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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. 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

  8. 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.

  9. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

    PubMed

    Shah, Kunal R; Patel, Dhaval K; Pappachan, Anju; Prabha, C Ratna; Singh, Desh Deepak

    2016-02-01

    Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

  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

    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.

  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

    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.

  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. 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

  20. 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

  1. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds.

    PubMed

    Batista, I F; Oliva, M L; Araujo, M S; Sampaio, M U; Richardson, M; Fritz, H; Sampaio, C A

    1996-03-01

    A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.

  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. 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

  8. 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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  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. 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

  12. 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.

  13. Tryptogalinin Is a Tick Kunitz Serine Protease Inhibitor with a Unique Intrinsic Disorder

    PubMed Central

    Valdés, James J.; Schwarz, Alexandra; Cabeza de Vaca, Israel; Calvo, Eric; Pedra, Joao H. F.

    2013-01-01

    Background A salivary proteome-transcriptome project on the hard tick Ixodes scapularis revealed that Kunitz peptides are the most abundant salivary proteins. Ticks use Kunitz peptides (among other salivary proteins) to combat host defense mechanisms and to obtain a blood meal. Most of these Kunitz peptides, however, remain functionally uncharacterized, thus limiting our knowledge about their biochemical interactions. Results We discovered an unusual cysteine motif in a Kunitz peptide. This peptide inhibits several serine proteases with high affinity and was named tryptogalinin due to its high affinity for β-tryptase. Compared with other functionally described peptides from the Acari subclass, we showed that tryptogalinin is phylogenetically related to a Kunitz peptide from Rhipicephalus appendiculatus, also reported to have a high affinity for β-tryptase. Using homology-based modeling (and other protein prediction programs) we were able to model and explain the multifaceted function of tryptogalinin. The N-terminus of the modeled tryptogalinin is detached from the rest of the peptide and exhibits intrinsic disorder allowing an increased flexibility for its high affinity with its inhibiting partners (i.e., serine proteases). Conclusions By incorporating experimental and computational methods our data not only describes the function of a Kunitz peptide from Ixodes scapularis, but also allows us to hypothesize about the molecular basis of this function at the atomic level. PMID:23658744

  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. 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.

  16. 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.

  17. 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.

  18. Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill].

    PubMed

    Lee, K J; Kim, J-B; Ha, B-K; Kim, S H; Kang, S-Y; Lee, B-M; Kim, D S

    2012-07-01

    The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5 kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4-7) in comparison with Tia (pI 4-5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

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

    PubMed

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

    2016-01-01

    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. 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. 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. 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 present in the reactive sites. Therefore, ASPI

  6. 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

  7. 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.

  8. [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.

  9. 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.

  10. Understanding the molecular basis of stability in Kunitz (STI) family of inhibitors in terms of a conserved core tryptophan residue: A theoretical investigation.

    PubMed

    Datta Sharma, Ravi; Goswami, Nabajyoti; Ghosh, Debasree; Majumder, Sudip

    2017-08-01

    β-trefoil is one of the superfolds among proteins. Important classes of proteins like Interleukins (ILs), FibroblastGrowth Factors (FGFs), Kunitz (STI) family of inhibitors etc. belong to this fold. Kunitz (STI) family of inhibitors of proteins possess a highly conserved and structurally important Trytophan 91 (W91) residue, which stitches the top layer of the barrel with the lid. In this article we have investigated the molecular insights of the involvement of this W91 residue in the stability and folding pathway of Kunitz (STI) family. Winged bean Chymotrypsin inhibitor (WCI), a member of Kunitz (STI) family was chosen as a model system for carrying out the work. Molecular dynamics (MD) simulations were run with a set of total six proteins, including wild type WCI (WT) & five mutants namely W91F, W91M, W91A, W91H and W91I. Among all of them the coordinates of four proteins were taken from their crystal structures deposited in the Protein Data Bank (PDB), where as the coordinates for the rest two was generated using in-silico modelling. Our results suggest that truly this W91 residue plays a determining role in stability and folding pathway of Kunitz (STI) family. The mutants are less stable and more susceptible to quicker unfolding at higher temperatures compared to the wild type WCI. These effects are most pronounced for the smallest mutants namely W91H and W91A, indicating more is the cavity created by mutation at W91 position more the proteins becomes unstable. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. 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

  12. 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.

  13. Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls.

    PubMed

    Khalf, Moustafa; Goulet, Charles; Vorster, Juan; Brunelle, France; Anguenot, Raphaël; Fliss, Ismaïl; Michaud, Dominique

    2010-02-01

    Recombinant protease inhibitors represent useful tools for the development of insect-resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad-spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI-expressing and control (comparator) lines, followed by a two-dimensional gel electrophoresis (2-DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI-expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2-DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2-DE showed unaltered deposition patterns in SlCDI-expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI-expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel-generation transgenic plants.

  14. 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

  15. 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.

  16. 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. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  17. Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.

    PubMed

    De Paula, V S; Silva, F H S; Francischetti, I M B; Monteiro, R Q; Valente, A P

    2017-11-01

    Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D (1)H and 2D (15)N-(1)H NMR analysis yielded high quality 2D (15)N-(1)H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. A tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula.

    PubMed

    Rech, Stefanie S; Heidt, Sven; Requena, Natalia

    2013-09-01

    Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza-specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two-hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  19. Biochemical characterization of a Kunitz type inhibitor similar to dendrotoxins produced by Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) hemocytes.

    PubMed

    Lima, Cássia A; Torquato, Ricardo J S; Sasaki, Sergio D; Justo, Giselle Z; Tanaka, Aparecida S

    2010-02-10

    A novel chymotrypsin inhibitor identified in fat body and hemocyte cDNA libraries of Boophilus microplus was named BmCI (B. microplus Chymotrypsin Inhibitor) (Genbank EU636772). The putative BmCI amino acid sequence presented a 22-residue-signal peptide and 58-residue-mature protein. BmCI amino acid sequence analysis allowed its classification as a Kunitz-BPTI inhibitor with six cysteine residues, a theoretical pI of 7.8, and the presence of Tyr at P1 position in the putative reactive site, suggesting inhibitory activity toward chymotrypsin. In this work, we reported the biochemical characterization of BmCI. The recombinant BmCI expressed in yeast Pichia pastoris was purified by size exclusion and reverse phase chromatographies. rBmCI expression yield was of 1mgL(-1) of culture. Purified rBmCI confirmed its chymotrypsin inhibitory activity with a low K(i) (6.2pM). The BmCI gene expression analysis by semi-quantitative RT-PCR indicated its transcription in the hemocytes, salivary gland and ovary. The cytotoxic activity of purified rBmCI was demonstrated in BALB/c 3T3 mouse fibroblasts. As assessed by the MTT reduction assay, rBMCI induced a dose-dependent decrease in 3T3 fibroblasts viability (IC(50)=8microM). Moreover, flow cytometry analysis revealed that rBmCI is able to induce apoptosis, whereas no effect was observed on cell cycle progression. In conclusion, we demonstrated that rBmCI is cytotoxic against mammalian cells and obtained evidence that this growth inhibition is caused by an apoptosis-inducing activity.

  20. 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.

  1. Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies.

    PubMed

    Araújo, Ana Paula Ulian; Hansen, Daiane; Vieira, Debora F; Oliveira, Cleide; Santana, Lucimeire A; Beltramini, Leila M; Sampaio, Claudio A M; Sampaio, Misako U; Oliva, Maria Luiza V

    2005-06-01

    Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.

  2. 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.

  3. 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

  4. Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz-type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor.

    PubMed

    Hansen, Lene; Petersen, Lars Christian; Lauritzen, Brian; Clausen, Jes Thorn; Grell, Susanne Nedergaard; Agersø, Henrik; Sørensen, Brit Binow; Hilden, Ida; Almholt, Kasper

    2014-03-01

    A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab. Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats. Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. cDNA cloning, structural, and functional analyses of venom phospholipases A₂ and a Kunitz-type protease inhibitor from steppe viper Vipera ursinii renardi.

    PubMed

    Tsai, Inn-Ho; Wang, Ying-Ming; Cheng, An Chun; Starkov, Vladislav; Osipov, Alexey; Nikitin, Ilya; Makarova, Yana; Ziganshin, Rustam; Utkin, Yuri

    2011-02-01

    Snake venom phospholipases A₂ (PLA₂s) display a wide array of biological activities and are each characteristic to the venom. Here, we report on the cDNA cloning and characterization of PLA₂s from the steppe viper Vipera ursinii renardi venom glands. Among the five distinct PLA₂ cDNAs cloned and sequenced, the most common were the clones encoding a basic Ser-49 containing PLA₂ (Vur-S49). Other clones encoded either ammodytin analogs I1, I2d and I2a (designated as Vur-PL1, Vur-PL2 and Vur-PL3, respectively) or an ammodytoxin-like PLA₂ (Vurtoxin). Additionally, a novel Kunitz-type trypsin inhibitor for this venom species was cloned and sequenced. Comparison of these PLA₂ and Kunitz inhibitor sequences with those in the sequence data banks suggests that the viper V. u. renardi is closely related to Vipera ammodytes and Vipera aspis. Separation of V. u. renardi venom components by gel-filtration and ion-exchange chromatography showed the presence of many PLA₂ isoforms. Remarkably, the most abundant PLA₂ isolated was Vur-PL2 while Vur-S49 analog was in very low yield. There are great differences between the proportion of cDNA clones and that of the proteins isolated. Two Vur-PL2 isoforms (designated as Vur-PL2A and Vur-PL2B) indistinguishable by masses, peptide mass fingerprinting, N-terminal sequences and CD spectroscopy were purified from the pooled venom. However, when rechromatographed on cation-exchanger, Vur-PL2A showed only one peak corresponding to Vur-PL2B, suggesting the existence of conformers for Vur-PL2. Vur-PL2B was weakly cytotoxic to rat pheochromocytoma PC12 cells and showed both strong anticoagulant and anti-platelet activities. This is the first case of a strong anticoagulating ammodytin I analog in Vipera venom. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Defense response in non-genomic model species: methyl jasmonate exposure reveals the passion fruit leaves' ability to assemble a cocktail of functionally diversified Kunitz-type trypsin inhibitors and recruit two of them against papain.

