidice; Chávez, María A.; Tytgat, Jan

    2016-01-01

    The bovine pancreatic trypsin inhibitor (BPTI)-Kunitz-type protein ShPI-1 (UniProt: P31713) is the major protease inhibitor from the sea anemone Stichodactyla helianthus. This molecule is used in biotechnology and has biomedical potential related to its anti-parasitic effect. A pseudo wild-type variant, rShPI-1A, with additional residues at the N- and C-terminal, has a similar three-dimensional structure and comparable trypsin inhibition strength. Further insights into the structure-function relationship of rShPI-1A are required in order to obtain a better understanding of the mechanism of action of this sea anemone peptide. Using enzyme kinetics, we now investigated its activity against other serine proteases. Considering previous reports of bifunctional Kunitz-type proteins from anemones, we also studied the effect of rShPI-1A on voltage-gated potassium (Kv) channels. rShPI-1A binds Kv1.1, Kv1.2, and Kv1.6 channels with IC50 values in the nM range. Hence, ShPI-1 is the first member of the sea anemone type 2 potassium channel toxins family with tight-binding potency against several proteases and different Kv1 channels. In depth sequence analysis and structural comparison of ShPI-1 with similar protease inhibitors and Kv channel toxins showed apparent non-sequence conservation for known key residues. However, we detected two subtle patterns of coordinated amino acid substitutions flanking the conserved cysteine residues at the N- and C-terminal ends. PMID:27089366

  4. Growing location has a pronounced effect on the accumulation of cancer chemopreventive agent Bowman-Birk inhibitor in soybean seeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybeans contain several health promoting compounds including phytosterols, isoflavones, phytic acid, and protease inhibitors. The two abundant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor and the Bowman-Birk inhibitor (BBI). BBI is a serine protease inhibitor that can inhi...

  5. Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

    NASA Astrophysics Data System (ADS)

    Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-04-01

    Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.

  6. Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

    PubMed Central

    Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

    2016-01-01

    Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors. PMID:27125351

  7. MmpL3 Inhibitors: Diverse Chemical Scaffolds Inhibit the Same Target.

    PubMed

    Poce, Giovanna; Consalvi, Sara; Biava, Mariangela

    2016-01-01

    MmpL3 belongs to the Resistance, Nodulation and Division (RND) superfamily whose role in mycobacteria is the formation of the outer membrane. Indeed, it has been shown that MmpL3 is associated with the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or the outer membrane. In the last few years several whole cell-based screenings of compound libraries brought by a number of diverse chemical scaffolds active against M. tuberculosis (Mtb) that surprisingly share MmpL3 as target. The diverse identified pharmacophores owe important differences among each other, in fact while some of them display inhibitory activity against pathogens that are devoid of mycolic acids and are active against non-replicating Mtb bacilli, some others specifically target mycobacteria and do not kill non-replicating bacilli. The scope of this review is to provide the recent advances in MmpL3 inhibitor discovery with a special focus on structure activity relationship (SAR) studies in order to provide information that could help in developing novel membrane-active anti- TB agents. Moreover, this review will provide the most recent insights into the modes of action of the MmpL3 inhibitors.

  8. Kunitz-Type Peptide HCRG21 from the Sea Anemone Heteractis crispa Is a Full Antagonist of the TRPV1 Receptor

    PubMed Central

    Monastyrnaya, Margarita; Peigneur, Steve; Zelepuga, Elena; Sintsova, Oksana; Gladkikh, Irina; Leychenko, Elena; Isaeva, Marina; Tytgat, Jan; Kozlovskaya, Emma

    2016-01-01

    Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides. PMID:27983679

  9. Discovery of structurally-diverse inhibitor scaffolds by high-throughput screening of a fragment library with dimethylarginine dimethylaminohydrolase.

    PubMed

    Linsky, Thomas W; Fast, Walter

    2012-09-15

    Potent and selective inhibitors of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) are useful as molecular probes to better understand cellular regulation of nitric oxide. Inhibitors are also potential therapeutic agents for treatment of pathological states associated with the inappropriate overproduction of nitric oxide, such as septic shock, selected types of cancer, and other conditions. Inhibitors with structures dissimilar to substrate may overcome limitations inherent to substrate analogs. Therefore, to identify structurally-diverse inhibitor scaffolds, high-throughput screening (HTS) of a 4000-member library of fragment-sized molecules was completed using the Pseudomonas aeruginosa DDAH and human DDAH-1 isoforms. Use of a substrate concentration equal to its K(M) value during the primary screen allowed for the detection of inhibitors with different modes of inhibition. A series of validation tests were designed and implemented in the identification of four inhibitors of human DDAH-1 that were unknown prior to the screen. Two inhibitors share a 4-halopyridine scaffold and act as quiescent affinity labels that selectively and covalently modify the active-site Cys residue. Two inhibitors are benzimidazole-like compounds that reversibly and competitively inhibit human DDAH-1 with Ligand Efficiency values ≥0.3 kcal/mol/heavy (non-hydrogen) atom, indicating their suitability for further development. Both inhibitor scaffolds have available sites to derivatize for further optimization. Therefore, use of this fragment-based HTS approach is demonstrated to successfully identify two novel scaffolds for development of DDAH-1 inhibitors.

  10. Discovery of diverse human dihydroorotate dehydrogenase inhibitors as immunosuppressive agents by structure-based virtual screening.

    PubMed

    Diao, Yanyan; Lu, Weiqiang; Jin, Huangtao; Zhu, Junsheng; Han, Le; Xu, Minghao; Gao, Rui; Shen, Xu; Zhao, Zhenjiang; Liu, Xiaofeng; Xu, Yufang; Huang, Jin; Li, Honglin

    2012-10-11

    This study applied an efficient virtual screening strategy integrating molecular docking with MM-GBSA rescoring to identify diverse human dihydroorotate dehydrogenase (hDHODH) inhibitors. Eighteen compounds with IC(50) values ranging from 0.11 to 18.8 μM were identified as novel hDHODH inhibitors that exhibited overall species-selectivity over Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH). Compound 8, the most potent one, showed low micromolar inhibitory activity against hDHODH with an IC(50) value of 0.11 μM. Moreover, lipopolysaccharide-induced B-cell assay and mixed lymphocyte reaction assay revealed that most of the hits showed potent antiproliferative activity against B and T cells, which demonstrates their potential application as immunosuppressive agents. In particular, compound 18 exhibited potent B-cell inhibitory activity (IC(50) = 1.78 μM) and presents a B-cell-specific profile with 17- and 26-fold selectivities toward T and Jurkat cells, respectively.

  11. Understanding the evolutionary structural variability and target specificity of tick salivary Kunitz peptides using next generation transcriptome data

    PubMed Central

    2014-01-01

    Background Ticks are blood-sucking arthropods and a primary function of tick salivary proteins is to counteract the host’s immune response. Tick salivary Kunitz-domain proteins perform multiple functions within the feeding lesion and have been classified as venoms; thereby, constituting them as one of the important elements in the arms race with the host. The two main mechanisms advocated to explain the functional heterogeneity of tick salivary Kunitz-domain proteins are gene sharing and gene duplication. Both do not, however, elucidate the evolution of the Kunitz family in ticks from a structural dynamic point of view. The Red Queen hypothesis offers a fruitful theoretical framework to give a dynamic explanation for host-parasite interactions. Using the recent salivary gland Ixodes ricinus transcriptome we analyze, for the first time, single Kunitz-domain encoding transcripts by means of computational, structural bioinformatics and phylogenetic approaches to improve our understanding of the structural evolution of this important multigenic protein family. Results Organizing the I. ricinus single Kunitz-domain peptides based on their cysteine motif allowed us to specify a putative target and to relate this target specificity to Illumina transcript reads during tick feeding. We observe that several of these Kunitz peptide groups vary in their translated amino acid sequence, secondary structure, antigenicity, and intrinsic disorder, and that the majority of these groups are subject to a purifying (negative) selection. We finalize by describing the evolution and emergence of these Kunitz peptides. The overall interpretation of our analyses discloses a rapidly emerging Kunitz group with a distinct disulfide bond pattern from the I. ricinus salivary gland transcriptome. Conclusions We propose a model to explain the structural and functional evolution of tick salivary Kunitz peptides that we call target-oriented evolution. Our study reveals that combining analytical

  12. Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).

    PubMed

    Venkatraman, Janani; Bhat, Jyothi; Solapure, Suresh M; Sandesh, Jatheendranath; Sarkar, Debasmita; Aishwarya, Sundaram; Mukherjee, Kakoli; Datta, Santanu; Malolanarasimhan, Krishnan; Bandodkar, Balachandra; Das, Kaveri S

    2012-03-01

    The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.

  13. Using Trypsin & Soybean Trypsin Inhibitor to Teach Principles of Enzyme Kinetics

    ERIC Educational Resources Information Center

    Howard, David R.; Herr, Julie; Hollister, Rhiannon

    2006-01-01

    Trypsin and soybean trypsin inhibitor (Kunitz inhibitor) can be used in a relatively simple and inexpensive student exercise to demonstrate the usefulness of enzyme kinetics. The study of enzyme kinetics is essential to biology because enzymes play such a crucial role in the biochemical pathways of all living organisms. The data from enzyme…

  14. Evaluation of a diverse set of potential P1 carboxylic acid bioisosteres in hepatitis C virus NS3 protease inhibitors.

    PubMed

    Rönn, Robert; Gossas, Thomas; Sabnis, Yogesh A; Daoud, Hanna; Kerblom, Eva; Danielson, U Helena; Sandström, Anja

    2007-06-15

    There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P(1) C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.

  15. Acetyl-lysine Binding Site of Bromodomain-Containing Protein 4 (BRD4) Interacts with Diverse Kinase Inhibitors

    PubMed Central

    2014-01-01

    Members of the bromodomain and extra terminal (BET) family of proteins are essential for the recognition of acetylated lysine (KAc) residues in histones and have emerged as promising drug targets in cancer, inflammation, and contraception research. In co-crystallization screening campaigns using the first bromodomain of BRD4 (BRD4-1) against kinase inhibitor libraries, we identified and characterized 14 kinase inhibitors (10 distinct chemical scaffolds) as ligands of the KAc binding site. Among these, the PLK1 inhibitor BI2536 and the JAK2 inhibitor TG101209 displayed strongest inhibitory potential against BRD4 (IC50 = 25 nM and 130 nM, respectively) and high selectivity for BET bromodomains. Comparative structural analysis revealed markedly different binding modes of kinase hinge-binding scaffolds in the KAc binding site, suggesting that BET proteins are potential off-targets of diverse kinase inhibitors. Combined, these findings provide a new structural framework for the rational design of next-generation BET-selective and dual-activity BET-kinase inhibitors. PMID:24568369

  16. [1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains.

    PubMed

    Fedorov, Oleg; Lingard, Hannah; Wells, Chris; Monteiro, Octovia P; Picaud, Sarah; Keates, Tracy; Yapp, Clarence; Philpott, Martin; Martin, Sarah J; Felletar, Ildiko; Marsden, Brian D; Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan; Brennan, Paul E

    2014-01-23

    Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology.

  17. [1,2,4]Triazolo[4,3-a]phthalazines: Inhibitors of Diverse Bromodomains

    PubMed Central

    2013-01-01

    Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology. PMID:24313754

  18. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    SciTech Connect

    Tamm, Christoffer Galito, Sara Pijuan Anneren, Cecilia

    2012-02-15

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  19. Purification and characterization of a chymotrypsin inhibitor from the venom of Ophiophagus hannah (King Cobra).

    PubMed

    Chang, L; Chung, C; Huang, H B; Lin, S

    2001-05-18

    A chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 microM) with chymotrypsin. Phylogenetic analysis suggests that the functional variations of the chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.

  20. Progress in HIV-1 Integrase Inhibitors: A Review of their Chemical Structure Diversity

    PubMed Central

    Hajimahdi, Zahra; Zarghi, Afshin

    2016-01-01

    HIV-1 integrase (IN) enzyme, one of the three main enzymes of HIV-1, catalyzed the insertion of the viral DNA into the genome of host cells. Because of the lack of its homologue in human cells and its essential role in HIV-1 replication, IN inhibition represents an attractive therapeutic target for HIV-1 treatment. Since identification of IN as a promising therapeutic target, a major progress has been made, which has facilitated and led to the approval of three drugs. This review focused on the structural features of the most important IN inhibitors and categorized them structurally in 10 scaffolds. We also briefly discussed the structural and functional properties of HIV-1 IN and binding modes of IN inhibitors. The SAR analysis of the known IN inhibitors provides some useful clues to the possible future discovery of novel IN inhibitors. PMID:28243261

  1. Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors*

    PubMed Central

    Møller, Marie S.; Vester-Christensen, Malene B.; Jensen, Johanne M.; Hachem, Maher Abou; Henriksen, Anette; Svensson, Birte

    2015-01-01

    Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs. PMID:25792743

  2. Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors

    PubMed Central

    Brannigan, James A.; Roberts, Shirley M.; Bell, Andrew S.; Hutton, Jennie A.; Hodgkinson, Michael R.; Tate, Edward W.; Leatherbarrow, Robin J.; Smith, Deborah F.; Wilkinson, Anthony J.

    2014-01-01

    The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed. PMID:25075346

  3. A novel serine protease inhibitor from Bungarus fasciatus venom.

    PubMed

    Lu, Jia; Yang, Hailong; Yu, Haining; Gao, Weikai; Lai, Ren; Liu, Jingze; Liang, Xingcai

    2008-03-01

    By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.

  4. Demethoxycurcumin Is A Potent Inhibitor of P-Type ATPases from Diverse Kingdoms of Life

    PubMed Central

    Dao, Trong Tuan; Sehgal, Pankaj; Tung, Truong Thanh; Møller, Jesper Vuust; Nielsen, John; Palmgren, Michael; Christensen, Søren Brøgger

    2016-01-01

    P-type ATPases catalyze the active transport of cations and phospholipids across biological membranes. Members of this large family are involved in a range of fundamental cellular processes. To date, a substantial number of P-type ATPase inhibitors have been characterized, some of which are used as drugs. In this work a library of natural compounds was screened and we first identified curcuminoids as plasma membrane H+-ATPases inhibitors in plant and fungal cells. We also found that some of the commercial curcumins contain several curcuminoids. Three of these were purified and, among the curcuminoids, demethoxycurcumin was the most potent inhibitor of all tested P-type ATPases from fungal (Pma1p; H+-ATPase), plant (AHA2; H+-ATPase) and animal (SERCA; Ca2+-ATPase) cells. All three curcuminoids acted as non-competitive antagonist to ATP and hence may bind to a highly conserved allosteric site of these pumps. Future research on biological effects of commercial preparations of curcumin should consider the heterogeneity of the material. PMID:27644036

  5. Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

    PubMed Central

    Reddy, B. K. Kishore; Landge, Sudhir; Ravishankar, Sudha; Patil, Vikas; Shinde, Vikas; Tantry, Subramanyam; Kale, Manoj; Raichurkar, Anandkumar; Menasinakai, Sreenivasaiah; Mudugal, Naina Vinay; Ambady, Anisha; Ghosh, Anirban; Tunduguru, Ragadeepthi; Kaur, Parvinder; Singh, Ragini; Kumar, Naveen; Bharath, Sowmya; Sundaram, Aishwarya; Bhat, Jyothi; Sambandamurthy, Vasan K.; Björkelid, Christofer; Jones, T. Alwyn; Das, Kaveri; Bandodkar, Balachandra; Malolanarasimhan, Krishnan; Mukherjee, Kakoli

    2014-01-01

    Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis. PMID:24687493

  6. The tissue inhibitors of metalloproteinases (TIMPs): An ancient family with structural and functional diversity

    PubMed Central

    Brew, Keith; Nagase, Hideaki

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are widely distributed in the animal kingdom and the human genome contains four paralogous genes encoding TIMPs 1 to 4. TIMPs were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has now been found to be broader as it includes the inhibition of several of the disintegrin-metalloproteinases, ADAMs and ADAMTSs. TIMPs are therefore key regulators of the metalloproteinases that degrade the extracellular matrix and shed cell surface molecules. Structural studies of TIMP–MMP complexes have elucidated the inhibition mechanism of TIMPs and the multiple sites through which they interact with target enzymes, allowing the generation of TIMP variants that selectively inhibit different groups of metalloproteinases. Engineering such variants is complicated by the fact that TIMPs can undergo changes in molecular dynamics induced by their interactions with proteases. TIMPs also have biological activities that are independent of metalloproteinases; these include effects on cell growth and differentiation, cell migration, anti-angiogenesis, anti- and pro-apoptosis, and synaptic plasticity. Receptors responsible for some of these activities have been identified and their signaling pathways have been investigated. A series of studies using mice with specific TIMP gene deletions has illuminated the importance of these molecules in biology and pathology. PMID:20080133

  7. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    PubMed Central

    Dhonukshe, Pankaj; Grigoriev, Ilya; Fischer, Rainer; Tominaga, Motoki; Robinson, David G.; Hašek, Jiří; Paciorek, Tomasz; Petrášek, Jan; Seifertová, Daniela; Tejos, Ricardo; Meisel, Lee A.; Zažímalová, Eva; Gadella, Theodorus W. J.; Stierhof, York-Dieter; Ueda, Takashi; Oiwa, Kazuhiro; Akhmanova, Anna; Brock, Roland; Spang, Anne; Friml, Jiří

    2008-01-01

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role. PMID:18337510

  8. Bifunctional apoptosis inhibitor (BAR) protects neurons from diverse cell death pathways.

    PubMed

    Roth, W; Kermer, P; Krajewska, M; Welsh, K; Davis, S; Krajewski, S; Reed, J C

    2003-10-01

    The bifunctional apoptosis regulator (BAR) is a multidomain protein that was originally identified as an inhibitor of Bax-induced apoptosis. Immunoblot analysis of normal human tissues demonstrated high BAR expression in the brain, compared to low or absent expression in other organs. Immunohistochemical staining of human adult tissues revealed that the BAR protein is predominantly expressed by neurons in the central nervous system. Immunofluorescence microscopy indicated that BAR localizes mainly to the endoplasmic reticulum (ER) of cells. Overexpression of BAR in CSM 14.1 neuronal cells resulted in significant protection from a broad range of cell death stimuli, including agents that activate apoptotic pathways involving mitochondria, TNF-family death receptors, and ER stress. Downregulation of BAR by antisense oligonucleotides sensitized neuronal cells to induction of apoptosis. Moreover, the search for novel interaction partners of BAR identified several candidate proteins that might contribute to the regulation of neuronal apoptosis (HIP1, Hippi, and Bap31). Taken together, the expression pattern and functional data suggest that the BAR protein is involved in the regulation of neuronal survival.

  9. Identification of a Pyridopyrimidinone Inhibitor of Orthopoxviruses from a Diversity-Oriented Synthesis Library

    PubMed Central

    Dower, Ken; Filone, Claire Marie; Hodges, Erin N.; Bjornson, Zach B.; Rubins, Kathleen H.; Brown, Lauren E.; Schaus, Scott; Hensley, Lisa E.

    2012-01-01

    Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals. PMID:22205744

  10. Elucidation of crystal form diversity of the HIV protease inhibitor ritonavir by high-throughput crystallization

    PubMed Central

    Morissette, Sherry L.; Soukasene, Stephen; Levinson, Douglas; Cima, Michael J.; Almarsson, Örn

    2003-01-01

    Pharmaceutical compounds are molecular solids that frequently exhibit polymorphism of crystal form. One high profile case of polymorphism was ritonavir, a peptidomimetic drug used to treat HIV-1 infection and introduced in 1996. In 1998, a lower energy, more stable polymorph (form II) appeared, causing slowed dissolution of the marketed dosage form and compromising the oral bioavailability of the drug. This event forced the removal of the oral capsule formulation from the market. We have carried out high-throughput crystallization experiments to comprehensively explore ritonavir form diversity. A total of five forms were found: both known forms and three previously unknown forms. The novel forms include a metastable polymorph, a hydrate phase, and a formamide solvate. The solvate was converted to form I via the hydrate phase by using a simple washing procedure, providing an unusual route to prepare the form I “disappearing polymorph” [Dunitz, J. D. & Bernstein, J. (1995) Acc. Chem. Res. 28, 193–200]. Crystals of form I prepared by using this method retained the small needle morphology of the solvate and thus offer a potential strategy for particle size and morphology control. PMID:12604798

  11. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    PubMed Central

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; Norton, Malgorzata G.; Teckman, Jeffrey H.; Marszal, Ewa; Osmulski, Pawel A.

    2016-01-01

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo

  12. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    DOE PAGES

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; ...

    2016-03-23

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemicalmore » data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo

  13. AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor

    SciTech Connect

    Gaczynska, Maria; Karpowicz, Przemyslaw; Stuart, Christine E.; Norton, Malgorzata G.; Teckman, Jeffrey H.; Marszal, Ewa; Osmulski, Pawel A.

    2016-03-23

    α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found inWT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in

  14. Structure based virtual screening of MDPI database: discovery of structurally diverse and novel DPP-IV inhibitors.

    PubMed

    Tanwar, Omprakash; Tanwar, Lalima; Shaquiquzzaman, Md; Alam, Md Mumtaz; Akhter, Mymoona

    2014-08-01

    Inhibition of dipeptidyl peptidase IV (DPP-IV) has been emerged as a promising approach for the treatment of type 2 diabetes (T2D). Structure based virtual screening (SBVS) of Molecular Diversity Preservation International (MDPI) database was performed using Glide and Gold against DPP-IV enzyme. Six promising hits were identified and tested for DPP-IV inhibition. Three compounds were found to be active at low micromolar concentration. The 3-(1-hydrazinyl-1-(phenylamino)ethyl)-4-hydroxy-1-methylquinolin-2(1H)-one (compound A) was found to be the most potent hit with an IC50 of 0.73 μM. These three compounds (A, B and D) were then assessed for their glucose lowering effects in glucose fed hyperglycemic female Wistar rats. The glucose lowering effects of compounds also confirms their potential as anti-diabetic agents. The present study demonstrates a successful utilization of in silico SBVS tools in identification of novel and potential DPP-IV inhibitor.

  15. Post-translational modification and conformational state of Heat Shock Protein 90 differentially affect binding of chemically diverse small molecule inhibitors

    PubMed Central

    Beebe, Kristin; Mollapour, Mehdi; Scroggins, Bradley; Prodromou, Chrisostomos; Xu, Wanping; Tokita, Mari; Taldone, Tony; Pullen, Lester; Zierer, Bettina K.; Lee, Min-Jung; Trepel, Jane; Buchner, Johannes; Bolon, Daniel; Chiosis, Gabriela; Neckers, Leonard

    2013-01-01

    Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic. PMID:23867252

  16. Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED

    PubMed Central

    Zhang, Man; Zhao, Mengxi; Feng, Lijian; Luo, Xiao; Gao, Zhenting; Huang, Ying; Ardayfio, Ophelia; Zhang, Ji-Hu; Lin, Ying; Fan, Hong; Mi, Yuan; Li, Guobin; Liu, Lei; Feng, Leying; Luo, Fangjun; Teng, Lin; Qi, Wei; Ottl, Johannes; Lingel, Andreas; Bussiere, Dirksen E.; Yu, Zhengtian; Atadja, Peter; Lu, Chris; Li, En; Gu, Justin; Zhao, Kehao

    2017-01-01

    Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity. PMID:28072869

  17. Fasciola hepatica Kunitz type molecule decreases dendritic cell activation and their ability to induce inflammatory responses.

    PubMed

    Falcón, Cristian R; Masih, Diana; Gatti, Gerardo; Sanchez, María Cecilia; Motrán, Claudia C; Cervi, Laura

    2014-01-01

    The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite.

  18. Fasciola hepatica Kunitz Type Molecule Decreases Dendritic Cell Activation and Their Ability to Induce Inflammatory Responses

    PubMed Central

    Falcón, Cristian R.; Masih, Diana; Gatti, Gerardo; Sanchez, María Cecilia; Motrán, Claudia C.; Cervi, Laura

    2014-01-01

    The complete repertoire of proteins with immunomodulatory activity in Fasciola hepatica (Fh) has not yet been fully described. Here, we demonstrated that Fh total extract (TE) reduced LPS-induced DC maturation, and the DC ability to induce allogeneic responses. After TE fractionating, a fraction lower than 10 kDa (F<10 kDa) was able to maintain the TE properties to modulate the DC pro- and anti-inflammatory cytokine production induced by LPS. In addition, TE or F<10 kDa treatment decreased the ability of immature DC to stimulate the allogeneic responses and induced a novo allogeneic CD4+CD25+Foxp3+ T cells. In contrast, treatment of DC with T/L or F<10 kDa plus LPS (F<10/L) induced a regulatory IL-27 dependent mechanism that diminished the proliferative and Th1 and Th17 allogeneic responses. Finally, we showed that a Kunitz type molecule (Fh-KTM), present in F<10 kDa, was responsible for suppressing pro-inflammatory cytokine production in LPS-activated DC, by printing tolerogenic features on DC that impaired their ability to induce inflammatory responses. These results suggest a modulatory role for this protein, which may be involved in the immune evasion mechanisms of the parasite. PMID:25486609

  19. In silico screening reveals structurally diverse, nanomolar inhibitors of NQO2 that are functionally active in cells and can modulate NFκB signalling

    PubMed Central

    Nolan, Karen A.; Dunstan, Mark S.; Caraher, Mary C.; Scott, Katherine A.; Leys, David; Stratford, Ian J.

    2011-01-01

    The NCI chemical database has been screened using in silico docking to identify novel nanomolar inhibitors of NRH:quinone oxidoreductase 2 (NQO2). The inhibitors identified from the screen exhibit a diverse range of scaffolds and the structure of one of the inhibitors, NSC13000 co-crystalized with NQO2, has been solved. This has been used to aid the generation of a structure/activity relationship between the computationally derived binding affinity and experimentally measured enzyme inhibitory potency. Many of the compounds are functionally active as inhibitors of NQO2 in cells at non toxic concentrations. To demonstrate this, advantage was taken of the NQO2-mediated toxicity of the chemotherapeutic drug CB1954. The toxicity of this drug is substantially reduced when the function of NQO2 is inhibited and many of the compounds achieve this in cells at nanomolar concentrations. The NQO2 inhibitors also attenuated TNFα-mediated, NFκB-driven transcriptional activity. The link between NQO2 and the regulation of NFκB was confirmed by using siRNA to NQO2 and by the observation that NRH, the cofactor for NQO2 enzyme activity, could regulate NFκB activity in an NQO2 dependent manner. NFκB is a potential therapeutic target and this study reveals an underlying mechanism that may exploitable for developing new anti-cancer drugs. PMID:22090421

  20. Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

    PubMed

    He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

    2008-10-01

    Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

  1. Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.

    PubMed Central

    Scheidig, A. J.; Hynes, T. R.; Pelletier, L. A.; Wells, J. A.; Kossiakoff, A. A.

    1997-01-01

    The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites

  2. The amino acid sequence of a weak trypsin inhibitor B from Dendroaspis Polylepis polylepis (black mamba) venom.

    PubMed

    Strydom, D J; Joubert, F J

    1981-10-01

    The sequence of protein B, a weak trypsin inhibitor from black mamba venom was determined. The sequence differs much from other proteinase inhibitors of snake venom, bovine pancreas, snail and turtle egg. The phylogenetic relationship of B and its homologues, the basic pancreatic trypsin inhibitor (Kunitz-type group, was investigated. The elapid snake proteins are grouped on a separate branch from the turtle egg - bovine - snail group, the viper inhibitor and the B-chain of beta-bungarotoxin each being a unique position.

  3. In vivo sequence diversity of the protease of human immunodeficiency virus type 1: presence of protease inhibitor-resistant variants in untreated subjects.

    PubMed Central

    Lech, W J; Wang, G; Yang, Y L; Chee, Y; Dorman, K; McCrae, D; Lazzeroni, L C; Erickson, J W; Sinsheimer, J S; Kaplan, A H

    1996-01-01

    We have evaluated the sequence diversity of the protease human immunodeficiency virus type 1 in vivo. Our analysis of 246 protease coding domain sequences obtained from 12 subjects indicates that amino acid substitutions predicted to give rise to protease inhibitor resistance may be present in patients who have not received protease inhibitors. In addition, we demonstrated that amino acid residues directly involved in enzyme-substrate interactions may be varied in infected individuals. Several of these substitutions occurred in combination either more or less frequently than would be expected if their appearance was independent, suggesting that one substitution may compensate for the effects of another. Taken together, our analysis indicates that the human immunodeficiency virus type 1 protease has flexibility sufficient to vary critical subsites in vivo, thereby retaining enzyme function and viral pathogenicity. PMID:8627733

  4. Purification and characterization of a trypsin-papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests.

    PubMed

    Oliveira, Adeliana S; Migliolo, Ludovico; Aquino, Rodrigo O; Ribeiro, Jannison K C; Macedo, Leonardo L P; Andrade, Lucia B S; Bemquerer, Marcelo P; Santos, Elizeu A; Kiyota, Sumika; de Sales, Maurício P

    2007-01-01

    A novel trypsin-papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin-Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an M(r) of 18.1 kDa as determined by SDS-PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2-10, temperature of 50 degrees C and had a K(i) value of 1.65 x 10(-8)M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and K(i) value of 5.1 x 10(-7)M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.

  5. Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    Ji, Haitao; Stanton, Benjamin Z.; Igarashi, Jotaro; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early “ADME/Tox” considerations and provides a basic platform for medicinal chemistry-driven efforts. PMID:18321097

  6. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

    PubMed

    Göckeritz, Elisa; Kerwien, Susan; Baumann, Michael; Wigger, Marion; Vondey, Verena; Neumann, Lars; Landwehr, Thomas; Wendtner, Clemens M; Klein, Christian; Liu, Ningshu; Hallek, Michael; Frenzel, Lukas P; Krause, Günter

    2015-11-01

    Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

  7. Stepwise development of structure-activity relationship of diverse PARP-1 inhibitors through comparative and validated in silico modeling techniques and molecular dynamics simulation.

    PubMed

    Halder, Amit K; Saha, Achintya; Saha, Krishna Das; Jha, Tarun

    2015-01-01

    Inhibitors of poly (ADP-ribose) polymerase-1 (PARP-1) enzyme are useful for the treatment of various diseases including cancer. Comparative in silico studies were performed on different ligand-based (2D-QSAR, Kernel-based partial least square (KPLS) analysis, Pharmacophore Search Engine (PHASE) pharmacophore mapping), and structure-based (molecular docking, MM-GBSA analyses, Gaussian-based 3D-QSAR analyses on docked poses) modeling techniques to explore the structure-activity relationship of a diverse set of PARP-1 inhibitors. Two-dimensional (2D)-QSAR highlighted the importance of charge topological index (JGI7), fractional polar surface area (JursFPSA3), and connectivity index (CIC2) along with different molecular fragments. Favorable and unfavorable fingerprints were demonstrated in KPLS analysis, whereas important pharmacophore features (one acceptor, one donor, and two ring aromatic) along with favorable and unfavorable field effects were demonstrated in PHASE-based pharmacophore model. MM-GBSA analyses revealed significance of different polar, non-polar, and solvation energies. Docking-based alignment of ligands was used to perform Gaussian-based 3D-QSAR study that further demonstrated importance of different field effects. Overall, it was found that polar interactions (hydrogen bonding, bridged hydrogen bonding, and pi-cation) play major roles for higher activity. Steric groups increase the total contact surface area but it should have higher fractional polar surface area to adjust solvation energy. Structure-based pharmacophore mapping spotted the positive ionizable feature of ligands as the most important feature for discriminating highly active compounds from inactives. Molecular dynamics simulation, conducted on highly active ligands, described the dynamic behaviors of the protein complexes and supported the interpretations obtained from other modeling analyses. The current study may be useful for designing PARP-1 inhibitors.

  8. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

    PubMed Central

    Demaeght, Peter; Osborne, Edward J.; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans

    2014-01-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a T. urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  9. Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis.

    PubMed

    Koehler, Angela N; Shamji, Alykhan F; Schreiber, Stuart L

    2003-07-16

    Small molecule microarrays were screened to identify a small molecule ligand for Hap3p, a subunit of the yeast Hap2/3/4/5p transcription factor complex. The compound, named haptamide A, was determined to have a KD of 5.03 muM for binding to Hap3p using surface plasmon resonance analysis. Haptamide A also inhibited activation of a GDH1-lacZ reporter gene in a dose-dependent fashion. To explore structure-activity relationships, 11 derivatives of haptamide A were prepared using the same synthetic route that was developed for the original library synthesis. Analysis of dissociation constants and IC50 values for the reporter gene assay revealed a more potent inhibitor, haptamide B, with a KD of 330 nM. Whole-genome transcriptional profiling was used to compare effects of haptamide B with a hap3Delta yeast strain. Treatment with haptamide B, like the deletion mutant, reduced lactate-induced transcription of several genes from wild-type levels. Profiling the genetic "knockout" and the chemical genetic "knockdown" led to the identification of several genes that are regulated by Hap3p under nonfermentative conditions. These results demonstrate that a small molecule discovered using the small molecule microarray binding assay can permeate yeast cells and reach its target transcription factor protein in cells.

  10. Spatial and temporal expression patterns of diverse Pin-II proteinase inhibitor genes in Capsicum annuum Linn.

    PubMed

    Tamhane, Vaijayanti A; Giri, Ashok P; Kumar, Pavan; Gupta, Vidya S

    2009-08-01

    Pin-II type proteinase inhibitor (PI) genes were cloned from fruit and stem tissues of Capsicum annuum L. var Phule Jyoti using primers designed from reported CanPI gene sequence (AF039398). In total, 21 novel CanPIs, members of the Pin-II PI family, were identified in the study, with three isoforms of 1-inhibitory repeat domain (IRD), eight isoforms of 2-IRD, three isoforms of 3-IRD, five isoforms of 4-IRD and two partial CanPI sequences. Most of the sequences showed variation (2 to 20%) in the deduced AA sequences which were pronounced close to the reactive site loop. Expression patterns of CanPIs in the fruit and stem tissues of mature C. annuum plants were shown to vary qualitatively and quantitatively using semi-quantitative RT-PCR expression analysis. In the fruit tissue, CanPIs with different IRDs (from 1 to 4) were expressed simultaneously. In stem tissue, 1- and 2-IRD CanPIs were strongly expressed along moderate expression of 3- and 4-IRD genes. Analysis of CanPI protein activity showed a range of active forms across the tissues. CanPI expression was differentially up-regulated upon wounding and insect attack. Although infestation by aphids (Myzus persicae) and lepidopteran pests (Spodoptera litura) specifically induced 4-IRD CanPIs, virus-infected leaves did not affect CanPI expression. Analysis of CanPI protein activity indicated that the up-regulation in CanPI expression was not always correlated with increase in PI activity. Our results demonstrated that CanPI expression is regulated spatially, temporally as well as qualitatively and quantitatively.

  11. Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

    PubMed Central

    Zhou, Dongwen; Lobo, Yara A.; Batista, Isabel F. C.; Marques-Porto, Rafael; Gustchina, Alla; Oliva, Maria L. V.; Wlodawer, Alexander

    2013-01-01

    A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1′ pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes. PMID:23626794

  12. Cystatins, serpins and other families of protease inhibitors in plants.

    PubMed

    Volpicella, Mariateresa; Leoni, Claudia; Costanza, Alessandra; De Leo, Francesca; Gallerani, Raffaele; Ceci, Luigi R

    2011-08-01

    Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman-Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics.

  13. Subtilisin protein inhibitor from potato tubers.

    PubMed

    Revina, T A; Speranskaya, A S; Kladnitskaya, G V; Shevelev, A B; Valueva, T A

    2004-10-01

    A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.

  14. Identification of a novel potent, selective and cell permeable inhibitor of protein kinase CK2 from the NIH/NCI Diversity Set Library.

    PubMed

    Guerra, Barbara; Hochscherf, Jennifer; Jensen, Nina Bjelkerup; Issinger, Olaf-Georg

    2015-08-01

    The anti-apoptotic protein kinase CK2 increasingly becomes an attractive target in cancer research with great therapeutic potential. Here, we have performed an in vitro screening of the Diversity Set III of the DTP program from the NCI/NIH, comprising 1600 compounds. We have identified 1,3-Dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl] dibenzo(b,d) furan-2,7-diol (referred to as D11) to be a potent and selective inhibitor of protein kinase CK2. The D11 compound was tested against 354 eukaryotic protein kinases. By setting the threshold for inhibition to <2% remaining kinase activity, only DYRK1B, IRAK1 and PIM3 were inhibited to an extent as the tetrameric CK2 holoenzyme and its catalytic subunits α and α'. The IC50 values for the CK2α and CK2α' were on average 1-2 nM in comparison to the DYRK1B, IRAK1 and PIM3 kinases, which ranged from 18 to 49 nM. Cell permeability and efficacy of D11 were tested with cells in culture. In MIA PaCa-2 cells (human pancreatic carcinoma cell line), the phosphorylation of the CK2 biomarker CDC37 at S13 was almost completely inhibited in the presence of D11. This was observed both under normoxia and hypoxia. In the case of the human non-small cell lung carcinoma cell line, H1299, increasing amounts of D11 led to an inhibition of S380/T382/383 phosphorylation in PTEN, another biomarker for CK2 activity.

  15. Changes in midgut endopeptidase activity of Spodoptera frugiperda (Lepidoptera: Noctuidae) are responsible for adaptation to soybean proteinase inhibitors.