    PubMed

    Botelho-Júnior, Sylvio; Machado, Olga L T; Fernandes, Kátia V S; Lemos, Francisco J A; Perdizio, Viviane A; Oliveira, Antônia E A; Monteiro, Leandro R; Filho, Mauri L; Jacinto, Tânia

    2014-08-01

    Multiplicity of protease inhibitors induced by predators may increase the understanding of a plant's intelligent behavior toward environmental challenges. Information about defense mechanisms of non-genomic model plant passion fruit (Passiflora edulis Sims) in response to predator attack is still limited. Here, via biochemical approaches, we showed its flexibility to build-up a broad repertoire of potent Kunitz-type trypsin inhibitors (KTIs) in response to methyl jasmonate. Seven inhibitors (20-25 kDa) were purified from exposed leaves by chromatographic techniques. Interestingly, the KTIs possessed truncated Kunitz motif in their N-terminus and some of them also presented non-consensus residues. Gelatin-Native-PAGE established multiple isoforms for each inhibitor. Significant differences regarding inhibitors' activity toward trypsin and chymotrypsin were observed, indicating functional polymorphism. Despite its rarity, two of them also inhibited papain, and such bifunctionality suggests a recruiting process onto another mechanistic class of target protease (cysteine-type). All inhibitors acted strongly on midgut proteases from sugarcane borer, Diatraea saccharalis (a lepidopteran insect) while in vivo assays supported their insecticide properties. Moreover, the bifunctional inhibitors displayed activity toward midgut proteases from cowpea weevil, Callosobruchus maculatus (a coleopteran insect). Unexpectedly, all inhibitors were highly effective against midgut proteases from Aedes aegypti a dipteran insect (vector of neglected tropical diseases) opening new avenues for plant-derived PIs for vector control-oriented research. Our results reflect the KTIs' complexities in passion fruit which could be wisely exploited by influencing plant defense conditions. Therefore, the potential of passion fruit as source of bioactive compounds with diversified biotechnological application was strengthened.

  19. 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.

  20. Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor.

    PubMed

    Mine, Shouhei; Yamazaki, Toshio; Miyata, Toshiyuki; Hara, Saburo; Kato, Hisao

    2002-01-08

    Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factor VIIa and Xa through its K1 and K2 domain, respectively, and the inhibitory activity is enhanced by heparin. The function of the K3 domain of TFPI has not been established, but the domain probably harbors a heparin binding site (HBS-2). We determined the three-dimensional solution structure of the TFPI K3 domain (Glu182-Gly242) by heteronuclear multidimensional NMR. The results showed that the molecule is composed of one antiparallel beta-sheet and one alpha-helix, and in overall structure is very similar to the K2 domain, with the rms deviation of 1.55 A for the 58 defined C(alpha) positions. However, the surface electrostatic properties of both domains are different each other. The lack of inhibitory activity of the K3 domain is explained by the absence of electrostatic interaction with factor Xa over a large surface area. A titration experiment with size-fractionated heparin showed that a heparin binding site was located in the vicinity of the alpha-helix. In this region, a positively charged cluster is formed by Lys213, Lys232, and Lys240, and the negatively charged sulfate groups of heparin bind there. The enhancement of inhibitory activity by heparin probably was not due to a conformational change to TFPI itself. It is likely that heparin simply increases the local concentration of TFPI on the cell surface and stabilizes the initial complex that forms.

  1. Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris.

    PubMed

    Gil, Dayrom F; García-Fernández, Rossana; Alonso-del-Rivero, Maday; Lamazares, Emilio; Pérez, Mariela; Varas, Laura; Díaz, Joaquín; Chávez, María A; González-González, Yamile; Mansur, Manuel

    2011-11-01

    Pichia pastoris is a highly successful system for the large-scale expression of heterologous proteins, with the added capability of performing most eukaryotic post-translational modifications. However, this system has one significant disadvantage - frequent proteolytic degradation by P. pastoris proteases of heterologously expressed proteins. Several methods have been proposed to address this problem, but none has proven fully effective. We tested the effectiveness of a broad specificity protease inhibitor to control proteolysis. A recombinant variant of the BPTI-Kunitz protease inhibitor ShPI-1 isolated from the sea anemone Stichodactyla helianthus, was expressed in P. pastoris. The recombinant inhibitor (rShPI-1A), containing four additional amino acids (EAEA) at the N-terminus, was folded similarly to the natural inhibitor, as assessed by circular dichroism. rShPI-1A had broad protease specificity, inhibiting serine, aspartic, and cysteine proteases similarly to the natural inhibitor. rShPI-1A protected a model protein, recombinant human miniproinsulin (rhMPI), from proteolytic degradation during expression in P. pastoris. The addition of purified rShPI-1A at the beginning of the induction phase significantly protected rhMPI from proteolysis in culture broth. The results suggest that a broad specificity protease inhibitor such as rShPI-1A can be used to improve the yield of recombinant proteins secreted from P. pastoris. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

    PubMed Central

    Mourão, Caroline B.F.; Schwartz, Elisabeth F.

    2013-01-01

    The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K+ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K+ channel blocking activity was also compared. PMID:23771044

  9. Expanding the chemical diversity of CK2 inhibitors.

    PubMed

    Prudent, Renaud; Moucadel, Virginie; López-Ramos, Miriam; Aci, Samia; Laudet, Beatrice; Mouawad, Liliane; Barette, Caroline; Einhorn, Jacques; Einhorn, Cathy; Denis, Jean-Noel; Bisson, Gilles; Schmidt, Frédéric; Roy, Sylvaine; Lafanechere, Laurence; Florent, Jean-Claude; Cochet, Claude

    2008-09-01

    None of the already described CK2 inhibitors did fulfill the requirements for successful clinical settings. In order to find innovative CK2 inhibitors based on new scaffolds, we have performed a high-throughput screening of diverse chemical libraries. We report here the identification and characterization of several classes of new inhibitors. Whereas some share characteristics of previously known CK2 inhibitors, others are chemically unrelated and may represent new opportunities for the development of better CK2 inhibitors. By combining structure-activity relationships with a docking procedure, we were able to determine the binding mode of these inhibitors. Interestingly, beside the identification of several nanomolar ATP-competitive inhibitors, one class of chemical inhibitors displays a non-ATP competitive mode of inhibition, a feature that suggests that CK2 possess distinct druggable binding sites. For the most promising inhibitors, selectivity profiling was performed. We also provide evidence that some chemical compounds are inhibiting CK2 in living cells. Finally, the collected data allowed us to draw the rules about the chemical requirements for CK2 inhibition both in vitro and in a cellular context.

  10. New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

    PubMed

    Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

    2015-09-24

    Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

  11. Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain

    PubMed Central

    2015-01-01

    Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes. PMID:24383758

  12. 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.

  13. Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors.

    PubMed

    Fischer, Matthias; Kuckenberg, Markus; Kastilan, Robin; Muth, Jost; Gebhardt, Christiane

    2015-02-01

    Plant protease inhibitors are a structurally highly diverse and ubiquitous class of small proteins, which play various roles in plant development and defense against pests and pathogens. Particular isoforms inhibit in vitro proteases and other enzymes that are not their natural substrates, for example proteases that have roles in human diseases. Mature potato tubers are a rich source of several protease inhibitor families. Different cultivars have different inhibitor profiles. With the objective to explore the functional diversity of the natural diversity of potato protease inhibitors, we randomly selected and sequenced 9,600 cDNA clones originated from mature tubers of ten potato cultivars. Among these, 120 unique inhibitor cDNA clones were identified by homology searches. Eighty-eight inhibitors represented novel sequence variants of known plant protease inhibitor families. Most frequent were Kunitz-type inhibitors (KTI), potato protease inhibitors I and II (PIN), pectin methylesterase inhibitors, metallocarboxypeptidase inhibitors and defensins. Twenty-three inhibitors were functionally characterized after heterologous expression in the yeast Pichia pastoris. The purified recombinant proteins were tested for inhibitory activity on trypsin, eleven pharmacological relevant proteases and the non-proteolytic enzyme 5-lipoxygenase. Members of the KTI and PIN families inhibited pig pancreas elastase, β-Secretase, Cathepsin K, HIV-1 protease and potato 5-lipoxygenase. Our results demonstrate in vitro inhibitory diversity of small potato tuber proteins commonly known as protease inhibitors, which might have biotechnological or medical applications.

  14. 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

  15. 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.

  16. 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.

  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

    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

  19. Evolution, expansion and expression of the Kunitz/BPTI gene family associated with long-term blood feeding in Ixodes Scapularis

    PubMed Central

    2012-01-01

    Background Recent studies of the tick saliva transcriptome have revealed the profound role of salivary proteins in blood feeding. Kunitz/BPTI proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding, such as inhibiting blood coagulation, regulating host blood supply and disrupting host angiogenesis. However, Kunitz/BPTI proteins in soft and hard ticks have different functions and molecular mechanisms. How these differences emerged and whether they are associated with the evolution of long-term blood feeding in hard ticks remain unknown. Results In this study, the evolution, expansion and expression of Kunitz/BPTI family in Ixodes scapularis were investigated. Single- and multi-domain Kunitz/BPTI proteins have similar gene structures. Single-domain proteins were classified into three groups (groups I, II and III) based on their cysteine patterns. Group I represents the ancestral branch of the Kunitz/BPTI family, and members of this group function as serine protease inhibitors. The group I domain was used as a module to create multi-domain proteins in hard ticks after the split between hard and soft ticks. However, groups II and III, which evolved from group I, are only present and expanded in the genus Ixodes. These lineage-specific expanded genes exhibit significantly higher expression during long-term blood feeding in Ixodes scapularis. Interestingly, functional site analysis suggested that group II proteins lost the ability to inhibit serine proteases and evolved a new function of modulating ion channels. Finally, evolutionary analyses revealed that the expansion and diversification of the Kunitz/BPTI family in the genus Ixodes were driven by positive selection. Conclusions These results suggest that the differences in the Kunitz/BPTI family between soft and hard ticks may be linked to the evolution of long-term blood feeding in hard ticks. In Ixodes, the lineage-specific expanded genes (Group II and III) lost the

  20. 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

  1. 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.

  2. [Trypsin inhibitor from Gleditsia triacanthos L. seeds].

    PubMed

    Mosolov, V V; Kolosova, G V; Valueva, T A; Dronova, L A

    1982-05-01

    The trypsin inhibitor from Gleditsia triacanthos (L.) seeds was purified by affinity chromatography on a column with trypsin-Sepharose 4B. The isolated inhibitor is a single-chain protein with molecular weight of about 20 000. The inhibitor suppresses bovine trypsin at a molar rate of 1 : 1, but weakly inhibits chymotrypsin in a non-stoichiometric manner. Some properties of the isolated inhibitor closely resembled those of soybean trypsin inhibitor (Kunitz).

  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. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. 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.

  7. 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

  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. 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.

  10. 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

  11. 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.

  12. 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.

  13. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

    PubMed

    Chen, Zongyun; Wang, Bin; Hu, Jun; Yang, Weishan; Cao, Zhijian; Zhuo, Renxi; Li, Wenxin; Wu, Yingliang

    2013-01-01

    Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target

  14. 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…

  15. 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…

  16. 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.

  17. Molecular evolution and diversity of dimeric alpha-amylase inhibitor gene in Kengyilia species (Triticeae: Poaceae).

    PubMed

    Zeng, Jian; Fan, Xing; Sha, Li-Na; Kang, Hou-Yang; Wang, Yi; Zhang, Hai-Qin; Zhou, Yong-Hong

    2013-10-25

    Kengyilia Yen et J. L. Yang is a group of allohexaploid species with StYP genomic constitutions in the wheat tribe. To investigate the evolution and diversity of dimeric alpha-amylase inhibitor genes in the Kengyilia, forty-five homoeologous DAAI gene sequences were isolated from sampled Kengyilia species and analyzed together with those of its close relatives. These results suggested that (1) Kengyilia species from Central Asia and the Qinghai-Tibetan Plateau had different origins from those of the geographically differentiated P genome; (2) the St and P genomes of Kengyilia were donated by Pseudoroegneria and Agropyron, respectively, and the Y genome had an independent origin and showed an affinity with the St genome; (3) purifying selection dominated the DAAI gene members and the St-DAAI gene was evolving at faster rate than the P- and Y-DAAI genes in Kengyilia; and (4) natural selection was the main factor on the codon usage pattern of the DAAI gene in Kengyilia.

  18. 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.

  19. 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

  20. [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.

  1. [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

  2. 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.

  3. 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. Copyright 2001 Academic Press.

  4. Diversity-Oriented Synthesis Probe Targets Plasmodium falciparum Cytochrome b Ubiquinone Reduction Site and Synergizes With Oxidation Site Inhibitors

    PubMed Central

    Lukens, Amanda K.; Heidebrecht, Richard W.; Mulrooney, Carol; Beaudoin, Jennifer A.; Comer, Eamon; Duvall, Jeremy R.; Fitzgerald, Mark E.; Masi, Daniela; Galinsky, Kevin; Scherer, Christina A.; Palmer, Michelle; Munoz, Benito; Foley, Michael; Schreiber, Stuart L.; Wiegand, Roger C.; Wirth, Dyann F.

    2015-01-01

    Background. The emergence and spread of drug resistance to current antimalarial therapies remains a pressing concern, escalating the need for compounds that demonstrate novel modes of action. Diversity-Oriented Synthesis (DOS) libraries bridge the gap between conventional small molecule and natural product libraries, allowing the interrogation of more diverse chemical space in efforts to identify probes of novel parasite pathways. Methods. We screened and optimized a probe from a DOS library using whole-cell phenotypic assays. Resistance selection and whole-genome sequencing approaches were employed to identify the cellular target of the compounds. Results. We identified a novel macrocyclic inhibitor of Plasmodium falciparum with nanomolar potency and identified the reduction site of cytochrome b as its cellular target. Combination experiments with reduction and oxidation site inhibitors showed synergistic inhibition of the parasite. Conclusions. The cytochrome b oxidation center is a validated antimalarial target. We show that the reduction site of cytochrome b is also a druggable target. Our results demonstrating a synergistic relationship between oxidation and reduction site inhibitors suggests a future strategy for new combination therapies in the treatment of malaria. PMID:25336726

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Molecular and biologic analysis of histone deacetylase inhibitors with diverse specificities.

    PubMed

    Newbold, Andrea; Matthews, Geoffrey M; Bots, Michael; Cluse, Leonie A; Clarke, Christopher J P; Banks, Kellie-Marie; Cullinane, Carleen; Bolden, Jessica E; Christiansen, Ailsa J; Dickins, Ross A; Miccolo, Claudia; Chiocca, Susanna; Kral, Astrid M; Ozerova, Nicole D; Miller, Thomas A; Methot, Joey L; Richon, Victoria M; Secrist, J Paul; Minucci, Saverio; Johnstone, Ricky W

    2013-12-01

    Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited. ©2013 AACR.

  11. 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

  12. 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

  13. Transferable scoring function based on semiempirical quantum mechanical PM6-DH2 method: CDK2 with 15 structurally diverse inhibitors

    NASA Astrophysics Data System (ADS)

    Dobeš, Petr; Fanfrlík, Jindřich; Řezáč, Jan; Otyepka, Michal; Hobza, Pavel

    2011-03-01

    A semiempirical quantum mechanical PM6-DH2 method accurately covering the dispersion interaction and H-bonding was used to score fifteen structurally diverse CDK2 inhibitors. The geometries of all the complexes were taken from the X-ray structures and were reoptimised by the PM6-DH2 method in continuum water. The total scoring function was constructed as an estimate of the binding free energy, i.e., as a sum of the interaction enthalpy, interaction entropy and the corrections for the inhibitor desolvation and deformation energies. The applied scoring function contains a clear thermodynamical terms and does not involve any adjustable empirical parameter. The best correlations with the experimental inhibition constants (ln K i) were found for bare interaction enthalpy ( r 2 = 0.87) and interaction enthalpy corrected for ligand desolvation and deformation energies ( r 2 = 0.77); when the entropic term was considered, however, the correlation becomes worse but still acceptable ( r 2 = 0.52). The resulting correlation based on the PM6-DH2 scoring function is better than previously published function based on various docking/scoring, SAR studies or advanced QM/MM approach, however, the robustness is limited by number of available experimental data used in the correlation. Since a very similar correlation between the experimental and theoretical results was found also for a different system of the HIV-1 protease, the suggested scoring function based on the PM6-DH2 method seems to be applicable in drug design, even if diverse protein-ligand complexes have to be ranked.

  14. HIV Type 1 genetic diversity and naturally occurring polymorphisms in HIV type 1 Kenyan isolates: implications for integrase inhibitors.

    PubMed

    Nyamache, Anthony Kebira; Muigai, Anne W T; Nganga, Zipporah; Khamadi, Samoel A

    2012-08-01

    Little is known about the extent and predictors of polymorphisms potentially influencing susceptibility to HIV integrase inhibitors. HIV-1 genetic diversity and drug resistance are major challenges in patient management globally. To evaluate HIV genetic diversity and drug resistance-associated mutations within a Nairobi cohort, genetic analysis of the HIV-1 pol-integrase gene regions was performed on samples collected from 42 subjects and 113 Kenyan intergrase sequences deposited in the Los Alamos HIV database. From the partial pol-integrase sequences analyzed phylogenetically, it was shown that 26 (61.9%) were subtype A1, 9 (21.4%) were subtype D, 5 (11.9%) were subtype C, 1 (2.4%) was subtype A2 and 1 (2.4%) was subtype CRF. Integrase-associated mutations were found in 12 patients, and in all 113 sequences already deposited in GenBank. One sample from this study and five from previous Kenyan integrase sequences had mutations at T97A, which is associated with reduced susceptibility to raltegravir.

  15. 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

  16. 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

  17. 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.