    PubMed

    Paulillo, L C; Lopes, A R; Cristofoletti, P T; Parra, J R; Terra, W R; Silva-Filho, M C

    2000-06-01

    The development of transgenic maize plants expressing soybean proteinase inhibitors could reduce the economic damage of one of the major maize pests in Brazil, the fall armyworm, Spodoptera frugiperda (J.E. Smith, 1797). We examined the influence of soybean proteinase inhibitors on digestive enzyme properties and development of S. frugiperda larvae. The inhibition of trypsin and chymotrypsin activities in vitro by soybean proteinase inhibitors suggested that either Kunitz (SBTI) or Bowman-Birk (SBBI) would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of semipurified soybean inhibitors did not result in a significant reduction of growth and development of fall armyworm. Therefore, digestive serine proteinase activities (trypsin and chymotrypsin) of fall armyworm larvae were characterized. The results suggest that S. frugiperda was able to physiologically adapt to dietary proteinase inhibitors by altering the complement of proteolytic enzymes in the insect midguts.

  16. Relative increase in Alzheimer's disease of soluble forms of cerebral Abeta amyloid protein precursor containing the Kunitz protease inhibitory domain.

    PubMed

    Moir, R D; Lynch, T; Bush, A I; Whyte, S; Henry, A; Portbury, S; Multhaup, G; Small, D H; Tanzi, R E; Beyreuther, K; Masters, C L

    1998-02-27

    Although a number of studies have examined amyloid precursor protein (APP) mRNA levels in Alzheimer's disease (AD), no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a Kunitz protease inhibitory (KPI)-encoded region are increased or decreased in AD. Here we compare AD and control brain for the relative amounts of APP protein containing KPI to APP protein lacking this domain. APP protein was purified from the soluble subcellular fraction and Triton X-100 membrane pellet extract of one hemisphere of AD (n = 10), normal (n = 7), and neurological control (n = 5) brains. The amount of KPI-containing APP in the purified protein samples was determined using two independent assay methods. The first assay exploited the inhibitory action of KPI-containing APP on trypsin. The second assay employed reflectance analysis of Western blots. The proportion of KPI-containing forms of APP in the soluble subcellular fraction of AD brains is significantly elevated (p < 0.01) compared with controls. Species containing a KPI domain comprise 32-41 and 76-77% of purified soluble APP from control and AD brains, respectively. For purified membrane-associated APP, 72-77 and 65-82% of control and AD samples, respectively, contain a KPI domain. Since KPI-containing species of APP may be more amyloidogenic (Ho, L., Fukuchi, K., and Yonkin, S. G. (1996) J. Biol. Chem. 271, 30929-30934), our findings support an imbalance of isoforms as one possible mechanism for amyloid deposition in sporadic AD.

  17. Crystallization and preliminary crystallographic studies of Schizolobium parahyba chymotrypsin inhibitor (SPCI) at 1.8 Å resolution

    SciTech Connect

    Teles, Rozeni Chagas Lima; Esteves, Gisele Ferreira; Araújo, Marcus Aurélio Miranda; Bloch, Carlos Jr; Barbosa, João Alexandre Ribeiro Gonçalves; Freitas, Sonia Maria de

    2007-11-01

    Crystallization and preliminary crystallographic studies of Schizolobium parahyba chymotrypsin inhibitor (SPCI) at 1.8 Å resolution. SPCI, a Kunitz-type chymotrypsin inhibitor, is a 180-amino-acid polypeptide isolated from Schizolobium parahyba seeds. This inhibitor has been characterized as a highly stable protein over a broad pH and temperature range. SPCI was crystallized using a solution containing 0.1 M sodium acetate trihydrate buffer pH 4.6, 33%(v/v) PEG 2000 and 0.2 M ammonium sulfate. Data were collected to 1.80 Å resolution from a single crystal of SPCI under cryogenic conditions. The protein crystallized in space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 40.01, b = 71.58, c = 108.68 Å and an R{sub merge} of 0.052. The structure of SPCI has been solved by molecular replacement using the known structure of the Kunitz-type trypsin inhibitor from Delonix regia (PDB code) as the search model.

  18. [Chymotrypsin and trypsin inhibitor isolated from potato tubers].

    PubMed

    Revina, T A; Parfenov, I A; Gvozdeva, E L; Gerasimova, N G; Valueva, T A

    2011-01-01

    Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., Zhukov's Jubilee breed) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-Sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for Carlsberg subtilisin. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.

  19. Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis.

    PubMed

    Liao, Min; Zhou, Jinlin; Gong, Haiyan; Boldbaatar, Damdinsuren; Shirafuji, Rika; Battur, Banzragch; Nishikawa, Yoshifumi; Fujisaki, Kozo

    2009-02-01

    A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.

  20. Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica*

    PubMed Central

    Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.

    2012-01-01

    This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

  1. Rapid Release of Protease Inhibitors from Soybeans

    PubMed Central

    Hwang, David L.; Yang, Wen-Kuang; Foard, Donald E.; Lin, K.-T. -Davis

    1978-01-01

    Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors

  2. The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

    PubMed

    Di Ciero, L; Oliva, M L; Torquato, R; Köhler, P; Weder, J K; Camillo Novello, J; Sampaio, C A; Oliveira, B; Marangoni, S

    1998-11-01

    Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).

  3. Bauhinia variegata var. variegata trypsin inhibitor: from isolation to potential medicinal applications.

    PubMed

    Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

    2010-06-11

    Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K(i), 0.1 x 10(-9)M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI.

  4. Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications

    SciTech Connect

    Fang, Evandro Fei; Wong, Jack Ho; Bah, Clara Shui Fern; Lin, Peng; Tsao, Sai Wah; Ng, Tzi Bun

    2010-06-11

    Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel's foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (K{sub i}, 0.1 x 10{sup -9} M) of BvvTI ranks the highest among them. Besides anti-HIV-1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI.

  5. Protease inhibitors from several classes work synergistically against Callosobruchus maculatus.

    PubMed

    Amirhusin, Bahagiawati; Shade, Richard E; Koiwa, Hisashi; Hasegawa, Paul M; Bressan, Ray A; Murdock, Larry L; Zhu-Salzman, Keyan

    2007-07-01

    Targeting multiple digestive proteases may be more effective in insect pest control than inhibition of a single enzyme class. We therefore explored possible interactions of three antimetabolic protease inhibitors fed to cowpea bruchids in artificial diets, using a recombinant soybean cysteine protease inhibitor scN, an aspartic protease inhibitor pepstatin A, and soybean Kunitz trypsin inhibitor KI. scN and pepstatin, inhibiting major digestive cysteine and aspartic proteases, respectively, significantly prolonged the developmental time of cowpea bruchids individually. When combined, the anti-insect effect was synergistic, i.e., the toxicity of the mixture was markedly greater than that of scN or pepstatin alone. KI alone did not impact insect development even at relatively high concentrations, but its anti-insect properties became apparent when acting jointly with scN or scN plus pepstatin. Incubating KI with bruchid midgut extract showed that it was partially degraded. This instability may explain its lack of anti-insect activity. However, this proteolytic degradation was inhibited by scN and/or pepstatin. Protection of KI from proteolysis in the insect digestive tract thus could be the basis for the synergistic effect. These observations support the concept that cowpea bruchid gut proteases play a dual role; digesting protein for nutrient needs and protecting insects by inactivating dietary proteins that may otherwise be toxic. Our results also suggest that transgenic resistance strategies that involve multigene products are likely to have enhanced efficacy and durability.

  6. Synthesis, biological evaluation and molecular modelling of diversely functionalized heterocyclic derivatives as inhibitors of acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+ channels and nicotinic receptors.

    PubMed

    Marco, José L; de los Ríos, Cristóbal; García, Antonio G; Villarroya, Mercedes; Carreiras, M Carmo; Martins, Carla; Eleutério, Ana; Morreale, Antonio; Orozco, M; Luque, F Javier

    2004-05-01

    The synthesis and the biological activity of compounds 5-40 as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), as well as modulators of voltage-dependent Ca(2+) channels and nicotinic receptors, are described. These molecules are tacrine analogues, which have been prepared from polyfunctionalized 6-amino-5-cyano-4H-pyrans, 6-amino-5-cyano-pyridines and 5-amino-2-aryl-3-cyano-1,3-oxazoles via Friedländer reaction with selected cycloalkanones. These compounds are moderate acetylcholinesterase and butyrylcholinesterase inhibitors, the BuChE/AChE selectivity of the most active molecules ranges from 10.0 (compound 29) to 76.9 (compound 16). Interestingly, the 'oxazolo-tacrine' derivatives are devoid of any activity. All compounds showed an important inhibitory effect on the nicotinic acetylcholine receptor. Most of them also blocked L-type Ca(2+) channels, and three of them, 64, 19 and 67, the non-L type of Ca(2+) channels. Molecular modelling studies suggest that these compounds might bind at the peripheral binding site of AChE, which opens the possibility to design inhibitors able to bind at both, the catalytic and peripheral binding sites of the enzyme.

  7. Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya

    SciTech Connect

    Azarkan, Mohamed; Garcia-Pino, Abel; Dibiani, Rachid; Wyns, Lode; Loris, Remy; Baeyens-Volant, Danielle

    2006-12-01

    The Kunitz-type trypsin/chymotrypsin inhibitor isolated from C. papaya latex has been crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms are observed, diffracting to 2.6 and 1.7 Å. A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 Å and belongs to space group P3{sub 1} or P3{sub 2}. This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = b = 74.70, c = 78.97 Å. Crystal form II, which was grown in the presence of Co{sup 2+}, diffracts to a resolution of 1.7 Å and belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 Å.

  8. Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β-lactam antibiotic resistance.

    PubMed

    Vadlamani, Grishma; Stubbs, Keith A; Désiré, Jérôme; Blériot, Yves; Vocadlo, David J; Mark, Brian L

    2017-03-28

    NagZ is an N-acetyl-β-d-glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram-negative bacteria by removing N-acetyl-glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6-anhydromuramoyl-peptide products generated by NagZ activate β-lactam resistance in many Gram-negative bacteria by inducing the expression of AmpC β-lactamase. Blocking NagZ activity can thereby suppress β-lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X-ray structures of NagZ bound to the potent yet non-selective N-acetyl-β-glucosaminidase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate), and two NagZ-selective inhibitors - EtBuPUG, a PUGNAc derivative bearing a 2-N-ethylbutyryl group, and MM-156, a 3-N-butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM-156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N-acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.

  9. Seed transmission rates of Bean pod mottle virus and Soybean mosaic virus in soybean may be affected by mixed infection or expression of the Kunitz trypsin inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To facilitate their spread, plant viruses have developed several methods for dispersal including insect and seed transmission. While insect transmission requires virus stability against insect digestion, seed-transmitted viruses have to overcome barriers to entry into embryos. Bean pod mottle virus ...

  10. Novel Injury Site Targeted Fusion Protein Comprising Annexin V and Kunitz Inhibitor Domains Ameliorates Ischemia-Reperfusion Injury and Promotes Survival of Ischemic Rat Abdominal Skin Flaps.

    PubMed

    Shyu, Victor Bong-Hang; Hsu, Chung En; Wen, Chih-Jen; Wun, Tze-Chein; Tang, Rui; Achilefu, Samuel; Wei, Fu-Chan; Cheng, Hui-Yun

    2017-03-01

    Appropriate antithrombotic therapy is critical for successful outcomes in reconstructive microsurgical procedures involving free tissue transfer. The annexin V-6L15 (ANV-6L15) fusion protein was developed as a targeted antithrombotic reagent. Annexin V specifically binds to exposed phosphatidylserine on apoptotic or injured cells, and prevents coagulation and cell adhesion, whereas 6L15 inhibits tissue factor-VIIa pathway within the coagulation cascade. The treatment efficacy of ANV-6L15 on rat island muscle and pedicled abdominal fasciocutaneous flaps following ischemic injury and ischemia-reperfusion injury (IRI) was evaluated.

  11. Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

    PubMed

    Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

    2016-08-01

    It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.

  12. Three-dimensional quantitative structure-activity relationships and docking studies of some structurally diverse flavonoids and design of new aldose reductase inhibitors

    PubMed Central

    Chandra De, Utpal; Debnath, Tanusree; Sen, Debanjan; Debnath, Sudhan

    2015-01-01

    Aldose reductase (AR) plays an important role in the development of several long-term diabetic complications. Inhibition of AR activities is a strategy for controlling complications arising from chronic diabetes. Several AR inhibitors have been reported in the literature. Flavonoid type compounds are shown to have significant AR inhibition. The objective of this study was to perform a computational work to get an idea about structural insight of flavonoid type compounds for developing as well as for searching new flavonoid based AR inhibitors. The data-set comprising 68 flavones along with their pIC50 values ranging from 0.44 to 4.59 have been collected from literature. Structure of all the flavonoids were drawn in Chembiodraw Ultra 11.0, converted into corresponding three-dimensional structure, saved as mole file and then imported to maestro project table. Imported ligands were prepared using LigPrep option of maestro 9.6 version. Three-dimensional quantitative structure-activity relationships and docking studies were performed with appropriate options of maestro 9.6 version installed in HP Z820 workstation with CentOS 6.3 (Linux). A model with partial least squares factor 5, standard deviation 0.2482, R2 = 0.9502 and variance ratio of regression 122 has been found as the best statistical model. PMID:25709964

  13. Diverse Small Molecule Inhibitors of Human Apurinic/Apyrimidinic Endonuclease APE1 Identified from a Screen of a Large Public Collection

    PubMed Central

    Dorjsuren, Dorjbal; Kim, Daemyung; Vyjayanti, Vaddadi N.; Maloney, David J.; Jadhav, Ajit; Wilson, David M.; Simeonov, Anton

    2012-01-01

    The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment. PMID:23110144

  14. Diverse combinatorial design, synthesis and in vitro evaluation of new HEPT analogues as potential non-nucleoside HIV-1 reverse transcription inhibitors.

    PubMed

    Puig-de-la-Bellacasa, Raimon; Giménez, Laura; Pettersson, Sofia; Pascual, Rosalia; Gonzalo, Encarna; Esté, José A; Clotet, Bonaventura; Borrell, José I; Teixidó, Jordi

    2012-08-01

    New analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) were synthesized and evaluated for their in vitro activities against HIV-1 in MT-4 cell cultures. Chemical diversity was introduced in 4 of the six positions of the core and the influence of each substituent was studied. This library was built on the basis of a rational diversity analysis with the objective of maximizing diversity and thus, the activity range with a minimum number of synthesized compounds. Among them, 2{1,2,3,1} and 2{1,2,3,4} exhibited the most potent anti-HIV-1 activities (EC(50)=0.015 μg/mL; 0.046 μM, SI >1667) and (EC(50)=0.025 μg/mL; 0.086 μM, SI >1000), respectively, which were about 71-fold and 38-fold more active than the reference compound HEPT (EC(50)=1.01 μg/mL; 3.27 μM, SI >25).

  15. The 3D-QSAR study of 110 diverse, dual binding, acetylcholinesterase inhibitors based on alignment independent descriptors (GRIND-2). The effects of conformation on predictive power and interpretability of the models.

    PubMed

    Vitorović-Todorović, Maja D; Cvijetić, Ilija N; Juranić, Ivan O; Drakulić, Branko J

    2012-09-01

    The 3D-QSAR analysis based on alignment independent descriptors (GRIND-2) was performed on the set of 110 structurally diverse, dual binding AChE reversible inhibitors. Three separate models were built, based on different conformations, generated following next criteria: (i) minimum energy conformations, (ii) conformation most similar to the co-crystalized ligand conformation, and (iii) docked conformation. We found that regardless on conformation used, all the three models had good statistic and predictivity. The models revealed the importance of protonated pyridine nitrogen of tacrine moiety for anti AChE activity, and recognized HBA and HBD interactions as highly important for the potency. This was revealed by the variables associated with protonated pyridinium nitrogen, and the two amino groups of the linker. MIFs calculated with the N1 (pyridinium nitrogen) and the DRY GRID probes in the AChE active site enabled us to establish the relationship between amino acid residues within AChE active site and the variables having high impact on models. External predictive power of the models was tested on the set of 40 AChE reversible inhibitors, most of them structurally different from the training set. Some of those compounds were tested on the different enzyme source. We found that external predictivity was highly sensitive on conformations used. Model based on docked conformations had superior predictive ability, emphasizing the need for the employment of conformations built by taking into account geometrical restrictions of AChE active site gorge.

  16. A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

    PubMed

    Pharo, Elizabeth A; Cane, Kylie N; McCoey, Julia; Buckle, Ashley M; Oosthuizen, W H; Guinet, Christophe; Arnould, John P Y

    2016-03-01

    The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

  17. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  18. Structure-based design of diverse inhibitors of Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase: combined molecular docking, dynamic simulation, and biological activity.

    PubMed

    Soni, Vijay; Suryadevara, Priyanka; Sriram, Dharmarajan; Kumar, Santhosh; Nandicoori, Vinay Kumar; Yogeeswari, Perumal

    2015-07-01

    Persistent nature of Mycobacterium tuberculosis is one of the major factors which make the drug development process monotonous against this organism. The highly lipophilic cell wall, which constituting outer mycolic acid and inner peptidoglycan layers, acts as a barrier for the drugs to enter the bacteria. The rigidity of the cell wall is imparted by the peptidoglycan layer, which is covalently linked to mycolic acid by arabinogalactan. Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) serves as the starting material in the biosynthesis of this peptidoglycan layers. This UDP-GlcNAc is synthesized by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU(Mtb)), a bi-functional enzyme with two functional sites, acetyltransferase site and uridyltransferase site. Here, we report design and screening of nine inhibitors against UTP and NAcGlc-1-P of uridyltransferase active site of glmU(Mtb). Compound 4 was showing good inhibition and was selected for further analysis. The isothermal titration calorimetry (ITC) experiments showed the binding energy pattern of compound 4 to the uridyltransferase active site is similar to that of substrate UTP. In silico molecular dynamics (MD) simulation studies, for compound 4, carried out for 10 ns showed the protein-compound complex to be stable throughout the simulation with relative rmsd in acceptable range. Hence, these compounds can serve as a starting point in the drug discovery processes against Mycobacterium tuberculosis.

  19. Primary Structure of a Trypsin Inhibitor (Copaifera langsdorffii Trypsin Inhibitor-1) Obtained from C. langsdorffii Seeds

    PubMed Central

    Silva, José A.; Pompeu, Dávia G.; Smolka, Marcus B.; Gozzo, Fabio C.; Comar, Moacyr; Eberlin, Marcos N.; Marangoni, Sérgio

    2015-01-01

    In this study, the aim was to determine the complete sequence of the Copaifera langsdorffii trypsin inhibitor (CTI)-1 using 2-dimensional (2D)-PAGE, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), and quadrupole time-of-flight (QTOF) spectrometry. Spots A (CTI-1) and F (CTI-2) were submitted to enzymatic digestions with trypsin, SV8, and clostripain. The accurate mass of the peptide obtained from each digest was determined by mass spectrometry (MS) using MALDI-TOF. The most abundant peptides were purified and sequenced in a liquid chromatograph connected to an electrospray ionization-QTOF MS. When the purified trypsin inhibitor was submitted to 2D electrophoresis, different spots were observed, suggesting that the protein is composed of 2 subunits with microheterogeneity. Isoelectric points of 8.0, 8.5, and 9.0 were determined for the 11 kDa subunit and of 4.7, 4.6, and 4.3 for the 9 kDa subunit. The primary structure of CTI-1, determined from the mass of the peptide of the enzymatic digestions and the sequence obtained by MS, indicated 180 shared amino acid residues and a high degree of similarity with other Kunitz (KTI)-type inhibitors. The peptide also contained an Arg residue at the reactive site position. Its 3-dimensional structure revealed that this is because the structural discrepancies do not affect the canonical conformation of the reactive loop of the peptide. Results demonstrate that a detailed investigation of the structural particularities of CTI-1 could provide a better understanding of the mechanism of action of these proteins, as well as clarify its biologic function in the seeds. CTI-1 belongs to the KTI family and is composed of 2 polypeptide chains and only 1 disulfide bridge. PMID:26207098

  20. The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity

    SciTech Connect

    Salameh, Moh'd A.; Soares, Alexei S.; Hockla, Alexandra; Radisky, Derek C.; Radisky, Evette S.

    2011-11-15

    PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P2' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P1 and P2' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant Ki of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

  1. The P2’ residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity

    SciTech Connect

    Salameh, M.A.; Soares, A.; Hockla, A.; Radisky, D. C.; Radisky, E. S.

    2011-11-15

    PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumor progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P'{sub 2} position. We find that bulky and charged residues strongly disfavor binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P{sub 1} and P'{sub 2} residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K{sub i} of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

  2. Conserved Patterns of Microbial Immune Escape: Pathogenic Microbes of Diverse Origin Target the Human Terminal Complement Inhibitor Vitronectin via a Single Common Motif

    PubMed Central

    Kraiczy, Peter; Hammerschmidt, Sven; Skerka, Christine; Zipfel, Peter F.; Riesbeck, Kristian

    2016-01-01

    Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13) bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352–374). This specific interaction leaves the terminal complement complex (TCC) regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response. PMID:26808444

  3. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

    SciTech Connect

    Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; Hockla, Alexandra; Soares, Alexei S.; Radisky, Evette S.

    2015-07-14

    Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.

  4. Mesotrypsin has evolved four unique residues to cleave trypsin inhibitors as substrates [Mesotrypsin has evolved to cleave trypsin inhibitors as substrates using four unique residues

    DOE PAGES

    Alloy, Alexandre P.; Kayode, Olumide; Wang, Ruiying; ...

    2015-07-14

    Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursormore » protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. As a result, these findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.« less

  5. Diverse Thinking about Diversity

    ERIC Educational Resources Information Center

    Kaplan, Sandra N.

    2013-01-01

    This article focuses on the concept of diversity in educational decision making. It is noted that the differences that distinguish the needs, interests and abilities are identified by educators. It lists misconceptions resulting from not attending to within-group diversity, and states that a "loss of self" for individual members of…

  6. Comparative effects of ohmic, induction cooker, and electric stove heating on soymilk trypsin inhibitor inactivation.

    PubMed

    Lu, Lu; Zhao, Luping; Zhang, Caimeng; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2015-03-01

    During thermal treatment of soymilk, a rapid incorporation of Kunitz trypsin inhibitor (KTI) into protein aggregates by covalent (disulfide bond, SS) and/or noncovalent interactions with other proteins is responsible for its fast inactivation of trypsin inhibitor activity (TIA). In contrast, the slow cleavage of a single Bowman-Birk inhibitor (BBI) peptide bond is responsible for its slow inactivation of TIA and chymotrypsin inhibitor activity (CIA). In this study, the effects of Ohmic heating (220 V, 50 Hz) on soymilk TIA and CIA inactivation were examined and compared to induction cooker and electric stove heating with similar thermal histories. It was found that: (1) TIA and CIA inactivation was slower from 0 to 3 min, and faster after 3 min as compared to induction cooker and electric stove. (2) The thiol (SH) loss rate was slower from 0 to 3 min, and similar to induction cooker and electric stove after 3 min. (3) Ohmic heating slightly increased protein aggregate formation. (4) In addition to the cleavage of one BBI peptide bond, an additional reaction might occur to enhance BBI inactivation. (5) Ohmic heating was more energy-efficient for TIA and CIA inactivation. (6) TIA and CIA inactivation was accelerated with increasing electric voltage (110, 165, and 220 V) of Ohmic heating. It is likely that the enhanced inactivation of TIA by Ohmic heating is due to its combined electrochemical and thermal effects.

  7. The tissue factor pathway inhibitor 1 of Sciaenops ocellatus possesses antimicrobial activity and is involved in the immune response against bacterial infection.

    PubMed

    Zhang, Min; Sun, Li

    2011-03-01

    Tissue factor pathway inhibitor 1 (TFPI-1) is a Kunitz-type serine protease inhibitor that regulates the activation of tissue factor-induced coagulation. In teleosts, TFPI-1-like sequences have been found to exist in two species (Danio rerio and Cyprinus carpio); however, the potential function of fish TFPI-1 has not been investigated. In this study, we identified and analyzed a TFPI-1 homologue, SoTFPI-1, from red drum (Sciaenops ocellatus). The deduced amino acid sequence of SoTFPI-1 is 284 residues in length and contains three Kunitz domains, an acidic N-terminus, and a basic C-terminus. SoTFPI-1 shares 49.5% and 46.9% overall sequence identities with the TFPI-1 of D. rerio and C. carpio, respectively. Quantitative real time RT-PCR analysis showed that constitutive SoTFPI-1 expression occurred, in increasing order, in kidney, brain, liver, gill, blood, spleen, muscle, and heart. Bacterial infection and lipopolysaccharide exposure upregulated SoTFPI-1 expression in kidney in time-dependent manners. Recombinant SoTFPI-1 (rSoTFPI-1) purified from Escherichia coli exhibits not only serine protease inhibitor activity but also bactericidal activity in a manner that is independent of any host factors. A synthetic peptide, TO17, corresponding to the C-terminal basic region of SoTFPI-1 also possesses antibacterial effect that is more potent than that of the full-length rSoTFPI-1. Taken together, these results demonstrate that (i) SoTFPI-1 is a biologically active serine protease inhibitor endowed with bactericidal property; (ii) provide the first indication that teleost TFPI-1 is likely to be involved in anti-microbial infection and thus is linked to innate immune defense.

  8. Crystallization and preliminary X-ray analysis of a protease inhibitor from the latex of Carica papaya

    PubMed Central

    Azarkan, Mohamed; Garcia-Pino, Abel; Dibiani, Rachid; Wyns, Lode; Loris, Remy; Baeyens-Volant, Danielle

    2006-01-01

    A Kunitz-type protease inhibitor purified from the latex of green papaya (Carica papaya) fruits was crystallized in the presence and absence of divalent metal ions. Crystal form I, which is devoid of divalent cations, diffracts to a resolution of 2.6 Å and belongs to space group P31 or P32. This crystal form is a merohedral twin with two molecules in the asymmetric unit and unit-cell parameters a = b = 74.70, c = 78.97 Å. Crystal form II, which was grown in the presence of Co2+, diffracts to a resolution of 1.7 Å and belongs to space group P212121, with unit-cell parameters a = 44.26, b = 81.99, c = 140.89 Å. PMID:17142906

  9. A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice.

    PubMed

    Theodoro-Júnior, Osmar Aparecido; Righetti, Renato Fraga; Almeida-Reis, Rafael; Martins-Oliveira, Bruno Tadeu; Oliva, Leandro Vilela; Prado, Carla Máximo; Saraiva-Romanholo, Beatriz Mangueira; Leick, Edna Aparecida; Pinheiro, Nathalia Montouro; Lobo, Yara Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Tibério, Iolanda de Fátima Lopes Calvo

    2017-02-14

    Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management.

  10. A Plant Proteinase Inhibitor from Enterolobium contortisiliquum Attenuates Pulmonary Mechanics, Inflammation and Remodeling Induced by Elastase in Mice

    PubMed Central

    Theodoro-Júnior, Osmar Aparecido; Righetti, Renato Fraga; Almeida-Reis, Rafael; Martins-Oliveira, Bruno Tadeu; Oliva, Leandro Vilela; Prado, Carla Máximo; Saraiva-Romanholo, Beatriz Mangueira; Leick, Edna Aparecida; Pinheiro, Nathalia Montouro; Lobo, Yara Aparecida; Martins, Mílton de Arruda; Oliva, Maria Luiza Vilela; Tibério, Iolanda de Fátima Lopes Calvo

    2017-01-01

    Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for emphysema. Our aim was to evaluate the effects of a plant Kunitz proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on several aspects of experimental elastase-induced pulmonary inflammation in mice. C57/Bl6 mice were intratracheally administered elastase (ELA) or saline (SAL) and were treated intraperitoneally with EcTI (ELA-EcTI, SAL-EcTI) on days 1, 14 and 21. On day 28, pulmonary mechanics, exhaled nitric oxide (ENO) and number leucocytes in the bronchoalveolar lavage fluid (BALF) were evaluated. Subsequently, lung immunohistochemical staining was submitted to morphometry. EcTI treatment reduced responses of the mechanical respiratory system, number of cells in the BALF, and reduced tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-12 (MMP-12), tissue inhibitor of matrix metalloproteinase (TIMP-1), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-positive cells and volume proportion of isoprostane, collagen and elastic fibers in the airways and alveolar walls compared with the ELA group. EcTI treatment reduced elastase induced pulmonary inflammation, remodeling, oxidative stress and mechanical alterations, suggesting that this inhibitor may be a potential therapeutic tool for chronic obstructive pulmonary disease (COPD) management. PMID:28216579

  11. Selective Inhibitors of Protein Methyltransferases

    PubMed Central

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  12. Identification and analysis of the tissue factor pathway inhibitor 2 of Sciaenops ocellatus.

    PubMed

    Zhang, Min; Xiao, Zhi-zhong; Sun, Li

    2011-01-01

    Tissue factor pathway inhibitor 2 (TFPI-2) is a structural homologue of TFPI, a potent inhibitor of tissue factor (TF)-mediated coagulation. Although TFPI-2 has been identified at sequence level in several fish species, no study on piscine TFPI-2 has been documented. In this report, we identified and analyzed a TFPI-2 homologue, SoTFPI2, from red drum Sciaenops ocellatus. The open reading frame of SoTFPI2 is 681 bp, which encodes a 226-residue protein that shares 59.2-82.3% overall sequence identities with known fish TFPI-2. SoTFPI2 possesses three tandem Kunitz domains and is negatively charged at the N-terminus and positively charged at the C-terminus. Expression of SoTFPI2 was detected, in increasing order, in spleen, muscle, gill, brain, liver, kidney, blood, and heart. Bacterial challenge and lipopolysaccharide treatment significantly upregulated SoTFPI2 expression in kidney in time-dependent manners. Recombinant SoTFPI2 purified from Escherichia coli inhibits the proteolytic activity of trypsin and exhibits bactericidal effect on a fish pathogen. Take together, these results indicate that SoTFPI2 is a biologically active serine protease inhibitor with antibacterial property and is likely to play a role in anti-bacterial infection.

  13. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  14. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  15. Purification and characterization of a trypsin inhibitor from the seeds of Artocarpus heterophyllus Lam.

    PubMed

    Lyu, Junchen; Liu, Yuan; An, Tianchen; Liu, Yujun; Wang, Manchuriga; Song, Yanting; Zheng, Feifei; Wu, Dan; Zhang, Yingxia; Deng, Shiming

    2015-05-01

    A proteinaceous inhibitor against trypsin was isolated from the seeds of Artocarpus heterophyllus Lam. by successive ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The trypsin inhibitor, named as AHLTI (A. heterophyllus Lam. trypsin inhibitor), consisted of a single polypeptide chain with a molecular weight of 28.5 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel-filtration chromatography. The N-terminal sequence of AHLTI was DEPPSELDAS, which showed no similarity to other known trypsin inhibitor sequence. AHLTI completely inhibited bovine trypsin at a molar ratio of 1:2 (AHLTI:trypsin) analyzed by native polyacrylamide gel electrophoresis, inhibition activity assay, and gel-filtration chromatography. Moreover, kinetic enzymatic studies were carried out to understand the inhibition mechanism of AHLTI against trypsin. Results showed that AHLTI was a competitive inhibitor with an equilibrium dissociation constant (Ki) of 3.7 × 10(-8) M. However, AHLTI showed weak inhibitory activity toward chymotrypsin and elastase. AHLTI was stable over a broad range of pH 4-8 and temperature 20-80°C. The reduction agent, dithiothreitol, had no obvious effect on AHLTI. The trypsin inhibition assays of AHLTI toward digestive enzymes from insect pest guts in vitro demonstrated that AHLTI was effective against enzymes from Locusta migratoria manilensis (Meyen). These results suggested that AHLTI might be a novel trypsin inhibitor from A. heterophyllus Lam. belonging to Kunitz family, and play an important role in protecting from insect pest.

  16. Molecular cloning and characterization of a novel bi-functional α-amylase/subtilisin inhibitor from Hevea brasiliensis.

    PubMed

    Bunyatang, Orawan; Chirapongsatonkul, Nion; Bangrak, Phuwadol; Henry, Robert; Churngchow, Nunta

    2016-04-01

    A novel cDNA encoding a bi-functional α-amylase/subtilisin inhibitor (HbASI) was isolated from rubber (Hevea brasiliensis) leaves cultivar RRIM600. The HbASI had strong homology with the soybean trypsin inhibitor (Kunitz) family of protease inhibitors. Its putative amino acid sequence was similar to that of the α-amylase/subtilisin inhibitor from Ricinus communis (72% identity). Genomic sequencing indicated that the HbASI gene contained no introns. The messenger RNA of HbASI was detected in leaf, hypocotyl and root. The recombinant HbASI expressed extracellularly in Pichia pastoris exhibited inhibitory activity against α-amylase from Aspergillus oryzae, trypsin and subtilisin A. The HbASI gene was induced in the rubber leaves infected with a rubber tree pathogen, Phytophthora palmivora. It was also enhanced by salicylic acid (SA) treatment and mechanical wounding. In addition, the biological activity of the HbASI protein involving in the plant defence responses was also investigated. The HbASI at a concentration of 0.16 mg mL(-1) could inhibit the mycelium growth of P. palmivora. These data suggested that the HbASI protein might play a crucial role in defence against pathogen of rubber trees.

  17. The Kallikrein Inhibitor from Bauhinia bauhinioides (BbKI) shows antithrombotic properties in venous and arterial thrombosis models.

    PubMed

    Brito, Marlon V; de Oliveira, Cleide; Salu, Bruno R; Andrade, Sonia A; Malloy, Paula M D; Sato, Ana C; Vicente, Cristina P; Sampaio, Misako U; Maffei, Francisco H A; Oliva, Maria Luiza V

    2014-05-01

    The Bauhinia bauhinioides Kallikrein Inhibitor (BbKI) is a Kunitz-type serine peptidase inhibitor of plant origin that has been shown to impair the viability of some tumor cells and to feature a potent inhibitory activity against human and rat plasma kallikrein (Kiapp 2.4 nmol/L and 5.2 nmol/L, respectively). This inhibitory activity is possibly responsible for an effect on hemostasis by prolonging activated partial thromboplastin time (aPTT). Because the association between cancer and thrombosis is well established, we evaluated the possible antithrombotic activity of this protein in venous and arterial thrombosis models. Vein thrombosis was studied in the vena cava ligature model in Wistar rats, and arterial thrombosis in the photochemical induced endothelium lesion model in the carotid artery of C57 black 6 mice. BbKI at a concentration of 2.0 mg/kg reduced the venous thrombus weight by 65% in treated rats in comparison to rats in the control group. The inhibitor prolonged the time for total artery occlusion in the carotid artery model mice indicating that this potent plasma kallikrein inhibitor prevented thrombosis.

  18. Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group.

    PubMed

    Carrión, M Dora; Chayah, Mariem; Entrena, Antonio; López, Ana; Gallo, Miguel A; Acuña-Castroviejo, Darío; Camacho, M Encarnación

    2013-07-15

    In a preliminary article, we reported a series of 4,5-dihydro-1H-pyrazole derivatives as neuronal nitric oxide synthase (nNOS) inhibitors. Here we present the data about the inhibition of inducible nitric oxide synthase (iNOS) of these compounds. In general, we can confirm that these pyrazoles are nNOS selective inhibitors. In addition, taking these compounds as a reference, we have designed and synthesized a series of new derivatives by modification of the heterocycle in 1-position, and by introduction of electron-donating or electron-withdrawing substituents in the aromatic ring. These derivatives have been evaluated as nNOS and iNOS inhibitors in order to identify new compounds with improved activity and selectivity. Compound 3r, with three methoxy electron-donating groups in the phenyl moiety, is the most potent nNOS inhibitor, showing good selectivity nNOS/iNOS.

  19. Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI.

    PubMed

    de Souza, Adriana F; Torquato, Ricardo J S; Tanaka, Aparecida S; Sampaio, Claudio A M

    2005-11-01

    A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. The clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. The deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. The BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.

  20. Kinase Inhibitors from Marine Sponges

    PubMed Central

    Skropeta, Danielle; Pastro, Natalie; Zivanovic, Ana

    2011-01-01

    Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included. PMID:22073013

  1. Rethinking Diversity.

    ERIC Educational Resources Information Center

    Gordon, Jack

    1992-01-01

    Managing diversity is about coping with unassimilated differences, about building systems and a culture that unite different people in a common pursuit without undermining their diversity. The goal of diversity training is a high performance organization rather than a climate in which no one's feathers are ruffled. (SK)

  2. Rethinking Diversity.

    ERIC Educational Resources Information Center

    1996

    These three papers were presented at a symposium on rethinking diversity in human resource development (HRD) moderated by Neal Chalofsky at the 1996 conference of the Academy of Human Resource Development. "Diversity: A Double-Edged Sword" (Sally F. Angus) presents the notion of work force diversity through two differing perspectives in order to…

  3. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  4. Peanut Seed Cultivars with Contrasting Resistance to Aspergillus parasiticus Colonization Display Differential Temporal Response of Protease Inhibitors.