  18. Amino acid sequence of winged bean (Psophocarpus tetragonolobus (L.) DC.) chymotrypsin inhibitor, WCI-3.

    PubMed

    Shibata, H; Hara, S; Ikenaka, T

    1988-10-01

    The complete amino acid sequence of winged bean chymotrypsin inhibitor 3 (WCI-3) was determined by the conventional methods. WCI-3 consisted of 183 amino acid residues, but was heterogeneous in the carboxyl terminal region owing to the loss of one to four carboxyl terminal amino acid residues. The sequence of WCI-3 was highly homologous with those of soybean trypsin inhibitor Tia, winged bean trypsin inhibitor WTI-1, and Erythrina latissima trypsin inhibitor DE-3. One of the reactive site peptide bonds of WCI-3 was identified as Leu(65)-Ser(66), which was located at the same position as those of the other Kunitz-family leguminous proteinase inhibitors.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. Three-dimensional structure-activity relationship study of belactosin A and its stereo- and regioisomers: development of potent proteasome inhibitors by a stereochemical diversity-oriented strategy.

    PubMed

    Yoshida, Keisuke; Yamaguchi, Kazuya; Mizuno, Akira; Unno, Yuka; Asai, Akira; Sone, Takayuki; Yokosawa, Hideyoshi; Matsuda, Akira; Arisawa, Mitsuhiro; Shuto, Satoshi

    2009-05-07

    The development of potent proteasome inhibitors based on the stereochemical diversity-oriented strategy using a conformationally rigid cyclopropane structure was investigated. Thus, a series of stereo- and regioisomeric analogs of belactosin A (2), a cyclopropane amino acid (methanoamino acid)-containing tripeptidic proteasome inhibitor, were designed, in which the central cyclopropane amino acid part was replaced with the corresponding stereo- or regioisomer. Using a series of stereoisomeric cyclopropane amino acid equivalents with the cis/trans, D/L, and syn/anti stereochemical diversity, which were previously developed by us, as key units, the target compounds were successfully synthesized. Biological evaluation showed that, as expected, compound activity changed depending on the stereochemistry of the central cyclopropane amino acid part: the trans/L-syn-isomer 7 and the cis/L-anti-isomer 9 were more than twice as potent as natural belactosin A (trans/L-anti-isomer). Furthermore, the tripeptidic compound 13, the synthetic precursor for the unnatural cis/L-anti-isomer 9, was identified as a highly potent proteasome inhibitor. This compound, which is 20 times as potent as belactosin A and is even more potent than the well-known inhibitor lactacystin (4), may be an effective lead for developing clinically useful anticancer drugs. These results show that the stereochemical diversity-oriented approach can be a powerful strategy for the development of highly active compounds in medicinal chemical studies.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors.

    PubMed

    Machado, Suzy Wider; de Oliveira, Caio Fernando Ramalho; Zério, Neide Graciano; Parra, José Roberto Postali; Macedo, Maria Lígia Rodrigues

    2017-08-01

    Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI-induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI-induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs. © 2017 Wiley Periodicals, Inc.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. High-throughput screening against ~6.1 million structurally diverse, lead-like compounds to discover novel ROCK inhibitors for cerebral injury recovery.

    PubMed

    Gong, Haoling; Yuan, Zhicheng; Zhan, Liping

    2016-05-01

    Rho-associated protein kinase (ROCK) has been recognized as an attractive therapeutic target to promote neurogenesis, neuroregeneration, and neurorecovery after cerebral injury. Here, a high-throughput screening protocol was described to discover novel ROCK inhibitors from a large chemical library containing ~6.1 million structurally diverse, lead-like compounds. The protocol employed empirical rules such as ADMET evaluation and chemical similarity analysis to exclude those of drug-unlike candidates, and then molecular docking and binding affinity predictions were performed to suggest few promising candidates with high scores. Consequently, five compounds were successfully identified to have satisfactory activity profile with IC50 values at nanomolar level. In order to elucidate the molecular mechanism of inhibitor binding to target, the complex structures of ROCK kinase domain with the five identified compounds were modeled and examined in detail. A number of polar chemical forces such as hydrogen bonds and cation-π interactions as well as nonpolar contacts such as π-π stacking and hydrophobic forces were revealed at the complex interface, conferring high affinity and strong specificity to inhibitor binding. In addition, several key residues around the kinase active site, including Val90, Lys105, Asn203, and Phe368, were found to play an important role in binding.

  17. 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.

  18. 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

  19. Purification and characterization of proteinase inhibitors from winged bean (Psophocarpus tetragonolobus (L.) DC.) seeds.

    PubMed

    Shibata, H; Hara, S; Ikenaka, T; Abe, J

    1986-04-01

    Seven proteinase inhibitors were isolated from winged bean seeds by ion-exchange chromatographies. These inhibitors had molecular weights of around 20,000, included four half-cystine residues, and were Kunitz-type inhibitors. Two (WTI-2 and 3) inhibited bovine trypsin strongly and four (WCI-1, 2, 3, and 4) inhibited bovine alpha-chymotrypsin, but in different ways. One mole of WCI-2 or -3 could inhibit 2 mol of alpha-chymotrypsin. The remaining inhibitor (WTCI-1) could bind both bovine trypsin and alpha-chymotrypsin at the molar ratio of 1:1, but not simultaneously. All four chymotrypsin inhibitors cross-reacted with rabbit anti-WCI-3 serum, while the other inhibitors did not.

  20. 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.

  1. 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

  2. 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.

  3. Crystal structures of a plant trypsin inhibitor from Enterolobium contortisiliquum (EcTI) and of its complex with bovine trypsin.

    PubMed

    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.

  4. 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

  5. Diverse inhibitory actions of quaternary ammonium cholinesterase inhibitors on Torpedo nicotinic ACh receptors transplanted to Xenopus oocytes

    PubMed Central

    Olivera-Bravo, Silvia; Ivorra, Isabel; Morales, Andrés

    2007-01-01

    Background and purpose: This work was aimed at comparing and analysing the effects and mechanisms of action of the quaternary ammonium cholinesterase inhibitors (QChEIs) BW284c51, decamethonium and edrophonium, on nicotinic ACh receptor (nAChR) function. Experimental approach: nAChRs purified from Torpedo electroplax were transplanted to oocytes and currents elicited by ACh (IACh) either alone or in presence of these QChEIs were recorded. Key results: None of the QChEIs, by itself, elicited changes in membrane conductance; however, when co-applied with ACh, all of them decreased IACh in a concentration-dependent way. The mechanisms of nAChR inhibition were different for these QChEIs. BW284c51 blockade was non-competitive and voltage-dependent, although it also affected the nH of the dose-response curve. By contrast, decamethonium and edrophonium inhibition, at –60 mV, was apparently competitive and did not modify either desensitisation or nH. Decamethonium effects were voltage-independent and washed out slowly after its removal; by contrast, edrophonium blockade had strong voltage dependence and its effects disappeared quickly after its withdrawal. Analysis of the voltage-dependent blockade indicated that BW284c51 bound to a shallow site into the channel pore, whereas edrophonium bound to a deeper locus. Accordingly, additive inhibitory effects on IACh were found among any pairs of these QChEIs. Conclusions and implications: The tested QChEIs bound to the nAChR at several and different loci, which might account for their complex inhibitory behaviour, acting both as allosteric effectors and, in the case of BW284c51 and edrophonium, as open channel blockers. PMID:17572698

  6. 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.

  7. Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.).

    PubMed

    Yamamoto, M; Hara, S; Ikenaka, T

    1983-09-01

    The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.

  8. Anexelekto /MER Tyrosine Kinase inhibitor ONO-7475 growth arrests and kills FMS-Like Tyrosine Kinase 3-Internal Tandem Duplication Mutant Acute Myeloid Leukemia cells by diverse mechanisms.

    PubMed

    Ruvolo, Peter P; Ma, Huaxian; Ruvolo, Vivian R; Zhang, Xiaorui; Mu, Hong; Schober, Wendy; Hernandez, Ivonne; Gallardo, Miguel; Khoury, Joseph; Cortes, Jorge; Andreeff, Michael; Post, Sean M

    2017-09-14

    Nearly one-third of patients with acute myeloid leukemia have FMS-Like Tyrosine Kinase 3 mutations and thus have poor survival prospects. Receptor tyrosine kinase Anexelekto is critical for FMS-Like Tyrosine Kinase 3 signaling and participates in FMS-Like Tyrosine Kinase 3 inhibitor resistance mechanisms. Thus, strategies targeting Anexelekto could prove useful for acute myeloid leukemia therapy. ONO-7475 is an inhibitor with high specificity for Anexelekto and MER Tyrosine Kinase. Here we report that ONO-7475 potently arrested growth and induced apoptosis in acute myeloid leukemia with internal tandem duplication mutation of FMS-Like Tyrosine Kinase 3. MER Tyrosine Kinase-lacking MOLM13 cells were sensitive to ONO-7475 while MER Tyrosine Kinase -expressing OCI-AML3 cells were resistant, suggesting that the drug acts via Anexelekto in acute myeloid leukemia cells. Reverse phase protein analysis of ONO-7475 treated cells revealed that cell cycle regulators like Cyclin Dependent Kinase 1, Cyclin B1, Polo-like Kinase 1, and Retinoblastoma were suppressed. ONO-7475 suppressed Cyclin Dependent Kinase 1, Cyclin B1, Polo-like Kinase 1gene expression suggesting that Anexelekto may regulate the cell cycle at least in part via transcriptional mechanisms. Importantly, ONO-7475 was effective in a human FMS-Like Tyrosine Kinase 3 with Internal Tandem Duplication mutant murine xenograft model. Mice fed a diet containing ONO-7475 exhibited significantly longer survival and, interestingly, blocked leukemia cell infiltration in the liver. In summary, ONO-7475 effectively kills acute myeloid leukemia cells in vitro and in vivo by mechanisms that involve disruption of diverse survival and proliferation pathways. Copyright © 2017, Ferrata Storti Foundation.

  9. Development of pharmacophore similarity-based quantitative activity hypothesis and its applicability domain: applied on a diverse data-set of HIV-1 integrase inhibitors.