    PubMed

    Müller, Virginia; Bonacci, Gustavo; Batthyany, Carlos; Amé, María V; Carrari, Fernando; Gieco, Jorge; Asis, Ramón

    2017-02-08

    Significant efforts are being made to minimize aflatoxin contamination in peanut seeds and one possible strategy is to understand and exploit the mechanisms of plant defense against fungal infection. In this study we have identified and characterized, at biochemical and molecular levels, plant protease inhibitors (PPIs) produced in peanut seeds of the resistant PI 337394 and the susceptible Forman cultivar during Aspergillus parasiticus colonization. With chromatographic methods and 2D-electrophoresis-mass spectrometry we have isolated and identified four variants of Bowman-Birk trypsin inhibitor (BBTI) and a novel Kunitz-type protease inhibitor (KPI) produced in response to A. parasiticus colonization. KPI was detected only in the resistant cultivar, while BBTI was produced in the resistant cultivar in a higher concentration than susceptible cultivar and with different isoforms. The kinetic expression of KPI and BBTI genes along with trypsin inhibitory activity was analyzed in both cultivars during infection. In the susceptible cultivar an early PPI activity response was associated with BBTI occurrence. Meanwhile, in the resistant cultivar a later response with a larger increase in PPI activity was associated with BBTI and KPI occurrence. The biological significance of PPI in seed defense against fungal infection was analyzed and linked to inhibitory properties on enzymes released by the fungus during infection, and to the antifungal effect of KPI.

  5. Analgesic Compound from Sea Anemone Heteractis crispa Is the First Polypeptide Inhibitor of Vanilloid Receptor 1 (TRPV1)*

    PubMed Central

    Andreev, Yaroslav A.; Kozlov, Sergey A.; Koshelev, Sergey G.; Ivanova, Ekaterina A.; Monastyrnaya, Margarita M.; Kozlovskaya, Emma P.; Grishin, Eugene V.

    2008-01-01

    Venomous animals from distinct phyla such as spiders, scorpions, snakes, cone snails, or sea anemones produce small toxic proteins interacting with a variety of cell targets. Their bites often cause pain. One of the ways of pain generation is the activation of TRPV1 channels. Screening of 30 different venoms from spiders and sea anemones for modulation of TRPV1 activity revealed inhibitors in tropical sea anemone Heteractis crispa venom. Several separation steps resulted in isolation of an inhibiting compound. This is a 56-residue-long polypeptide named APHC1 that has a Bos taurus trypsin inhibitor (BPTI)/Kunitz-type fold, mostly represented by serine protease inhibitors and ion channel blockers. APHC1 acted as a partial antagonist of capsaicin-induced currents (32 ± 9% inhibition) with half-maximal effective concentration (EC50) 54 ± 4 nm. In vivo, a 0.1 mg/kg dose of APHC1 significantly prolonged tail-flick latency and reduced capsaicin-induced acute pain. Therefore, our results can make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of overactivity of this receptor during a number of pathological processes in the organism. PMID:18579526

  6. Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents

    PubMed Central

    Ververis, Katherine; Hiong, Alison; Karagiannis, Tom C; Licciardi, Paul V

    2013-01-01

    Histone deacetylase (HDAC) inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents) as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax). More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the advancement of these drugs, especially to facilitate the rational design of HDAC inhibitors that are effective as antineoplastic agents. This review will discuss the use of HDAC inhibitors as multitargeted therapies for malignancy. Further, we outline the pharmacology and mechanisms of action of HDAC inhibitors while

  7. Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs.

    PubMed

    Chen, Xiaole; Wang, He; Shen, Yue; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2016-08-19

    Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

  8. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

    PubMed Central

    Tucker, Erik I.; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D.; Gailani, Dave; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  9. Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis

    PubMed Central

    Decrem, Yves; Rath, Géraldine; Blasioli, Virginie; Cauchie, Philippe; Robert, Séverine; Beaufays, Jérôme; Frère, Jean-Marie; Feron, Olivier; Dogné, Jean-Michel; Dessy, Chantal; Vanhamme, Luc

    2009-01-01

    Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo. PMID:19808248

  10. Inhibitors of the Metalloproteinase Anthrax Lethal Factor

    PubMed Central

    Goldberg, Allison B.; Turk, Benjamin E.

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LF-inhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and high-throughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  11. Endogenous tissue factor pathway inhibitor has a limited effect on host defence in murine pneumococcal pneumonia.

    PubMed

    van den Boogaard, Florry E; van 't Veer, Cornelis; Roelofs, Joris J T H; Meijers, Joost C M; Schultz, Marcus J; Broze, George J; van der Poll, Tom

    2015-07-01

    Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.

  12. Natural products as aromatase inhibitors.

    PubMed

    Balunas, Marcy J; Su, Bin; Brueggemeier, Robert W; Kinghorn, A Douglas

    2008-08-01

    With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always available through current synthetic chemistry techniques. Natural products that have been used traditionally for nutritional or medicinal purposes (e.g., botanical dietary supplements) may also afford AIs with reduced side effects. A thorough review of the literature regarding natural product extracts and secondary metabolites of plant, microbial, and marine origin that have been shown to exhibit aromatase inhibitory activity is presented herein.

  13. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  14. Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease.

    PubMed

    de Almeida Nogueira, Natália Pereira; Morgado-Díaz, José Andrés; Menna-Barreto, Rubem Figueiredo Sadok; Paes, Marcia Cristina; da Silva-López, Raquel Elisa

    2013-10-01

    It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.

  15. Astrocyte Mitogen Inhibitor Related to Epidermal Growth Factor Receptor

    NASA Astrophysics Data System (ADS)

    Nieto-Sampedro, Manuel

    1988-06-01

    Epidermal growth factor (EGF) is a well-characterized polypeptide hormone with diverse biological activities, including stimulation of astrocyte division. A soluble astrocyte mitogen inhibitor, immunologically related to the EGF receptor, is present in rat brain. Injury to the brain causes a time-dependent reduction in the levels of this inhibitor and the concomitant appearance of EGF receptor on the astrocyte surface. Intracerebral injection of antibody capable of binding the inhibitor caused the appearance of numerous reactive astrocytes. EGF receptor-related inhibitors may play a key role in the control of glial cell division in both normal and injured brain.

  16. Evolutionary mechanisms acting on proteinase inhibitor variability.

    PubMed

    Christeller, John T

    2005-11-01

    The interaction of proteinase inhibitors produced, in most cases, by host organisms and the invasive proteinases of pathogens or parasites or the dietary proteinases of predators, results in an evolutionary 'arms race' of rapid and ongoing change in both interacting proteins. The importance of these interactions in pathogenicity and predation is indicated by the high level and diversity of observable evolutionary activity that has been found. At the initial level of evolutionary change, recruitment of other functional protein-folding families has occurred, with the more recent evolution of one class of proteinase inhibitor from another, using the same mechanism and proteinase contact residues. The combination of different inhibitor domains into a single molecule is also observed. The basis from which variation is possible is shown by the high rate of retention of gene duplication events and by the associated process of inhibitory domain multiplication. At this level of reorganization, mutually exclusive splicing is also observed. Finally, the major mechanism by which variation is achieved rapidly is hypervariation of contact residues, an almost ubiquitous feature of proteinase inhibitors. The diversity of evolutionary mechanisms in a single class of proteins is unlikely to be common, because few systems are under similar pressure to create variation. Proteinase inhibitors are therefore a potential model system in which to study basic evolutionary process such as functional diversification.

  17. Discovering Diversity.

    ERIC Educational Resources Information Center

    Manner, Barbara M.; Hattler, Jean Anne

    2000-01-01

    Introduces a preservice teacher field trip to the rain forests and coastal areas. This experience develops an awareness for different cultures among preservice teachers by experiencing biological and cultural diversity in Costa Rica. Presents students' own ideas on this experience. (YDS)

  18. Diversity's Calling

    ERIC Educational Resources Information Center

    Cooper, Kenneth J.

    2011-01-01

    This article discusses how a Harvard-educated scholar of English and poetry, Dr. M. Lee Pelton puts a prominent face on changes that are underway at Boston's Emerson College. Faced with a public controversy over its limited faculty diversity, Emerson College has responded with a spate of hirings and promotions of minorities, capped by the…

  19. PLANT DIVERSITY

    EPA Science Inventory

    Habitat change statistics and species-area curves were used to estimate the effects of alternative future scenarios for agriculture on plant diversity in Iowa farmlands. Study areas were two watersheds in central Iowa of about 50 and 90 square kilometers, respectively. Future s...

  20. Generational diversity.

    PubMed

    Kramer, Linda W

    2010-01-01

    Generational diversity has proven challenges for nurse leaders, and generational values may influence ideas about work and career planning. This article discusses generational gaps, influencing factors and support, and the various generational groups present in today's workplace as well as the consequences of need addressing these issues. The article ends with a discussion of possible solutions.

  1. Epididymal protein targets: a brief history of the development of epididymal protease inhibitor as a contraceptive.

    PubMed

    O'Rand, Michael G; Widgren, Esther E; Hamil, Katherine G; Silva, Erick J; Richardson, Richard T

    2011-01-01

    The Laboratories for Reproductive Biology at the University of North Carolina at Chapel Hill began collaboration with Human Genome Sciences (Rockville, Maryland) to sequence a human epididymal library and identify epididymal-specific genes. Among the first clones obtained from Human Genome Sciences was a clone for EPPIN (official symbol, SPINLW1). Our laboratory has described EPPIN (epididymal protease inhibitor) as a novel gene on human chromosome 20q12-13.2 that encodes a cysteine-rich protein containing both Kunitz-type and WAP-type 4-disulfide core consensus sequences that characterize it as a protease inhibitor. EPPIN expresses 3 mRNA splice variants that encode 2 protein isoforms found in the testis and epididymis. Of the 2 isoforms, 1 is secreted and 1 lacks a secretory signal piece. EPPIN is predominantly a dimer, although multiples often exist, and in its native form, EPPIN is found on the sperm surface complexed with lactotransferrin and clusterin. During ejaculation, semenogelin from the seminal vesicles is bound to the EPPIN protein complex, initiating a series of events that define EPPIN's function: modulating prostate-specific antigen (PSA) activity, providing antimicrobial protection, and binding semenogelin, thereby inhibiting sperm motility. As PSA hydrolyzes semenogelin in the ejaculate coagulum, spermatozoa gain progressive motility. Using immunization as a tool to study antigen function, we demonstrated that EPPIN is essential for fertility because immunization of male monkeys with recombinant EPPIN results in complete, but reversible, contraception. To exploit our understanding of EPPIN's function, we have developed a high-throughput screen to look for compounds that inhibit EPPIN-semenogelin interaction and mimic anti-EPPIN, inhibiting sperm motility. These compounds are now being developed into a nonhormonal male contraceptive.

  2. Molecular mechanism of enzyme inhibition: prediction of the three-dimensional structure of the dimeric trypsin inhibitor from Leucaena leucocephala by homology modelling.

    PubMed

    Sattar, Rabia; Ali, Syed Abid; Kamal, Mustafa; Khan, Aftab Ahmed; Abbasi, Atiya

    2004-02-13

    Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymotrypsin. These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against the attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the families Leguminosae, Graminae, and Solanaceae. Leucaena leucocephala belongs to the family Leguminosae. It is widely used both as an ornamental tree as well as cattle food. We have constructed a three-dimensional model of a serine proteinase inhibitor from L. leucocephala seeds (LTI) complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTI shows structural features characteristic of the Kunitz type trypsin inhibitor and shows 39% residue identity with STI. LTI consists of 172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI and LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. We have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique features of LTI.

  3. Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema.

    PubMed

    Cruz-Silva, Ilana; Neuhof, Christiane; Gozzo, Andrezza Justino; Nunes, Viviane Abreu; Hirata, Izaura Yoshico; Sampaio, Misako Uemura; Figueiredo-Ribeiro, Rita de Cássia; Neuhof, Heinz; Araújo, Mariana da Silva

    2013-12-01

    Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

  4. Interaction proteins of invertase and invertase inhibitor in cold-stored potato tubers suggested a protein complex underlying post-translational regulation of invertase.

    PubMed

    Lin, Yuan; Liu, Jun; Liu, Xun; Ou, Yongbin; Li, Meng; Zhang, Huiling; Song, Botao; Xie, Conghua

    2013-12-01

    The activity of vacuolar invertase (VI) is vital to potato cold-induced sweetening (CIS). A post-translational regulation of VI activity has been proposed which involves invertase inhibitor (VIH), but the mechanism for the interaction between VI and VIH has not been fully understood. To identify the potential partners of VI and VIH, two cDNA libraries were respectively constructed from CIS-resistant wild potato species Solanum berthaultii and CIS-sensitive potato cultivar AC035-01 for the yeast two-hybrid analysis. The StvacINV1 (one of the potato VIs) and StInvInh2B (one of the potato VIHs), previously identified to be associated with potato CIS, were used as baits to screen the two libraries. Through positive selection and sequencing, 27 potential target proteins of StvacINV1 and eight of StInvInh2B were clarified. The Kunitz-type protein inhibitors were captured by StvacINV1 in both libraries and the interaction between them was confirmed by bimolecular fluorescence complementation assay in tobacco cells, reinforcing a fundamental interaction between VI and VIH. Notably, a sucrose non-fermenting-1-related protein kinase 1 was captured by both the baits, suggesting that a protein complex could be necessary for fine turning of the invertase activity. The target proteins clarified in present research provide a route to elucidate the mechanism by which the VI activity can be subtly modulated.

  5. Response of the digestive system of Helicoverpa zea to ingestion of potato carboxypeptidase inhibitor and characterization of an uninhibited carboxypeptidase B.

    PubMed

    Bayés, Alex; de la Vega, Mónica Rodríguez; Vendrell, Josep; Aviles, Francesc X; Jongsma, Maarten A; Beekwilder, Jules

    2006-08-01

    Carboxypeptidase activity participates in the protein digestion process in the gut of lepidopteran insects, supplying free amino-acids to developing larvae. To study the role of different carboxypeptidases in lepidopteran protein digestion, the effect of potato carboxypeptidase inhibitor (PCI) on the digestive system of larvae of the pest insect Helicoverpa zea was investigated, and compared to that of Soybean Kunitz Trypsin Inhibitor. Analysis of carboxypeptidase activity in the guts showed that ingested PCI remained active in the gut, and completely inhibited the activity of carboxypeptidases A and O. Interestingly, carboxypeptidase B activity was not affected by PCI. All previously described enzymes from the same family, both from insect or mammalian origin, have been found to be very sensitive to PCI. Analysis of several lepidopteran species showed the presence of carboxypeptidase B activity resistant to PCI in most of them. The H. zea carboxypeptidase B enzyme (CPBHz) was purified from gut content by affinity chromatography. N-terminal sequence information was used to isolate its corresponding full-length cDNA, and recombinant expression of the zymogen of CPBHz in Pichia pastoris was achieved. The substrate specificity of recombinant CPBHz was tested using peptides. Unlike other CPB enzymes, the enzyme appeared to be highly selective for C-terminal lysine residues. Inhibition by PCI appeared to be pH-dependent.

  6. Genome-wide identification and immune response analysis of serine protease inhibitor genes in the silkworm, Bombyx mori.

    PubMed

    Zhao, Ping; Dong, Zhaoming; Duan, Jun; Wang, Genhong; Wang, Lingyan; Li, Youshan; Xiang, Zhonghuai; Xia, Qingyou

    2012-01-01

    In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein.

  7. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  8. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  9. Natural Products as Aromatase Inhibitors

    PubMed Central

    Balunas, Marcy J.; Su, Bin; Brueggemeier, Robert W.; Kinghorn, A. Douglas

    2010-01-01

    With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating also the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always available through current synthetic chemistry techniques. Natural products that have been used traditionally for nutritional or medicinal purposes (e.g., botanical dietary supplements) may also afford AIs with reduced side effects. A thorough review of the literature regarding natural product extracts and secondary metabolites of plant, microbial, and marine origin that have been shown to exhibit aromatase inhibitory activity is presented herein. PMID:18690828

  10. Blood and Diversity

    MedlinePlus

    ... Blood > Blood and Diversity Printable Version Blood and Diversity People come in all different shapes, sizes and ... groups. Therefore it is essential that the donor diversity match the patient diversity. For example, U-negative ...

  11. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  12. Genotype-dependent expression of specific members of potato protease inhibitor gene families in different tissues and in response to wounding and nematode infection.

    PubMed

    Turrà, David; Bellin, Diana; Lorito, Matteo; Gebhardt, Christiane

    2009-05-01

    Protease inhibitors (PIs) are small ubiquitous proteins with a variety of biological functions in plants, including protein stabilization, modulation of apoptosis and defense against pathogens. Kunitz-like inhibitors (PKPIs) and proteinase inhibitors 1 (PI-1) are abundant in storage organs of potato plants and are up-regulated in other tissues in response to biotic and abiotic stress. However, little information is available on genotype-dependent regulation of individual PKPI group- and PI-1 genes. We isolated, sequenced and characterized four novel full-length PI-1 cDNAs (PPI3A2, PPI3A4, PPI2C4 and PPI2C1A) from Solanum tuberosum cv. Desirée. Specific primers were developed for PI-1 genes PPI3A2, PPI3B2 and PPI2C4 and the three PKPI homology groups A, B and C. Their expression profiles were studied by semi-quantitative RT-PCR in comparison with transcripts of the PI-1, Pin2 and PR1 gene families in various tissues, after wounding and Globodera rostochiensis infection of nematode-resistant genotypes P40 and LB7/4/c-I-7, and susceptible cv. Desirée. Individual PI-1 genes and PKPI homology groups were expressed in a tissue- and genotype-dependent manner after wounding and nematode infection. The differences in PI expression patterns were related to the intensity, type of inhibitors produced, and the kinetics of induction. Therefore, different genotype-environment combinations produce different sets of PI transcripts. Potato plants reacted to G. rostochiensis infection by modulating PKPI, PI-1 and Pin2, but not PR1 gene expression, suggesting that the jasmonic acid but not the salicylic acid defense signaling pathway is activated. PI expression profiles were not correlated with the resistance status of the potato genotype infected with G. rostochiensis.

  13. 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.

  14. Purification and Partial Characterization of Trypsin-Specific Proteinase Inhibitors from Pigeonpea Wild Relative Cajanus platycarpus L. (Fabaceae) Active against Gut Proteases of Lepidopteran Pest Helicoverpa armigera

    PubMed Central

    Swathi, Marri; Mishra, Prashant K.; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Dutta-Gupta, Aparna; Padmasree, Kollipara

    2016-01-01

    Proteinase inhibitors (PIs) are natural defense proteins of plants found to be active against gut proteases of various insects. A pigeonpea wild relative Cajanus platycarpus was identified as a source of resistance against Helicoverpa armigera, a most devastating pest of several crops including pigeonpea. In the light of earlier studies, trypsin-specific PIs (CpPI 63) were purified from mature dry seeds of C. platycarpus (ICPW-63) and characterized their biochemical properties in contributing to H. armigera resistance. CpPI 63 possessed significant H. armigera gut trypsin-like proteinase inhibitor (HGPI) activity than trypsin inhibitor (TI) activity. Analysis of CpPI 63 using two-dimensional (2-D) electrophoresis and matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that it contained several isoinhibitors and small oligomers with masses ranging between 6 and 58 kDa. The gelatin activity staining studies suggest that these isoinhibitors and oligomers possessed strong inhibitory activity against H. armigera gut trypsin-like proteases (HGPs). The N-terminal sequence of the isoinhibitors (pI 6.6 and pI 5.6) of CpPI 63 exhibited 80% homology with several Kunitz trypsin inhibitors (KTIs) as well as miraculin-like proteins (MLPs). Further, modification of lysine residue(s) lead to 80% loss in both TI and HGPI activities of CpPI 63. In contrast, the TI and HGPI activities of CpPI 63 were stable over a wide range of temperature and pH conditions. The reported results provide a biochemical basis for pod borer resistance in C. platycarpus. PMID:27656149

  15. Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom

    PubMed Central

    Vivas, Jeilyn; Ibarra, Carlos; Salazar, Ana M.; Neves-Ferreira, Ana G.C.; Sánchez, Elda E.; Perales, Jonás; Rodríguez-Acosta, Alexis; Guerrero, Belsy

    2015-01-01

    A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542 Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5 nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1 nM). Aprotinin (2 nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05 nM) was inhibited by 67% following incubation with TP1 (0.1 nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2 nM) acts like aprotinin (0.4 nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed. PMID:26419785

  16. Conformational Lability in Serine Protease Active Sites: Structures of Hepatocyte Growth Factor Activator (HGFA) Alone and with the Inhibitory Domain from HGFA Inhibitor-1B

    SciTech Connect

    Shia, Steven; Stamos, Jennifer; Kirchhofer, Daniel; Fan, Bin; Wu, Judy; Corpuz, Raquel T.; Santell, Lydia; Lazarus, Robert A.; Eigenbrot, Charles

    2010-07-20

    Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 {angstrom} resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 {angstrom} resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.

  17. Structure based design of 11β-HSD1 inhibitors.

    PubMed

    Singh, Suresh; Tice, Colin

    2010-11-01

    Controlling elevated tissue-specific levels of cortisol may provide a novel therapeutic approach for treating metabolic syndrome. This concept has spurred large scale medicinal chemistry efforts in the pharmaceutical industry for the design of 11β-HSD1 inhibitors. High resolution X-ray crystal structures of inhibitors in complex with the enzyme have facilitated the structure-based design of diverse classes of molecules. A summary of binding modes, trends in structure-activity relationships, and the pharmacodynamic data of inhibitors from each class is presented.

  18. Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

    PubMed

    Okuzumi, Tatsuya; Ducker, Gregory S; Zhang, Chao; Aizenstein, Brian; Hoffman, Randy; Shokat, Kevan M

    2010-08-01

    The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

  19. Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition.

    PubMed

    de Veer, Simon J; Swedberg, Joakim E; Akcan, Muharrem; Rosengren, K Johan; Brattsand, Maria; Craik, David J; Harris, Jonathan M

    2015-07-15

    Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

  20. CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR

    PubMed Central

    Kunitz, M.

    1947-01-01

    A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ. PMID:19873496

  1. Diverse Classrooms, Diverse Curriculum, Diverse Complications: Three Teacher Perspectives

    ERIC Educational Resources Information Center

    Ungemah, Lori D.

    2015-01-01

    Racial, ethnic, linguistic, and religious diversity continues to increase in classrooms. Many call for a more diverse curriculum, but curricular diversity brings its own challenges to both teachers and students. These three vignettes are drawn from my ethnographic data at Atlantic High School in Brooklyn, New York, where I worked for ten years as…

  2. Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

    PubMed Central

    Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

    2014-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

  3. Footprinting of Inhibitor Interactions of In Silico Identified Inhibitors of Trypanothione Reductase of Leishmania Parasite

    PubMed Central

    Venkatesan, Santhosh K.; Dubey, Vikash Kumar

    2012-01-01

    Structure-based virtual screening of NCI Diversity set II compounds was performed to indentify novel inhibitor scaffolds of trypanothione reductase (TR) from Leishmania infantum. The top 50 ranked hits were clustered using the AuPoSOM tool. Majority of the top-ranked compounds were Tricyclic. Clustering of hits yielded four major clusters each comprising varying number of subclusters differing in their mode of binding and orientation in the active site. Moreover, for the first time, we report selected alkaloids and dibenzothiazepines as inhibitors of Leishmania infantum TR. The mode of binding observed among the clusters also potentiates the probable in vitro inhibition kinetics and aids in defining key interaction which might contribute to the inhibition of enzymatic reduction of T[S] 2. The method provides scope for automation and integration into the virtual screening process employing docking softwares, for clustering the small molecule inhibitors based upon protein-ligand interactions. PMID:22550471

  4. Aminodeoxychorismate synthase inhibitors from one-bead one-compound combinatorial libraries: "staged" inhibitor design.

    PubMed

    Dixon, Seth; Ziebart, Kristin T; He, Ze; Jeddeloh, Melissa; Yoo, Choong Leol; Wang, Xiaobing; Lehman, Alan; Lam, Kit S; Toney, Michael D; Kurth, Mark J

    2006-12-14

    4-Amino-4-deoxychorismate synthase (ADCS) catalyzes the first step in the conversion of chorismate into p-aminobenzoate, which is incorporated into folic acid. We aim to discover compounds that inhibit ADCS and serve as leads for a new class of antimicrobial compounds. This report presents (1) synthesis of a mass-tag encoded library based on a "staged" design, (2) massively parallel fluorescence-based on-bead screening, (3) rapid structural identification of hits, and (4) full kinetic analysis of ADCS. All inhibitors are competitive against chorismate and Mg(2+). The most potent ADCS inhibitor identified has a K(i) of 360 microM. We show that the combinatorial diversity elements add substantial binding affinity by interacting with residues outside of but proximal to the active site. The methods presented here constitute a paradigm for inhibitor discovery through active site targeting, enabled by rapid library synthesis, facile massively parallel screening, and straightforward hit identification.

  5. Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: insight into structural and antimalarial features.

    PubMed

    Bhattacharyya, Arindam; Babu, Cherukuri R

    2009-04-01

    A potent serine proteinase inhibitor was isolated and characterized from the seeds of the tropical legume liana, Derris trifoliata (DtTCI) by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. SDS-PAGE as well as MALDI-TOF analysis showed that DtTCI is a single polypeptide chain with a molecular mass of approximately 20 kDa. DtTCI has three isoinhibitors (pI: 4.55, 5.34 and 5.72) and, inhibited both trypsin and chymotrypsin in a 1:1 molar ratio. Both Dixon plots and Lineweaver-Burk double reciprocal plots revealed a competitive inhibition of trypsin and chymotrypsin activity, with inhibition constants (K(i)) of 1.7x10(-10) and 1.25x10(-10) M, respectively. N-terminal sequence of DtTCI showed over 50% similarity with numerous Kunitz-type inhibitors of the Papilionoideae subfamily. High pH amplitude and broad temperature optima were noted for DtTCI, and time course experiments indicated a gradual loss in inhibitory potency on treatment with dithiothreitol (DTT). Circular Dichroism (CD) spectrum of native DtTCI revealed an unordered structure whereas exposure to thermal-pH extremes, DTT and guanidine hydrochloride (Gdn HCl) suggested that an abundance of beta-sheets along with intramolecular disulfide bonds provide conformational stability to the active site of DtTCI, and that severity of denaturants cause structural modifications promoting inhibitory inactivity. Antimalarial studies of DtTCI indicate it to be a potent antiparasitic agent.

  6. Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.

    PubMed

    Oliva, M L; Souza-Pinto, J C; Batista, I F; Araujo, M S; Silveira, V F; Auerswald, E A; Mentele, R; Eckerskorn, C; Sampaio, M U; Sampaio, C A

    2000-03-07

    A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

  7. Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

    PubMed Central

    Chang, Hsiang-Hua D.; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J.; Pan, Yu; Zhou, Emily; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells. PMID:25786220

  8. Differential subcellular localization renders HAI-2 a matriptase inhibitor in breast cancer cells but not in mammary epithelial cells.

    PubMed

    Chang, Hsiang-Hua D; Xu, Yuan; Lai, Hongyu; Yang, Xiaoyu; Tseng, Chun-Che; Lai, Ying-Jung J; Pan, Yu; Zhou, Emily; Johnson, Michael D; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.

  9. The Chief Diversity Officer

    ERIC Educational Resources Information Center

    Williams, Damon; Wade-Golden, Katrina

    2007-01-01

    Numerous institutions are moving toward the chief diversity officer model of leading and managing diversity in higher education. These officers carry formal administrative titles and ranks that range from vice president for institutional diversity to associate vice chancellor for diversity and climate and dean of diversity and academic engagement.…

  10. Therapeutic potential of monoacylglycerol lipase inhibitors.

    PubMed

    Mulvihill, Melinda M; Nomura, Daniel K

    2013-03-19

    Marijuana and aspirin have been used for millennia to treat a wide range of maladies including pain and inflammation. Both cannabinoids, like marijuana, that exert anti-inflammatory action through stimulating cannabinoid receptors, and cyclooxygenase (COX) inhibitors, like aspirin, that suppress pro-inflammatory eicosanoid production have shown beneficial outcomes in mouse models of neurodegenerative diseases and cancer. Both cannabinoids and COX inhibitors, however, have untoward effects that discourage their chronic usage, including cognitive deficits and gastrointestinal toxicity, respectively. Recent studies have uncovered that the serine hydrolase monoacylglycerol lipase (MAGL) links the endocannabinoid and eicosanoid systems together through hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) to provide the major arachidonic acid (AA) precursor pools for pro-inflammatory eicosanoid synthesis in specific tissues. Studies in recent years have shown that MAGL inhibitors elicit anti-nociceptive, anxiolytic, and anti-emetic responses and attenuate precipitated withdrawal symptoms in addiction paradigms through enhancing endocannabinoid signaling. MAGL inhibitors have also been shown to exert anti-inflammatory action in the brain and protect against neurodegeneration through lowering eicosanoid production. In cancer, MAGL inhibitors have been shown to have anti-cancer properties not only through modulating the endocannabinoid-eicosanoid network, but also by controlling fatty acid release for the synthesis of protumorigenic signaling lipids. Thus, MAGL serves as a critical node in simultaneously coordinating multiple lipid signaling pathways in both physiological and disease contexts. This review will discuss the diverse (patho)physiological roles of MAGL and the therapeutic potential of MAGL inhibitors in treating a vast array of complex human diseases.

  11. The diamondback moth, Plutella xylostella, specifically inactivates Mustard Trypsin Inhibitor 2 (MTI2) to overcome host plant defence.

    PubMed

    Yang, Limei; Fang, Zhiyuan; Dicke, Marcel; van Loon, Joop J A; Jongsma, Maarten A

    2009-01-01

    The mustard trypsin inhibitor family has so far only been described among cruciferous species which represent the host plants for the specialist diamondback moth (DBM), Plutella xylostella. The performance of a Dutch and Chinese strain of DBM was assessed on transgenic Arabidopsis expressing Mustard Trypsin Inhibitor 2 (MTI2) at a level of 84 microg/g fresh weight equivalent to 12 microM. No significant differences in larval mortality or development were found relative to the control. Trypsin activity in gut extracts from larvae feeding on either control or transgenic plants were titrated with MTI2 and SKTI (Soybean Kunitz Trypsin Inhibitor) to assess the basis of the insensitivity to MTI2. The specific trypsin activity per gut of larvae reared on MTI2 plants was not significantly higher compared to the control, and ca. 80% of trypsin activity could be inhibited by both inhibitors in both treatments, suggesting no specific induction of PI-insensitive activity in response to MTI2 in the diet. On the basis of the apparent equilibrium dissociation constant of Plutella trypsins for MTI2 (80 nM), the gut trypsin concentration (4.8 microM), and the MTI2 concentration in the leaves (12 microM) it was calculated that 99% of the gut trypsin activity sensitive to MTI2 should be inhibited in vivo, unless MTI2 was degraded. Indeed, we found that a pre-incubation of MTI2 and SKTI with gut proteases for 3 h resulted in complete loss of inhibitory activity of MTI2, but not of SKTI, at the concentration ratios found in planta. This process was enzymatic as it was inactivated by heat. Gut extracts of larvae reared on control or MTI2 leaves were equally well capable of this degradation indicating that the inactivating enzymes are constitutively expressed. In conclusion, it appears that the insensitivity of the diamondback moth to MTI2 can be sufficiently explained by the specific degradation of MTI2, thereby protecting itself against this protease inhibitor which is part of the

  12. Calmodulin inhibitors from natural sources: an update.

    PubMed

    Mata, Rachel; Figueroa, Mario; González-Andrade, Martín; Rivera-Chávez, José Alberto; Madariaga-Mazón, Abraham; Del Valle, Paulina

    2015-03-27

    Calmodulin (CaM) plays a central role in regulating a myriad of cellular functions in physiological and pathophysiological processes, thus representing an important drug target. In previous reviews, our group has reported relevant information regarding natural anti-CaM compounds up to 2009. Natural sources continue to provide a diverse and unique reservoir of CaM inhibitors for drug and research tool discovery. This review provides an update of natural products with reported CaM inhibitory properties, which includes around 70 natural products and some synthetic analogues, belonging to different structural classes. Most of these natural inhibitors were isolated from fungi and plants and belong to the stilbenoid, polyketide, alkaloid, and peptide structural classes. These products were discovered mainly using a fluorescence-based method on rationally designed biosensors, which are highly specific, low-cost, and selective and have short reaction times. The effect of several antimitotic drugs on Ca(2+)-hCaM is also described.

  13. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM.

  14. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  15. Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds.

    PubMed

    Oliva, M L; Andrade, S A; Batista, I F; Sampaio, M U; Juliano, M; Fritz, H; Auerswald, E A; Sampaio, C A

    1999-12-01

    Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki = 80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km = 0.68 microM, k(cat)/Km = 1.3 x 10(6) M(-1) s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km = 0.66 microM, k(cat)/Km = 2.2 x 10(3) M(-1) s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

  16. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  17. Understanding the mechanisms of aromatase inhibitor resistance

    PubMed Central

    2012-01-01

    Aromatase inhibitors (AIs) have a central role in the treatment of breast cancer; however, resistance is a major obstacle to optimal management. Evidence from endocrine, molecular and pathological measurements in clinical material taken before and after therapy with AIs and data from clinical trials in which AIs have been given as treatment either alone or in combination with other targeted agents suggest diverse causes for resistance. These include inherent tumour insensitivity to oestrogen, ineffective inhibition of aromatase, sources of oestrogenic hormones independent of aromatase, activation of signalling by non-endocrine pathways, enhanced cell survival and selection of hormone-insensitive cellular clones during treatment. PMID:22277572

  18. 10 Diversity Champions II

    ERIC Educational Resources Information Center

    Nealy, Michelle J.; Pluviose, David; Roach, Ronald

    2008-01-01

    Introducing the "Champions of Diversity" in the Academic Kickoff issue proved a timely reminder of the mission of Diverse during the lead up to the 25th anniversary of Cox, Matthews and Associates, the founder of the former Black Issues in Higher Education and publisher of Diverse. In this edition, the editors at Diverse unveil its second slate of…

  19. Concepts of Diversity.

    ERIC Educational Resources Information Center

    Jacklin, Phil

    This paper attempts to establish a theory of communication essential to democratic diversity. Twelve kinds of diversity, divided into two classes, are described. One class relates to the way in which diverse things differ, the other class relates to the kinds of things which are diverse. The criteria for evaluating the importance of a certain kind…

  20. DNA Methyltransferases Inhibitors from Natural Sources.

    PubMed

    Zwergel, Clemens; Valente, Sergio; Mai, Antonello

    2016-01-01

    DNA methyltransferases (DNMTs) catalyze the methylation at cytosine-C5 mainly in a CpG dinucleotide context. Although DNA methylation is essential for fundamental processes like embryonic development or differentiation, aberrant expression and/or activities of DNMTs are involved in several pathologies, from neurodegeneration to cancer. DNMTs inhibition can arrest tumor growth, cells invasiveness and induce differentiation, whereas their increased expression is shown in numerous cancer types. Moreover, hypermethylated promoters of tumor suppressor genes lead to their silencing. Hence, the use of specific inhibitors of DNMT might reactivate those genes and stop or even reverse the aberrant cell processes. To date, the only approved DNMTs inhibitors for therapy belong to the nucleoside-based family of drugs, but they display relevant side effects as well as high chemical instability. Thus, there is a keen interest actually exists to develop novel, potent and safe inhibitors possessing a nonnucleoside structure. Increasing literature evidence is highlighting that natural sources could help the researchers to achieve this goal. Indeed, several polyphenols, flavonoids, antraquinones, and others are described able to inhibit DNMTs activity and/or expression, thus decreasing the methylation/silencing of different genes involved in tumorigenesis. These events can lead to re-expression of such genes and to cell death in diverse cancer cell lines. Epigallocatechin-3-gallate (1) and laccaic acid A (11) resulted the most effective DNMT1 inhibitors with submicromolar IC50 values, acting as competitive inhibitors. Compound 1 and 11 both displayed gene demethylation and re-activation in several cancers. However, all of the natural compounds described in this review showed important results, from gene reactivation to cell growth inhibition. Moreover, some of them displayed interesting activity even in rodent cancer models and very recently entered clinical trials.

  1. Thrombin inhibitor design.

    PubMed

    Sanderson, P E; Naylor-Olsen, A M

    1998-08-01

    Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.

  2. Diversity Statements: How Faculty Applicants Address Diversity

    ERIC Educational Resources Information Center

    Schmaling, Karen B.; Trevino, Amira Y.; Lind, Justin R.; Blume, Arthur W.; Baker, Dana L.

    2015-01-01

    The purpose of the present study was to examine application materials for assistant professor positions in 3 academic disciplines. Applicants were asked to write a diversity statement describing how they would advance diversity through their research, teaching, and service. The sample included application materials submitted by 191 candidates for…

  3. Does Staff Diversity Imply Openness to Diversity?

    ERIC Educational Resources Information Center

    Lauring, Jakob; Selmer, Jan

    2013-01-01

    Purpose: Post-secondary educational organizations are currently some of the most diverse settings to be found. However, few educational studies have dealt with staff diversity and hardly any has looked outside the USA. The purpose of this paper is to present a study of members of international university departments in Denmark. The authors set out…

  4. Capturing the Diversity in Lexical Diversity

    ERIC Educational Resources Information Center

    Jarvis, Scott

    2013-01-01

    The range, variety, or diversity of words found in learners' language use is believed to reflect the complexity of their vocabulary knowledge as well as the level of their language proficiency. Many indices of lexical diversity have been proposed, most of which involve statistical relationships between types and tokens, and which ultimately…

  5. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  6. [SGLT2 inhibitor].

    PubMed

    Kubota, Naoto; Kadowaki, Takashi

    2015-12-01

    SGLT2 is a glucose transporter which plays an important role for reabsorption of urinary glucose depending on the sodium concentration gradient. SGLT2 is mainly present in apical site of S1 segment of renal proximal tubule and accounts for approximately 90% of total urinary glucose reabsorption. SLC5a2, which codes SGLT2, is also known as the causative gene of familial renal glucosuria. SGLT2 inhibitors are attracting attention as newly developed oral anti-diabetic agents which improve glucose intolerance and also have an anti-obese effect by promoting urinary glucose excretion (UGE), which is a different pharmacological effect from other conventional anti-diabetic agents. In this review, we will discuss the effect of SGLT2 inhibitor on the regulation of glucose and lipid metabolism in type 2 diabetes.

  7. A shrimp pacifastin light chain-like inhibitor: molecular identification and role in the control of the prophenoloxidase system.

    PubMed

    Sangsuriya, Pakkakul; Charoensapsri, Walaiporn; Chomwong, Sudarat; Senapin, Saengchan; Tassanakajon, Anchalee; Amparyup, Piti

    2016-01-01

    Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp.

  8. Development of scale inhibitors

    SciTech Connect

    Gill, J.S.

    1996-12-01

    During the last fifty years, scale inhibition has gone from an art to a science. Scale inhibition has changed from simple pH adjustment to the use of optimized dose of designer polymers from multiple monomers. The water-treatment industry faces many challenges due to the need to conserve water, availability of only low quality water, increasing environmental regulations of the water discharge, and concern for human safety when using acid. Natural materials such as starch, lignin, tannin, etc., have been replaced with hydrolytically stable organic phosphates and synthetic polymers. Most progress in scale inhibition has come from the use of synergistic mixtures and copolymerizing different functionalities to achieve specific goals. Development of scale inhibitors requires an understanding of the mechanism of crystal growth and its inhibition. This paper discusses the historic perspective of scale inhibition and the development of new inhibitors based on the understanding of the mechanism of crystal growth and the use of powerful tools like molecular modeling to visualize crystal-inhibitor interactions.

  9. Piperazine and piperidine carboxamides and carbamates as inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Korhonen, Jani; Kuusisto, Anne; van Bruchem, John; Patel, Jayendra Z; Laitinen, Tuomo; Navia-Paldanius, Dina; Laitinen, Jarmo T; Savinainen, Juha R; Parkkari, Teija; Nevalainen, Tapio J

    2014-12-01

    The key hydrolytic enzymes of the endocannabinoid system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), are potential targets for various therapeutic applications. In this paper, we present more extensively the results of our previous work on piperazine and piperidine carboxamides and carbamates as FAAH and MAGL inhibitors. The best compounds of these series function as potent and selective MAGL/FAAH inhibitors or as dual FAAH/MAGL inhibitors at nanomolar concentrations. This study revealed that MAGL inhibitors should comprise leaving-groups with a conjugate acid pKa of 8-10, while diverse leaving groups are tolerated for FAAH inhibitors.

  10. Molecular cloning, bioinformatics analysis and functional characterization of HWTX-XI toxin superfamily from the spider Ornithoctonus huwena.

    PubMed

    Jiang, Liping; Deng, Meichun; Duan, Zhigui; Tang, Xing; Liang, Songping

    2014-04-01

    Spider venom contains a very valuable repertoire of natural resources to discover novel components for molecular diversity analyses and therapeutic applications. In this study, HWTX-XI toxins from the spider venom glands of Ornithoctonus huwena which are Kunitz-type toxins (KTTs) and were directly cloned, analyzed and functionally characterized. To date, the HWTX-XI superfamily consists of 38 members deduced from 121 high-quality expressed sequence tags, which is the largest spider KTT superfamily with significant molecular diversity mainly resulted from cDNA tandem repeats as well as focal hypermutation. Among them, HW11c40 and HW11c50 may be intermediate variants between native Kunitz toxins and sub-Kunitz toxins based on evolutionary analyses. In order to elucidate their biological activities, recombinant HW11c4, HW11c24, HW11c27 and HW11c39 were successfully expressed, further purified and functionally characterized. Both HW11c4 and HW11c27 display inhibitory activities against trypsin, chymotrypsin and kallikrein. Moreover, HW11c4 is also an inhibitor relatively specific for Kv1.1 channels. HW11c24 and HW11c39 are found to be inactive on chymotrysin, trypsin, kallikrein, thrombin and ion channels. These findings provide molecular evidence for toxin diversification of the HWTX-XI superfamily and useful molecular templates of serine protease inhibitors and ion channel blockers for the development of potentially clinical applications.

  11. NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

    PubMed

    Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

    2013-01-01

    In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

  12. Leadership and Diversity

    ERIC Educational Resources Information Center

    Coleman, Marianne

    2012-01-01

    As part of the special edition recognizing the 40th anniversary of "Educational Management Administration & Leadership" this article reviews the coverage of leadership and diversity issues in the journal. The majority of articles concerning diversity have focused on gender, with attention turning to the wider concept of diversity since the year…

  13. Insights on Diversity.

    ERIC Educational Resources Information Center

    Bloom, Carol, Ed.; And Others

    This state-of-the-art report presents a series of essays on the topic of diversity. Essays include: (1) "Committing to Diversity" (George L. Mehaffy); (2) "Serving the Community by Serving Our Members" (Michael P. Wolfe); (3) "How Diversity Matters" (Asa G. Hilliard, III); (4) "A Prerequisite to Teaching Multiculturally" (Mary Louise Gomez); (5)…

  14. BioDiversity.

    ERIC Educational Resources Information Center

    Wilson, E. O., Ed.; Peter, Frances M., Ed.

    The diversity of life forms is one of the greatest wonders of the planet earth. The biosphere is an intricate tapestry of interwoven life forms. This book offers an overall view of this biological diversity and carries an urgent warning about the rapid alteration and destruction of the environments that have fostered the diversity of life forms…

  15. Multilevel and Diverse Classrooms

    ERIC Educational Resources Information Center

    Baurain, Bradley, Ed.; Ha, Phan Le, Ed.

    2010-01-01

    The benefits and advantages of classroom practices incorporating unity-in-diversity and diversity-in-unity are what "Multilevel and Diverse Classrooms" is all about. Multilevel classrooms--also known as mixed-ability or heterogeneous classrooms--are a fact of life in ESOL programs around the world. These classrooms are often not only…

  16. ACAT inhibitors: the search for novel cholesterol lowering agents.

    PubMed

    Pal, Palash; Gandhi, Hardik; Giridhar, Rajani; Yadav, Mange Ram

    2013-06-01

    Increased level of serum cholesterol (hyperlipidemia) is the most significant risk factor for the development of atherosclerosis. Cholesterol levels are affected by factors such as rate of endogenous cholesterol synthesis, biliary cholesterol excretion and dietary cholesterol absorption. Acyl CoA: Cholesterol O-acyl transferases (ACAT) are a small family of enzymes that catalyze cholesterol esterification and cholesterol absorption in intestinal mucosal cells and maintain the cholesterol homeostasis in the blood. Inhibition of the ACAT enzymes is one of the attractive targets to treat hyperlipidemia. Literature survey shows that structurally diverse compounds possess ACAT inhibitory properties. In this review, a comprehensive presentation of the literature on diverse ACAT inhibitors has been given.

  17. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  18. Discovery of potent, selective chymase inhibitors via fragment linking strategies.

    PubMed

    Taylor, Steven J; Padyana, Anil K; Abeywardane, Asitha; Liang, Shuang; Hao, Ming-Hong; De Lombaert, Stéphane; Proudfoot, John; Farmer, Bennett S; Li, Xiang; Collins, Brandon; Martin, Leslie; Albaugh, Daniel R; Hill-Drzewi, Melissa; Pullen, Steven S; Takahashi, Hidenori

    2013-06-13

    Chymase plays an important and diverse role in the homeostasis of a number of cardiovascular processes. Herein, we describe the identification of potent, selective chymase inhibitors, developed using fragment-based, structure-guided linking and optimization techniques. High-concentration biophysical screening methods followed by high-throughput crystallography identified an oxindole fragment bound to the S1 pocket of the protein exhibiting a novel interaction pattern hitherto not observed in chymase inhibitors. X-ray crystallographic structures were used to guide the elaboration/linking of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over cathepsin G and that mitigated a number of liabilities associated with poor physicochemical properties of the series it was derived from.

  19. Structure and mechanism of action of tau aggregation inhibitors

    PubMed Central

    Cisek, Katryna; Cooper, Grace L.; Huseby, Carol J.; Kuret, Jeff

    2015-01-01

    Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimer’s disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects. PMID:25387336

  20. Histone deacetylase inhibitor trichostatin A and proteasome inhibitor PS-341 synergistically induce apoptosis in pancreatic cancer cells

    SciTech Connect

    Bai Jirong . E-mail: jbai@bidmc.harvard.edu; Demirjian, Aram; Sui Jianhua; Marasco, Wayne; Callery, Mark P. . E-mail: mcallery@bidmc.harvard.ede

    2006-10-06

    Pancreatic cancer is a common and lethal malignancy. Pancreatic cancer cells overexpress multiple anti-apoptotic factors and death receptor decoys, and are strongly resistant to radiation and to 5-fluorouracil (5-FU)- or gemcitabine (Gem)-based chemotherapy regimens. We have found that low-dose proteasome inhibitor PS-341 and histone deacetylase inhibitor trichostatin A (TSA) synergistically induce cytotoxicity in a panel of eight diverse pancreatic cancer cell lines. Combining TSA with PS-341 effectively inactivated NF{kappa}B signaling, downregulated the predominant endogenous anti-apoptotic factor Bcl-XL overexpression, and disrupted MAP kinase pathway. The combined drug regimen effectively inflicted an average of 71.5% apoptotic cell death (55.2-80%) in diverse pancreatic cancer cell lines by activating the intrinsic apoptotic pathway. Conclusion: the TSA/PS-341 regimen may represent a potential novel therapeutic strategy for pancreatic cancer.

  1. Combined Inhibitor Free-Energy Landscape and Structural Analysis Reports on the Mannosidase Conformational Coordinate**

    PubMed Central

    Williams, Rohan J; Iglesias-Fernández, Javier; Stepper, Judith; Jackson, Adam; Thompson, Andrew J; Lowe, Elisabeth C; White, Jonathan M; Gilbert, Harry J; Rovira, Carme; Davies, Gideon J; Williams, Spencer J

    2014-01-01

    Mannosidases catalyze the hydrolysis of a diverse range of polysaccharides and glycoconjugates, and the various sequence-based mannosidase families have evolved ingenious strategies to overcome the stereoelectronic challenges of mannoside chemistry. Using a combination of computational chemistry, inhibitor design and synthesis, and X-ray crystallography of inhibitor/enzyme complexes, it is demonstrated that mannoimidazole-type inhibitors are energetically poised to report faithfully on mannosidase transition-state conformation, and provide direct evidence for the conformational itinerary used by diverse mannosidases, including β-mannanases from families GH26 and GH113. Isofagomine-type inhibitors are poor mimics of transition-state conformation, owing to the high energy barriers that must be crossed to attain mechanistically relevant conformations, however, these sugar-shaped heterocycles allow the acquisition of ternary complexes that span the active site, thus providing valuable insight into active-site residues involved in substrate recognition. PMID:24339341

  2. Sequencing of aromatase inhibitors

    PubMed Central

    Bertelli, G

    2005-01-01

    Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain prolonged clinical benefit. Many questions remain, however, as to the best sequence of the two types of AIs and of the other available agents, including tamoxifen and fulvestrant, in different patient groups. PMID:16100523

  3. Sirtuin activators and inhibitors

    PubMed Central

    Villalba, José M.; Alcaín, Francisco J.

    2012-01-01

    Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD+ for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activity. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin-based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging-related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex®, SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases. PMID:22730114

  4. Proteinase inhibitor homologues as potassium channel blockers.

    PubMed

    Lancelin, J M; Foray, M F; Poncin, M; Hollecker, M; Marion, D

    1994-04-01

    We report here the NMR structure of dendrotoxin I, a powerful potassium channel blocker from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (black mamba), calculated from an experimentally-derived set of 719 geometric restraints. The backbone of the toxin superimposes on bovine pancreatic trypsin inhibitor (BPTI) with a root-mean-square deviation of < 1.7 A. The surface electrostatic potential calculated for dendrotoxin I and BPTI, reveal an important difference which might account for the differences in function of the two proteins. These proteins may provide examples of adaptation for specific and diverse biological functions while at the same time maintaining the overall three-dimensional structure of a common ancestor.

  5. Biological abatement of cellulase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  6. Fragment-based discovery of BACE1 inhibitors using functional assays.

    PubMed

    Godemann, Robert; Madden, James; Krämer, Joachim; Smith, Myron; Fritz, Ulrike; Hesterkamp, Thomas; Barker, John; Höppner, Sabine; Hallett, David; Cesura, Andrea; Ebneth, Andreas; Kemp, John

    2009-11-17

    Novel nonpeptidic inhibitors of beta-secretase (BACE1) have been discovered by employing a fragment-based biochemical screening approach. A diverse library of 20000 low-molecular weight compounds were screened and yielded 26 novel hits that were confirmed by biochemical and surface plasmon resonance secondary assays. We describe here fragment inhibitors cocrystallized with BACE1 in a flap open and flap closed conformation as determined by X-ray crystallography.

  7. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  8. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

    PubMed

    Bai, Fengwei; Asojo, Oluwatoyin A; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A; Leng, Lin; Koski, Raymond A; Powell, Thomas J; Bucala, Richard; Anthony, Karen G

    2012-08-31

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

  9. Platelet aggregation Inhibitors from Hametophagous Animals

    PubMed Central

    Francischetti, Ivo M. B.

    2010-01-01

    Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sandflies, fleas, ticks, leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis in a redundant and synergistic manner. This review discusses recent progress in the identification of salivary inhibitors of platelet aggregation, their molecular characterization, and detailed mechanism of action. Diversity of inhibitors is remarkable, with distinct families of proteins characterized as apyrases that enzymatically degrade ADP or as collagen-binding proteins that prevent its interaction with vWF, or platelet integrin α2β1 or GPVI. Molecules that bind ADP, TXA2, epinephrine, or serotonin with high affinity have also been cloned, expressed, and their structure determined. In addition, a repertoire of antithrombins and an increasingly number of RGD and non-RGD disintegrins targeting platelet αIIbβ3 have been reported. Moreover, metalloproteases with fibrinogen(olytic) activity and PAF phosphorylcholine hydrolase are enzymes that have been recruited to the salivary gland to block platelet aggregation. Platelet inhibitory prostaglandins, lysophosphatydilcholine, adenosine, and nitric oxide (NO)-carrying proteins are other notable examples of molecules from hematophagous salivary secretions (herein named sialogenins) with antihemostatic properties. Sialogenins have been employed as tools in biochemistry and cell biology and also display potential therapeutic applications. PMID:20035779

  10. On the interaction of bovine pancreatic trypsin inhibitor with maxi Ca(2+)-activated K+ channels. A model system for analysis of peptide- induced subconductance states

    PubMed Central

    1991-01-01

    Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue basic peptide that is a representative member of a widely distributed class of serine protease inhibitors known as Kunitz inhibitors. BPTI is also homologous to dendrotoxin peptides from mamba snake venom that have been characterized as inhibitors of various types of voltage-dependent K+ channels. In this study we compared the effect of DTX-I, a dendrotoxin peptide, and BPTI on large conductance Ca(2+)-activated K+ channels from rat skeletal muscle using planar bilayer methodology. As previously found for DTX-I (1990. Neuron. 2:141-148), BPTI induces the appearance of distinct subconductance events when present on the internal side of maxi K(Ca) channels. The single channel kinetics of substate formation follow the predictions of reversible binding of the peptide to a single site or class of sites with a Kd of 4.6 microM at 0 mV and 50 mM symmetrical KCl. The apparent association rate of BPTI binding decreases approximately 1,000-fold per 10-fold increase in ionic strength, suggestive of a strong electrostatic interaction between the basic peptide and negative surface charge in the vicinity of the binding site. The equilibrium Kd for BPTI and DTX-I is also voltage dependent, decreasing e-fold per 30 mV of depolarization. The unitary subconductance current produced by BPTI binding exhibits strong inward rectification in the presence of symmetrical KCl, corresponding to 15% of open channel current at +60 mV and 70% of open state at -40 mV. In competition experiments, the internal pore-blocking ions, Ba2+ and TEA+, readily block the substate with the same affinity as that for blocking the normal open state. These results suggest that BPTI does not bind near the inner mouth of the channel so as to directly interfere with cation entry to the channel. Rather, the mechanism of substate production appears to involve a conformational change that affects the energetics of K+ permeation. PMID:1714938

  11. A Diversity Visionary

    ERIC Educational Resources Information Center

    Smith, Susan

    2012-01-01

    Today's chief diversity officer could be tomorrow's university president, says Dr. Damon Williams. The author profiles Damon Williams who shines as sought-after expert on issues surrounding higher education inclusion. As head of a diversity division with an eight-figure budget at Wisconsin's flagship state university, Williams oversees four…

  12. Dissecting Diversity Part II

    ERIC Educational Resources Information Center

    Matthews, Frank

    2005-01-01

    This article presents "Dissecting Diversity, Part II," the conclusion of a wide-ranging two-part roundtable discussion on diversity in higher education. The participants were as follows: Lezli Baskerville, J.D., President and CEO of the National Association for Equal Opportunity (NAFEO); Dr. Gerald E. Gipp, Executive Director of the…

  13. Past Planktonic Diversity

    NASA Astrophysics Data System (ADS)

    Rufino, M. M.; Salgueiro, E.; Voelker, A. H. L.; Abrantes, F. F. G.

    2014-12-01

    Planktonic organisms have been extensively used in paleoceanographic studies as proxies for most marine environmental variables (temperature, salinity, currents, frontal zones, upwelling, etc.), both directly by species occurrences and indirectly through particular chemical components produced (e.g. Mg/Ca, stable isotopes, alkanones). In 1965 Stehli pioneered by suggesting the use of planktonic organisms diversity to decipher ancient oceanic circulation, instead of the traditional approaches based on particular indicator species or assemblages composition (transfer functions). The use of species diversity has two main advantages. First, it is not restricted to a temporal epoch where the species existed and second, it does not assume that the species ecology is the same as in the present. In the current work, we compare planktonic organisms diversity on the Atlantic Ocean, obtained from surface samples, with the main satellite measured oceanographic variables, i.e. SST (Sea Surface Temperature), CHL (as an indicator of primary productivity) and the main currents in the area. Three indices were used to quantify diversity: Shannon-Weaver diversity (H), specific richness (S) and Hulbert's probability of interspecific encounter index of species evenness (PIE). Diversity was then modelled spatially using geostatistical tools at two scales: Atlantic Ocean oceanographic scale and the Iberian margin regional scale. The main conclusions will then be used to interpret measured down core diversity, on a paleo perspective. This work will understand how did diversity reacted to major climatic events, and how long it took to recover - system resilience.

  14. Global Diversity and Leadership.

    ERIC Educational Resources Information Center

    Ruiz, Art

    2003-01-01

    Argues that global diversity has become a business imperative in today's business climate. Global diversity is of core importance even for companies that are considered domestic. Suggests community colleges need help in understanding their customer base and their shifting values in order to meet their needs and win customer loyalty. (NB)

  15. Evolution & Diversity in Plants.

    ERIC Educational Resources Information Center

    Pearson, Lorentz C.

    1988-01-01

    Summarizes recent findings that help in understanding how evolution has brought about the diversity of plant life that presently exists. Discusses basic concepts of evolution, diversity and classification, the three-line hypothesis of plant evolution, the origin of fungi, and the geologic time table. Included are 31 references. (CW)

  16. Voices for Diversity.

    ERIC Educational Resources Information Center

    Future Teacher, 1995

    1995-01-01

    Prominent Americans were asked to reflect on the diversity challenge facing America's teacher workforce. The following leaders from several fields voiced their support of teachers and their beliefs America needs more diverse and culturally responsive teachers: (1) Mary Hatwood Futrell, President of Education International; (2) Carol Moseley-Braun,…

  17. Reconsidering the Diversity Rationale

    ERIC Educational Resources Information Center

    Chang, Mitchell J.

    2005-01-01

    The concept of diversity has come a long way in U.S. higher education, and its impact has been far reaching. Over the last three and a half decades, diversity and its related interventions have evolved to encompass a broad set of purposes, issues, and initiatives on college campuses. The earliest initiatives to increase minority access on…

  18. Diversity and Social Cohesion

    ERIC Educational Resources Information Center

    Pagani, Camilla

    2014-01-01

    The issue of diversity, in its broadest sense, is discussed here in its relation to social cohesion, cross-cultural relations, ingroup-outgroup relations and educational interventions. The main thesis of the paper is that real social cohesion in an ingroup rests on the acknowledgment of and the dialog with the diversities of the members of the…

  19. Diversity in Leadership

    ERIC Educational Resources Information Center

    Beer, Janet

    2015-01-01

    This paper presents a lecture given at the 17th Annual Lecture of the Association of University Administrators (AUA). The subject of the lecture is equality and diversity in higher education (HE) leadership, or possibly the absence of equality and diversity. The author focuses on what can be done to ensure that capable women enter HE leadership…

  20. Issue Brief on Diversity

    ERIC Educational Resources Information Center

    Division on Developmental Disabilities, Council for Exceptional Children (NJ1), 2013

    2013-01-01

    During the past year, the Diversity Committee of the Division of Developmental Disabilities (DDD) Board worked with the Board and the Issues Committee Chair to develop an issue brief addressing diversity, its impact on the membership and the wider community that is served by the work of DDD, resulting in recommendations that will influence policy…

  1. Advancing Diversity in STEM

    ERIC Educational Resources Information Center

    Hill, Paul L.; Shaw, Rose A.; Taylor, Jan R.; Hallar, Brittan L.

    2011-01-01

    Although progress has been made, greater efforts are needed to promote faculty diversity at the college and university levels, especially in STEM fields. Thus, it is important to elucidate best practices both for increasing awareness of diversity issues pertaining to higher education and for implementing change. This article focuses on the…

  2. Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli.

    PubMed

    Bønsager, Birgit C; Praetorius-Ibba, Mette; Nielsen, Peter K; Svensson, Birte

    2003-08-01

    Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.

  3. Cathepsin B Inhibitors: Combining Dipeptide Nitriles with an Occluding Loop Recognition Element by Click Chemistry

    PubMed Central

    2015-01-01

    An active site mapping of human cathepsin B with dipeptide nitrile inhibitors was performed for a combinatorial approach by introducing several points of diversity and stepwise optimizing the inhibitor structure. To address the occluding loop of cathepsin B by a carboxylate moiety, click chemistry to generate linker-connected molecules was applied. Inhibitor 17 exhibited Ki values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of the assay. Kinetic data were discussed with respect to the conformational selection and induced fit models. PMID:26985300

  4. Beyond the Diversity Crisis Model: Decentralized Diversity Planning and Implementation

    ERIC Educational Resources Information Center

    Williams, Damon A.

    2008-01-01

    This article critiques the diversity crises model of diversity planning in higher education and presents a decentralized diversity planning model. The model is based on interviews with the nation's leading diversity officers, a review of the literature and the authors own experiences leading diversity change initiatives in higher education. The…

  5. Climate, energy and diversity

    PubMed Central

    Clarke, Andrew; Gaston, Kevin J

    2006-01-01

    In recent years, a number of species–energy hypotheses have been developed to explain global patterns in plant and animal diversity. These hypotheses frequently fail to distinguish between fundamentally different forms of energy which influence diversity in dissimilar ways. Photosynthetically active radiation (PAR) can be utilized only by plants, though their abundance and growth rate is also greatly influenced by water. The Gibbs free energy (chemical energy) retained in the reduced organic compounds of tissue can be utilized by all heterotrophic organisms. Neither PAR nor chemical energy influences diversity directly. Both, however, influence biomass and/or abundance; diversity may then increase as a result of secondary population dynamic or evolutionary processes. Temperature is not a form of energy, though it is often used loosely by ecologists as a proxy for energy; it does, however, influence the rate of utilization of chemical energy by organisms. It may also influence diversity by allowing a greater range of energetic lifestyles at warmer temperatures (the metabolic niche hypothesis). We conclude that there is no single species/energy mechanism; fundamentally different processes link energy to abundance in plants and animals, and diversity is affected secondarily. If we are to make progress in elucidating these mechanisms, it is important to distinguish climatic effects on species' distribution and abundance from processes linking energy supply to plant and animal diversity. PMID:16928626

  6. Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock-key system to protect Acacia food bodies from exploitation.

    PubMed

    Orona-Tamayo, Domancar; Wielsch, Natalie; Blanco-Labra, Alejandro; Svatos, Ales; Farías-Rodríguez, Rodolfo; Heil, Martin

    2013-08-01

    Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two-dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz-type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin-like and elastase-like proteolytic activity in the guts of seed-feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase-like and to a lower degree chymotrypsin 1-like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term 'exclusive rewards' can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.

  7. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  8. Gender diversity in STEM

    NASA Astrophysics Data System (ADS)

    Beijerinck, Herman C. W.

    2017-03-01

    There is a strong business case for the value of diversity. Research by the World Economic Forum shows a 36% higher return on equity (ROE) for companies having a workforce with strong gender diversity1. Also growth is influenced in a positive way: in 2009 - 2012 companies with a strong female leadership have increased their ROE by 10.1% as compared to an average of 7.4% for the rest. Diversity is not a problem but a solution!2

  9. 2. Salmon Creek Diversion Dam, overview, diversion weir center foreground, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. Salmon Creek Diversion Dam, overview, diversion weir center foreground, headworks overflow weir to center left, view to east - Salmon Creek Diversion Dam, Salmon Creek, Okanogan, Okanogan County, WA

  10. [ACE inhibitors and the kidney].

    PubMed

    Hörl, W H

    1996-01-01

    Treatment with ACE inhibitors results in kidney protection due to reduction of systemic blood pressure, intraglomerular pressure, an antiproliferative effect, reduction of proteinuria and a lipid-lowering effect in proteinuric patients (secondary due to reduction of protein excretion). Elderly patients with diabetes melitus, coronary heart disease or peripheral vascular occlusion are at risk for deterioration of kidney function due to a high frequency of renal artery stenosis in these patients. In patients with renal insufficiency dose reduction of ACE inhibitors is necessary (exception: fosinopril) but more important is the risk for development of hyperkalemia. Patients at risk for renal artery stenosis and patients pretreated with diuretics should receive a low ACE inhibitor dosage initially ("start low - go slow"). For compliance reasons once daily ACE inhibitor dosage is recommended.

  11. Managing biological diversity

    USGS Publications Warehouse

    Samson, Fred B.; Knopf, Fritz L.

    1993-01-01

    Biological diversity is the variety of life and accompanying ecological processes (Off. Technol. Assess. 1987, Wilcove and Samson 1987, Keystone 1991). Conservation of biological diversity is a major environmental issue (Wilson 1988, Counc. Environ. Quality 1991). The health and future of the earth's ecological systems (Lubchenco et al. 1991), global climate change (Botkin 1990), and an ever-increasing rate in loss of species, communities, and ecological systems (Myers 1990) are among issues drawing biological diversity to the mainstream of conservation worldwide (Int. Union Conserv. Nat. and Nat. Resour. [IUCN] et al. 1991). The legal mandate for conserving biological diversity is now in place (Carlson 1988, Doremus 1991). More than 19 federal laws govern the use of biological resources in the United States (Rein 1991). The proposed National Biological Diversity Conservation and Environmental Research Act (H.R. 585 and S.58) notes the need for a national biological diversity policy, would create a national center for biological diversity research, and recommends a federal interagency strategy for ecosystem conservation. There are, however, hard choices ahead for the conservation of biological diversity, and biologists are grappling with how to set priorities in research and management (Roberts 1988). We sense disillusion among field biologists and managers relative to how to operationally approach the seemingly overwhelming charge of conserving biological diversity. Biologists also need to respond to critics like Hunt (1991) who suggest a tree farm has more biological diversity than an equal area of old-growth forest. At present, science has played only a minor role in the conservation of biological diversity (Weston 1992) with no unified approach available to evaluate strategies and programs that address the quality and quantity of biological diversity (Murphy 1990, Erwin 1992). Although actions to conserve biological diversity need to be clearly defined by

  12. Does Adult Diversion Work?

    ERIC Educational Resources Information Center

    Roesch, Ronald

    1978-01-01

    The author argues that criminal justice professionals have failed in most cases to critically evaluate programs introduced into the criminal justice system. This problem is discussed in the context of pretrial diversion, an increasingly popular innovation in criminal justice. (Author)

  13. Examining Correlates of Diversity.

    ERIC Educational Resources Information Center

    Raudenbush, Stephen W.; Bryk, Anthony S.

    1987-01-01

    Statistical methods are presented for studying "correlates of diversity," defined as characteristics of educational organizations that predict dispersion on the dependent variable. Strategies based on exact distribution theory and asymptotic normal approximation are considered. (TJH)

  14. Addressing diversion effects.

    PubMed

    Resnik, David B

    2015-07-01

    Alan Wertheimer argues that those who promulgate principles of research ethics have a responsibility to take into account the diversion effects of those principles. In this commentary, I argue that Wertheimer's proposal that diversion effects should be considered when promulgating principles of research ethics makes sense, but it often may be best to deal with these effects once a principle has been accepted and implemented, rather than focusing on them at the outset.

  15. Addressing diversion effects

    PubMed Central

    Resnik, David B.

    2015-01-01

    Alan Wertheimer argues that those who promulgate principles of research ethics have a responsibility to take into account the diversion effects of those principles. In this commentary, I argue that Wertheimer's proposal that diversion effects should be considered when promulgating principles of research ethics makes sense, but it often may be best to deal with these effects once a principle has been accepted and implemented, rather than focusing on them at the outset. PMID:27774202

  16. Inhibitors of pig kidney trehalase.

    PubMed

    Kyosseva, S V; Kyossev, Z N; Elbein, A D

    1995-02-01

    Trehazolin, a new trehalase inhibitor isolated from the culture broth of Micromonospora, was reported to be a highly specific inhibitor for porcine and silk worm trehalases with IC50 values of 5.5 x 10(-9) and 3.7 x 10(-9) M, respectively (O. Ando, H. Satake, K. Itoi, A. Sato, M. Nakajima, S. Takashi, H. Haruyama, Y. Ohkuma, T. Kinoshita, and R. Enokita (1991) J. Antibiot. 44, 1165-1168). We also found that trehazolin is a very powerful and quite specific inhibitor against purified pig kidney trehalase, giving an IC50 value of 1.9 x 10(-8) M. Lineweaver-Burk plots showed that this compound was a competitive inhibitor of the trehalase. However, even at concentrations of 200 micrograms/ml, trehazolin did not inhibit the rat intestinal maltase or sucrase, yeast alpha-glucosidase or almond beta-glucosidase. Validoxylamine A and validamycin A, two other trehalase inhibitors, showed potent competitive inhibition against purified pig kidney trehalase, with IC50 values of 2.4 x 10(-9) and 2.5 x 10(-4) M, respectively. On the other hand, validoxylamine A was almost inactive against rat intestinal sucrase and maltase, with some inhibition being observed at millimolar concentration. A number of other glucosidase inhibitors, such as MDL 25637, castanospermine, and deoxynojirimycin were also tested against the purified trehalase and showed reasonable inhibitory activity.

  17. Understanding the Language of Diversity.

    ERIC Educational Resources Information Center

    Powell, Gary C.

    1997-01-01

    Provides definitions for the diversity-related terminology used in this issue that deals with diversity in instructional design. Topics include culture, cross cultural awareness or sensitivity, cultural pluralism, diversity, ethnic group, and race. (LRW)

  18. Leadership in diversity.

    PubMed

    Hunt, P L

    1994-12-01

    As principal change agents, healthcare leaders are well positioned to integrate diversity into their institutions' organizational structure. Thus healthcare leaders must be competent in handling diversity issues. Diversity refers to any characteristic that helps shape a person's attitudes, behaviors, perspective, and interpretation of what is "normal." In the healthcare ministry, diversity encompasses the cultural differences that can be found across functions or among organizations when they merge or partner. Managers and supervisors will have to be familiar with the nuances of diversity if they are to be effective. Those managers who are not adept at incorporating diversity into human resource management may incorrectly evaluate subordinates' capabilities and provide inappropriate training or supervision. As a result, some employees may be underutilized. Others may resist needed direction, overlook instructions, or hide problems such as a language barrier. If executives, marketers, and strategic planners are to develop relevant healthcare services that take into account the needs of their constituencies, they will need to determine how different groups understand and access healthcare. Healthcare leaders who know how to uncover cultural dynamics and challenge cultural assumptions will go far in enabling their staff and managers to confront personal attitudes about community residents. Ultimately, quality of service delivery will be improved.

  19. Arginine mimetic structures in biologically active antagonists and inhibitors.

    PubMed

    Masic, Lucija Peterlin

    2006-01-01

    Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the

  20. Structure-based lead discovery for protein kinase C zeta inhibitor design by exploiting kinase-inhibitor complex crystal structure data and potential therapeutics for preterm labour.

    PubMed

    Shao, Qing-Chun; Zhang, Cui-Juan; Li, Jie

    2014-10-14

    The protein kinase C (PKC) is a family of serine/threonine kinases with a broad range of cellular targets. Members of the PKC family participate at the diverse biological events involved in cellular proliferation, differentiation and survival. The PKC isoform zeta (PKCζ) is an atypical member that has recently been found to play an essential role in promoting human uterine contractility and thus been raised as a new target for treating preterm labour and other tocolytic diseases. In this study, an integrative protocol was described to graft hundreds of inhibitor ligands from their complex crystal structures with cognate kinases into the active pocket of PKCζ and, based on the modeled structures, to evaluate the binding strength of these inhibitors to the non-cognate PKCζ receptor by using a consensus scoring strategy. A total of 32 inhibitors with top score were compiled, and eight out of them were tested for inhibitory potency against PKCζ. Consequently, five compounds, i.e. CDK6 inhibitor fisetin, PIM1 inhibitor myricetin, CDK9 inhibitor flavopiridol and PknB inhibitor mitoxantrone as well as the promiscuous kinase inhibitor staurosporine showed high or moderate inhibitory activity on PKCζ, with IC50 values of 58 ± 9, 1.7 ± 0.4, 108 ± 17, 280 ± 47 and 0.019 ± 0.004 μM, respectively, while other three compounds, including two marketed drugs dasatinib and sunitinib as well as the Rho inhibitor fasudil, have not been detected to possess observable activity. Next, based on the modeled structure data we modified three flavonoid kinase inhibitors, i.e. fisetin, myricetin and flavopiridol, to generate a number of more potential molecular entities, two of which were found to have a moderately improved activity as compared to their parent compounds.

  1. Juggling jobs: roles and mechanisms of multifunctional protease inhibitors in plants.

    PubMed

    Grosse-Holz, Friederike M; van der Hoorn, Renier A L

    2016-05-01

    Multifunctional protease inhibitors juggle jobs by targeting different enzymes and thereby often controlling more than one biological process. Here, we discuss the biological functions, mechanisms and evolution of three types of multifunctional protease inhibitors in plants. The first type is double-headed inhibitors, which feature two inhibitory sites targeting proteases with different specificities (e.g. Bowman-Birk inhibitors) or even different hydrolases (e.g. α-amylase/protease inhibitors preventing both early germination and seed predation). The second type consists of multidomain inhibitors which evolved by intragenic duplication and are released by processing (e.g. multicystatins and potato inhibitor II, implicated in tuber dormancy and defence, respectively). The third type consists of promiscuous inhibitory folds which resemble mouse traps that can inhibit different proteases cleaving the bait they offer (e.g. serpins, regulating cell death, and α-macroglobulins). Understanding how multifunctional inhibitors juggle biological jobs increases our knowledge of the connections between the networks they regulate. These examples show that multifunctionality evolved independently from a remarkable diversity of molecular mechanisms that can be exploited for crop improvement and provide concepts for protein design.

  2. Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors.

    PubMed

    Golkowski, Martin; Vidadala, Rama Subba Rao; Lombard, Chloe K; Suh, Hyong Won; Maly, Dustin J; Ong, Shao-En

    2017-03-03

    ATP-competitive protein kinase inhibitors are important research tools and therapeutic agents. Because there are >500 human kinases that contain highly conserved active sites, the development of selective inhibitors is extremely challenging. Methods to rapidly and efficiently profile kinase inhibitor targets in cell lysates are urgently needed to discover selective compounds and to elucidate the mechanisms of action for polypharmacological inhibitors. Here, we describe a protocol for microgram-scale chemoproteomic profiling of ATP-competitive kinase inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography-mass spectrometry to profile ∼200 kinases in single analytical runs using as little as 5 μL of kinobeads and 300 μg of protein. With our kinobead reagents, we obtained broad coverage of the kinome, monitoring the relative expression levels of 312 kinases in a diverse panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors in competition-binding experiments with label-free quantification, leading to the discovery of a novel selective and potent inhibitor of protein kinase D (PKD) 1, 2, and 3. Our protocol is useful for rapid and sensitive profiling of kinase expression levels and ATP-competitive kinase inhibitor selectivity in native proteomes.

  3. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2

    DOE PAGES

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; ...

    2014-07-31

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, themore » high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.« less

  4. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2

    SciTech Connect

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P.; Pai, Emil F.; Rottapel, Robert; Chirgadze, Nickolay Y.

    2014-07-31

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

  5. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2.

    PubMed

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P; Pai, Emil F; Rottapel, Robert; Chirgadze, Nickolay Y

    2014-10-01

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

  6. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2

    PubMed Central

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P.; Pai, Emil F.; Rottapel, Robert; Chirgadze, Nickolay Y.