    PubMed

    Kumar, Sivakumar Prasanth; Jasrai, Yogesh T; Mehta, Vijay P; Pandya, Himanshu A

    2015-01-01

    Quantitative pharmacophore hypothesis combines the 3D spatial arrangement of pharmacophore features with biological activities of the ligand data-set and predicts the activities of geometrically and/or pharmacophoric similar ligands. Most pharmacophore discovery programs face difficulties in conformational flexibility, molecular alignment, pharmacophore features sampling, and feature selection to score models if the data-set constitutes diverse ligands. Towards this focus, we describe a ligand-based computational procedure to introduce flexibility in aligning the small molecules and generating a pharmacophore hypothesis without geometrical constraints to define pharmacophore space, enriched with chemical features necessary to elucidate common pharmacophore hypotheses (CPHs). Maximal common substructure (MCS)-based alignment method was adopted to guide the alignment of carbon molecules, deciphered the MCS atom connectivity to cluster molecules in bins and subsequently, calculated the pharmacophore similarity matrix with the bin-specific reference molecules. After alignment, the carbon molecules were enriched with original atoms in their respective positions and conventional pharmacophore features were perceived. Distance-based pharmacophoric descriptors were enumerated by computing the interdistance between perceived features and MCS-aligned 'centroid' position. The descriptor set and biological activities were used to develop support vector machine models to predict the activities of the external test set. Finally, fitness score was estimated based on pharmacophore similarity with its bin-specific reference molecules to recognize the best and poor alignments and, also with each reference molecule to predict outliers of the quantitative hypothesis model. We applied this procedure to a diverse data-set of 40 HIV-1 integrase inhibitors and discussed its effectiveness with the reported CPH model.

  10. 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.

  11. 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.

  12. 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.

  13. Preliminary crystallographic studies of EcTI, a serine proteinase inhibitor from Enterolobium contortisiliquum seeds.

    PubMed

    Batista, I F; Nonato, M C; Bonfadini, M R; Beltramini, L M; Oliva, M L; Sampaio, M U; Sampaio, C A; Garratt, R C

    2001-04-01

    Enterolobium contortisiliquum trypsin inhibitor (EcTI) belongs to the Kunitz family of plant inhibitors, which are widely distributed in nature, especially in plant seeds. EcTI is composed of two polypeptide chains with a total of 174 residues, homologous to other inhibitors from the same family. EcTI crystals, which were obtained with the acupuncture-gel technique, diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters a = 37.12, b = 38.42, c = 54.08 A, beta = 98.08 degrees. Molecular-replacement techniques using Erythrina caffra trypsin inhibitor (PDB code 1tie) as the search model indicate one monomer in the asymmetric unit. The secondary-structure content of EcTI was determined by circular dichroism spectroscopy, yielding values compatible with the expected topology.

  14. Protective immunity against tick infestation in cattle vaccinated with recombinant trypsin inhibitor of Rhipicephalus microplus.

    PubMed

    Andreotti, Renato; Cunha, Rodrigo Casquero; Soares, Mariana Aparecida; Guerrero, Felix D; Leite, Fábio P Leivas; de León, Adalberto A Pérez

    2012-10-19

    The cattle tick, Rhipicephalus microplus, is regarded as the most economically important ectoparasite of livestock globally. Control is achieved primarily through the use of acaricides. This approach is hampered by the development of resistance to commercial acaricides among cattle tick populations. Vaccination against R. microplus infestation is another technology that can be integrated for effective cattle tick control. Proteins belonging to the Kunitz-BPTI family are abundant in cattle tick salivary glands, midgut, and ovaries. These organs are attractive targets for the development of a novel cattle tick vaccine. Efficacy assessment against cattle tick infestation in bovines using a vaccine containing the recombinant form of a member of the Kunitz family from R. microplus produced in a yeast expression system is reported for the first time here. The yeast Pichia pastoris was bioengineered to produce the recombinant version of a trypsin inhibitor that is expressed in cattle tick larvae (rRmLTI). Immunization with rRmLTI afforded 32% efficacy against R. microplus. The estimated molecular weight of rRmLTI was 46 kDa. Structural homology to the native form of the larval trypsin inhibitor was documented by recognition of rRmLTI in Western-blots using polyclonal antibodies from mice immunized with cattle tick larval extract or rRmLTI. Bioinformatics analysis of the partial nucleotide and deduced amino acid sequences indicated that the rRmLTI closely resembles BmTI-6, which is a three-headed Kunitz protein present in cattle tick ovary and fat tissue. Published by Elsevier Ltd.

  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. 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.

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

    PubMed Central

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

    2007-01-01

    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 P21212, with unit-cell parameters a = 40.01, b = 71.58, c = 108.68 Å and an R 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 1r8n) as the search model. PMID:18007042

  20. 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.

  1. 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

  2. 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.

  3. 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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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).

  9. Detection of novel trypsin inhibitors in the cotyledons of Phaseolus vulgaris seeds.

    PubMed

    Alves, Marta; Chaves, Inês; Carrilho, Dina; Veloso, Manuela; Ricardo, Cândido Pinto

    2010-07-01

    Protease inhibitors play important roles in plants in association with stress. Trypsin inhibitors (TIs) in particular are known to act as protective agents against insect and pathogen attacks. The growing relevance of these inhibitors requires expedited techniques for their detection. By using the two-dimensional electrophoresis (2-DE) reverse zymography technique, we identified, from the crude extract of bean seeds, nine novel polypeptides that showed trypsin inhibitor activity. One of these polypeptide inhibitors yielded no homology in the database, which can be an indication that we are found a new protein with unique TI properties. The remaining showed homology with proteins annotated in the UniProt database and form, together with a Kunitz type inhibitor, a new TI cluster for Phaseolus spp. Three of these polypeptides showed additional high homology with lectins, likely indicating that they have lectin properties, while the other five showed high homology with alpha-amylase inhibitors, indicating that they probably have a dual inhibitory effect against trypsin and the alpha-amylase enzyme. These bifunctional inhibitors can be highly useful for crop management, since the two inhibitory activities are important for plants when coping with pathogen and pest attacks. 2010 Elsevier GmbH. All rights reserved.

  10. 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.

  11. 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.

  12. Inhibitors of the Histone Methyltransferases EZH2/1 Induce a Potent Antiviral State and Suppress Infection by Diverse Viral Pathogens.

    PubMed

    Arbuckle, Jesse H; Gardina, Paul J; Gordon, David N; Hickman, Heather D; Yewdell, Jonathan W; Pierson, Theodore C; Myers, Timothy G; Kristie, Thomas M

    2017-08-15

    Epigenetic regulation is based on a network of complexes that modulate the chromatin character and structure of the genome to impact gene expression, cell fate, and development. Thus, epigenetic modulators represent novel therapeutic targets used to treat a range of diseases, including malignancies. Infectious pathogens such as herpesviruses are also regulated by cellular epigenetic machinery, and epigenetic therapeutics represent a novel approach used to control infection, persistence, and the resulting recurrent disease. The histone H3K27 methyltransferases EZH2 and EZH1 (EZH2/1) are epigenetic repressors that suppress gene transcription via propagation of repressive H3K27me3-enriched chromatin domains. However, while EZH2/1 are implicated in the repression of herpesviral gene expression, inhibitors of these enzymes suppressed primary herpes simplex virus (HSV) infection in vitro and in vivo Furthermore, these compounds blocked lytic viral replication following induction of HSV reactivation in latently infected sensory ganglia. Suppression correlated with the induction of multiple inflammatory, stress, and antipathogen pathways, as well as enhanced recruitment of immune cells to in vivo infection sites. Importantly, EZH2/1 inhibitors induced a cellular antiviral state that also suppressed infection with DNA (human cytomegalovirus, adenovirus) and RNA (Zika virus) viruses. Thus, EZH2/1 inhibitors have considerable potential as general antivirals through the activation of cellular antiviral and immune responses.IMPORTANCE A significant proportion of the world's population is infected with herpes simplex virus. Primary infection and subsequent recurrent reactivation can result in diseases ranging from mild lesions to severe ocular or neurological damage. Herpesviruses are subject to epigenetic regulation that modulates viral gene expression, lytic replication, and latency-reactivation cycles. Thus, epigenetic pharmaceuticals have the potential to alter the course of

  13. 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.

  14. 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. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  15. Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.

    PubMed

    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.

  16. 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

  17. 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

  18. Efficiency of urease and nitrification inhibitors in reducing ammonia volatilization from diverse nitrogen fertilizers applied to different soil types and wheat straw mulching.

    PubMed

    San Francisco, Sara; Urrutia, Oscar; Martin, Vincent; Peristeropoulos, Angelos; Garcia-Mina, Jose Maria

    2011-07-01

    Some authors suggest that the absence of tillage in agricultural soils might have an influence on the efficiency of nitrogen applied in the soil surface. In this study we investigate the influence of no-tillage and soil characteristics on the efficiency of a urease inhibitor (N-(n-butyl)thiophosphoric triamide, NBPT) and a nitrification inhibitor (diciandiamide, DCD) in decreasing ammonia volatilization from urea and ammonium nitrate (AN), respectively. The results indicate that ammonia volatilization in soils amended with urea was significantly higher than in those fertilized with AN. Likewise, the main soil factors affecting ammonia volatilization from urea are clay and sand soil contents. While clay impedes ammonia volatilization, sand favours it. The presence of organic residues on soil surface (no-tillage) tends to increase ammonia volatilization from urea, although this fact depended on soil type. The presence of NBPT in urea fertilizer significantly reduced soil ammonia volatilization. This action of NBPT was negatively affected by acid soil pH and favoured by soil clay content. The presence of organic residues on soil surface amended with urea increased ammonia volatilization, and was particularly high in sandy compared with clay soils. Application of NBPT reduced ammonia volatilization although its efficiency is reduced in acid soils. Concerning AN fertilization, there were no differences in ammonia volatilization with or without DCD in no-tillage soils. Copyright © 2011 Society of Chemical Industry.

  19. 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).

  20. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. 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.

  2. ACE inhibitors

    MedlinePlus

    ... ACE inhibitors There are many different names and brands of ACE inhibitors. Most work as well as ... urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. follows ...

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae).

    PubMed

    Rodrigues Macedo, Maria Lígia; Machado Freire, Maria das Graças; Cabrini, Elaine Cristina; Toyama, Marcos H; Novello, José Camillo; Marangoni, Sérgio

    2003-05-02

    A novel trypsin inhibitor was purified from the seeds of Peltophorum dubium (Spreng.). SDS-PAGE under reducing conditions showed that the inhibitor consisted of a single polypeptide chain (ca. 20 kDa). The dissociation constants of 4 x 10(-10) and 1.6 x 10(-10) M were obtained with bovine and porcine trypsin, respectively. This constant was lower (2.6 x 10(-7) M) for chymotrypsin. The inhibitory activity was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the P. dubium inhibitor showed a high degree of homology with other Kunitz-type inhibitors. When fed to the insect Anagasta kuehniella, in an artificial diet (inhibitor concentration 1.6%), the inhibitor produced approximately 56% and delayed the development of this lepidopteran. The concentration of inhibitor in the diet necessary to cause a 50% reduction in the weight (ED50) of fourth instar larvae was approximately 1%. The action of the P. dubium trypsin inhibitor (PDTI) on A. kuehniella may involve inhibition of the trypsin-like activity present in the larval midgut, resistance of the inhibitor to digestion by midgut enzymes and bovine trypsin, and association of the inhibitor with a chitin column and chitinous structures in the peritrophic membrane and/or midgut of the insect.

  9. 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

  10. 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.

  11. 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

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

    PubMed

    Hallström, Teresia; Singh, Birendra; 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.

  13. IrSPI, a Tick Serine Protease Inhibitor Involved in Tick Feeding and Bartonella henselae Infection

    PubMed Central

    Liu, Xiang Ye; de la Fuente, Jose; Cote, Martine; Galindo, Ruth C.; Moutailler, Sara; Vayssier-Taussat, Muriel; Bonnet, Sarah I.

    2014-01-01

    Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its

  14. Homology modeling and structural validation of tissue factor pathway inhibitor

    PubMed Central

    Agrawal, Piyush; Thakur, Zoozeal; Kulharia, Mahesh

    2013-01-01

    Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either “Tissue Dependent Pathway” (also known as extrinsic pathway) or by “contact activation pathway” (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it's functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism. PMID:24143050

  15. Synthesis of influenza neuraminidase inhibitors.

    PubMed

    Abdel-Magid, A F; Maryanoff, C A; Mehrman, S J

    2001-11-01

    Influenza neuraminidase inhibitors provide a means to combat flu, a potentially very serious disease. For the first time, there is a way to treat influenza by blocking the influenza enzyme neuraminidase to stop or slow the progression of infection. The diverse structures and synthesis of several influenza neuraminidase inhibitors are discussed. Contributions from chemical process development groups are highlighted for those compounds that have reached the market, such as zanamivir and oseltamivir phosphate.

  16. 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…

  17. 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…

  18. 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.

  19. Structure of BbKI, a disulfide-free plasma kallikrein inhibitor.

    PubMed

    Zhou, Dongwen; Hansen, Daiane; Shabalin, Ivan G; Gustchina, Alla; Vieira, Debora F; de Brito, Marlon V; Araújo, Ana Paula U; Oliva, Maria Luiza V; Wlodawer, Alexander

    2015-08-01

    A serine protease inhibitor from Bauhinia bauhinioides (BbKI) belongs to the Kunitz family of plant inhibitors, which are common in plant seeds. BbKI does not contain any disulfides, unlike most other members of this family. It is a potent inhibitor of plasma kallikrein, in addition to other serine proteases, and thus exhibits antithrombotic activity. A high-resolution crystal structure of recombinantly expressed BbKI was determined (at 1.4 Å resolution) and was compared with the structures of other members of the family. Modeling of a complex of BbKI with plasma kallikrein indicates that changes in the local structure of the reactive loop that includes the specificity-determining Arg64 are necessary in order to explain the tight binding. An R64A mutant of BbKI was found to be a weaker inhibitor of plasma kallikrein, but was much more potent against plasmin, suggesting that this mutant may be useful for preventing the breakup of fibrin and maintaining clot stability, thus preventing excessive bleeding.

  20. Structure of BbKI, a disulfide-free plasma kallikrein inhibitor

    PubMed Central

    Zhou, Dongwen; Hansen, Daiane; Shabalin, Ivan G.; Gustchina, Alla; Vieira, Debora F.; de Brito, Marlon V.; Araújo, Ana Paula U.; Oliva, Maria Luiza V.; Wlodawer, Alexander

    2015-01-01

    A serine protease inhibitor from Bauhinia bauhinioides (BbKI) belongs to the Kunitz family of plant inhibitors, which are common in plant seeds. BbKI does not contain any disulfides, unlike most other members of this family. It is a potent inhibitor of plasma kallikrein, in addition to other serine proteases, and thus exhibits antithrombotic activity. A high-resolution crystal structure of recombinantly expressed BbKI was determined (at 1.4 Å resolution) and was compared with the structures of other members of the family. Modeling of a complex of BbKI with plasma kallikrein indicates that changes in the local structure of the reactive loop that includes the specificity-determining Arg64 are necessary in order to explain the tight binding. An R64A mutant of BbKI was found to be a weaker inhibitor of plasma kallikrein, but was much more potent against plasmin, suggesting that this mutant may be useful for preventing the breakup of fibrin and maintaining clot stability, thus preventing excessive bleeding. PMID:26249699

  1. 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.

  2. An advance for removing antinutritional protease inhibitors: Soybean whey purification of Bowman-Birk chymotrypsin inhibitor by combination of two oppositely charged polysaccharides.

    PubMed

    Li, Xingfei; Hua, Yufei; Chen, Yeming; Kong, Xiangzhen; Zhang, Caimeng; Yu, Xiaobin

    2017-05-15

    Two successive and selective coacervations induced by chitosan (Ch) and carrageenan (CG) were applied to remove antinutritional protease inhibitors and purify Bowman-Birk protease inhibitor (BBI) from soybean whey. At the first coacervation induced by Ch (66.7, 200, and 510kDa), only Kunitz trypsin inhibitor (KTI) and BBI complexed with Ch were extracted, while β-amylase and soybean agglutinin remained in supernatant. The binding constants for the interaction increased on the order Ch-66.7

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

    Lysine methyltransferase which catalyze methylation of histone and non-histone 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. PMID:26258118

  9. 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

  10. 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. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  11. 1,2,3-Triazolylalkylribitol derivatives as nucleoside hydrolase inhibitors.

    PubMed

    Goeminne, A; McNaughton, M; Bal, G; Surpateanu, G; Van der Veken, P; De Prol, S; Versées, W; Steyaert, J; Apers, S; Haemers, A; Augustyns, K

    2007-05-01

    A range of novel 1,2,3-triazolylalkylribitol derivatives were synthesized and evaluated as nucleoside hydrolase inhibitors. The most active compound (11a) has low micromolar potency and is structurally diverse from previously reported nucleoside hydrolase inhibitors, which, along with the simplicity of the chemistry involved in its synthesis, makes it a good lead for the further development of novel nucleoside hydrolase inhibitors.

  12. Trypsin inhibitors in passion fruit (Passiflora f. edulis flavicarpa) leaves: accumulation in response to methyl jasmonate, mechanical wounding, and herbivory.

    PubMed

    Botelho-Júnior, Sylvio; Siqueira-Júnior, César L; Jardim, Bruno C; Machado, Olga L T; Neves-Ferreira, Ana G C; Perales, Jonas; Jacinto, Tânia

    2008-10-22

    This work investigates the effect of methyl jasmonte (MeJa), mechanical wounding, and herbivory caused by larval feeding of a specialist insect ( Agraulis vanillae vanillae) upon trypsin inhibitory activity in passion fruit leaves. Despite the fact that all treatments caused accumulation of trypsin inhibitors (TIs), higher levels were observed in MeJa treated leaves when plants were assayed 24 and 48 h after stimulus. Concerning both mechanically injured plants and attacked ones, a systemic induction was observed. Partially purified inhibitors from MeJa exposed plants were further characterized by X-ray film contact print technique and N-terminal sequence. Such analysis indicated that the TIs identified belong to the Kunitz family. Moreover, the partially purified inhibitors strongly inhibited trypsin-like digestive enzymes from sugar cane stalk borer ( Diatraea saccharalis) in vitro. Our results further support the protective function of wound-inducible trypsin inhibitors and their potential as tools to improve important crop species against insect predation through genetic engineering.

  13. 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.

  14. 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.

  15. 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.

  16. Managing diversity.

    PubMed

    Epting, L A; Glover, S H; Boyd, S D

    1994-06-01

    The U.S. work force is becoming increasingly diverse as the 20th century approaches. Statistics prove that most organizations are experiencing gender, culture, and age diversity within their labor forces. All managers and leaders must accept this diversity and work to handle it effectively. This article examines the current literature concerning management of diversity and its implications for the health care profession. Gender, culture, and age diversity and the potential problems that may arise with each are also addressed. Reasons to manage diversity are offered, as well as methods of managing diversity for both the manager and the chief executive officer.