    2014-01-01

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors. PMID:25286857

  7. The marine diversity spectrum.

    PubMed

    Reuman, Daniel C; Gislason, Henrik; Barnes, Carolyn; Mélin, Frédéric; Jennings, Simon

    2014-07-01

    Distributions of species body sizes within a taxonomic group, for example, mammals, are widely studied and important because they help illuminate the evolutionary processes that produced these distributions. Distributions of the sizes of species within an assemblage delineated by geography instead of taxonomy (all the species in a region regardless of clade) are much less studied but are equally important and will illuminate a different set of ecological and evolutionary processes. We develop and test a mechanistic model of how diversity varies with body mass in marine ecosystems. The model predicts the form of the 'diversity spectrum', which quantifies the distribution of species' asymptotic body masses, is a species analogue of the classic size spectrum of individuals, and which we have found to be a new and widely applicable description of diversity patterns. The marine diversity spectrum is predicted to be approximately linear across an asymptotic mass range spanning seven orders of magnitude. Slope -0.5 is predicted for the global marine diversity spectrum for all combined pelagic zones of continental shelf seas, and slopes for large regions are predicted to lie between -0.5 and -0.1. Slopes of -0.5 and -0.1 represent markedly different communities: a slope of -0.5 depicts a 10-fold reduction in diversity for every 100-fold increase in asymptotic mass; a slope of -0.1 depicts a 1.6-fold reduction. Steeper slopes are predicted for larger or colder regions, meaning fewer large species per small species for such regions. Predictions were largely validated by a global empirical analysis. Results explain for the first time a new and widespread phenomenon of biodiversity. Results have implications for estimating numbers of species of small asymptotic mass, where taxonomic inventories are far from complete. Results show that the relationship between diversity and body mass can be explained from the dependence of predation behaviour, dispersal, and life history on

  8. Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments

    PubMed Central

    King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Eléonore

    2013-01-01

    Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

  9. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    PubMed Central

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

    Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of the human degradome composed of 561 proteases and homologs. This increased complexity of rat proteases mainly derives from the expansion of several families, including placental cathepsins, testases, kallikreins, and hematopoietic serine proteases, involved in reproductive or immunological functions. These protease families have also evolved differently in rat and mouse and may contribute to explain some functional differences between these closely related species. Likewise, genomic analysis of rat protease inhibitors has shown some differences with mouse protease inhibitors and the expansion of families of cysteine and serine protease inhibitors in rodents with respect to human. These comparative analyses may provide new views on the functional diversity of proteases and inhibitors and contribute to the development of innovative strategies for treating proteolysis diseases. PMID:15060002

  10. Novel and specific inhibitors of a poxvirus type I topoisomerase.

    PubMed

    Bond, Alexis; Reichert, Zachary; Stivers, James T

    2006-02-01

    Vaccinia DNA topoisomerase (vTopo) is a prototypic pox virus family topoisomerase that shares extensive structural and mechanistic properties with the human type IB enzyme (hTopo) and is important for viral replication. Despite their far-reaching similarities, vTopo and hTopo have surprisingly distinct pharmacological properties. To further exploit these differences, we have developed recently the first high-throughput screen for vTopo, which has allowed rapid screening of a 1990-member small-molecule library for inhibitors. Using this approach, 21 compounds were identified with IC(90) values less than 10 muM, and 19 of these were also found to inhibit DNA supercoil relaxation by vTopo. Four of the most potent compounds were completely characterized and are structurally novel topo I inhibitors with efficacies at nanomolar concentrations. These inhibitors were highly specific for vTopo, showing no inhibition of the human enzyme even at 500- to 2000-fold greater concentrations. We describe a battery of efficient experiments to characterize the unique mechanisms of these vTopo inhibitors and discuss the surprising promiscuity of this enzyme to inhibition by structurally diverse small molecules.

  11. Deubiquitinases (DUBs) and DUB inhibitors: a patent review

    PubMed Central

    Nwankwo, Joseph O.; Arkwright, Richard T.; Cvek, Boris; Liu, Jinbao; Dou, Q. Ping

    2016-01-01

    Introduction Deubiquitinating-enzymes (DUBs) are key components of the ubiquitin-proteasome-system (UPS). The fundamental role of DUBs is specific removal of ubiquitin from substrates. DUBs contribute to activation/deactivation, recycling and localization of numerous regulatory-proteins, thus playing major roles in diverse cellular-processes. Altered DUB activity is associated with multitudes of pathologies including cancer. Therefore, DUBs represent novel candidates for target-directed drug development. Areas covered The article is a thorough review/accounting of patented compounds targeting DUBs stratifying/classifying the patented compounds based on: chemical-structures, nucleic-acid compositions, modes-of-action and targeting-sites. The review provides a brief background on the UPS and DUBs involvement. Furthermore, methods for assessing efficacy and potential pharmacological utility of DUB inhibitor (DUBi) are discussed. Expert opinion The FDA’s approval of the 20S proteasome inhibitors: bortezomib and carfilzomib for treatment of hematological malignancies established the UPS as an anti-cancer target. Unfortunately, many patients are inherently resistant or develop resistance to proteasome inhibitors (PIs). One potential strategy to combat PI resistance is targeting upstream components of the UPS such as DUBs. DUBs represent a promising potential therapeutic target due to their critical roles in various cellular processes including protein-turnover, localization and cellular homeostasis. While considerable efforts have been undertaken to develop DUB modulators, significant advancement is necessary move DUB inhibitors into the clinic. PMID:26077642

  12. Engineering trypsin for inhibitor resistance.

    PubMed

    Batt, Anna R; St Germain, Commodore P; Gokey, Trevor; Guliaev, Anton B; Baird, Teaster

    2015-09-01

    The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM  = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

  13. NASFAA Diversity and Inclusion: Recommendations of the Professional Diversity Caucus

    ERIC Educational Resources Information Center

    National Association of Student Financial Aid Administrators, 2015

    2015-01-01

    NASFAA's Diversity and Inclusion Report emphasizes the importance of diversity and inclusivity to NASFAA. Included in this report is a diversity statement developed by NASFAA's Professional Diversity Caucus, and approved by NASFAA's Board in March of 2015. The Caucus convened in the summer of 2014 to better understand issues related to diversity…

  14. Teaching for Diversity: Addressing Diversity Issues in Responsive ESL Instruction

    ERIC Educational Resources Information Center

    Fu, Jing

    2013-01-01

    Student diversity has become a typical phenomenon in American public schools. The impact of increasing diversity on literacy instruction is unchallenged. Teachers reinforce this message by often citing ESL student diversity as a barrier for literacy teaching. In order to better understand the complexity of diversity issues, I explored two ESL…

  15. Reshaping Antibody Diversity

    PubMed Central

    Wang, Feng; Ekiert, Damian C.; Ahmad, Insha; Yu, Wenli; Zhang, Yong; Bazirgan, Omar; Torkamani, Ali; Raudsepp, Terje; Mwangi, Waithaka; Criscitiello, Michael F.; Wilson, Ian A.; Schultz, Peter G.; Smider, Vaughn V.

    2014-01-01

    Summary Unlike humans or mice, some species have limited genome encoded combinatorial diversity potential, yet mount a robust antibody response. Cows are unusual in having exceptionally long CDR H3 loops and few V-regions, but the mechanism for creating diversity is not understood. Deep sequencing revealed that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded mini-domains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a β-strand “stalk” that supports a structurally diverse, disulfide-bonded, “knob” domain. Sequence analysis suggests that diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias towards mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of CDR H3s of unprecedented length that fold into a diversity of mini-domains generated through combinations of somatically generated disulfides. PMID:23746848

  16. Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics

    PubMed Central

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate diverse cellular processes including proliferation, adhesion, survival, and motility. Dysregulated PI3K pathway signaling occurs in one-third of human tumors. Aberrantly activated PI3K signaling also confers sensitivity and resistance to conventional therapies. PI3K has been recognized as an attractive molecular target for novel anti-cancer molecules. In the last few years, several classes of potent and selective small molecule PI3K inhibitors have been developed, and at least fifteen compounds have progressed into clinical trials as new anticancer drugs. Among these, idelalisib has advanced to phase III trials in patients with advanced indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. In this review, we summarized the major molecules of PI3K signaling pathway, and discussed the preclinical models and clinical trials of potent small-molecule PI3K inhibitors. PMID:24261963

  17. Identification and characterization of small-molecule inhibitors of hepsin

    PubMed Central

    Chevillet, John R.; Park, Gemma J.; Bedalov, Antonio; Simon, Julian A.; Vasioukhin, Valeri I.

    2009-01-01

    Hepsin is a type-II transmembrane serine protease overexpressed in the majority of human prostate cancers. We recently demonstrated that hepsin promotes prostate cancer progression and metastasis and thus represents a potential therapeutic target. Here we report the identification of novel small-molecule inhibitors of hepsin catalytic activity. We utilized purified human hepsin for high-throughput screening of established drug and chemical diversity libraries and identified sixteen inhibitory compounds with IC50 values against hepsin ranging from 0.23–2.31μM and relative selectivity of up to 86-fold or greater. Two compounds are orally administered drugs established for human use. Four compounds attenuated hepsin-dependent pericellular serine protease activity in a dose dependent manner with limited or no cytotoxicity to a range of cell types. These compounds may be used as leads to develop even more potent and specific inhibitors of hepsin to prevent prostate cancer progression and metastasis. PMID:18852137

  18. The Ubiquitin-Proteasome Pathway and Proteasome Inhibitors

    PubMed Central

    Myung, Jayhyuk; Kim, Kyung Bo

    2008-01-01

    The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases. PMID:11410931

  19. Virtual High-Throughput Screening for Matrix Metalloproteinase Inhibitors.

    PubMed

    Choi, Jun Yong; Fuerst, Rita

    2017-01-01

    Structure-based virtual screening (SBVS) is a common method for the fast identification of hit structures at the beginning of a medicinal chemistry program in drug discovery. The SBVS, described in this manuscript, is focused on finding small molecule hits that can be further utilized as a starting point for the development of inhibitors of matrix metalloproteinase 13 (MMP-13) via structure-based molecular design. We intended to identify a set of structurally diverse hits, which occupy all subsites (S1'-S3', S2, and S3) centering the zinc containing binding site of MMP-13, by the virtual screening of a chemical library comprising more than ten million commercially available compounds. In total, 23 compounds were found as potential MMP-13 inhibitors using Glide docking followed by the analysis of the structural interaction fingerprints (SIFt) of the docked structures.

  20. [New anticoagulants - direct thrombin inhibitors].

    PubMed

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

  1. Diversity of Poissonian populations

    NASA Astrophysics Data System (ADS)

    Eliazar, Iddo I.; Sokolov, Igor M.

    2010-01-01

    Populations represented by collections of points scattered randomly on the real line are ubiquitous in science and engineering. The statistical modeling of such populations leads naturally to Poissonian populations—Poisson processes on the real line with a distinguished maximal point. Poissonian populations are infinite objects underlying key issues in statistical physics, probability theory, and random fractals. Due to their infiniteness, measuring the diversity of Poissonian populations depends on the lower-bound cut-off applied. This research characterizes the classes of Poissonian populations whose diversities are invariant with respect to the cut-off level applied and establishes an elemental connection between these classes and extreme-value theory. The measures of diversity considered are variance and dispersion, Simpson’s index and inverse participation ratio, Shannon’s entropy and Rényi’s entropy, and Gini’s index.

  2. Diversity as strategy.

    PubMed

    Thomas, David A

    2004-09-01

    IBM's turnaround in the last decade is an impressive and well-documented business story. But behind that success is a less told people story, which explains how the corporation dramatically altered its already diverse composition and created millions of dollars in new business. By the time Lou Gerstner took the helm in 1993, IBM had a long history of progressive management when it came to civil rights and equal-opportunity employment. But Gerstner felt IBM wasn't taking full advantage of a diverse market for talent, nor was it maximizing the potential of its diverse customer and employee base. So in 1995, he launched a diversity task force initiative to uncover and understand differences among people within the organization and find ways to appeal to an even broader set of employees and customers. Gerstner established a task force for each of eight constituencies: Asians; blacks; the gay, lesbian, bisexual, transgendered community; Hispanics; white men; Native Americans; people with disabilities; and women. He asked the task forces to research four questions: What does your constituency need to feel welcome and valued at IBM? What can the corporation do, in partnership with your group, to maximize your constituency's productivity? What can the corporation do to influence your constituency's buying decisions so that IBM is seen as a preferred solution provider? And with which external organizations should IBM form relationships to better understand the needs of your constituency? The answers to these questions became the basis for IBM's diversity strategy. Thomas stresses that four factors are key to implementing any major change initiative: strong support from company leaders, an employee base that is fully engaged with the initiative, management practices that are integrated and aligned with the effort, and a strong and well-articulated business case for action. All four elements have helped IBM make diversity a key corporate strategy tied to real growth.

  3. Authoritarian Disbeliefs in Diversity.

    PubMed

    Asbrock, Frank; Kauff, Mathias

    2015-01-01

    Ethnic diversity poses a threat to authoritarians, as it indicates non-conformism to group norms and poses a threat to group conformity. According to authoritarian dynamic theory, threats elicit authoritarian reactions in people with authoritarian predispositions. In the present article we tested a mediation model derived from authoritarian dynamic theory in a sample of 171 students. As expected, authoritarian predisposition negatively predicted diversity beliefs. This effect was fully mediated by an authoritarian manifestation, that is, authoritarian aggression. The two other components of right-wing authoritarianism, authoritarian submission and conventionalism, did not mediate the effect. Results confirm contemporary research on authoritarianism and the differentiation of authoritarian predispositions and its manifestations.

  4. The interplay of diversity training and diversity beliefs on team creativity in nationality diverse teams.

    PubMed

    Homan, Astrid C; Buengeler, Claudia; Eckhoff, Robert A; van Ginkel, Wendy P; Voelpel, Sven C

    2015-09-01

    Attaining value from nationality diversity requires active diversity management, which organizations often employ in the form of diversity training programs. Interestingly, however, the previously reported effects of diversity training are often weak and, sometimes, even negative. This situation calls for research on the conditions under which diversity training helps or harms teams. We propose that diversity training can increase team creativity, but only for teams with less positive pretraining diversity beliefs (i.e., teams with a greater need for such training) and that are sufficiently diverse in nationality. Comparing the creativity of teams that attended nationality diversity training versus control training, we found that for teams with less positive diversity beliefs, diversity training increased creative performance when the team's nationality diversity was high, but undermined creativity when the team's nationality diversity was low. Diversity training had less impact on teams with more positive diversity beliefs, and training effects were not contingent upon these teams' diversity. Speaking to the underlying process, we showed that these interactive effects were driven by the experienced team efficacy of the team members. We discuss theoretical and practical implications for nationality diversity management.

  5. Corrosion inhibitors from expired drugs.

    PubMed

    Vaszilcsin, Nicolae; Ordodi, Valentin; Borza, Alexandra

    2012-07-15

    This paper presents a method of expired or unused drugs valorization as corrosion inhibitors for metals in various media. Cyclic voltammograms were drawn on platinum in order to assess the stability of pharmaceutically active substances from drugs at the metal-corrosive environment interface. Tafel slope method was used to determine corrosion rates of steel in the absence and presence of inhibitors. Expired Carbamazepine and Paracetamol tablets were used to obtain corrosion inhibitors. For the former, the corrosion inhibition of carbon steel in 0.1 mol L(-1) sulfuric acid solution was about 90%, whereas for the latter, the corrosion inhibition efficiency of the same material in the 0.25 mol L(-1) acetic acid-0.25 mol L(-1) sodium acetate buffer solution was about 85%.

  6. Electrochemical studies of corrosion inhibitors

    NASA Technical Reports Server (NTRS)

    Danford, M. D.

    1990-01-01

    The effect of single salts, as well as multicomponent mixtures, on corrosion inhibition was studied for type 1010 steel; for 5052, 1100, and 2219-T87 aluminum alloys; and for copper. Molybdate-containing inhibitors exhibit an immediate, positive effect for steel corrosion, but an incubation period may be required for aluminum before the effect of a given inhibitor can be determined. The absence of oxygen was found to provide a positive effect (smaller corrosion rate) for steel and copper, but a negative effect for aluminum. This is attributed to the two possible mechanisms by which aluminum can oxidize. Corrosion inhibition is generally similar for oxygen-rich and oxygen-free environments. The results show that the electrochemical method is an effective means of screening inhibitors for the corrosion of single metals, with caution to be exercised in the case of aluminum.

  7. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  8. An environmentally friendly scale inhibitor

    SciTech Connect

    Dobbs, J.B.; Brown, J.M.

    1999-11-01

    This paper describes a method of inhibiting the formation of scales such as barium and strontium sulfate in low pH aqueous systems, and calcium carbonate in systems containing high concentrations of dissolved iron. The solution, chemically, involves treating the aqueous system with an inhibitor designed to replace organic-phosphonates. Typical low pH aqueous systems where the inhibitor is particularly useful are oilfield produced-water, resin bed water softeners that form scale during low pH, acid regeneration operations. Downhole applications are recommended where high concentrations of dissolved iron are present in the produced water. This new approach to inhibition replaces typical organic phosphonates and polymers with a non-toxic, biodegradable scale inhibitor that performs in harsh environments.

  9. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  10. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  11. Landslides as agents of diversity

    NASA Astrophysics Data System (ADS)

    Geertsema, Marten

    2016-04-01

    Landslides, often destructive and damaging, are also agents of change that introduce diversity to landscapes. I discuss landslide diversity at three levels: site diversity, soil diversity, and habitat diversity. There are many landslide types involving different materials and rates and styles of movement. Landscape diversity varies with different types of landslides. Landslides, at the same time depositional and erosional agents, influence sites by redistributing materials and changing microtopography. Eroded portions of landslides, with exposed parent material, revert to the initial stages of soil development and ecological succession. Landslides can also alter soil properties including, surface texture, chemistry and porosity. Landslides influence habitat diversity by creating ecosystem mosaics.

  12. STAT inhibitors for cancer therapy

    PubMed Central

    2013-01-01

    Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds. PMID:24308725

  13. [Kinase inhibitors against hematological malignancies].

    PubMed

    Tojo, Arinobu

    2014-06-01

    Dysregulation of protein phosphorylation, especially on tyrosine residues, plays a crucial role in development and progression of hematological malignancies. Since remarkable success in imatinib therapy of CML and Ph+ALL, extensive efforts have made to explore candidate molecular targets and next breakthrough drugs. Now that next generation ABL kinase inhibitors are available for CML, the therapeutic algorithm has been revolutionized. As for AML and lymphoid malignancies, many kinase inhibitors targeting FLT3, BTK and aurora-A are on early and late clinical trials, and a number of promising drugs including ibrutinib are picked up for further evaluation.

  14. HIV envelope: challenges and opportunities for development of entry inhibitors

    PubMed Central

    Caffrey, Michael

    2011-01-01

    The HIV envelope proteins gp120 and gp41 play critical roles in HIV entry and thus are of extreme interest for the development of novel therapeutics. Study by diverse methods, including structural biology and mutagenesis, has resulted in a detailed model for envelope-mediated entry, which consists of multiple conformations, each a potential target for therapeutic intervention. In this review we discuss the challenges, strategies and progress to date for developing novel entry inhibitors directed at disrupting HIV gp120 and gp41 function. PMID:21377881

  15. The potential of histone deacetylase inhibitors in lung cancer.

    PubMed

    Aparicio, Ana

    2006-03-01

    In the nucleus, DNA is wrapped around octamers of histone proteins. Histones, like other proteins, are posttranslationally modified by the addition of an array of chemical groups that affect their interactions with surrounding structures. Histone acetyltransferases and histone deacetylases (HDACs) are the enzymes involved in the addition and removal, respectively, of acetyl groups from the aminoterminal tails of histones. A number of structurally diverse compounds are capable of inhibiting HDACs and exert a variety of biologic effects on cancer cells in preclinical models. Early clinical trials with the first generation of HDAC inhibitors (HDACIs) have demonstrated promising therapeutic activity, and HDACs have become one of the hottest targets in drug development today.

  16. EGFR inhibitors and autophagy in cancer treatment.

    PubMed

    Cui, Jie; Hu, Yun-Feng; Feng, Xie-Min; Tian, Tao; Guo, Ya-Huan; Ma, Jun-Wei; Nan, Ke-Jun; Zhang, Hong-Yi

    2014-12-01

    Epidermal growth factor receptor (EGFR) inhibitor treatment is a strategy for cancer therapy. However, innate and acquired resistance is a major obstacle of the efficacy. Autophagy is a self-digesting process in cells, which is considered to be associated with anti-cancer drug resistance. The activation of EGFR can regulate autophagy through multiple signal pathways. EGFR inhibitors can induce autophagy, but the specific function of the induction of autophagy by EGFR inhibitors remains biphasic. On the one hand, autophagy induced by EGFR inhibitors acts as a cytoprotective response in cancer cells, and autophagy inhibitors can enhance the cytotoxic effects of EGFR inhibitors. On the other hand, a high level of autophagy after treatment of EGFR inhibitors can also result in autophagic cell death lacking features of apoptosis, and the combination of EGFR inhibitors with an autophagy inducer might be beneficial. Thus, autophagy regulation represents a promising approach for improving the efficacy of EGFR inhibitors in the treatment of cancer patients.

  17. "An Engine of Diversity"

    ERIC Educational Resources Information Center

    Galuszka, Peter

    2008-01-01

    This article features North Carolina's Research Triangle Park (RTP), which provides research and career opportunities for the region and creates a diverse work force. The convergence of higher education and research at the famed RTP has been all but idyllic for years. What happened there is a strong example of how regions can start their own…

  18. Modeling Antibody Diversity.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy Ronstadt

    1998-01-01

    Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)

  19. Diversity: A Corporate Campaign

    ERIC Educational Resources Information Center

    Akiyama, Diana D.

    2008-01-01

    In this article, the author calls for a "campaign" because she believes there is a need to build upon the successes of diversity initiatives with renewed commitment, in much the same way as capital campaigns build upon past successes and refocus campuses on their work. Just as a capital campaign invests in financial stability by stimulating…

  20. Banking on Diversity

    ERIC Educational Resources Information Center

    Roach, Ronald

    2010-01-01

    Few organizations have as racially and culturally diverse a work force as the organizations that make up the World Bank Group. Of its 13,000 employees, nearly 60 percent of whom are located in downtown Washington, D.C., and the rest scattered across 160 offices around the globe, nearly every nation in the world is represented in the World Bank…

  1. Diversity and International.

    ERIC Educational Resources Information Center

    Justice, Madeline, Ed.

    This document contains the following papers on diversity and international issues in technology and teacher education: (1) "'At-Risk' Learners and the 'Digital Divide': Exploring the Equity in Access Issue" (Jeanne M. Foster and Sharla L. Snider); (2) "Integrating Standards-Based Instructional Technology" (Nicole M. Snow); (3)…

  2. Supply and Demand Diversity

    ERIC Educational Resources Information Center

    Galuszka, Peter

    2007-01-01

    Public universities in Virginia, as in many states, have generally not paid much attention to diversity among their suppliers. For years, state expenditures for outside contracts went to the usual suspects--White contractors from well-established companies. Four years ago, former Governor Mark Warner, a progressive Democrat from the high…

  3. Teaching for Diversity.

    ERIC Educational Resources Information Center

    Jones, Nancy Baker

    1994-01-01

    The summer 1993 Southwest Educational Development Laboratory (SEDL) "Networkshop" focused on the need for teacher education programs to prepare future teachers to work with and teach effectively increasingly diverse student populations, and the need to increase the number of minority teachers. A major focus was on how policy and…

  4. A Diversity Grab Bag.

    ERIC Educational Resources Information Center

    Moore, Thomas

    1999-01-01

    Suggests 15 strategies for introducing the concept of diversity to children including: (1) promoting conversation; (2) making the familiar different; (3) exploring music; (4) learning about celebrations; (5) showing objects; (6) taking field trips; (7) introducing foods; (8) encouraging empathy; (9) collaborating with different people; and (10)…

  5. What Is Diversity Pedagogy?

    ERIC Educational Resources Information Center

    Sheets, Rosa Hernandez

    2009-01-01

    Diversity Pedagogy Theory (DPT) is a set of principles that point out the natural and inseparable connection between culture and cognition. In other words, to be effective as a teacher, he/she must understand and acknowledge the critical role culture plays in the teaching-learning process. DPT maintains that culturally inclusive teachers (a)…

  6. Diversity and Adolescent Literature.

    ERIC Educational Resources Information Center

    Ediger, Marlow

    This paper expresses the opinion that reading about different minority groups is a must in a quality literature program. Each student should learn as much as possible about diverse minority groups, and literature on minority groups needs adequate curriculum emphasis. Some books which can be a real value for African-American students are…

  7. Academies and School Diversity

    ERIC Educational Resources Information Center

    Curtis, Andrew

    2009-01-01

    This article considers the implications of Academies for the diversity of schooling in England. It seeks to establish the extent to which Academies are distinctive compared to other types of state secondary schools and whether this has been affected by a number of recent reforms. Different types of Academies are also be examined. Previous work in…

  8. How Symbiosis Creates Diversity

    ERIC Educational Resources Information Center

    Lord, Joshua

    2010-01-01

    Diversity in habitats on Earth is astounding--whether on land or in the sea--and this is in part due to symbiosis. The lesson described in this article helps students understand how symbiosis affects different organisms through a fun and engaging game where they match hosts and symbionts based on their respective needs. This 45-minute lesson is…

  9. National Testing and Diversity.

    ERIC Educational Resources Information Center

    Hajj-Bahous, Jocelyne

    This paper examines the direct relationship between curriculum, instruction, and evaluation, suggesting that following a national curriculum and preparing students to take national examinations requires diverse teaching materials, teaching methodologies, and testing techniques to train students to apply their cognitive skills to thinking,…

  10. Campus Diversity Climate Survey.

    ERIC Educational Resources Information Center

    Hindes, Victoria

    In order to be responsive to the changing demographics of the emerging community college student population, and in an attempt to answer the Chancellor's Office's call to discover how community colleges serve the diverse needs of students, Shasta College, California, collaborated with Feather River College, California, to conduct a study that…

  11. Education and Diversity

    ERIC Educational Resources Information Center

    Banks, James A.; Cookson, Peter; Gay, Geneva; Hawley, Willis D.; Irvine, Jacqueline Jordan; Nieto, Sonia; Schofield, Janet Ward; Stephan, Walter G.

    2005-01-01

    What do we know about education and diversity, and how do we know it? This two-part question guided the work of the Multicultural Education Consensus Panel, which included the eight scholars named above. The panel's work was sponsored by the Center for Multicultural Education at the University of Washington and the Common Destiny Alliance at the…

  12. Diversity Networking Reception

    NASA Astrophysics Data System (ADS)

    2014-03-01

    Join us at the APS Diversity Reception to relax, network with colleagues, and learn about programs and initiatives for women, underrepresented minorities, and LGBT physicists. You'll have a great time meeting friends in a supportive environment and making connections.

  13. Workplace Diversity Issues.

    ERIC Educational Resources Information Center

    1999

    This document contains three symposium papers on workplace diversity issues. "Expanding Theories of Career Development: Adding the Voices of African American Women in the White Academy" (Mary V. Alfred) questions the validity of existing career development models for women and minority groups and examines the professional development of…

  14. Computational Approaches for the Discovery of Human Proteasome Inhibitors: An Overview.

    PubMed

    Guedes, Romina A; Serra, Patrícia; Salvador, Jorge A R; Guedes, Rita C

    2016-07-16

    Proteasome emerged as an important target in recent pharmacological research due to its pivotal role in degrading proteins in the cytoplasm and nucleus of eukaryotic cells, regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription, immune response, and signaling processes. The last two decades witnessed intensive efforts to discover 20S proteasome inhibitors with significant chemical diversity and efficacy. To date, the US FDA approved to market three proteasome inhibitors: bortezomib, carfilzomib, and ixazomib. However new, safer and more efficient drugs are still required. Computer-aided drug discovery has long being used in drug discovery campaigns targeting the human proteasome. The aim of this review is to illustrate selected in silico methods like homology modeling, molecular docking, pharmacophore modeling, virtual screening, and combined methods that have been used in proteasome inhibitors discovery. Applications of these methods to proteasome inhibitors discovery will also be presented and discussed to raise improvements in this particular field.

  15. Shaping development of autophagy inhibitors with the structure of the lipid kinase Vps34.

    PubMed

    Miller, Simon; Tavshanjian, Brandon; Oleksy, Arkadiusz; Perisic, Olga; Houseman, Benjamin T; Shokat, Kevan M; Williams, Roger L

    2010-03-26

    Phosphoinositide 3-kinases (PI3Ks) are lipid kinases with diverse roles in health and disease. The primordial PI3K, Vps34, is present in all eukaryotes and has essential roles in autophagy, membrane trafficking, and cell signaling. We solved the crystal structure of Vps34 at 2.9 angstrom resolution, which revealed a constricted adenine-binding pocket, suggesting the reason that specific inhibitors of this class of PI3K have proven elusive. Both the phosphoinositide-binding loop and the carboxyl-terminal helix of Vps34 mediate catalysis on membranes and suppress futile adenosine triphosphatase cycles. Vps34 appears to alternate between a closed cytosolic form and an open form on the membrane. Structures of Vps34 complexes with a series of inhibitors reveal the reason that an autophagy inhibitor preferentially inhibits Vps34 and underpin the development of new potent and specific Vps34 inhibitors.

  16. The marine diversity spectrum

    PubMed Central

    Reuman, Daniel C; Gislason, Henrik; Barnes, Carolyn; Mélin, Frédéric; Jennings, Simon

    2014-01-01

    Distributions of species body sizes within a taxonomic group, for example, mammals, are widely studied and important because they help illuminate the evolutionary processes that produced these distributions. Distributions of the sizes of species within an assemblage delineated by geography instead of taxonomy (all the species in a region regardless of clade) are much less studied but are equally important and will illuminate a different set of ecological and evolutionary processes. We develop and test a mechanistic model of how diversity varies with body mass in marine ecosystems. The model predicts the form of the ‘diversity spectrum’, which quantifies the distribution of species' asymptotic body masses, is a species analogue of the classic size spectrum of individuals, and which we have found to be a new and widely applicable description of diversity patterns. The marine diversity spectrum is predicted to be approximately linear across an asymptotic mass range spanning seven orders of magnitude. Slope −0·5 is predicted for the global marine diversity spectrum for all combined pelagic zones of continental shelf seas, and slopes for large regions are predicted to lie between −0·5 and −0·1. Slopes of −0·5 and −0·1 represent markedly different communities: a slope of −0·5 depicts a 10-fold reduction in diversity for every 100-fold increase in asymptotic mass; a slope of −0·1 depicts a 1·6-fold reduction. Steeper slopes are predicted for larger or colder regions, meaning fewer large species per small species for such regions. Predictions were largely validated by a global empirical analysis. Results explain for the first time a new and widespread phenomenon of biodiversity. Results have implications for estimating numbers of species of small asymptotic mass, where taxonomic inventories are far from complete. Results show that the relationship between diversity and body mass can be explained from the dependence of predation behaviour

  17. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    PubMed Central

    Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466

  18. Benzimidazole derivatives as kinase inhibitors.

    PubMed

    Garuti, Laura; Roberti, Marinella; Bottegoni, Giovanni

    2014-01-01

    Benzimidazole is a common kinase inhibitor scaffold and benzimidazole-based compounds interact with enzymes by multiple binding modes. In some cases, the benzimidazole acts as part of the hinge-binding motif, in others it has a scaffolding role without evidence for direct hinge binding. Several of these compounds are ATP-competitive inhibitors and show high selectivity by exploiting unique structural properties that distinguish one kinase from the majority of other kinases. However, the high specificity for a single target is not always sufficient. Thus another approach, called multi-target therapy, has been developed over the last few years. The simultaneous inhibition of various kinases may be useful because the disease is attacked at several relevant targets. Moreover, if a kinase becomes drug-resistant, a multitargeted drug can act on the other kinases. Some benzimidazole derivatives are multi-target inhibitors. In this article benzimidazole inhibitors are reported with their mechanisms of action, structure-activity relationship (SAR) and biological properties.

  19. Biocatalysts with enhanced inhibitor tolerance

    DOEpatents

    Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min

    2015-12-08

    Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.

  20. Azidoblebbistatin, a photoreactive myosin inhibitor

    PubMed Central

    Képiró, Miklós; Várkuti, Boglárka H.; Bodor, Andrea; Hegyi, György; Drahos, László; Kovács, Mihály; Málnási-Csizmadia, András

    2012-01-01

    Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. PMID:22647605

  1. Inhibitor Discovery by Convolution ABPP.

    PubMed

    Chandrasekar, Balakumaran; Hong, Tram Ngoc; van der Hoorn, Renier A L

    2017-01-01

    Activity-based protein profiling (ABPP) has emerged as a powerful proteomic approach to study the active proteins in their native environment by using chemical probes that label active site residues in proteins. Traditionally, ABPP is classified as either comparative or competitive ABPP. In this protocol, we describe a simple method called convolution ABPP, which takes benefit from both the competitive and comparative ABPP. Convolution ABPP allows one to detect if a reduced signal observed during comparative ABPP could be due to the presence of inhibitors. In convolution ABPP, the proteomes are analyzed by comparing labeling intensities in two mixed proteomes that were labeled either before or after mixing. A reduction of labeling in the mix-and-label sample when compared to the label-and-mix sample indicates the presence of an inhibitor excess in one of the proteomes. This method is broadly applicable to detect inhibitors in proteomes against any proteome containing protein activities of interest. As a proof of concept, we applied convolution ABPP to analyze secreted proteomes from Pseudomonas syringae-infected Nicotiana benthamiana leaves to display the presence of a beta-galactosidase inhibitor.

  2. An Ethylene-Protected Achilles’ Heel of Etiolated Seedlings for Arthropod Deterrence

    PubMed Central

    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Pollmann, Stephan; Reinbothe, Steffen; Reinbothe, Christiane

    2016-01-01

    A small family of Kunitz protease inhibitors exists in Arabidopsis thaliana, a member of which (encoded by At1g72290) accomplishes highly specific roles during plant development. Arabidopsis Kunitz-protease inhibitor 1 (Kunitz-PI;1), as we dubbed this protein here, is operative as cysteine PI. Activity measurements revealed that despite the presence of the conserved Kunitz-motif the bacterially expressed Kunitz-PI;1 was unable to inhibit serine proteases such as trypsin and chymotrypsin, but very efficiently inhibited the cysteine protease RESPONSIVE TO DESICCATION 21. Western blotting and cytolocalization studies using mono-specific antibodies recalled Kunitz-PI;1 protein expression in flowers, young siliques and etiolated seedlings. In dark-grown seedlings, maximum Kunitz-PI;1 promoter activity was detected in the apical hook region and apical parts of the hypocotyls. Immunolocalization confirmed Kunitz-PI;1 expression in these organs and tissues. No transmitting tract (NTT) and HECATE 1 (HEC1), two transcription factors previously implicated in the formation of the female reproductive tract in flowers of Arabidopsis, were identified to regulate Kunitz-PI;1 expression in the dark and during greening, with NTT acting negatively and HEC1 acting positively. Laboratory feeding experiments with isopod crustaceans such as Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug) pinpointed the apical hook as ethylene-protected Achilles’ heel of etiolated seedlings. Because exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and mechanical stress (wounding) strongly up-regulated HEC1-dependent Kunitz-PI;1 gene expression, our results identify a new circuit controlling herbivore deterrence of etiolated plants in which Kunitz-PI;1 is involved. PMID:27625656

  3. 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 IC50 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.

  4. Identification of Chemical Inhibitors to Human Tissue Transglutaminase by Screening Existing Drug Libraries

    PubMed Central

    Lai, Thung-S.; Liu, Yusha; Tucker, Tim; Daniel, Kurt R.; Sane, David C.; Toone, Eric; Burke, James R.; Strittmatter, Warren J.; Greenberg, Charles S.

    2008-01-01

    Human tissue transglutaminase (TGM2) is a calcium-dependent crosslinking enzyme involved in the post-translational modification of intra- and extra-cellular proteins and involved in several neurodegenerative diseases. To find specific inhibitors to TGM2, two structurally diverse chemical libraries (Lopac and Prestwick) were screened. We found that ZM39923, a Janus kinase inhibitor and its metabolite ZM449829 were the most potent inhibitors with IC50 of 10 and 5 nM, respectively. In addition, two other inhibitors including Tyrphostin 47 and Vitamin K3 were found to have an IC50 in the µM range. These agents utilized in part a thiol-dependent mechanism to inhibit TGM2, consistent with the activation of TGM2 by reduction of an intra-molecular disulfide bond. These inhibitors were tested in a polyglutamine-expressing Drosophila model of neurodegeneration and found to improve survival. The TGM2 inhibitors we discovered may serve as valuable lead compounds for the development of orally active TGM2 inhibitors to treat human diseases. PMID:18804034

  5. Diversity within the Profession. Part Two: Initiatives Promoting Diversity.

    ERIC Educational Resources Information Center

    Spafford, Marlee M.; Sharma, Neepun; Nygaard, Vicki L.; Kahlou, Christina

    2002-01-01

    Examines the literature on minority experiences in optometry and other health professions, describing programs geared either toward increasing diversity or facilitating acceptance of diversity within the optometric profession, including affirmative action and other institutional support structures. (EV)

  6. Cathepsin D inhibitor from Vicia sativa L.

    PubMed

    Roszkowska-Jakimiec, W; Bańkowska, A

    1998-01-01

    Specific inhibitor of cathepsin D has been shown in the extract of Vicia sativa L. seeds. This inhibitor does not inhibit the activity of other aspartic proteases. Also it does not inhibit the activity of cysteine proteases and serine proteases.