  17. Developmentally regulated transcription of the four liver-specific genes for inter-alpha-inhibitor family in mouse.

    PubMed

    Salier, J P; Chan, P; Raguenez, G; Zwingman, T; Erickson, R P

    1993-11-15

    The inter-alpha-inhibitor family is composed of the plasma-protease inhibitors inter-alpha-inhibitor, pre-alpha-inhibitor and bikunin. Inter-alpha-inhibitor and pre-alpha-inhibitor are distinct assemblies of bikunin with distinct sets from three heavy (H) chains designated H1, H2 and H3. These H chains are encoded by a set of three evolutionarily related H genes, and bikunin by an alpha-1-microglobulin/bikunin precursor gene (AMBP). This precursor is cleaved to yield bikunin, a member of the Kunitz-type protease-inhibitor superfamily, and alpha-1-microglobulin, which belongs to the lipocalin superfamily. Northern-blot experiments with RNAs obtained from various tissues in fetal and in adult mice indicated that the transcription of the four AMBP and H genes is liver-restricted, although there is expression of H3 in brain. An analysis of the H1, H2, H3 and AMBP transcripts, as well as of transcripts for other control genes, in liver during development showed a progressive increase in the amounts of the H1, H2, H3 and AMBP RNAs, which all peak transiently at day 5 after birth. This was shown by a nuclear run-on experiment to originate from a change in transcription rate. The transient and postnatal increase in transcription could be explained neither by the liver-restricted expression nor by a common origin of these four genes, nor by a perinatal requirement for many lipocalins or protease inhibitors. This suggests that all four genes are perinatally triggered at the level of similar elements in their transcriptional regulatory regions, a conclusion strengthened by the weak expression of the four genes that is seen in a mutant mouse strain (albino) that is deficient in some liver-specific transcription factors.

  18. Developmentally regulated transcription of the four liver-specific genes for inter-alpha-inhibitor family in mouse.

    PubMed Central

    Salier, J P; Chan, P; Raguenez, G; Zwingman, T; Erickson, R P

    1993-01-01

    The inter-alpha-inhibitor family is composed of the plasma-protease inhibitors inter-alpha-inhibitor, pre-alpha-inhibitor and bikunin. Inter-alpha-inhibitor and pre-alpha-inhibitor are distinct assemblies of bikunin with distinct sets from three heavy (H) chains designated H1, H2 and H3. These H chains are encoded by a set of three evolutionarily related H genes, and bikunin by an alpha-1-microglobulin/bikunin precursor gene (AMBP). This precursor is cleaved to yield bikunin, a member of the Kunitz-type protease-inhibitor superfamily, and alpha-1-microglobulin, which belongs to the lipocalin superfamily. Northern-blot experiments with RNAs obtained from various tissues in fetal and in adult mice indicated that the transcription of the four AMBP and H genes is liver-restricted, although there is expression of H3 in brain. An analysis of the H1, H2, H3 and AMBP transcripts, as well as of transcripts for other control genes, in liver during development showed a progressive increase in the amounts of the H1, H2, H3 and AMBP RNAs, which all peak transiently at day 5 after birth. This was shown by a nuclear run-on experiment to originate from a change in transcription rate. The transient and postnatal increase in transcription could be explained neither by the liver-restricted expression nor by a common origin of these four genes, nor by a perinatal requirement for many lipocalins or protease inhibitors. This suggests that all four genes are perinatally triggered at the level of similar elements in their transcriptional regulatory regions, a conclusion strengthened by the weak expression of the four genes that is seen in a mutant mouse strain (albino) that is deficient in some liver-specific transcription factors. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7504460

  19. (1)H, (15)N and (13)C resonance assignments of Ixolaris, a tissue factor pathway inhibitor from the tick salivary gland.

    PubMed

    De Paula, V S; Silva, F H S; Francischetti, I M B; Monteiro, R Q; Valente, A P

    2017-08-30

    Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents sequence homology to TFPI (tissue factor pathway inhibitor). It binds to the coagulation enzyme factor Xa (FXa) or to its zymogen form, FX, and further inhibits tissue factor/FVIIa complex (extrinsic Xnase compex). Differently from TFPI, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. The Ixolaris-FXa/FX complex formation has been characterized by using a combination of biophysical and biochemical technics although no structural data is currently available. In this study, we reported the NMR chemical shift assignment of Ixolaris, as a first step to further establishing the structure, dynamics and function relationship for this protein.

  20. 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.

  1. Managing Diversity.

    ERIC Educational Resources Information Center

    Geber, Beverly

    1990-01-01

    Demographic trends imply that organizations must learn to manage a diverse work force. Ways to change organizational systems, structures, and practices to eliminate subtle barriers are awareness training, attitude change, and valuing diversity. (SK)

  2. 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

  3. Diversity & Dartmouth.

    ERIC Educational Resources Information Center

    Freedman, James O.

    1991-01-01

    The president of Dartmouth College (New Hampshire) discusses campus cultural pluralism, the need for diversity in higher education, overcoming resistance to change, techniques for supporting a diverse student population, monitoring diversity through institutional research, and the issue of "political correctness" in higher education…

  4. 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)

  5. 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…

  6. 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…

  7. 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)

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.

    PubMed

    Bao, Rui; Zhou, Cong-Zhao; Jiang, Chunhui; Lin, Sheng-Xiang; Chi, Cheng-Wu; Chen, Yuxing

    2009-09-25

    The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5' Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5' Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

  13. Embracing Diversity

    ERIC Educational Resources Information Center

    Roeck, Kathryn T.

    2009-01-01

    The high school art unit "Embracing Diversity" was the author's principal work towards the completion of a Masters thesis. The objective was to learn whether or not teaching an art unit that focused on sexual diversity could have a positive impact on the current culture one finds in high schools. The unit was found to have a positive…

  14. 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.

  15. 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.

  16. The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice.

    PubMed

    Chand, Hitendra Singh; Du, Xin; Ma, Duan; Inzunza, Hector David; Kamei, Shintaro; Foster, Donald; Brodie, Steven; Kisiel, Walter

    2004-02-01

    Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits the plasmin- and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion, and metastasis. To directly assess its role in tumor growth and metastasis in vivo, we stably transfected HT-1080 fibrosarcoma cells expressing either fully active wild-type human TFPI-2 (WT) or inactive R24Q TFPI-2 (QT) and examined their ability to form tumors and metastasize in athymic mice in comparison to mock-transfected cells (MT). MT and QT fibrosarcoma tumors grew 2 to 3 times larger than WT tumors. Tumor metastasis was confined to the lung and was observed in 75% of mice treated with either MT or QT cells, whereas only 42% of mice treated with WT cells developed lung metastases. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of each tumor group revealed 3- to 6-fold lower levels of murine vascular endothelial growth factor gene expression in WT tumors in relation to either MT or QT tumors. Comparative tumor gene expression analysis revealed that several human genes implicated in oncogenesis, invasion, metastasis, apoptosis, and angiogenesis had significantly altered levels of expression in WT tumors. Our collective data demonstrate that secretion of inhibitory TFPI-2 by a highly metastatic tumor cell markedly inhibits its growth and metastasis in vivo by regulating pericellular extracellular matrix (ECM) remodeling and angiogenesis.

  17. 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

  18. 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

  19. Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety "Golden Valley".

    PubMed

    Kim, Mi-Hyun; Park, Seong-Cheol; Kim, Jin-Young; Lee, Sun Young; Lim, Hak-Tae; Cheong, Hyeonsook; Hahm, Kyung-Soo; Park, Yoonkyung

    2006-08-04

    Potide-G, a small (5578.9 Da) antimicrobial peptide, was isolated from potato tubers (Solanum tuberosum L. cv. Golden Valley) through extraction of the water-soluble fraction, dialysis, ultrafiltration and DEAE-cellulose and C18 reverse-phase high performance liquid chromatography. This antimicrobial peptide was heat-stable and almost completely suppressed the proteolytic activity of trypsin, chymotrypsin and papain, with no hemolytic activity. In addition, potide-G potently inhibited growth of a variety of bacterial (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Clavibacter michiganense subsp. michiganinse) and fungal (Candida albicans and Rhizoctonia solani) strains. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the N-terminal sequence (residues from 1 to 11) of the protein is identical to that of potato proteinase inhibitor, a member of the Kunitz superfamily. And like other members of this class of protease inhibitor, potide-G may have a number of beneficial and therapeutic uses.

  20. 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

  1. 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.

  2. 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.

  3. 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 ...