  7. DEVELOPMENTAL DIVERSITY OF AMPHIBIANS

    PubMed Central

    Elinson, Richard P.; del Pino, Eugenia M.

    2011-01-01

    The current model amphibian, Xenopus laevis, develops rapidly in water to a tadpole which metamorphoses into a frog. Many amphibians deviate from the X. laevis developmental pattern. Among other adaptations, their embryos develop in foam nests on land or in pouches on their mother’s back or on a leaf guarded by a parent. The diversity of developmental patterns includes multinucleated oogenesis, lack of RNA localization, huge non-pigmented eggs, and asynchronous, irregular early cleavages. Variations in patterns of gastrulation highlight the modularity of this critical developmental period. Many species have eliminated the larva or tadpole and directly develop to the adult. The wealth of developmental diversity among amphibians coupled with the wealth of mechanistic information from X. laevis permit comparisons that provide deeper insights into developmental processes. PMID:22662314

  8. NSF announces diversity programme

    NASA Astrophysics Data System (ADS)

    Kruesi, Liz

    2016-04-01

    The US National Science Foundation (NSF) has initiated a new funding programme that will create schemes to increase diversity in science, technology, engineering and mathematics (STEM). The initiative - Inclusion across the Nation of Communities of Learners of Underrepresented Discoverers in Engineering and Science (INCLUDES) - aims to increase the participation of women, those with a low socioeconomic status, people with disabilities and those from minority racial backgrounds.

  9. Spectrum of large copy number variations in 26 diverse Indian populations: potential involvement in phenotypic diversity.

    PubMed

    Gautam, Pramod; Jha, Pankaj; Kumar, Dhirendra; Tyagi, Shivani; Varma, Binuja; Dash, Debasis; Mukhopadhyay, Arijit; Mukerji, Mitali

    2012-01-01

    Copy number variations (CNVs) have provided a dynamic aspect to the apparently static human genome. We have analyzed CNVs larger than 100 kb in 477 healthy individuals from 26 diverse Indian populations of different linguistic, ethnic and geographic backgrounds. These CNVRs were identified using the Affymetrix 50K Xba 240 Array. We observed 1,425 and 1,337 CNVRs in the deletion and amplification sets, respectively, after pooling data from all the populations. More than 50% of the genes encompassed entirely in CNVs had both deletions and amplifications. There was wide variability across populations not only with respect to CNV extent (ranging from 0.04-1.14% of genome under deletion and 0.11-0.86% under amplification) but also in terms of functional enrichments of processes like keratinization, serine proteases and their inhibitors, cadherins, homeobox, olfactory receptors etc. These did not correlate with linguistic, ethnic, geographic backgrounds and size of populations. Certain processes were near exclusive to deletion (serine proteases, keratinization, olfactory receptors, GPCRs) or duplication (homeobox, serine protease inhibitors, embryonic limb morphogenesis) datasets. Populations having same enriched processes were observed to contain genes from different genomic loci. Comparison of polymorphic CNVRs (5% or more) with those cataloged in Database of Genomic Variants revealed that 78% (2473) of the genes in CNVRs in Indian populations are novel. Validation of CNVs using Sequenom MassARRAY revealed extensive heterogeneity in CNV boundaries. Exploration of CNV profiles in such diverse populations would provide a widely valuable resource for understanding diversity in phenotypes and disease.

  10. Diversity: The Business Case?

    NASA Astrophysics Data System (ADS)

    Jones, B.

    2013-12-01

    Understanding perceptions and managing expectations are learnable skills that do not necessarily come with project funding. Finding life balance as one moves through a STEM career path poses unique challenges that require a certain skill set that is not always intuitive. Some of those challenges include: selecting grad or post doc positions; balancing work and family commitments; and dealing with employer/advisor perceptions and expectations. As in nature, the STEM enterprise requires multiple perspectives to flourish (necessity of peer review), and in a changing environment (e.g., budget, generations, technology, etc.), embracing diversity in thought and application may help drive the evolution of STEM in the U.S. Many Agencies and organizations have ';workforce development' programs that focus on preparing the next generation of scientists and engineers at the graduate and undergraduate level that focus on preparing students in the diverse disciplines that are unique to those Agency and organizational missions. While financial support certainly is critical to assist students in Science Technology Engineering and Mathematics (STEM) and other fields, professional development is just as important to equip students with a balanced arsenal of tactics to be successful professionals in the STEM workforce of today. Success in these efforts requires an honest look at the issue of inequality in the STEM ecosystem... meaning, what initiatives have been successful in addressing the imbalance in sources of thought and application, therefore promoting the importance of diversity.

  11. Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness

    PubMed Central

    Scott, Sarah A; Selvy, Paige E; Buck, Jason R; Cho, Hyekyung P; Criswell, Tracy L; Thomas, Ashley L; Armstrong, Michelle D; Arteaga, Carlos L; Lindsley, Craig W; Brown, H Alex

    2013-01-01

    Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with > 100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors—a new class of antimetastatic agents. PMID:19136975

  12. The Business Case for Diversity.

    ERIC Educational Resources Information Center

    Miller, Joanne

    1995-01-01

    The business case for diversity is tied to competition, demand for labor, and imperatives to reorganize work. Through diversity, talent can be mobilized into teams, adding value and relying on differences for competitive advantage. (SK)

  13. Algorithm Diversity for Resilent Systems

    DTIC Science & Technology

    2016-06-27

    4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Algorithm Diversity for Resilent Systems N/A 5b. GRANT NUMBER NOOO 141512208 5c. PROGRAM ELEMENT NUMBER...changes to a prograrn’s state during execution. Specifically, the project aims to develop techniques to introduce algorithm -level diversity, in contrast...to existing work on execution-level diversity. Algorithm -level diversity can introduce larger differences between variants than execution-level

  14. Small Molecule Deubiquitinase Inhibitors Promote Macrophage Anti-Infective Capacity

    PubMed Central

    Charbonneau, Marie-Eve; Gonzalez-Hernandez, Marta J.; Showalter, Hollis D.; Donato, Nicholas J.; Wobus, Christiane E.; O’Riordan, Mary X. D.

    2014-01-01

    The global spread of anti-microbial resistance requires urgent attention, and diverse alternative strategies have been suggested to address this public health concern. Host-directed immunomodulatory therapies represent one approach that could reduce selection for resistant bacterial strains. Recently, the small molecule deubiquitinase inhibitor WP1130 was reported as a potential anti-infective drug against important human food-borne pathogens, notably Listeria monocytogenes and noroviruses. Utilization of WP1130 itself is limited due to poor solubility, but given the potential of this new compound, we initiated an iterative rational design approach to synthesize new derivatives with increased solubility that retained anti-infective activity. Here, we test a small library of novel synthetic molecules based on the structure of the parent compound, WP1130, for anti-infective activity in vitro. Our studies identify a promising candidate, compound 9, which reduced intracellular growth of L. monocytogenes at concentrations that caused minimal cellular toxicity. Compound 9 itself had no bactericidal activity and only modestly slowed Listeria growth rate in liquid broth culture, suggesting that this drug acts as an anti-infective compound by modulating host-cell function. Moreover, this new compound also showed anti-infective activity against murine norovirus (MNV-1) and human norovirus, using the Norwalk virus replicon system. This small molecule inhibitor may provide a chemical platform for further development of therapeutic deubiquitinase inhibitors with broad-spectrum anti-infective activity. PMID:25093325

  15. Small molecule deubiquitinase inhibitors promote macrophage anti-infective capacity.

    PubMed

    Charbonneau, Marie-Eve; Gonzalez-Hernandez, Marta J; Showalter, Hollis D; Donato, Nicholas J; Wobus, Christiane E; O'Riordan, Mary X D

    2014-01-01

    The global spread of anti-microbial resistance requires urgent attention, and diverse alternative strategies have been suggested to address this public health concern. Host-directed immunomodulatory therapies represent one approach that could reduce selection for resistant bacterial strains. Recently, the small molecule deubiquitinase inhibitor WP1130 was reported as a potential anti-infective drug against important human food-borne pathogens, notably Listeria monocytogenes and noroviruses. Utilization of WP1130 itself is limited due to poor solubility, but given the potential of this new compound, we initiated an iterative rational design approach to synthesize new derivatives with increased solubility that retained anti-infective activity. Here, we test a small library of novel synthetic molecules based on the structure of the parent compound, WP1130, for anti-infective activity in vitro. Our studies identify a promising candidate, compound 9, which reduced intracellular growth of L. monocytogenes at concentrations that caused minimal cellular toxicity. Compound 9 itself had no bactericidal activity and only modestly slowed Listeria growth rate in liquid broth culture, suggesting that this drug acts as an anti-infective compound by modulating host-cell function. Moreover, this new compound also showed anti-infective activity against murine norovirus (MNV-1) and human norovirus, using the Norwalk virus replicon system. This small molecule inhibitor may provide a chemical platform for further development of therapeutic deubiquitinase inhibitors with broad-spectrum anti-infective activity.

  16. Indole based Tubulin Polymerization Inhibitors: An Update on Recent Developments.

    PubMed

    Sunil, Dhanya; Kamath, Pooja R

    2016-01-01

    The exploration of cancer microenvironment and its physiology have exposed a number of potential molecular targets for selective therapeutic intervention by anti-cancer agents. Microtubules are basic cell components formed by polymerization of αβ heterodimers which play a pivotal role in cellular functions as well as cell division. Drugs that can control the microtubule assembly either by hindering tubulin polymerization or by obstructing microtubule disassembly are of great importance in anti-cancer therapy. Diverse classes of naturally occurring as well as synthetic and semi-synthetic compounds with an indole nucleus induce microtubule polymerization and depolymerization and thereby change tubulin dynamics. Rapid development of several novel tubulin polymerization inhibitors has been observed over the past few years and some of them have associated vascular disrupting properties too. The present review starts with the structure, function and importance of microtubules in a eukaryotic cell. The well characterized tubulin binding domains and the corresponding inhibitors including their mechanism of action is also a part of this article. The report mainly focuses on the brief synthetic methodology with the relevant SAR studies of different indole derived molecules that have been reported in the past few years as potential inhibitors of tubulin polymerization is discussed. This review will provide the up-to-date evidence-base for synthetic chemists as well as biologists to design and synthesize new active molecules to inhibit tubulin polymerization.

  17. Small-molecule arginase inhibitors.

    PubMed

    Ivanenkov, Yan A; Chufarova, Nina V

    2014-01-01

    Arginase is an enzyme that metabolizes L-arginine to L-ornithine and urea. In addition to its fundamental role in the hepatic ornithine cycle, it also influences the immune systems in humans and mice. Arginase participates in many inflammatory disorders by decreasing the synthesis of nitric oxide and inducing fibrosis and tissue regeneration. L-arginine deficiency, which is modulated by myeloid cell arginase, suppresses T-cell immune response. This mechanism plays a fundamental role in inflammation-associated immunosuppression. Pathogens can synthesize their own arginase to elude immune reaction. Small-molecule arginase inhibitors are currently described as promising therapeutics for the treatment of several diseases, including allergic asthma, inflammatory bowel disease, ulcerative colitis, cardiovascular diseases (atherosclerosis and hypertension), diseases associated with pathogens (e.g., Helicobacter pylori, Trypanosoma cruzi, Leishmania, Mycobacterium tuberculosis and Salmonella), cancer and induced or spontaneous immune disorders. This article summarizes recent patents in the area of arginase inhibitors and discusses their properties.

  18. Salicylanilide Inhibitors of Toxoplasma gondii

    PubMed Central

    Fomovska, Alina; Wood, Richard D.; Mui, Ernest; Dubey, Jitenter P.; Ferriera, Leandra R.; Hickman, Mark R.; Lee, Patricia J.; Leed, Susan E.; Auschwitz, Jennifer M.; Welsh, William J.; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima

    2012-01-01

    Toxoplasma gondii(T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose anti-apicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles. PMID:22970937

  19. Salicylanilide inhibitors of Toxoplasma gondii.

    PubMed

    Fomovska, Alina; Wood, Richard D; Mui, Ernest; Dubey, Jitenter P; Ferreira, Leandra R; Hickman, Mark R; Lee, Patricia J; Leed, Susan E; Auschwitz, Jennifer M; Welsh, William J; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima

    2012-10-11

    Toxoplasma gondii (T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose antiapicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles.

  20. [Microbial metabolites that inhibit sterol biosynthesis, their chemical diversity and characteristics of mode of action].

    PubMed

    Trenin, A S

    2013-01-01

    Inhibitors of sterol biosynthesis (ISB) are widespread in nature and characterized by appreciable diversity both in their chemical structure and mode of action. Many of these inhibitors express noticeable biological activity and approved themselves in development of various pharmaceuticals. In this review there is a detailed description of biologically active microbial metabolites with revealed chemical structure that have ability to inhibit sterol biosynthesis. Inhibitors of mevalonate pathway in fungous and mammalian cells, exhibiting hypolipidemic or antifungal activity, as well as inhibitors of alternative non-mevalonate (pyruvate gliceraldehyde phosphate) isoprenoid pathway, which are promising in the development of affective antimicrobial or antiparasitic drugs, are under consideration in this review. Chemical formulas of the main natural inhibitors and their semi-synthetic derivatives are represented. Mechanism of their action at cellular and biochemical level is discussed. Special attention is given to inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase (group of lovastatin) and inhibitors of acyl-CoA-cholesterol-acyl transferase (ACAT) that possess hypolipidemic activity and could be affective in the treatment of atherosclerosis. In case of inhibitors of late stages of sterol biosynthesis (after squalene formation) special attention is paid to compounds possessing evident antifungal and antitumoral activity. Explanation of mechanism of anticancer and antiviral action of microbial ISB, as well as the description of their ability to induce apoptosis is given.

  1. Resisting HRD's Resistance to Diversity

    ERIC Educational Resources Information Center

    Bierema, Laura L.

    2010-01-01

    Purpose: The purpose of this paper is to empirically illustrate how human resource development (HRD) resists and omits issues of diversity in academic programs, textbooks, and research; analyze the research on HRD and diversity over a ten-year period; discuss HRD's resistance to diversity; and offer some recommendations for a more authentic…

  2. Valuing Diversity: The Primary Years.

    ERIC Educational Resources Information Center

    McCracken, Janet Brown

    Noting that children who learn to accept and value human diversity will develop the open, flexible approach to life that is needed in today's world, this book examines ways to help young children learn to appreciate cultural diversity in the classroom. Following introductory chapters on the value of diversity and a child's right to the valuing of…

  3. Religious Diversity in the Schools.

    ERIC Educational Resources Information Center

    Michel, George J.; Smith, William Gause; Vickers, Dianne Koenig; Brown, Elsie

    This document contains four papers that address constitutional issues of religious diversity in the schools. The first paper, "Religious Diversity in the Schools--The Overview" (George J. Michel), provides an overview of religious diversity in American public schools, with a focus on the long history of cooperation with Christian churches. It…

  4. Genetic diversity in Gossypium genus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The overall objectives of this paper are to report on cotton germplasm resources, morphobiological and agronomic diversity of Gossypium genus and review efforts on molecular genetic diversity of cotton gene pools as well as on the challenges and perspectives of exploiting genetic diversity in cotton...

  5. Recent progress on fucosyltransferase inhibitors.

    PubMed

    Merino, Pedro; Tejero, Tomás; Delso, Ignacio; Hurtado-Guerrero, Ramon; Gómez-SanJuan, Asier; Sádaba, David

    2012-12-01

    Fucosyltransferases (FucTs) are enzymes that transfer L-fucose from GDP-fucose to a glycoside or a peptide. They have important roles in a variety of diseases including cancer and autoimmune disorders, viral and bacterial infections and inflammatory processes, and thus they represent important drug targets for the development of agents for the treatment of such disorders. This review highlights recent developments regarding carbohydrate mimics as inhibitors of FucTs. The most recent and relevant synthetic strategies are described.

  6. Nelfinavir: fourth protease inhibitor approved.

    PubMed

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  7. Voglibose: An Alpha Glucosidase Inhibitor

    PubMed Central

    Dabhi, Ajay S.; Bhatt, Nikita R.; Shah, Mohit J.

    2013-01-01

    Diabetes Mellitus (DM) is a morbid disease worldwide, with increasing incidence as time passes. It has macro-vascular and micro-vascular complications. The main cause of these complications is poorly controlled postprandial hyperglycaemia. Alpha glucosidase inhibitors, namely acarbose, voglibose and miglitol, are available for therapy. Voglibose is well tolerated and effective in comparable doses among these drugs. This article highlights the important features of voglibose. PMID:24551718

  8. Resource availability controls fungal diversity across a plant diversity gradient

    USGS Publications Warehouse

    Waldrop, M.P.; Zak, D.R.; Blackwood, C.B.; Curtis, C.D.; Tilman, D.

    2006-01-01

    Despite decades of research, the ecological determinants of microbial diversity remain poorly understood. Here, we test two alternative hypotheses concerning the factors regulating fungal diversity in soil. The first states that higher levels of plant detritus production increase the supply of limiting resources (i.e. organic substrates) thereby increasing fungal diversity. Alternatively, greater plant diversity increases the range of organic substrates entering soil, thereby increasing the number of niches to be filled by a greater array of heterotrophic fungi. These two hypotheses were simultaneously examined in experimental plant communities consisting of one to 16 species that have been maintained for a decade. We used ribosomal intergenic spacer analysis (RISA), in combination with cloning and sequencing, to quantify fungal community composition and diversity within the experimental plant communities. We used soil microbial biomass as a temporally integrated measure of resource supply. Plant diversity was unrelated to fungal diversity, but fungal diversity was a unimodal function of resource supply. Canonical correspondence analysis (CCA) indicated that plant diversity showed a relationship to fungal community composition, although the occurrence of RISA bands and operational taxonomic units (OTUs) did not differ among the treatments. The relationship between fungal diversity and resource availability parallels similar relationships reported for grasslands, tropical forests, coral reefs, and other biotic communities, strongly suggesting that the same underlying mechanisms determine the diversity of organisms at multiple scales. ?? 2006 Blackwell Publishing Ltd/CNRS.

  9. Phytochemical diversity drives plant–insect community diversity

    PubMed Central

    Richards, Lora A.; Dyer, Lee A.; Forister, Matthew L.; Smilanich, Angela M.; Dodson, Craig D.; Leonard, Michael D.; Jeffrey, Christopher S.

    2015-01-01

    What are the ecological causes and consequences of variation in phytochemical diversity within and between plant taxa? Despite decades of natural products discovery by organic chemists and research by chemical ecologists, our understanding of phytochemically mediated ecological processes in natural communities has been restricted to studies of either broad classes of compounds or a small number of well-characterized molecules. Until now, no studies have assessed the ecological causes or consequences of rigorously quantified phytochemical diversity across taxa in natural systems. Consequently, hypotheses that attempt to explain variation in phytochemical diversity among plants remain largely untested. We use spectral data from crude plant extracts to characterize phytochemical diversity in a suite of co-occurring plants in the tropical genus Piper (Piperaceae). In combination with 20 years of data focused on Piper-associated insects, we find that phytochemical diversity has a direct and positive effect on the diversity of herbivores but also reduces overall herbivore damage. Elevated chemical diversity is associated with more specialized assemblages of herbivores, and the cascading positive effect of phytochemistry on herbivore enemies is stronger as herbivore diet breadth narrows. These results are consistent with traditional hypotheses that predict positive associations between plant chemical diversity, insect herbivore diversity, and trophic specialization. It is clear from these results that high phytochemical diversity not only enhances the diversity of plant-associated insects but also contributes to the ecological predominance of specialized insect herbivores. PMID:26283384

  10. Phytochemical diversity drives plant-insect community diversity.

    PubMed

    Richards, Lora A; Dyer, Lee A; Forister, Matthew L; Smilanich, Angela M; Dodson, Craig D; Leonard, Michael D; Jeffrey, Christopher S

    2015-09-01

    What are the ecological causes and consequences of variation in phytochemical diversity within and between plant taxa? Despite decades of natural products discovery by organic chemists and research by chemical ecologists, our understanding of phytochemically mediated ecological processes in natural communities has been restricted to studies of either broad classes of compounds or a small number of well-characterized molecules. Until now, no studies have assessed the ecological causes or consequences of rigorously quantified phytochemical diversity across taxa in natural systems. Consequently, hypotheses that attempt to explain variation in phytochemical diversity among plants remain largely untested. We use spectral data from crude plant extracts to characterize phytochemical diversity in a suite of co-occurring plants in the tropical genus Piper (Piperaceae). In combination with 20 years of data focused on Piper-associated insects, we find that phytochemical diversity has a direct and positive effect on the diversity of herbivores but also reduces overall herbivore damage. Elevated chemical diversity is associated with more specialized assemblages of herbivores, and the cascading positive effect of phytochemistry on herbivore enemies is stronger as herbivore diet breadth narrows. These results are consistent with traditional hypotheses that predict positive associations between plant chemical diversity, insect herbivore diversity, and trophic specialization. It is clear from these results that high phytochemical diversity not only enhances the diversity of plant-associated insects but also contributes to the ecological predominance of specialized insect herbivores.

  11. Carbonic anhydrase inhibitors drug design.

    PubMed

    McKenna, Robert; Supuran, Claudiu T

    2014-01-01

    Inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) has pharmacologic applications in the field of antiglaucoma, anticonvulsant, antiobesity, and anticancer agents but is also emerging for designing anti-infectives (antifungal and antibacterial agents) with a novel mechanism of action. As a consequence, the drug design of CA inhibitors (CAIs) is a very dynamic field. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion in the enzyme active site. Recently the dithiocarbamates, possessing a similar mechanism of action, were reported as a new class of inhibitors. Other families of CAIs possess a distinct mechanism of action: phenols, polyamines, some carboxylates, and sulfocoumarins anchor to the zinc-coordinated water molecule. Coumarins and five/six-membered lactones are prodrug inhibitors, binding in hydrolyzed form at the entrance of the active site cavity. Novel drug design strategies have been reported principally based on the tail approach for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Sugar-based tails as well as click chemistry were the most fruitful developments of the tail approach. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the dithiocarbamate, phenol and carboxylate types have also been reported.

  12. Substituted androstanes as aromatase inhibitors

    NASA Astrophysics Data System (ADS)

    Levina, Inna S.

    1998-11-01

    The synthesis and structure-activity relationships of inhibitors of steroid aromatase which catalyses the last stage of a multistep biotransformation of cholesterol into estrogens, viz., aromatisation of C19-steroids into C18-phenolic steroids, are discussed. Compounds of the androstane series which are structurally related to the natural substrate, viz., androst-4-ene-3,17-dione, are the subjects of consideration. The review encompasses problems of synthesis of various substituted androstanes and their aromatase-inhibiting activities and structural requirements for selective specific aromatase inhibitors based on in vitro and in vivo structure-activity studies of compounds synthesised, their biological properties and the results of clinical trials. Special attention is paid to practical applications of aromatase inhibitors in the treatment of hormone-dependent mammary and ovarian tumours as well as benign prostatic tumours. In writing this report, the author has used all the information currently available in the chemical, biochemical, endocrinological and medicinal literature as well as in patents. The bibliography includes 173 references.

  13. Design and synthesis of 3,3'-biscoumarin-based c-Met inhibitors.

    PubMed

    Xu, Jimin; Ai, Jing; Liu, Sheng; Peng, Xia; Yu, Linqian; Geng, Meiyu; Nan, Fajun

    2014-06-14

    A library of biscoumarin-based c-Met inhibitors was synthesized, based on optimization of 3,3'-biscoumarin hit 3, which was identified as a non-ATP competitive inhibitor of c-Met from a diverse library of coumarin derivatives. Among these compounds, 38 and 40 not only showed potent enzyme activities with IC50 values of 107 nM and 30 nM, respectively, but also inhibited c-Met phosphorylation in BaF3/TPR-Met and EBC-1 cells.

  14. Aromatase Inhibitors and Other Compounds for Lowering Breast Cancer Risk

    MedlinePlus

    ... Cancer Risk and Prevention Aromatase Inhibitors for Lowering Breast Cancer Risk Aromatase inhibitors (drugs that lower estrogen levels) ... day. Can aromatase inhibitors lower the risk of breast cancer? Aromatase inhibitors are used mainly to treat hormone ...

  15. Identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library.

    PubMed

    Shah, Falgun; Mukherjee, Prasenjit; Gut, Jiri; Legac, Jennifer; Rosenthal, Philip J; Tekwani, Babu L; Avery, Mitchell A

    2011-04-25

    Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

  16. Cycles in fossil diversity

    SciTech Connect

    Rohde, Robert A.; Muller, Richard A.

    2004-10-20

    It is well-known that the diversity of life appears to fluctuate during the course the Phanerozoic, the eon during which hard shells and skeletons left abundant fossils (0-542 Ma). Using Sepkoski's compendium of the first and last stratigraphic appearances of 36380 marine genera, we report a strong 62 {+-} 3 Myr cycle, which is particularly strong in the shorter-lived genera. The five great extinctions enumerated by Raup and Sepkoski may be an aspect of this cycle. Because of the high statistical significance, we also consider contributing environmental factors and possible causes.

  17. Diverse Rock Named Squash

    NASA Technical Reports Server (NTRS)

    1997-01-01

    This image from the Sojourner rover's right front camera was taken on Sol 27. The Pathfinder lander is seen at middle left. The large rock at right, nicknamed 'Squash', exhibits a diversity of textures. It looks very similar to a conglomerate, a type of rock found on Earth that forms from sedimentary processes.

    Mars Pathfinder is the second in NASA's Discovery program of low-cost spacecraft with highly focused science goals. The Jet Propulsion Laboratory, Pasadena, CA, developed and managed the Mars Pathfinder mission for NASA's Office of Space Science, Washington, D.C. JPL is a division of the California Institute of Technology (Caltech).

  18. Cycles in fossil diversity.

    PubMed

    Rohde, Robert A; Muller, Richard A

    2005-03-10

    It is well known that the diversity of life appears to fluctuate during the course of the Phanerozoic, the eon during which hard shells and skeletons left abundant fossils (0-542 million years ago). Here we show, using Sepkoski's compendium of the first and last stratigraphic appearances of 36,380 marine genera, a strong 62 +/- 3-million-year cycle, which is particularly evident in the shorter-lived genera. The five great extinctions enumerated by Raup and Sepkoski may be an aspect of this cycle. Because of the high statistical significance we also consider the contributions of environmental factors, and possible causes.

  19. Investigating the selectivity of metalloenzyme inhibitors.

    PubMed

    Day, Joshua A; Cohen, Seth M

    2013-10-24

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin to gauge the ability of the inhibitors to access Fe(3+) from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity.

  20. Structural Basis for Inhibition of Histamine N-Methyltransferase by Diverse Drugs

    SciTech Connect

    Horton,J.; Sawada, K.; Nishibori, M.; Cheng, X.

    2005-01-01

    In mammals, histamine action is terminated through metabolic inactivation by histamine N-methyltransferase (HNMT) and diamine oxidase. In addition to three well-studied pharmacological functions, smooth muscle contraction, increased vascular permeability, and stimulation of gastric acid secretion, histamine plays important roles in neurotransmission, immunomodulation, and regulation of cell proliferation. The histamine receptor H1 antagonist diphenhydramine, the antimalarial drug amodiaquine, the antifolate drug metoprine, and the anticholinesterase drug tacrine (an early drug for Alzheimer's disease) are surprisingly all potent HNMT inhibitors, having inhibition constants in the range of 10-100 nM. We have determined the structural mode of interaction of these four inhibitors with HNMT. Despite their structural diversity, they all occupy the histamine-binding site, thus blocking access to the enzyme's active site. Near the N terminus of HNMT, several aromatic residues (Phe9, Tyr15, and Phe19) adopt different rotamer conformations or become disordered in the enzyme-inhibitor complexes, accommodating the diverse, rigid hydrophobic groups of the inhibitors. The maximized shape complementarity between the protein aromatic side-chains and aromatic ring(s) of the inhibitors are responsible for the tight binding of these varied inhibitors.

  1. Inhibitors

    MedlinePlus

    ... Mutation Project (CHAMP) mutation list: a new online resource. Human Mutation. 2012; E2382-E2392. Li T, Miller CH, Payne AB, Hooper CW. The CDC Hemophilia B mutation project mutation list: a new online resource. Molecular Genetics and Genomic Medicine. 2013; 1(4): ...

  2. Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

    PubMed

    Shakespeare, William C; Metcalf, Chester A; Wang, Yihan; Sundaramoorthi, Raji; Keenan, Terence; Weigele, Manfred; Bohacek, Regine S; Dalgarno, David C; Sawyer, Tomi K

    2003-09-01

    Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone

  3. Do CDK4/6 inhibitors have potential as targeted therapeutics for squamous cell cancers?

    PubMed

    Kalu, Nene N; Johnson, Faye M

    2017-02-01

    Introduction Dysregulation of cell cycle progression has an established link to neoplasia and cancer progression. Components of the cyclin D-CDK4/6-INK4-Rb pathway are frequently altered in squamous cell carcinomas (SCCs) by diverse mechanisms, including viral oncogene-induced degradation, mutation, deletion, and amplification. Activation of the CDK4/6 pathway may predict response to CDK4/6 inhibitors and provide clinical biomarkers. Recently, the CDK4/6 inhibitor palbociclib showed clinical efficacy in combination with cetuximab in HNSCC patients. Areas covered This review focuses on the current research on the use of CDK4/6 inhibitors, comprising preclinical animal studies through phase II clinical trials across all SCCs. Expert opinion CDK4/6 inhibitors have a proven clinical benefit in breast cancer, but data on SCCs are sparse. Although frequent dysregulation of the cyclin D-CDK4/6-INK4-Rb pathway in SCCs suggests that targeting CDK4/6 may hold promise for improved clinical outcomes, single-agent activity has been modest in preclinical studies and absent in clinical studies. Combinations with immunotherapy or inhibitors of the PI3 K/mTOR or EGFR pathway may be effective. Given that SCCs caused by human papillomavirus have high levels of p16 and low levels of Rb, the CDK4/6 inhibitors are predicted to be ineffective in these cancers.

  4. Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors

    PubMed Central

    Hollensen, Anne Kruse; Bak, Rasmus O.; Haslund, Didde; Mikkelsen, Jacob Giehm

    2013-01-01

    MicroRNAs (miRNAs) are ubiquitous regulators of gene expression that contribute to almost any cellular process. Methods for managing of miRNA activity are attracting increasing attention in relation to diverse experimental and therapeutic applications. DNA-encoded miRNA inhibitors expressed from plasmid or virus-based vectors provide persistent miRNA suppression and options of tissue-directed micromanaging. In this report, we explore the potential of exploiting short, hairpin-shaped RNAs for simultaneous suppression of two or more miRNAs. Based on the “Tough Decoy” (TuD) design, we create dual-targeting hairpins carrying two miRNA recognition sites and demonstrate potent co-suppression of different pairs of unrelated miRNAs by a single DNA-encoded inhibitor RNA. In addition, enhanced miRNA suppression is achieved by expression of RNA polymerase II-transcribed inhibitors carrying clustered TuD hairpins with up to a total of eight miRNA recognition sites. Notably, by expressing clustered TuD inhibitors harboring a single recognition site for each of a total of six miRNAs, we document robust parallel suppression of multiple miRNAs by inhibitor RNA molecules encoded by a single expression cassette. These findings unveil a new potential of TuD-based miRNA inhibitors and pave the way for standardizing synchronized suppression of families or clusters of miRNAs. PMID:23324610

  5. MMP Inhibitors on Dentin Stability

    PubMed Central

    Montagner, A.F.; Sarkis-Onofre, R.; Pereira-Cenci, T.; Cenci, M.S.

    2014-01-01

    The aim of this study was to systematically review the literature for in vitro and ex vivo studies that evaluated the effect of matrix metalloproteinase (MMP) inhibitors during the adhesive procedure on the immediate and long-term resin-dentin bond strength. The search was conducted in 6 databases with no publication year or language limits, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. From 1,336 potentially eligible studies, 48 were selected for full-text analysis, and 30 were included for review, with 17 considered in the meta-analysis. Two reviewers independently selected the studies, extracted the data, and assessed the risk of bias. Pooled effect estimates were expressed as the weighted mean difference between groups. The most used MMP inhibitor was chlorhexidine (CHX). Immediate bond strength results showed no difference between 2% CHX and control; however, a difference was found between 0.2% CHX and control at baseline. After aging, CHX presented higher bond strength values compared to control groups (p < .05). However, this was not observed for longer periods of aging. High heterogeneity was found in some comparisons, especially for the water storage aging subgroup. Subgroup analyses showed that self-etching and etch-and-rinse adhesives are benefited by the CHX use. From the studies included, only 1 presented low risk of bias, while the others showed medium or high risk of bias. The use of MMP inhibitors did not affect the immediate bond strength overall, while it influenced the aged bond strength. Aging procedures influenced bond strength values of the dentin adhesion stability. PMID:24935066

  6. Monoamine Oxidase Inhibitors: Clinical Review

    PubMed Central

    Remick, Ronald A.; Froese, Colleen

    1990-01-01

    Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984

  7. Techniques for Screening Translation Inhibitors

    PubMed Central

    Osterman, Ilya A.; Bogdanov, Alexey A.; Dontsova, Olga A.; Sergiev, Petr V.

    2016-01-01

    The machinery of translation is one of the most common targets of antibiotics. The development and screening of new antibiotics usually proceeds by testing antimicrobial activity followed by laborious studies of the mechanism of action. High-throughput methods for new antibiotic screening based on antimicrobial activity have become routine; however, identification of molecular targets is usually a challenge. Therefore, it is highly beneficial to combine primary screening with the identification of the mechanism of action. In this review, we describe a collection of methods for screening translation inhibitors, with a special emphasis on methods which can be performed in a high-throughput manner. PMID:27348012

  8. Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

    PubMed

    Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A; Gupta, Piyush B; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

    2017-01-10

    The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

  9. Human phylogeography and diversity

    PubMed Central

    Harcourt, Alexander H.

    2016-01-01

    Homo sapiens phylogeography begins with the species’ origin nearly 200 kya in Africa. First signs of the species outside Africa (in Arabia) are from 125 kya. Earliest dates elsewhere are now 100 kya in China, 45 kya in Australia and southern Europe (maybe even 60 kya in Australia), 32 kya in northeast Siberia, and maybe 20 kya in the Americas. Humans reached arctic regions and oceanic islands last—arctic North America about 5 kya, mid- and eastern Pacific islands about 2–1 kya, and New Zealand about 700 y ago. Initial routes along coasts seem the most likely given abundant and easily harvested shellfish there as indicated by huge ancient oyster shell middens on all continents. Nevertheless, the effect of geographic barriers—mountains and oceans—is clear. The phylogeographic pattern of diasporas from several single origins—northeast Africa to Eurasia, southeast Eurasia to Australia, and northeast Siberia to the Americas—allows the equivalent of a repeat experiment on the relation between geography and phylogenetic and cultural diversity. On all continents, cultural diversity is high in productive low latitudes, presumably because such regions can support populations of sustainable size in a small area, therefore allowing a high density of cultures. Of course, other factors operate. South America has an unusually low density of cultures in its tropical latitudes. A likely factor is the phylogeographic movement of peoples from the Old World bringing novel and hence, lethal diseases to the New World, a foretaste, perhaps, of present day global transport of tropical diseases. PMID:27432967

  10. Plasmid diversity in neisseriae.

    PubMed

    van Passel, Mark W J; van der Ende, Arie; Bart, Aldert

    2006-08-01

    Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

  11. Human phylogeography and diversity.

    PubMed

    Harcourt, Alexander H

    2016-07-19

    Homo sapiens phylogeography begins with the species' origin nearly 200 kya in Africa. First signs of the species outside Africa (in Arabia) are from 125 kya. Earliest dates elsewhere are now 100 kya in China, 45 kya in Australia and southern Europe (maybe even 60 kya in Australia), 32 kya in northeast Siberia, and maybe 20 kya in the Americas. Humans reached arctic regions and oceanic islands last-arctic North America about 5 kya, mid- and eastern Pacific islands about 2-1 kya, and New Zealand about 700 y ago. Initial routes along coasts seem the most likely given abundant and easily harvested shellfish there as indicated by huge ancient oyster shell middens on all continents. Nevertheless, the effect of geographic barriers-mountains and oceans-is clear. The phylogeographic pattern of diasporas from several single origins-northeast Africa to Eurasia, southeast Eurasia to Australia, and northeast Siberia to the Americas-allows the equivalent of a repeat experiment on the relation between geography and phylogenetic and cultural diversity. On all continents, cultural diversity is high in productive low latitudes, presumably because such regions can support populations of sustainable size in a small area, therefore allowing a high density of cultures. Of course, other factors operate. South America has an unusually low density of cultures in its tropical latitudes. A likely factor is the phylogeographic movement of peoples from the Old World bringing novel and hence, lethal diseases to the New World, a foretaste, perhaps, of present day global transport of tropical diseases.

  12. Behavioral diversity as multiagent cooperation

    NASA Astrophysics Data System (ADS)

    Balch, Tucker

    1999-08-01

    In many cases cooperation between robots is implemented using explicit, perhaps complex, coordination protocols. However, research in behavior-based multirobot systems suggest that effective cooperative teams can be composed of agents using simple individual agent behaviors with limited or no communication. In this paper we prose behavioral diversity as an alternative cooperative strategy. Behavioral diversity refers to the extent to which agents for various components of the task. It is not always the case, however, that diversity is advantageous. Results of experiments in robotics soccer and multirobot foraging tasks indicate that the utility of diversity depends on the task. This paper describes behaviorally diverse solutions to these task and provides a comparison that suggests why some tasks are suited for behavioral diversity and others are not.

  13. C1 inhibitor: quantification and purification.

    PubMed

    Varga, Lilian; Dobó, József

    2014-01-01

    C1 inhibitor is a multipotent serpin capable of inhibiting the classical and the lectin pathways of complement, the fibrinolytic system, and contact/kinin system of coagulation. Deficiency of C1 inhibitor manifest as hereditary angioedema (HAE), an autosomal dominant hereditary disease. Measuring the C1 inhibitor level is of vital importance for the diagnosis of HAE and also for monitoring patients receiving C1 inhibitor for therapy. Determination of the antigenic C1 inhibitor level by the radial immunodiffusion (RID) technique is described in detail in this chapter. The presented purification method of plasma C1 inhibitor is primarily based on its high carbohydrate content and its affinity to the lectin jacalin.

  14. A Combination of Receptor-Based Pharmacophore Modeling & QM Techniques for Identification of Human Chymase Inhibitors

    PubMed Central

    Arooj, Mahreen; Sakkiah, Sugunadevi; Kim, Songmi; Arulalapperumal, Venkatesh; Lee, Keun Woo

    2013-01-01

    Inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, and fibrotic disorders. To find novel and potent chymase inhibitors and to provide a new idea for drug design, we used both ligand-based and structure-based methods to perform the virtual screening(VS) of commercially available databases. Different pharmacophore models generated from various crystal structures of enzyme may depict diverse inhibitor binding modes. Therefore, multiple pharmacophore-based approach is applied in this study. X-ray crystallographic data of chymase in complex with different inhibitors were used to generate four structure–based pharmacophore models. One ligand–based pharmacophore model was also developed from experimentally known inhibitors. After successful validation, all pharmacophore models were employed in database screening to retrieve hits with novel chemical scaffolds. Drug-like hit compounds were subjected to molecular docking using GOLD and AutoDock. Finally four structurally diverse compounds with high GOLD score and binding affinity for several crystal structures of chymase were selected as final hits. Identification of final hits by three different pharmacophore models necessitates the use of multiple pharmacophore-based approach in VS process. Quantum mechanical calculation is also conducted for analysis of electrostatic characteristics of compounds which illustrates their significant role in driving the inhibitor to adopt a suitable bioactive conformation oriented in the active site of enzyme. In general, this study is used as example to illustrate how multiple pharmacophore approach can be useful in identifying structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme. The strategy used in the current study could be appropriate to design drugs for other enzymes as well. PMID:23658661

  15. Tubulin inhibitors: a patent survey.

    PubMed

    Nepali, Kunal; Ojha, Ritu; Sharma, Sahil; Bedi, Preet M S; Dhar, Kanaya L

    2014-05-01

    Tubulin is one of the most useful and strategic molecular targets for anticancer drugs. The dynamic process of microtubule assembly and disassembly can be blocked by various agents that bind to distinct sites in the β-tubulin subunit. By interfering with microtubule function in vitro, these agents arrest cells in mitosis, eventually leading to cell death, by both apoptosis and necrosis. So far, three binding domains have been identified a) the colchicine site close to the α/β interface, b) the area where the vinca alkaloids bind, and c) the taxane-binding pocket. This review compiles the patent literature up to 2013 and offers a detailed account of all the advances on Tubulin inhibitors (lead molecules) along with in depth knowledge about the number of novel scaffolds, modified analogs and derivatives of the lead molecules. Colchicine binding site remains the most explored site indicated by the patent survey as majority of the patents revolves around phenstatin and combretastatin based molecules where the key structural feature for tubulin inhibition is an appropriate arrangement of the two aromatic rings at an appropriate distance and optimal dihedral angle maximizing interactions with tubulin. A brief account of promising tubulin inhibitors in stages of clinical development and some strategies for the development of potent molecules overcoming the problem of drug resistance have also been discussed.

  16. Aromatase inhibitors and bone loss.

    PubMed

    Perez, Edith A; Weilbaecher, Katherine

    2006-08-01

    The aromatase inhibitors (AIs) anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin) are significantly more effective than the selective estrogen-receptor modulator (SERM) tamoxifen in preventing recurrence in estrogen receptor-positive early breast cancer. Aromatase inhibitors are likely to replace SERMs as first-line adjuvant therapy for many patients. However, AIs are associated with significantly more osteoporotic fractures and greater bone mineral loss. As antiresorptive agents, oral and intravenous bisphosphonates such as alendronate (Fosamax), risedronate (Actonel), ibandronate (Boniva), pamidronate (Aredia), and zoledronic acid (Zometa) have efficacy in preventing postmenopausal osteoporosis, cancer treatment-related bone loss, or skeletal complications of metastatic disease. Clinical practice guidelines recommend baseline and annual follow-up bone density monitoring for all patients initiating AI therapy. Bisphosphonate therapy should be prescribed for patients with osteoporosis (T score < -2.5) and considered on an individual basis for those with osteopenia (T score < -1). Modifiable lifestyle behaviors including adequate calcium and vitamin D intake, weight-bearing exercise, and smoking cessation should be addressed. Adverse events associated with bisphosphonates include gastrointestinal toxicity, renal toxicity, and osteonecrosis of the jaw. These safety concerns should be balanced with the potential of bisphosphonates to minimize or prevent the debilitating effects of AI-associated bone loss in patients with early, hormone receptor-positive breast cancer.

  17. Computer tools in the discovery of HIV-I integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Nicklaus, Marc C

    2010-01-01

    Computer-aided drug design (CADD) methodologies have made great advances and contributed significantly to the discovery and/or optimization of many clinically used drugs in recent years. CADD tools have likewise been applied to the discovery of inhibitors of HIV-I integrase, a difficult and worthwhile target for the development of efficient anti-HIV drugs. This article reviews the application of CADD tools, including pharmacophore search, quantitative structure–activity relationships, model building of integrase complexed with viral DNA and quantum-chemical studies in the discovery of HIV-I integrase inhibitors. Different structurally diverse integrase inhibitors have been identified by, or with significant help from, various CADD tools. PMID:21426160

  18. Aurora Kinase Inhibitors in Oncology Clinical Trials: Current State of the Progress.

    PubMed

    Falchook, Gerald S; Bastida, Christel C; Kurzrock, Razelle

    2015-12-01

    The Aurora kinase family of kinases (Aurora A, B, and C) are involved in multiple mitotic events, and aberrant expression of these kinases is associated with tumorigenesis. Aurora A and Aurora B are validated anticancer targets, and the development of Aurora kinase inhibitors has progressed from preclinical to clinical studies. A variety of Aurora A, B and pan-Aurora kinase inhibitors have entered the clinic. The main side effects include febrile neutropenia, stomatitis, gastrointestinal toxicity, hypertension, and fatigue. Responses including complete remissions have been described in diverse, advanced malignancies, most notably ovarian cancer and acute myelogenous leukemia. This review highlights the biologic rationale for Aurora kinase as a target, and clinical trials involving Aurora kinase inhibitors, with particular emphasis on published early phase studies, and the observed anti-tumor activity of these agents.

  19. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain

    PubMed Central

    Picaud, Sarah; Wells, Christopher; Felletar, Ildiko; Brotherton, Deborah; Martin, Sarah; Savitsky, Pavel; Diez-Dacal, Beatriz; Philpott, Martin; Bountra, Chas; Lingard, Hannah; Fedorov, Oleg; Müller, Susanne; Brennan, Paul E.; Knapp, Stefan; Filippakopoulos, Panagis

    2013-01-01

    Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation. PMID:24248379

  20. RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain.

    PubMed

    Picaud, Sarah; Wells, Christopher; Felletar, Ildiko; Brotherton, Deborah; Martin, Sarah; Savitsky, Pavel; Diez-Dacal, Beatriz; Philpott, Martin; Bountra, Chas; Lingard, Hannah; Fedorov, Oleg; Müller, Susanne; Brennan, Paul E; Knapp, Stefan; Filippakopoulos, Panagis

    2013-12-03

    Bromodomains have emerged as attractive candidates for the development of inhibitors targeting gene transcription. Inhibitors of the bromo and extraterminal (BET) family recently showed promising activity in diverse disease models. However, the pleiotropic nature of BET proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. Here, we report that RVX-208, a compound currently in phase II clinical trials, is a BET bromodomain inhibitor specific for second bromodomains (BD2s). Cocrystal structures revealed binding modes of RVX-208 and its synthetic precursor, and fluorescent recovery after photobleaching demonstrated that RVX-208 displaces BET proteins from chromatin. However, gene-expression data showed that BD2 inhibition only modestly affects BET-dependent gene transcription. Our data demonstrate the feasibility of specific targeting within the BET family resulting in different transcriptional outcomes and highlight the importance of BD1 in transcriptional regulation.

  1. Identification and Validation of Human DNA Ligase Inhibitors Using Computer-Aided Drug Design

    PubMed Central

    Zhong, Shijun; Chen, Xi; Zhu, Xiao; Dziegielewska, Barbara; Bachman, Kurtis E.; Ellenberger, Tom; Ballin, Jeff D.; Wilson, Gerald M.; Tomkinson, Alan E.; MacKerell, Alexander D.

    2009-01-01

    Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies. PMID:18630893

  2. Hypotensive effect of hydroxylamine, an endogenous nitric oxide donor and SSAO inhibitor.

    PubMed

    Vidrio, H; Medina, M

    2007-01-01

    The endogenous compound hydroxylamine relaxes vascular smooth muscle in vitro, apparently through conversion to the vasodilator factor nitric oxide, but its effect on blood pressure has not been characterized. We found that in the anesthetized rat the amine elicits dose-related hypotension when administered by continuous iv infusion. In experiments designed to explore the mechanism of this effect, hydroxylamine was compared with the nitric oxide donor nitroprusside and the direct-acting vasodilator hydralazine, using pretreatments known to modify diverse mechanisms of vasodilation. Hydroxylamine hypotension was enhanced by the SSAO inhibitor isoniazid and the SSAO substrate methylamine, a pattern shared by hydralazine. Responses were blocked by the guanylate cyclase inhibitor methylene blue and were increased by the nitric oxide synthase inhibitor L-NAME, a pattern shared by nitroprusside. It was concluded that hydroxylamine exerts hypotension partly through conversion to nitric oxide and partly by a "hydralazine-like" mechanism involving SSAO inhibition.

  3. Knowledge, Skills, and Dispositions for Diversity

    ERIC Educational Resources Information Center

    Jones, Anne

    2011-01-01

    The purposes of this research are to explore how currently assessed diversity knowledge, diversity skills, and diversity dispositions of pre-service teachers (PST) relate to each other and further to surmise if the presence of diversity knowledge, diversity skills, and diversity dispositions manifests in cultural efficacy and a general cultural…

  4. Plant Biofilm Inhibitors to Discover Biofilm Genes

    DTIC Science & Technology

    2011-04-08

    REPORT Final Report for Plant Biofilm Inhibitors to Discover Biofilm Genes 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: To control biofilms , we have...synthesized the natural biofilm inhibitor (5Z)-4-bromo-5-(bromomethylene) -3-butyl-2(5H)-furanone from the red alga Delisea pulchra and determined that...Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS biofilms , biofilm inhibitors Thomas K. Wood Texas Engineering

  5. Quantitative High-Throughput Screening Identifies 8-Hydroxyquinolines as Cell-Active Histone Demethylase Inhibitors

    PubMed Central

    Kawamura, Akane; Rose, Nathan R.; Ng, Stanley S.; Quinn, Amy M.; Rai, Ganesha; Mott, Bryan T.; Beswick, Paul; Klose, Robert J.; Oppermann, Udo; Jadhav, Ajit; Heightman, Tom D.; Maloney, David J.; Schofield, Christopher J.; Simeonov, Anton

    2010-01-01

    Background Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. Nε-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. Nε-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors. Principal Findings High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay. Conclusions These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation. PMID:21124847

  6. Diversity in High Schools and Diversity Management: A Qualitative Study

    ERIC Educational Resources Information Center

    Ordu, Aydan

    2015-01-01

    The purpose of the present study is to present the diversities in high schools and opinions of teachers about management of these diversities. The sample of the study is from nine teachers working at the official high schools in the center of Denizli in Turkey. In this qualitative study, the data are collected with a semi-structured interview form…

  7. Diversity Management and Respect for Diversity at Schools

    ERIC Educational Resources Information Center

    Saylik, Ahmet; Polatcan, Mahmut; Saylik, Numan

    2016-01-01

    The purpose of the study is to examine employees' individual attitudes towards diversity management and respect for diversity in secondary education in views of secondary school administrators and teachers, and to explore the relationship between these concepts. According to the results of the study, administrators and teachers in secondary…

  8. Root exudate diversity regulates soil fungal community composition and diversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant diversity is thought to influence diversity of the soil microbial community, though how this occurs is poorly understood. We report that under greenhouse conditions, two model plant species (Arabidopsis thaliana and Medicago truncatula) show an inability to support the native soil fungal comm...

  9. Why Diversity for Diversity's Sake Won't Work

    ERIC Educational Resources Information Center

    Delton, Jennifer

    2007-01-01

    Proponents of "diversity hiring" insist that faculty members of color have a different perspective on issues of race and ethnicity that will increase students' understanding of the multiracial, multicultural world they will inhabit in the 21st century. The mere presence of "diverse" faculty members will prepare students for workplace realities,…

  10. WATER DIVERSION MODEL

    SciTech Connect

    J.B. Case

    1999-12-21

    The distribution of seepage in the proposed repository will be highly variable due in part to variations in the spatial distribution of percolations. The performance of the drip shield and the backfill system may divert the water flux around the waste packages to the invert. Diversion will occur along the drift surface, within the backfill, at the drip shield, and at the Waste Package (WP) surface, even after the drip shield and WP have been breached by corrosion. The purpose and objective of this Analysis and Modeling Report (AMR) are to develop a conceptual model and constitutive properties for bounding the volume and rate of seepage water that flows around the drip shield (CRWMS M&O 1999c). This analysis model is to be compatible with the selected repository conceptual design (Wilkins and Heath, 1999) and will be used to evaluate the performance of the Engineered Barrier System (EBS), and to provide input to the EBS Water Distribution and Removal Model. This model supports the Engineered Barrier System (EBS) postclosure performance assessment for the Site Recommendation (SR). This document characterizes the hydrological constitutive properties of the backfill and invert materials (Section 6.2) and a third material that represents a mixture of the two. These include the Overton Sand which is selected as a backfill (Section 5.2), crushed tuff which is selected as the invert (Section 5.1), and a combined material (Sections 5.9 and 5.10) which has retention and hydraulic conductivity properties intermediate to the selected materials for the backfill and the invert. The properties include the grain size distribution, the dry bulk density and porosity, the moisture retention, the intrinsic permeability, the relative permeability, and the material thermal properties. The van Genuchten relationships with curve fit parameters are used to define the basic retention relationship of moisture potential to volumetric moisture content, and the basic relationship of unsaturated

  11. General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor.

    PubMed

    Brioschi, Daniela; Nadalini, Larissa D; Bengtson, Mario H; Sogayar, Mari Cleide; Moura, Daniel S; Silva-Filho, Marcio C

    2007-12-01

    The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a "shotgun" approach, based on a general up regulation of a large set of endoproteinases.

  12. Combinatorial Pharmacophore-Based 3D-QSAR Analysis and Virtual Screening of FGFR1 Inhibitors

    PubMed Central

    Zhou, Nannan; Xu, Yuan; Liu, Xian; Wang, Yulan; Peng, Jianlong; Luo, Xiaomin; Zheng, Mingyue; Chen, Kaixian; Jiang, Hualiang

    2015-01-01

    The fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) signaling pathway plays crucial roles in cell proliferation, angiogenesis, migration, and survival. Aberration in FGFRs correlates with several malignancies and disorders. FGFRs have proved to be attractive targets for therapeutic intervention in cancer, and it is of high interest to find FGFR inhibitors with novel scaffolds. In this study, a combinatorial three-dimensional quantitative structure-activity relationship (3D-QSAR) model was developed based on previously reported FGFR1 inhibitors with diverse structural skeletons. This model was evaluated for its prediction performance on a diverse test set containing 232 FGFR inhibitors, and it yielded a SD value of 0.75 pIC50 units from measured inhibition affinities and a Pearson’s correlation coefficient R2 of 0.53. This result suggests that the combinatorial 3D-QSAR model could be used to search for new FGFR1 hit structures and predict their potential activity. To further evaluate the performance of the model, a decoy set validation was used to measure the efficiency of the model by calculating EF (enrichment factor). Based on the combinatorial pharmacophore model, a virtual screening against SPECS database was performed. Nineteen novel active compounds were successfully identified, which provide new chemical starting points for further structural optimization of FGFR1 inhibitors. PMID:26110383

  13. [Development of new antiatherosclerotic agents--ACAT inhibitors and CETP inhibitors].

    PubMed

    Miyazaki, A; Horiuchi, S

    1999-12-01

    Development of new antiatherosclerotic agents were reviewed focusing on ACAT inhibitors and CETP inhibitors. ACAT inhibitors enhance intracellular degradation of VLDL in hepatocytes. Cholesterol absorption in small intestine is inhibited by ACAT inhibitors. Thus, ACAT inhibitors reduce plasma cholesterol levels. In atherosclerotic lesions, ACAT inhibitors suppress foam cell formation (cholesteryl ester accumulation) in macrophages. Since ACAT inhibitors have multiple anti-atherogenic effects, they are considered future drugs controlling hypercholesterolemia and atherosclerosis. CETP inhibitors are expected to increase HDL and decrease LDL. Although the patients with CETP deficiency show high level of HDL, recent studies showed that they are not necessarily resistant to atherosclerosis. The strategy to inhibit CETP for suppressing atherosclerosis has not been established.

  14. An Integrated Model of RAF Inhibitor Action Predicts Inhibitor Activity against Oncogenic BRAF Signaling.

    PubMed

    Karoulia, Zoi; Wu, Yang; Ahmed, Tamer A; Xin, Qisheng; Bollard, Julien; Krepler, Clemens; Wu, Xuewei; Zhang, Chao; Bollag, Gideon; Herlyn, Meenhard; Fagin, James A; Lujambio, Amaia; Gavathiotis, Evripidis; Poulikakos, Poulikos I

    2016-09-12

    The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF(V600E) cancers, in which first-generation RAF inhibitors have been ineffective.

  15. Diversity of plant evolutionary lineages promotes arthropod diversity.

    PubMed

    Dinnage, Russell; Cadotte, Marc W; Haddad, Nick M; Crutsinger, Gregory M; Tilman, David

    2012-11-01

    Large-scale habitat destruction and climate change result in the non-random loss of evolutionary lineages, reducing the amount of evolutionary history represented in ecological communities. Yet, we have limited understanding of the consequences of evolutionary history on the structure of food webs and the services provided by biological communities. Drawing on 11 years of data from a long-term plant diversity experiment, we show that evolutionary history of plant communities - measured as phylogenetic diversity - strongly predicts diversity and abundance of herbivorous and predatory arthropods. Effects of plant species richness on arthropods become stronger when phylogenetic diversity is high. Plant phylogenetic diversity explains predator and parasitoid richness as strongly as it does herbivore richness. Our findings indicate that accounting for evolutionary relationships is critical to understanding the severity of species loss for food webs and ecosystems, and for developing conservation and restoration policies.

  16. Diversity among African Pygmies

    PubMed Central

    Ramírez Rozzi, Fernando V.; Sardi, Marina L.

    2010-01-01

    Although dissimilarities in cranial and post-cranial morphology among African pygmies groups have been recognized, comparative studies on skull morphology usually pull all pygmies together assuming that morphological characters are similar among them and different with respect to other populations. The main aim of this study is to compare cranial morphology between African pygmies and non-pygmies populations from Equatorial Africa derived from both the Eastern and the Western regions in order to test if the greatest morphological difference is obtained in the comparison between pygmies and non-pygmies. Thirty three-dimensional (3D) landmarks registered with Microscribe in four cranial samples (Western and Eastern pygmies and non-pygmies) were obtained. Multivariate analysis (generalized Procrustes analysis, Mahalanobis distances, multivariate regression) and complementary dimensions of size were evaluated with ANOVA and post hoc LSD. Results suggest that important cranial shape differentiation does occur between pygmies and non-pygmies but also between Eastern and Western populations and that size changes and allometries do not affect similarly Eastern and Western pygmies. Therefore, our findings raise serious doubt about the fact to consider African pygmies as a homogenous group in studies on skull morphology. Differences in cranial morphology among pygmies would suggest differentiation after divergence. Although not directly related to skull differentiation, the diversity among pygmies would probably suggest that the process responsible for reduced stature occurred after the split of the ancestors of modern Eastern and Western pygmies. PMID:21049030

  17. Diversity of Food Allergy.

    PubMed

    Moriyama, Tatsuya

    2015-01-01

    Food allergy is defined as an immune system-mediated adverse reaction to food components. Food allergic reactions are mostly IgE mediated and also known as immediate type hypersensitivity (type I reaction). There are several characteristic clinical types of food allergy, such as Anaphylaxis, Food-dependent exercise-induced anaphylaxis (FDEIA), and Oral allergy syndrome (OAS). In addition, food allergy is also classified into two types (class 1 and class 2) based on the pathophysiological mechanism. In the class 2 food allergy, pollen allergy causes plant food allergy; therefore this type of allergy is sometimes called Pollen-food allergy syndrome (PFAS). The risk of food allergy (allergenicity) may vary with the treatment of the food allergens. The formation or status of the causative food affects its allergenicity. Class 1 food allergens are generally heat-, enzyme-, and low pH-resistant glycoproteins ranging in size from 10 to 70 kD. Class 1 food allergens induce allergic sensitization via the gastrointestinal tract and are responsible for systemic reactions. Class 2 food allergens are generally heat-labile, susceptible to digestion, and highly homologous with pollen allergens. Taken together, it may be important to consider the diversity of food allergy in order to fight against food allergy.

  18. Functional diversity of laminins.

    PubMed

    Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl

    2012-01-01

    Laminins are a large family of conserved, multidomain trimeric basement membrane proteins that contribute to the structure of extracellular matrix and influence the behavior of associated cells, such as adhesion, differentiation, migration, phenotype stability, and resistance to anoikis. In lower organisms such as Hydra there is only one isoform of laminin, but higher organisms have at least 16 trimeric isoforms with varying degrees of cell/tissue specificity. In vitro protein and cell culture studies, gene manipulation in animals, and laminin gene mutations in human diseases have provided insight into the specific functions of some laminins, but the biological roles of many isoforms are still largely unexplored, mainly owing to difficulties in isolating them in pure form from tissues or cells. In this review, we elucidate the evolution of laminins, describe their molecular complexity, and explore the current knowledge of their diversity and functional aspects, including laminin-mediated signaling via membrane receptors, in vitro cell biology, and involvement in various tissues gained from animal model and human disease studies. The potential use of laminins in cell biology research and biotechnology is discussed.

  19. Particle fuel diversion structure

    SciTech Connect

    Eshleman, R. D.

    1985-07-30

    A particle fuel burning furnace has an upper combustion chamber for holding a pile of particle fuel and burning the same from the bottom thereof. The furnace also includes a lower combustion chamber for after-burning combustible gases given off by the burning of solid fuel in the upper chamber and a series of spaced apart vertically-extending passageways arranged in a row and interconnecting the upper and lower chambers for communicating the combustible gases from the upper to the lower chamber. A first improved feature relates to a particle fuel delivery control device which operates an auger for filling the upper chamber with particle fuel to a desired level. A beam of light is transmitted and reflected between a photoelectric cell and reflector respectively of the device. When the particle fuel pile has grown in height during filling to the desired level the light beam is interrupted and filling is terminated. A second improved feature relates to a particle fuel diversion structure positioned in spaced relationship above and overlying the row of passageways. The structure forms a horizontal slot which extends laterally from the passageways which prevents particles of fuel from falling through the passageways and relocates the flame which burns the particle fuel pile from the bottom to a region away from the passageways.

  20. Digital Diversity Combiner (DIDICOM) - An Applique for Obtaining Quadruple Diversity with Dual Diversity Drama Radio Terminals,

    DTIC Science & Technology

    1980-01-01

    AD-ARO 985 ROME AIR DEVELOPMENT CENTER BRIFFISS AF9 NY F/S 17/2.1 DIGITAL DIVERSITY COMBINER (DIDICOM3 - AN APPLIQUE FOR OBTAININ-ETC(U) JAN 80 F 0...In-Houw Report Jenuuuy 19 GO DIGITAL DIVERSITY COMBINER 0 (DIDICOM) - AN APPLIQUE FOR OBTAINING QUADRUPLE DIVERSITY WITH DUAL DIVERSITY DRAMA RADIO...APPROVED: g e14F.r{ FRED I. DIAOND Technical Director Comunications and Control Division FOR THE COHNDR Acting Chief, Plans Office If your address has

  1. Development of Antioxidant COX-2 Inhibitors as Radioprotective Agents for Radiation Therapy—A Hypothesis-Driven Review

    PubMed Central

    Laube, Markus; Kniess, Torsten; Pietzsch, Jens

    2016-01-01

    Radiation therapy (RT) evolved to be a primary treatment modality for cancer patients. Unfortunately, the cure or relief of symptoms is still accompanied by radiation-induced side effects with severe acute and late pathophysiological consequences. Inhibitors of cyclooxygenase-2 (COX-2) are potentially useful in this regard because radioprotection of normal tissue and/or radiosensitizing effects on tumor tissue have been described for several compounds of this structurally diverse class. This review aims to substantiate the hypothesis that antioxidant COX-2 inhibitors are promising radioprotectants because of intercepting radiation-induced oxidative stress and inflammation in normal tissue, especially the vascular system. For this, literature reporting on COX inhibitors exerting radioprotective and/or radiosensitizing action as well as on antioxidant COX inhibitors will be reviewed comprehensively with the aim to find cross-points of both and, by that, stimulate further research in the field of radioprotective agents. PMID:27104573

  2. Discovery of Novel Polo-Like Kinase 1 Polo-Box Domain Inhibitors to Induce Mitotic Arrest in Tumor Cells.

    PubMed

    Qin, Tan; Chen, Fangjin; Zhuo, Xiaolong; Guo, Xiao; Yun, Taikangxiang; Liu, Ying; Zhang, Chuanmao; Lai, Luhua

    2016-08-11

    Polo-like kinase 1(Plk1) is vital for cell mitosis and has been identified as anticancer target. Its polo-box domain (PBD) mediates substrate binding, blocking of which may offer selective Plk1 inhibition compared to kinase domain inhibitors. Although several PBD inhibitors were reported, most of them suffer from low cell activity. Here, we report the discovery of novel inhibitors to induce mitotic arrest in HeLa cells by virtual screening with Plk1 PBD and cellular activity testing. Of the 81 compounds tested in the cell assay, 10 molecules with diverse chemical scaffolds are potent to induce mitotic arrest of HeLa at low micromolar concentrations. The best compound induces mitotic arrest of HeLa cells with an EC50 of 4.4 μM. The cellular active inhibitors showed binding to Plk1 PBD and compete with PBD substrate in microscale thermophoresis analysis.

  3. Quorum sensing inhibitors: an overview.

    PubMed

    Kalia, Vipin Chandra

    2013-01-01

    Excessive and indiscriminate use of antibiotics to treat bacterial infections has lead to the emergence of multiple drug resistant strains. Most infectious diseases are caused by bacteria which proliferate within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. These molecules act primarily by quenching the QS system. The phenomenon is also termed as quorum quenching (QQ). In addition, synthetic compounds have also been found to be effective in QQ. This review focuses primarily on natural and synthetic quorum sensing inhibitors (QSIs) with the potential for treating bacterial infections. It has been opined that the most versatile prokaryotes to produce QSI are likely to be those, which are generally regarded as safe. Among the eukaryotes, certain legumes and traditional medicinal plants are likely to act as QSIs. Such findings are likely to lead to efficient treatments with much lower doses of drugs especially antibiotics than required at present.

  4. Protein farnesyltransferase inhibitors and progeria.

    PubMed

    Meta, Margarita; Yang, Shao H; Bergo, Martin O; Fong, Loren G; Young, Stephen G

    2006-10-01

    Genetic mutations that lead to an accumulation of farnesyl-prelamin A cause progeroid syndromes, including Hutchinson-Gilford progeria syndrome. It seemed possible that the farnesylated form of prelamin A might be toxic to mammalian cells, accounting for all the disease phenotypes that are characteristic of progeria. This concept led to the hypothesis that protein farnesyltransferase inhibitors (FTIs) might ameliorate the disease phenotypes of progeria in mouse models. Thus far, two different mouse models of progeria have been examined. In both models, FTIs improved progeria-like disease phenotypes. Here, prelamin A post-translational processing is discussed and several mutations underlying human progeroid syndromes are described. In addition, recent data showing that FTIs ameliorate disease phenotypes in a pair of mouse models of progeria are discussed.

  5. Macrocyclic Inhibitors of Hsp90

    PubMed Central

    Johnson, Victoria A.; Singh, Erinprit K.; Nazarova, Lidia A.; Alexander, Leslie D.; McAlpine, Shelli R.

    2011-01-01

    Heat shock proteins (HSP) are a family of highly conserved proteins, whose expression increases in response to stresses that may threaten cell survival. Over the past decade, heat shock protein 90 (Hsp90) has emerged as a potential therapeutic target for cancer as it plays a vital role in normal cell maturation and acts as a molecular chaperone for proper folding, assembly, and stabilization of many oncogenic proteins. To date, a majority of Hsp90 inhibitors that have been discovered are macrocycles. The relatively rigid conformation provided by the macrocyclic scaffold allows for a selective interaction with a biological target such as Hsp90. This review highlights the discovery and development of nine macro-cycles that inhibit the function of Hsp90, detailing their potency and the client proteins affected by Hsp90 inhibition. PMID:20536417

  6. Quinolone-based HDAC inhibitors.

    PubMed

    Balasubramanian, Gopalan; Kilambi, Narasimhan; Rathinasamy, Suresh; Rajendran, Praveen; Narayanan, Shridhar; Rajagopal, Sridharan

    2014-08-01

    HDAC inhibitors emerged as promising drug candidates in combating wide variety of cancers. At present, two of the compounds SAHA and Romidepsin were approved by FDA for cutaneous T-cell lymphoma and many are in various clinical phases. A new quinolone cap structure was explored with hydroxamic acid as zinc-binding group (ZBG). The pan HDAC inhibitory and antiproliferative activities against three human cancer cell lines HCT-116 (colon), NCI-H460 (lung) and U251 (glioblastoma) of the compounds (4a-4w) were evaluated. Introduction of heterocyclic amines in CAP region increased the enzyme inhibitory and antiproliferative activities and few of the compounds tested are metabolically stable in both MLM and HLM.

  7. Checkpoint inhibitors in Hodgkin's lymphoma.

    PubMed

    Jezeršek Novaković, Barbara

    2016-04-01

    Hodgkin's lymphoma is unusual among cancers in that it consists of a small number of malignant Hodgkin/Reed-Sternberg cells in a sea of immune system cells, including T cells. Most of these T cells are reversibly inactivated in different ways and their reactivation may induce a very strong immune response to cancer cells. One way of reactivation of T cells is with antibodies blocking the CTLA-4 and especially with antibodies directed against PD-1 or the PD-L1 ligand thereby reversing the tumor-induced downregulation of T-cell function and augmenting antitumor immune activity at the priming (CTLA-4) or tissue effector (PD-1) phase. Immune checkpoint inhibitors have been evidenced as an additional treatment option with substantial effectiveness and acceptable toxicity in heavily pretreated patients with Hodgkin's lymphoma. Particularly, PD-1 blockade with nivolumab and pembrolizumab has demonstrated significant single-agent activity in this select population.

  8. Glycine Transporters and Their Inhibitors

    NASA Astrophysics Data System (ADS)

    Gilfillan, Robert; Kerr, Jennifer; Walker, Glenn; Wishart, Grant

    Glycine plays a ubiquitous role in many biological processes. In the central nervous system it serves as an important neurotransmitter acting as an agonist at strychnine-sensitive glycine receptors and as an essential co-agonist with glutamate at the NMDA receptor complex. Control of glycine concentrations in the vicinity of these receptors is mediated by the specific glycine transporters, GlyT1 and GlyT2. Inhibition of these transporters has been postulated to be of potential benefit in several therapeutic indications including schizophrenia and pain. In this review we discuss our current knowledge of glycine transporters and focus on recent advances in the medicinal chemistry of GlyT1 and GlyT2 inhibitors.

  9. Loratadine analogues as MAGL inhibitors.

    PubMed

    Patel, Jayendra Z; Ahenkorah, Stephen; Vaara, Miia; Staszewski, Marek; Adams, Yahaya; Laitinen, Tuomo; Navia-Paldanius, Dina; Parkkari, Teija; Savinainen, Juha R; Walczyński, Krzysztof; Laitinen, Jarmo T; Nevalainen, Tapio J

    2015-04-01

    Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46 nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24 μM) and 35-fold higher selectivity over human α/β-hydrolase-6 (hABHD6, IC50=1.79 μM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations ⩽ 10 μM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.

  10. Citizenship, Diversity and National Identity

    ERIC Educational Resources Information Center

    Crick, Bernard

    2008-01-01

    This article explores the issues of citizenship, diversity and national identity in the context of the introduction of citizenship education in the UK. It considers the historical context of national identity in the UK and notes that the "British national identity has historically implied diversity". It also analyses the views of British…

  11. Plasmas: Diversity, pervasiveness and potential

    SciTech Connect

    Eastman, Timothy E.

    1998-01-01

    When considered inclusively, plasma science and technology encompass immense diversity, pervasiveness and potential: diversity through numerous topical areas (see list of nearly 200 in Table II); pervasiveness with examples covering the full range of energy, time and spatial scale; and potential through innumerable current and future applications.

  12. Brownian Motion, "Diverse and Undulating"

    NASA Astrophysics Data System (ADS)

    Duplantier, Bertrand

    Truly man is a marvelously vain, diverse, and undulating object. It is hard to found any constant and uniform judgment on him. Michel de Montaigne, Les Essais, Book I, Chapter 1: "By diverse means we arrive at the same end"; in The Complete Essays of Montaigne, Donald M. Frame transl., Stanford University Press (1958).

  13. Pathways to Tolerance: Student Diversity.

    ERIC Educational Resources Information Center

    Daugherty, Dorothy, Ed.; Stanhope, Victoria, Ed.

    Ideas for schools to support tolerance and celebrate student diversity are presented in this volume of reprinted articles. Titles include: (1) "One of Us, One of Them: Lessons in Diversity for a School Psychologist" (M. M. Chittooran); (2) "The Tolerance-in-Action Campaign" (H. M. Knoff); (3) "Immigrant Parents and the…

  14. Cultural Diversity: Implications for Collaboration.

    ERIC Educational Resources Information Center

    Jairrels, Veda

    1999-01-01

    Explores the implications of an increasingly diverse school population for the process of teacher collaboration. Focuses on the competencies for collaboration as pertinent to diverse exceptional learners, the role of the special education teacher, and the concept of collaboration across disciplines. (Author/CR)

  15. The Scoop on Board Diversity.

    ERIC Educational Resources Information Center

    Holland, Robert, Jr.

    1996-01-01

    In an interview, a prominent college trustee discusses strategies for increasing ethnic and cultural diversity on governing boards, the relationship between board diversity and institutional policies, effects on the student body, the responsibility of the board, and the challenges and rewards of trusteeship. (MSE)

  16. Genetic Diversity and Human Equality.

    ERIC Educational Resources Information Center

    Dobzhansky, Theodosius

    The idea of equality often, if not frequently, bogs down in confusion and apparent contradictions; equality is confused with identity, and diversity with inequality. It would seem that the easiest way to discredit the idea of equality is to show that people are innately, genetically, and, therefore, irremediably diverse and unlike. The snare is,…

  17. Working with Culturally Diverse Learners.

    ERIC Educational Resources Information Center

    Knott, Elizabeth S.

    1991-01-01

    Reviews demographic and economic trends promoting cultural diversity in postsecondary education. Urges educators to support cultural diversity and respect cultural differences, rather than forcing students to reject their culture of origin and adopt the dominant culture. Discusses instructional implications of ethnic/racial differences,…

  18. The Changing Face of Diversity

    ERIC Educational Resources Information Center

    Reis, Noni Mendoza; Mendez, Sylvia

    2009-01-01

    As the nation's schools strive to provide quality education for students most at risk for failure, the notion of diversity continues to lead the discussion. Revisiting understandings about diversity as a response to creating equitable learning opportunities to foster achievement for all students has become increasingly urgent given that, while the…

  19. Diversity, Disunity, and Campus Community.

    ERIC Educational Resources Information Center

    Terrell, Melvin C., Ed.

    This monograph offers a collection of nine papers demonstrating how the student affairs subculture in institutions of higher education can provide academic as well as managerial leadership in promoting cultural diversity and planned change. The papers are as follows: (1) "Achieving Cultural Diversity: Meeting the Challenges" by Barbara…

  20. Area and mammalian elevational diversity.

    PubMed

    McCain, Christy M

    2007-01-01

    Elevational gradients hold enormous potential for understanding general properties of biodiversity. Like latitudinal gradients, the hypotheses for diversity patterns can be grouped into historical explanations, climatic drivers, and spatial hypotheses. The spatial hypotheses include the species-area effect and spatial constraint (mid-domain effect null models). I test these two spatial hypotheses using regional diversity patterns for mammals (non-volant small mammals and bats) along 34 elevational gradients spanning 24.4 degrees S-40.4 degrees N latitude. There was high variability in the fit to the species-area hypothesis and the mid-domain effect. Both hypotheses can be eliminated as primary drivers of elevational diversity. Area and spatial constraint both represent sources of error rather than mechanisms underlying these mammalian diversity patterns. Similar results are expected for other vertebrate taxa, plants, and invertebrates since they show comparable distributions of elevational diversity patterns to mammalian patterns.

  1. New rubrolide analogues as inhibitors of photosynthesis light reactions.

    PubMed

    Varejão, Jodieh O S; Barbosa, Luiz C A; Ramos, Gabriela Álvarez; Varejão, Eduardo V V; King-Díaz, Beatriz; Lotina-Hennsen, Blas

    2015-04-01

    Natural products called rubrolides have been investigated as a model for the development of new herbicides that act on the photosynthesis apparatus. This study comprises a comprehensive analysis of the photosynthesis inhibitory ability of 27 new structurally diverse rubrolide analogues. In general, the results revealed that the compounds exhibited efficient inhibition of the photosynthetic process, but in some cases low water solubility may be a limiting factor. To elucidate their mode of action, the effects of the compounds on PSII and PSI, as well as their partial reaction on chloroplasts and the chlorophyll a fluorescence transients were measured. Our results showed that some of the most active rubrolide analogues act as a Hill reaction inhibitors at the QB level by interacting with the D1 protein at the reducing side of PSII. All of the active analogues follow Tice's rule of 5, which indicates that these compounds present physicochemical properties suitable for herbicides.

  2. Transglutaminase 2 Inhibitors and their Therapeutic Role in Disease States

    PubMed Central

    Siegel, Matthew; Khosla, Chaitan

    2007-01-01

    Transglutaminase 2 (TG2) is a multi-domain, multi-functional enzyme that post-translationally modifies proteins by catalyzing the formation of intermolecular isopeptide bonds between glutamine and lysine side-chains. It plays a role in diverse biological functions, including extracellular matrix formation, integrin mediated signaling, and signal transduction involving 7-transmembrane receptors. While some of the roles of TG2 under normal physiological conditions remain obscure, the protein is believed to participate in the pathogenesis of several unrelated diseases including celiac sprue, neurodegenerative diseases, and certain types of cancer. A variety of small molecule and peptidomimetic inhibitors of the TG2 active site have been identified. Here we summarize the biochemistry, biology, pharmacology and medicinal chemistry of human TG2. PMID:17582505

  3. Trypsin inhibitors for the treatment of pancreatitis.

    PubMed

    Brandl, Trixi; Simic, Oliver; Skaanderup, Philip R; Namoto, Kenji; Berst, Frederic; Ehrhardt, Claus; Schiering, Nikolaus; Mueller, Irene; Woelcke, Julian

    2016-09-01

    Proline-based trypsin inhibitors occupying the S1-S2-S1' region were identified by an HTS screening campaign. It was discovered that truncation of the P1' moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.

  4. Intellectual property issues of immune checkpoint inhibitors

    PubMed Central

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors. PMID:26466763

  5. Discovery of novel heterocyclic factor VIIa inhibitors.

    PubMed

    Rai, Roopa; Kolesnikov, Aleksandr; Sprengeler, Paul A; Torkelson, Steven; Ton, Tony; Katz, Bradley A; Yu, Christine; Hendrix, John; Shrader, William D; Stephens, Robin; Cabuslay, Ronnell; Sanford, Ellen; Young, Wendy B

    2006-04-15

    Structure-activity relationships and binding mode of novel heterocyclic factor VIIa inhibitors will be described. In these inhibitors, a highly basic 5-amidinoindole moiety has been successfully replaced with a less basic 5-aminopyrrolo[3,2-b]pyridine scaffold.

  6. Rust inhibitor and oil composition containing same

    SciTech Connect

    Bialy, J.J.; Cullen, W.P.; Dorn, P.; Nebzydoski, J.W.; Sung, R.L.

    1981-04-21

    A rust inhibitor comprising the reaction product of a hydrocarbylsuccinic anhydride in which the hydrocarbyl radical has from about 6 to 30 carbon atoms and an aminotriazole is provided. The rust inhibitor is effective in motor fuel and lubricating oil compositions.

  7. Wheat Landrace Genome Diversity.

    PubMed

    Wingen, Luzie U; West, Claire; Leverington-Waite, Michelle; Collier, Sarah; Orford, Simon; Goram, Richard; Yang, Cai-Yun; King, Julie; Allen, Alexandra M; Burridge, Amanda; Edwards, Keith J; Griffiths, Simon

    2017-04-01

    Understanding the genomic complexity of bread wheat (Triticum aestivum L.) is a cornerstone in the quest to unravel the processes of domestication and the following adaptation of domesticated wheat to a wide variety of environments across the globe. Additionally, it is of importance for future improvement of the crop, particularly in the light of climate change. Focusing on the adaptation after domestication, a nested association mapping (NAM) panel of 60 segregating biparental populations was developed, mainly involving landrace accessions from the core set of the Watkins hexaploid wheat collection optimized for genetic diversity. A modern spring elite variety, "Paragon," was used as common reference parent. Genetic maps were constructed following identical rules to make them comparable. In total, 1611 linkage groups were identified, based on recombination from an estimated 126,300 crossover events over the whole NAM panel. A consensus map, named landrace consensus map (LRC), was constructed and contained 2498 genetic loci. These newly developed genetics tools were used to investigate the rules underlying genome fluidity or rigidity, e.g., by comparing marker distances and marker orders. In general, marker order was highly correlated, which provides support for strong synteny between bread wheat accessions. However, many exceptional cases of incongruent linkage groups and increased marker distances were also found. Segregation distortion was detected for many markers, sometimes as hot spots present in different populations. Furthermore, evidence for translocations in at least 36 of the maps was found. These translocations fell, in general, into many different translocation classes, but a few translocation classes were found in several accessions, the most frequent one being the well-known T5B:7B translocation. Loci involved in recombination rate, which is an interesting trait for plant breeding, were identified by QTL analyses using the crossover counts as a trait

  8. Wheat Landrace Genome Diversity

    PubMed Central

    Wingen, Luzie U.; West, Claire; Leverington-Waite, Michelle; Collier, Sarah; Orford, Simon; Goram, Richard; Yang, Cai-Yun; King, Julie; Allen, Alexandra M.; Burridge, Amanda; Edwards, Keith J.; Griffiths, Simon

    2017-01-01

    Understanding the genomic complexity of bread wheat (Triticum aestivum L.) is a cornerstone in the quest to unravel the processes of domestication and the following adaptation of domesticated wheat to a wide variety of environments across the globe. Additionally, it is of importance for future improvement of the crop, particularly in the light of climate change. Focusing on the adaptation after domestication, a nested association mapping (NAM) panel of 60 segregating biparental populations was developed, mainly involving landrace accessions from the core set of the Watkins hexaploid wheat collection optimized for genetic diversity. A modern spring elite variety, “Paragon,” was used as common reference parent. Genetic maps were constructed following identical rules to make them comparable. In total, 1611 linkage groups were identified, based on recombination from an estimated 126,300 crossover events over the whole NAM panel. A consensus map, named landrace consensus map (LRC), was constructed and contained 2498 genetic loci. These newly developed genetics tools were used to investigate the rules underlying genome fluidity or rigidity, e.g., by comparing marker distances and marker orders. In general, marker order was highly correlated, which provides support for strong synteny between bread wheat accessions. However, many exceptional cases of incongruent linkage groups and increased marker distances were also found. Segregation distortion was detected for many markers, sometimes as hot spots present in different populations. Furthermore, evidence for translocations in at least 36 of the maps was found. These translocations fell, in general, into many different translocation classes, but a few translocation classes were found in several accessions, the most frequent one being the well-known T5B:7B translocation. Loci involved in recombination rate, which is an interesting trait for plant breeding, were identified by QTL analyses using the crossover counts as a

  9. Neuroblastoma stem cells - mechanisms of chemoresistance and histone deacetylase inhibitors.

    PubMed

    Khalil, M A; Hrabeta, J; Cipro, S; Stiborova, M; Vicha, A; Eckschlager, T

    2012-01-01

    Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: self-renewal capacity, the ability to develop into different lineages and proliferative potential. The interest in CSCs emerged from their expected role in initiation, progression and recurrence of many tumors. They are generally resistant to conventional chemotherapy and radiotherapy. There are two hypotheses about their origin: The first assumes that CSCs may arise from normal stem cells, and the second supposes that differentiated cells acquire the properties of CSCs. Both hypotheses are not mutually exclusive, as it is possible that CSCs have a diverse origin in different tumors. CD133+ cells (CD133 is marker of CSC in some tumors) isolated from NBL, osteosarcoma and Ewing sarcoma cell lines are resistant to cisplatin, carboplatin, etoposide and doxorubicin than the CD133- ones. Being resistant to chemotherapy, there were many attempts to target CSCs epigenetically including the use of histone deacetylase inhibitors. The diverse influence of valproic acid (histone deacetylase inhibitor) on normal and cancer stem cells was proved in different experiments. We have found an increase percentage of CD133+ NBL cells after their incubation with VPA in a dose that does not induce apoptosis. Further researches on CSCs and clinical application for their detection are necessary: (i) to define the CSC function in carcinogenesis, cancer development and their role in metastasis; (ii) to find a specific marker for CSCs in different tumors; (iii) to explain the role of different pathways that determine their behavior and (iv) to explain mechanisms of chemoresistance of CSCs.

  10. Exploring the scaffold universe of kinase inhibitors.

    PubMed

    Hu, Ye; Bajorath, Jürgen

    2015-01-08

    The scaffold concept was applied to systematically determine, analyze, and compare core structures of kinase inhibitors. From publicly available inhibitors of the human kinome, scaffolds and cyclic skeletons were systematically extracted and organized taking activity data, structural relationships, and retrosynthetic criteria into account. Scaffold coverage varied greatly across the kinome, and many scaffolds representing compounds with different activity profiles were identified. The majority of kinase inhibitor scaffolds were involved in well-defined yet distinct structural relationships, which had different consequences on compound activity. Scaffolds exclusively representing highly potent compounds were identified as well as structurally analogous scaffolds with very different degrees of promiscuity. Scaffold relationships presented herein suggest a variety of hypotheses for inhibitor design. Our detailed organization of the kinase inhibitor scaffold universe with respect to different activity and structural criteria, all scaffolds, and the original compound data assembled for our analysis are made freely available.

  11. Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Lauffenburger, Doug A; Chen, Chia-Hung

    2015-02-21

    Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

  12. Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes.

    PubMed

    Forsberg, Kevin J; Patel, Sanket; Witt, Evan; Wang, Bin; Ellison, Tyler D; Dantas, Gautam

    2015-11-06

    The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications.

  13. QSAR, molecular docking studies of thiophene and imidazopyridine derivatives as polo-like kinase 1 inhibitors

    NASA Astrophysics Data System (ADS)

    Cao, Shandong

    2012-08-01

    The purpose of the present study was to develop in silico models allowing for a reliable prediction of polo-like kinase inhibitors based on a large diverse dataset of 136 compounds. As an effective method, quantitative structure activity relationship (QSAR) was applied using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The proposed QSAR models showed reasonable predictivity of thiophene analogs (Rcv2=0.533, Rpred2=0.845) and included four molecular descriptors, namely IC3, RDF075m, Mor02m and R4e+. The optimal model for imidazopyridine derivatives (Rcv2=0.776, Rpred2=0.876) was shown to perform good in prediction accuracy, using GATS2m and BEHe1 descriptors. Analysis of the contour maps helped to identify structural requirements for the inhibitors and served as a basis for the design of the next generation of the inhibitor analogues. Docking studies were also employed to position the inhibitors into the polo-like kinase active site to determine the most probable binding mode. These studies may help to understand the factors influencing the binding affinity of chemicals and to develop alternative methods for prescreening and designing of polo-like kinase inhibitors.

  14. The bean. alpha. -amylase inhibitor is encoded by a lectin gene

    SciTech Connect

    Moreno, J.; Altabella, T.; Chrispeels, M.J. )

    1989-04-01

    The common bean, Phaseolus vulgaris, contains an inhibitor of insect and mammalian {alpha}-amylases that does not inhibit plant {alpha}-amylase. This inhibitor functions as an anti-feedant or seed-defense protein. We purified this inhibitor by affinity chromatography and found that it consists of a series of glycoforms of two polypeptides (Mr 14,000-19,000). Partial amino acid sequencing was carried out, and the sequences obtained are identical with portions of the derived amino acid sequence of a lectin-like gene. This lectin gene encodes a polypeptide of MW 28,000, and the primary in vitro translation product identified by antibodies to the {alpha}-amylase inhibitor has the same size. Co- and posttranslational processing of this polypeptide results in glycosylated polypeptides of 14-19 kDa. Our interpretation of these results is that the bean lectins constitute a gene family that encodes diverse plant defense proteins, including phytohemagglutinin, arcelin and {alpha}-amylase inhibitor.

  15. Identification of Polo-like kinase 1 interaction inhibitors using a novel cell-based assay

    PubMed Central

    Normandin, Karine; Lavallée, Jean-François; Futter, Marie; Beautrait, Alexandre; Duchaine, Jean; Guiral, Sébastien; Marinier, Anne; Archambault, Vincent

    2016-01-01

    Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries. PMID:27874094

  16. Identification of Genes Conferring Tolerance to Lignocellulose-Derived Inhibitors by Functional Selections in Soil Metagenomes

    PubMed Central

    Forsberg, Kevin J.; Patel, Sanket; Witt, Evan; Wang, Bin; Ellison, Tyler D.

    2015-01-01

    The production of fuels or chemicals from lignocellulose currently requires thermochemical pretreatment to release fermentable sugars. These harsh conditions also generate numerous small-molecule inhibitors of microbial growth and fermentation, limiting production. We applied small-insert functional metagenomic selections to discover genes that confer microbial tolerance to these inhibitors, identifying both individual genes and general biological processes associated with tolerance to multiple inhibitory compounds. Having screened over 248 Gb of DNA cloned from 16 diverse soil metagenomes, we describe gain-of-function tolerance against acid, alcohol, and aldehyde inhibitors derived from hemicellulose and lignin, demonstrating that uncultured soil microbial communities hold tremendous genetic potential to address the toxicity of pretreated lignocellulose. We recovered genes previously known to confer tolerance to lignocellulosic inhibitors as well as novel genes that confer tolerance via unknown functions. For instance, we implicated galactose metabolism in overcoming the toxicity of lignin monomers and identified a decarboxylase that confers tolerance to ferulic acid; this enzyme has been shown to catalyze the production of 4-vinyl guaiacol, a valuable precursor to vanillin production. These metagenomic tolerance genes can enable the flexible design of hardy microbial catalysts, customized to withstand inhibitors abundant in specific bioprocessing applications. PMID:26546427

  17. Discovery of 8-Membered Ring Sulfonamides as Inhibitors of Oncogenic Mutant Isocitrate Dehydrogenase 1.

    PubMed

    Law, Jason M; Stark, Sebastian C; Liu, Ke; Liang, Norah E; Hussain, Mahmud M; Leiendecker, Matthias; Ito, Daisuke; Verho, Oscar; Stern, Andrew M; Johnston, Stephen E; Zhang, Yan-Ling; Dunn, Gavin P; Shamji, Alykhan F; Schreiber, Stuart L

    2016-10-13

    Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress (R)-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo, the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.

  18. High-throughput screening to identify selective inhibitors of microbial sulfate reduction (and beyond)

    NASA Astrophysics Data System (ADS)

    Carlson, H. K.; Coates, J. D.; Deutschbauer, A. M.

    2015-12-01

    The selective perturbation of complex microbial ecosystems to predictably influence outcomes in engineered and industrial environments remains a grand challenge for geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and corrosive. Current strategies to selectively inhibit sulfidogenesis are based on non-specific biocide treatments, bio-competitive exclusion by alternative electron acceptors or sulfate-analogs which are competitive inhibitors or futile/alternative substrates of the sulfate reduction pathway. Despite the economic cost of sulfidogenesis, there has been minimal exploration of the chemical space of possible inhibitory compounds, and very little work has quantitatively assessed the selectivity of putative souring treatments. We have developed a high-throughput screening strategy to target SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and identified previously unrecognized SRM selective inhibitors and synergistic interactions between inhibitors. Once inhibitor selectivity is defined, high-throughput characterization of microbial community structure across compound gradients and identification of fitness determinants using isolate bar-coded transposon mutant libraries can give insights into the genetic mechanisms whereby compounds structure microbial communities. The high-throughput (HT) approach we present can be readily applied to target SRM in diverse environments and more broadly, could be used to identify and quantify the potency and selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach are relevant for engineering environmental ecosystems and also to understand the role of natural gradients in shaping microbial niche space.

  19. Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

    PubMed

    Nguyen, Phuong Q T; Luu, Thuy T; Bai, Yang; Nguyen, Giang K T; Pervushin, Konstantin; Tam, James P

    2015-04-24

    Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

  20. Selective Targeting of Extracellular Insulin-Degrading Enzyme by Quasi-Irreversible Thiol-Modifying Inhibitors.

    PubMed

    Abdul-Hay, Samer O; Bannister, Thomas D; Wang, Hui; Cameron, Michael D; Caulfield, Thomas R; Masson, Amandine; Bertrand, Juliette; Howard, Erin A; McGuire, Michael P; Crisafulli, Umberto; Rosenberry, Terrone R; Topper, Caitlyn L; Thompson, Caroline R; Schürer, Stephan C; Madoux, Franck; Hodder, Peter; Leissring, Malcolm A

    2015-12-18

    Many therapeutically important enzymes are present in multiple cellular compartments, where they can carry out markedly different functions; thus, there is a need for pharmacological strategies to selectively manipulate distinct pools of target enzymes. Insulin-degrading enzyme (IDE) is a thiol-sensitive zinc-metallopeptidase that hydrolyzes diverse peptide substrates in both the cytosol and the extracellular space, but current genetic and pharmacological approaches are incapable of selectively inhibiting the protease in specific subcellular compartments. Here, we describe the discovery, characterization, and kinetics-based optimization of potent benzoisothiazolone-based inhibitors that, by virtue of a unique quasi-irreversible mode of inhibition, exclusively inhibit extracellular IDE. The mechanism of inhibition involves nucleophilic attack by a specific active-site thiol of the enzyme on the inhibitors, which bear an isothiazolone ring that undergoes irreversible ring opening with the formation of a disulfide bond. Notably, binding of the inhibitors is reversible under reducing conditions, thus restricting inhibition to IDE present in the extracellular space. The identified inhibitors are highly potent (IC50(app) = 63 nM), nontoxic at concentrations up to 100 μM, and appear to preferentially target a specific cysteine residue within IDE. These novel inhibitors represent powerful new tools for clarifying the physiological and pathophysiological roles of this poorly understood protease, and their unusual mechanism of action should be applicable to other therapeutic targets.

  1. Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors.

    PubMed

    Bertrand, J A; Thieffine, S; Vulpetti, A; Cristiani, C; Valsasina, B; Knapp, S; Kalisz, H M; Flocco, M

    2003-10-17

    GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.

  2. Recent developments in tubulin polymerization inhibitors: An overview.

    PubMed

    Kaur, Ramandeep; Kaur, Gurneet; Gill, Rupinder Kaur; Soni, Richard; Bariwal, Jitender

    2014-11-24

    Microtubules are protein biopolymers formed through polymerization of heterodimers of α- and β-tubulins. Disruption of microtubules can induce cell cycle arrest in G2-M phase and formation of abnormal mitotic spindles. Their importance in mitosis and cell division makes microtubules an attractive target for anticancer drug discovery. A number of naturally occurring compounds such as paclitaxel, epothilones, vinblastine, combretastatin, and colchicines exert their effect by changing dynamics of tubulin such as polymerization and depolymerization. During past few years, rapid development of the novel tubulin polymerization inhibitors has been witnessed. Diverse classes of chemical compounds from the natural as well as from the synthetic origin have been extensively studied. This review highlights the various classes of synthetically derived chemical compounds those have been reported in last few years as potential tubulin polymerization inhibitors. A brief synthetic methodology to access these compounds has been highlighted along with the brief SAR studies. We strongly believe that this review will provide a platform to the synthetic chemists and biologists to design and synthesize new and potent compounds to inhibit the tubulin polymerization.

  3. INHIBITOR OF APOPTOSIS PROTEINS AS INTRACELLULAR SIGNALING INTERMEDIATES

    PubMed Central

    Kocab, Andrew J.; Duckett, Colin S.

    2015-01-01

    The inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early studies describing their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving for the vital roles played by the IAPs as transduction intermediates in a diverse set of signaling cascades that have been associated with functions ranging from the innate immune response to cell migration to cell cycle regulation. In this review, we discuss the functions of the IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in the TNF receptor superfamily signaling cascades, which include the activation of the NF-κB transcription factor family. Since these receptors can modulate cell proliferation and cell death, the roles of the c-IAPs in these pathways provide additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may impact c-IAP activity in intracellular signaling. Collectively, the multifaceted functions and complex regulation of the c-IAPs illustrate the importance of the c-IAPs as intracellular signaling intermediates. PMID:26462035

  4. Novel inhibitor cystine knot peptides from Momordica charantia.

    PubMed

    He, Wen-Jun; Chan, Lai Yue; Clark, Richard J; Tang, Jun; Zeng, Guang-Zhi; Franco, Octavio L; Cantacessi, Cinzia; Craik, David J; Daly, Norelle L; Tan, Ning-Hua

    2013-01-01

    Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

  5. Inhibitor of apoptosis proteins as intracellular signaling intermediates.

    PubMed

    Kocab, Andrew J; Duckett, Colin S

    2016-01-01

    Inhibitor of apoptosis (IAP) proteins have often been considered inhibitors of cell death due to early reports that described their ability to directly bind and inhibit caspases, the primary factors that implement apoptosis. However, a greater understanding is evolving regarding the vital roles played by IAPs as transduction intermediates in a diverse set of signaling cascades associated with functions ranging from the innate immune response to cell migration to cell-cycle regulation. In this review, we discuss the functions of IAPs in signaling, focusing primarily on the cellular IAP (c-IAP) proteins. The c-IAPs are important components in tumor necrosis factor receptor superfamily signaling cascades, which include activation of the NF-κB transcription factor family. As these receptors modulate cell proliferation and cell death, the involvement of the c-IAPs in these pathways provides an additional means of controlling cellular fate beyond simply inhibiting caspase activity. Additionally, IAP-binding proteins, such as Smac and caspases, which have been described as having cell death-independent roles, may affect c-IAP activity in intracellular signaling. Collectively, the multi-faceted functions and complex regulation of the c-IAPs illustrate their importance as intracellular signaling intermediates.

  6. Maximizing the Therapeutic Potential of Hsp90 Inhibitors

    PubMed Central

    Butler, Lisa M.; Ferraldeschi, Roberta; Armstrong, Heather K.; Centenera, Margaret M.; Workman, Paul

    2015-01-01

    Hsp90 is required for maintaining the stability and activity of a diverse group of client proteins, including protein kinases, transcription factors and steroid hormone receptors involved in cell signaling, proliferation, survival, oncogenesis and cancer progression. Inhibition of Hsp90 alters the Hsp90-client protein complex, leading to reduced activity, misfolding, ubiquitination and, ultimately, proteasomal degradation of client proteins. Hsp90 inhibitors have demonstrated significant antitumor activity in a wide variety of preclinical models with evidence of selectivity for cancer versus normal cells. In the clinic however, the efficacy of this class of therapeutic agents has been relatively limited to date, with promising responses mainly observed in breast and lung cancer, but no major activity seen in other tumor types. In addition, adverse events and some significant toxicities have been documented. Key to improving these clinical outcomes is a better understanding of the cellular consequences of inhibiting Hsp90 that may underlie treatment response or resistance. This review considers the recent progress that has been made in the study of Hsp90 and its inhibitors, and highlights new opportunities to maximize their therapeutic potential. PMID:26219697

  7. Diverse roles of strigolactones in plant development.

    PubMed

    Brewer, Philip B; Koltai, Hinanit; Beveridge, Christine A

    2013-01-01

    With the discovery of strigolactones as root exudate signals that trigger parasitic weed seed germination, and then as a branching inhibitor and plant hormone, the next phase of strigolactone research has quickly revealed this hormone class as a major player in optimizing plant growth and development. From the early stages of plant evolution, it seems that strigolactones were involved in enabling plants to modify growth in order to gain advantage in competition with neighboring organisms for limited resources. For example, a moss plant can alter its growth in response to strigolactones emanating from a neighbor. Within a higher plant, strigolactones appear to be involved in controlling the balance of resource distribution via strategic modification of growth and development. Most notably, higher plants that encounter phosphate deficiency increase strigolactone production, which changes root growth and promotes fungal symbiosis to enhance phosphate intake. The shoot also changes by channeling resources away from unessential leaves and branches and into the main stem and root system. This hormonal response is a key adaption that radically alters whole-plant architecture in order to optimize growth and development under diverse environmental conditions.

  8. The phylogenetic diversity of metagenomes.

    PubMed

    Kembel, Steven W; Eisen, Jonathan A; Pollard, Katherine S; Green, Jessica L

    2011-01-01

    Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.

  9. A novel inhibitor of Rho GDP-dissociation inhibitor α improves the therapeutic efficacy of paclitaxel in Lewis lung carcinoma.

    PubMed

    Peng, Xing Chen; Chen, Xu Xia; Zhang, Y U; Wang, Hai Jun; Feng, You

    2015-07-01

    Molecular-targeted therapies are considered a promising strategy for the treatment of most types of human cancer. Rho GDP-dissociation inhibitor α (RhoGDIα), which functions mainly by controlling the cellular distribution and activity of Rho GTPases and is associated with tumor progression and poor prognosis of cancer patients, has become a new promising target for anticancer treatment. Recently, a specific RhoGDIα inhibitor (no. SKLB-163) was developed via computer-aided drug design and de novo synthesis. Previous studies have shown that SKLB-163 had extremely good antitumor activities against diverse cancer cell lines. In the present study, SKLB-163 was used in combination with paclitaxel in order to determine the synergistic effect of the antitumor activity. The findings showed that the combination therapy clearly inhibited cell proliferation and induced apoptosis of LL/2 in vitro. The LL/2 mice model also showed that the combination therapy inhibited tumor growth in vivo. Proliferative cell nuclear antigen (PCNA) immunohistochmeistry and terminal deoxynucleotidyl transferase dUTP nick end-labeling showed that combination therapy inhibited cell proliferation and increased apoptosis compared to the treatment with SKLB-163 or paclitaxel alone. The data suggests that the combination therapy exerted synergistic antitumor effects, providing a novel way to augment the antitumor efficacy of cytotoxic chemotherapy.

  10. Designing Inhibitors of Anthrax Toxin

    PubMed Central

    Nestorovich, Ekaterina M.; Bezrukov, Sergey M.

    2014-01-01

    Introduction Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates, and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded “for the development of multiscale models for complex chemical systems” once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial and error approach to a minimum. The “rational drug design” term is rather comprehensive as it includes all contemporary methods of drug discovery where serendipity and screening are substituted by the information-guided search for new and existing compounds. Successful implementation of these innovative drug discovery approaches is inevitably preceded by learning the physics, chemistry, and physiology of functioning of biological structures under normal and pathological conditions. Areas covered This article provides an overview of the recent rational drug design approaches to discover inhibitors of anthrax toxin. Some of the examples include small-molecule and peptide-based post-exposure therapeutic agents as well as several polyvalent compounds. The review also directs the reader to the vast literature on the recognized advances and future possibilities in the field. Expert opinion Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (PA-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, in our view, the situation is still insecure. The FDA’s animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Besides, unlike PA, which is known to be unstable, LF remains active in cells and in animal tissues for days. Therefore, the effectiveness of the post-exposure treatment of the individuals

  11. Oligo-aspartic acid conjugates with benzo[c][2,6]naphthyridine-8-carboxylic acid scaffold as picomolar inhibitors of CK2.

    PubMed

    Vahter, Jürgen; Viht, Kaido; Uri, Asko; Enkvist, Erki

    2017-02-28

    Structurally diverse inhibitors of the protein kinase CK2 are required for regulation of this ubiquitous protein to establish biological roles of the enzyme which catalyzes the phosphorylation of a vast number of substrate proteins. In this article we disclose a series of new bisubstrate inhibitors of CK2 that are structurally represented by the oligo(l-Asp) peptide conjugates of benzo[c][2,6]naphthyridine-8-carboxylic acid. This fragment originated from CX-4945, the first in class inhibitor taken to clinical trials. The most potent conjugates possessed two-digit picomolar affinity and clear selectivity for CK2α in a panel of 140 protein kinases. Labeling of the inhibitors with a fluorescent dye yielded probes for a fluorescence anisotropy-based binding/displacement assay which can be used for analysis of CK2 and precise determination of affinity of the highly potent (tight-binding) CK2-targeting inhibitors.

  12. Discovery of highly selective CRAF inhibitors, 3-carboxamido-2H-indazole-6-arylamide: In silico FBLD design, synthesis and evaluation.

    PubMed

    Aman, Waqar; Lee, Junghun; Kim, Minjung; Yang, Songyi; Jung, Hoyong; Hah, Jung-Mi

    2016-02-15

    The recent success of vemurafenib shows the importance of selective BRAF V600E inhibition in melanoma. However, paradoxical activation by structurally diverse ATP-competitive RAF kinase inhibitors strongly suggests that selective CRAF inhibitors, not BRAF inhibitors, would be ideal for some Ras mutation cancer treatment. In this respect, we approached designing selective CRAF inhibitors starting from in silico fragment screening and synthesized a 3-carboxamido-2H-indazole-6-arylamide scaffold. Most of the compounds showed potent antiproliferative activity against the WM3629 melanoma cell line and the most promising compound, compound 10d, was found to be a potent and selective CRAF inhibitor with an IC50 value of 38.6 nM, which shows greater than 270-fold selectivity over BRAF kinase (9.45 μM).

  13. Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea.

    PubMed

    Clemente, Alfonso; Arques, Maria C; Dalmais, Marion; Le Signor, Christine; Chinoy, Catherine; Olias, Raquel; Rayner, Tracey; Isaac, Peter G; Lawson, David M; Bendahmane, Abdelhafid; Domoney, Claire

    2015-01-01

    Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving

  14. Eliminating Anti-Nutritional Plant Food Proteins: The Case of Seed Protease Inhibitors in Pea

    PubMed Central

    Clemente, Alfonso; Arques, Maria C.; Dalmais, Marion; Le Signor, Christine; Chinoy, Catherine; Olias, Raquel; Rayner, Tracey; Isaac, Peter G.; Lawson, David M.; Bendahmane, Abdelhafid; Domoney, Claire

    2015-01-01

    Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving

  15. Current use of phosphodiesterase inhibitors in urology

    PubMed Central

    Hakky, Tariq Said; Jain, Lakshay

    2015-01-01

    The causes of male erectile dysfunction (ED) are quite variable and are now commonly divided into etiologies such as ischemia, smooth muscle damage, or altered blood flow. Although varying rates of ED have been reported in literature, the number of men with ED is projected to increase worldwide by 2025 to approximately 322 million. Since the introduction of phosphodiesterase 5 (PDE5) inhibitors, there has been a paradigm shift in the treatment of ED because PDE5 inhibitors address a broad spectrum of etiologies for ED. Today, the American Urological Association recommends the use of three PDE5 inhibitors (sildenafil, tadalafil, and vardenafil) as a first-line therapy for the treatment of ED. This review evaluates the pharmacological mechanism of PDE5 inhibitors along with the impact and use of sildenafil, vardenafil, tadalafil, and avanafil. By increasing intracellular cGMP levels, PDE5 inhibitors have been shown to be effective in the treatment of ED. Through their effects on other cellular signaling pathways, PDE5 inhibitors have the potential for treating other urologic conditions as well. The use of PDE5 inhibitors can also be combined to produce a synergistic effect in conditions such as male hypogonadism and benign prostatic hyperplasia in addition to ED. PMID:26328208

  16. Leflunomide, a Reversible Monoamine Oxidase Inhibitor.

    PubMed

    Petzer, Jacobus P; Petzer, Anél

    2016-01-01

    A screening study aimed at identifying inhibitors of the enzyme, monoamine oxidase (MAO), among clinically used drugs have indicated that the antirheumatic drug, leflunomide, is an inhibitor of both MAO isoforms. Leflunomide inhibits human MAO-A and MAO-B and exhibits IC50 values of 19.1 μM and 13.7 μM, respectively. The corresponding Ki values are 17.7 μM (MAO-A) and 10.1 μM (MAO-B). Dialyses of mixtures of the MAO enzymes and leflunomide show that inhibition of the MAOs by leflunomide is reversible. The principal metabolite of leflunomide, teriflunomide (A77 1726), in contrast is not an MAO inhibitor. This study concludes that, although leflunomide is only moderately potent as an MAO inhibitor, isoxazole derivatives may represent a general class of MAO inhibitors and this heterocycle may find application in MAO inhibitor design. In this respect, MAO inhibitors are used in the clinic for the treatment of depressive illness and Parkinson's disease, and are under investigation as therapy for certain types of cancer, Alzheimer's disease and age-related impairment of cardiac function.

  17. Nonpeptide Macrocyclic Histone Deacetylase Inhibitors

    PubMed Central

    Oyelere, Adegboyega K.; Chen, Po C.; Guerrant, William; Mwakwari, Sandra C.; Hood, Rebecca; Zhang, Yunzhe; Fan, Yuhong

    2009-01-01

    Inhibition of Histone Deacetylases inhibitors (HDACi) hold great promise in cancer therapy due to their demonstrated ability to arrest proliferation of nearly all transformed cell types. Of the several structurally distinct small molecules HDACi reported, macrocyclic depsipeptides have the most complex recognition cap-group moieties and present an excellent opportunity for the modulation of the biological activities of HDACi. Unfortunately, the structure–activity relationship (SAR) studies for this class of compounds have been impaired largely because most macrocyclic HDACi known to date are comprised of complex peptide macrocycles. In addition to retaining the pharmacologically disadvantaged peptidyl-backbone, they offer only limited opportunity for side-chain modifications. Here we report the discovery of a new class of macrocyclic HDACi based on the macrolide antibiotics skeletons. SAR studies revealed that these compounds displayed both linker-length and macrolide-type dependent HDAC inhibition activities with IC50 in low nanomolar range. In addition, these nonpeptide macrocyclic HDACi are more selective against HDAC 1 and 2 relative to HDAC 8, another class I HDAC isoform, hence have sub-class HDAC isoform selectivity. PMID:19093884

  18. Inhibitors of specific ceramide synthases.

    PubMed

    Schiffmann, Susanne; Hartmann, Daniela; Fuchs, Sina; Birod, Kerstin; Ferreiròs, Nerea; Schreiber, Yannick; Zivkovic, Aleksandra; Geisslinger, Gerd; Grösch, Sabine; Stark, Holger

    2012-02-01

    Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed.

  19. COMT inhibitors and liver toxicity.

    PubMed

    Watkins, P

    2000-01-01

    This paper reviews the issue of hepatotoxicity with the use of the catechol-O-methly transferase (COMT) inhibitors tolcapone and entacapone. Neither drug caused hepatotoxicity in preclinical toxicity testing. However, in clinical trials of tolcapone, liver chemistry tests were elevated more than 3 times above the upper limit of normal in approximately 1% of patients who took the 100 mg dose and in approximately 3% of patients who took the 200 mg dose. These observations led to the recommendation that periodic monitoring of liver function be performed. Post-marketing surveillance studies noted 3 instances of acute liver failure with death after 60,000 patients had received tolcapone for a total of 40,000 patient-years. For this reason, the drug was withdrawn from the market in Europe and Canada, and a black box warning issued in the United States. In contrast, clinical trials with entacapone demonstrated no increase in liver enzymes above those observed with placebo. Further, no instances of acute liver failure or death attributed to the drug have been observed in post-marketing surveillance studies. Consequently, liver monitoring is not required with this agent. These data demonstrate that tolcapone is associated with a risk of hepatotoxicity but that no such risk has been detected with entacapone.

  20. Pharmacology of Proton Pump Inhibitors

    PubMed Central

    Shin, Jai Moo; Sachs, George

    2010-01-01

    The gastric H,K-ATPase is the primary target for the treatment of acid-related diseases. Proton pump inhibitors (PPIs) are weak bases composed of two moieties, a substituted pyridine with a primary pKa of about 4.0, which allows selective accumulation in the secretory canaliculus of the parietal cell, and a benzimidazole with a second pKa of about 1.0. PPIs are acid-activated prodrugs that convert to sulfenic acids or sulfenamides that react covalently with one or more cysteines accessible from the luminal surface of the ATPase. Because of covalent binding, their inhibitory effects last much longer than their plasma half-life. However, the short half-life of the drug in the blood and the requirement for acid activation impair their efficacy in acid suppression, particularly at night. PPIs with longer half-life promise to improve acid suppression. All PPIs give excellent healing of peptic ulcers and produce good results in reflux esophagitis. PPIs combined with antibiotics eradicate Helicobacter pylori. PMID:19006606