  4. Matrix metalloproteinase inhibitors.

    PubMed

    Wojtowicz-Praga, S M; Dickson, R B; Hawkins, M J

    1997-01-01

    The matrix metalloproteinases (MMPs) are a family of at least fifteen secreted and membrane-bound zinc-endopeptidases. Collectively, these enzymes can degrade all of the components of the extracellular matrix, including fibrallar and non-fibrallar collagens, fibronectin, laminin and basement membrane glycoproteins. MMPs are thought to be essential for the diverse invasive processes of angiogenesis and tumor metastasis. Numerous studies have shown that there is a close association between expression of various members of the MMP family by tumors and their proliferative and invasive behavior and metastatic potential. In some of human cancers a positive correlation has also been demonstrated between the intensity of new blood vessel growth (angiogenesis) and the likelihood of developing metastases. Thus, control of MMP activity in these two different contexts has generated considerable interest as a possible therapeutic target. The tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring proteins that specifically inhibit matrix metalloproteinases, thus maintaining balance between matrix destruction and formation. An imbalance between MMPs and the associated TIMPs may play a significant role in the invasive phenotype of malignant tumors. TIMP-1 has been shown to inhibit tumor-induced angiogenesis in experimental systems. These findings raised the possibility of using an agent that affects expression or activity of MMPs as an anti-cancer therapy. TIMPs are probably not suitable for pharmacologic applications due to their short half-life in vivo. Batimastat (BB-94) and marimastat (BB-2516) are synthetic, low-molecular weight MMP inhibitors. They have a collagen-mimicking hydroxamate structure, which facilitates chelation of the zinc ion in the active site of the MMPs. These compounds inhibit MMPs potently and specifically. Batimastat was the first synthetic MMP inhibitor studied in humans with advanced malignancies, but its usefulness has been limited by

  5. 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…

  6. 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...

  7. Diversity Trailblazer

    ERIC Educational Resources Information Center

    Stuart, Reginald

    2012-01-01

    When Dr. Kumea Shorter-Gooden took on her newly created job this month at the University of Maryland's flagship College Park campus, she assumed a challenge at the school with a lot riding on her shoulders--helping the University of Maryland strengthen its diversity efforts and, thus, its relevance to the state in the future and standing among the…

  8. 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...

  9. 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)

  10. 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.

  11. 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.

  12. 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.

  13. Inhibitors of the Metalloproteinase Anthrax Lethal Factor.

    PubMed

    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 LFinhibitor 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 highthroughput 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.

  14. Inhibition of tissue factor pathway inhibitor increases the sensitivity of thrombin generation assay to procoagulant microvesicles.

    PubMed

    Gheldof, Damien; Mullier, François; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian

    2013-07-01

    Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.

  15. Design and Synthesis of Human ABCB1 (P-Glycoprotein) Inhibitors by Peptide Coupling of Diverse Chemical Scaffolds on Carboxyl and Amino Termini of (S)-Valine-Derived Thiazole Amino Acid

    PubMed Central

    2015-01-01

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [125I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp. PMID:24773054

  16. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer.

    PubMed

    Jacobsen, Kirstine; Bertran-Alamillo, Jordi; Molina, Miguel Angel; Teixidó, Cristina; Karachaliou, Niki; Pedersen, Martin Haar; Castellví, Josep; Garzón, Mónica; Codony-Servat, Carles; Codony-Servat, Jordi; Giménez-Capitán, Ana; Drozdowskyj, Ana; Viteri, Santiago; Larsen, Martin R; Lassen, Ulrik; Felip, Enriqueta; Bivona, Trever G; Ditzel, Henrik J; Rosell, Rafael

    2017-09-04

    Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo.

  17. Managing diversity.

    PubMed

    Wagner, M

    1991-09-30

    One look at projections for the U.S. work force through the year 2000 shows why healthcare administrators will be facing some new challenges. With the majority of new workers belonging to minority groups, "managing diversity" has become the newest catch phrase as executives work to reduce tensions resulting from race, gender or culture-based differences among workers, while also learning to understand and value those differences.

  18. GSK-3 Inhibitors: Preclinical and Clinical Focus on CNS

    PubMed Central

    Eldar-Finkelman, Hagit; Martinez, Ana

    2011-01-01

    Inhibiting glycogen synthase kinase-3 (GSK-3) activity via pharmacological intervention has become an important strategy for treating neurodegenerative and psychiatric disorders. The known GSK-3 inhibitors are of diverse chemotypes and mechanisms of action and include compounds isolated from natural sources, cations, synthetic small-molecule ATP-competitive inhibitors, non-ATP-competitive inhibitors, and substrate–competitive inhibitors. Here we describe the variety of GSK-3 inhibitors with a specific emphasis on their biological activities in neurons and neurological disorders. We further highlight our current progress in the development of non-ATP-competitive inhibitors of GSK-3. The available data raise the hope that one or more of these drug design approaches will prove successful at stabilizing or even reversing the aberrant neuropathology and cognitive deficits of certain central nervous system disorders. PMID:22065134

  19. 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.

  20. 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.

  1. 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.

  2. 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. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  3. Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization.

    PubMed

    Murray, Andrew S; Varela, Fausto A; Hyland, Thomas E; Schoenbeck, Andrew J; White, Jordan M; Tanabe, Lauren M; Todi, Sokol V; List, Karin

    2017-09-08

    TMPRSS13 is a member of the type II transmembrane serine protease (TTSP) family. Although various TTSPs have been characterized in detail biochemically and functionally, the basic properties of TMPRSS13 remain unclear. Here, we investigate the activation, inhibition, post-translational modification, and localization of TMPRSS13. We show that TMPRSS13 is a glycosylated, active protease and that its own proteolytic activity mediates zymogen cleavage. Full-length, active TMPRSS13 exhibits impaired cell-surface expression in the absence of the cognate Kunitz-type serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 or HAI-2. Concomitant presence of TMPRSS13 with either HAI-1 or -2 mediates phosphorylation of residues in the intracellular domain of the protease, and it coincides with efficient transport of the protease to the cell surface and its subsequent shedding. Cell-surface labeling experiments indicate that the dominant form of TMPRSS13 on the cell surface is phosphorylated, whereas intracellular TMPRSS13 is predominantly non-phosphorylated. These data provide novel insight into the cellular properties of TMPRSS13 and highlight phosphorylation of TMPRSS13 as a novel post-translational modification of this TTSP family member and potentially other members of this family of proteases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. 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.

  5. Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

    PubMed Central

    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. PMID:22348050

  6. Valuing Diversity

    PubMed Central

    Fryer, Roland G.; Loury, Glenn C.

    2014-01-01

    This paper explores the economics of diversity-enhancing policies. A model is proposed in which heterogeneous agents, distinguished by skill level and social identity, purchase productive opportunities in a competitive market. We analyze policies designed to raise the status of a disadvantaged identity group. When agent identity is contractible, efficient policy grants preferred access to slots but offers no direct assistance for acquiring skills. When identity is not contractible, efficient policy provides universal subsidies to skill development when the fraction of the disadvantaged group at the skill development margin is larger than their share at the slot assignment margin. PMID:25525280

  7. 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

  8. 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

  9. HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

    PubMed

    Fun, Axel; van Maarseveen, Noortje M; Pokorná, Jana; Maas, Renée Em; Schipper, Pauline J; Konvalinka, Jan; Nijhuis, Monique

    2011-08-24

    Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different

  10. 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

  11. Blood and Diversity

    MedlinePlus

    ... Learn About Blood > Blood and Diversity 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 ...

  12. Surface Diversity

    NASA Image and Video Library

    2016-03-17

    This enhanced color view of Pluto's surface diversity was created by merging Ralph/Multispectral Visible Imaging Camera (MVIC) color imagery (650 meters per pixel) with Long Range Reconnaissance Imager panchromatic imagery (230 meters per pixel). At lower right, ancient, heavily cratered terrain is coated with dark, reddish tholins. At upper right, volatile ices filling the informally named Sputnik Planum have modified the surface, creating a chaos-like array of blocky mountains. Volatile ice also occupies a few nearby deep craters, and in some areas the volatile ice is pocked with arrays of small sublimation pits. At left, and across the bottom of the scene, gray-white CH4 ice deposits modify tectonic ridges, the rims of craters, and north-facing slopes. The scene in this image is 260 miles (420 kilometers) wide and 140 miles (225 kilometers) from top to bottom; north is to the upper left. http://photojournal.jpl.nasa.gov/catalog/PIA20534

  13. 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.

  14. Proteasome inhibitor patents (2010 - present).

    PubMed

    Metcalf, Rainer; Scott, Latanya M; Daniel, Kenyon G; Dou, Q Ping

    2014-04-01

    Over the past 3 years, numerous patents and patent applications have been submitted and published involving compounds designed to inhibit the proteasome. Proteasome inhibition has been of great interest in cancer research since disruption of proteolysis leads to a significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells. The current standards in proteasome inhibition are the only FDA-approved inhibitors, bortezomib and carfilzomib. Although these drugs are quite effective in treating multiple myeloma and other blood tumors, there are shortcomings, including toxicities and resistance. Most of the current patents attempt to improve on existing compounds, by increasing bioavailability and selectivity, while attempting to reduce toxicity. A general categorization of similar compounds was employed to evaluate and compare drug design strategies. This review focuses on novel compounds and subsequent analogs developed for proteasome inhibition, used in preventing and treating human cancers. A comprehensive description and categorization of patents related to each type of compound and its derivatives, as well as their uses and efficacies as anticancer agents is included. A review of combination therapy patents has also been included. Although there are many diverse chemical scaffolds being published, there are few patented proteasome inhibitors whose method of inhibition is genuinely novel. Most patents utilize a destructive chemical warhead to attack the catalytic threonine residue of the proteasome active sites. Few patents try to depart from this, emphasizing the need for developing new mechanisms of action and specific targeting.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors.

    PubMed

    Brown, C R; Andani, Z; Hartree, E F

    1975-07-01

    1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-alpha-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca(2+), and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris-HCl buffer (pH8.2) activation by Ca(2+) did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca(2+) no longer exerts an activating effect and may become inhibitory. Ca(2+) is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca(2+) on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca(2+). Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca(2+). 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-alpha-benzoyl-dl-arginine 2-naphthylamide. 8. K(m) values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and k(cat.) values are slightly higher than those for

  4. Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors

    PubMed Central

    Brown, Colin R.; Andani, Zarina; Hartree, Edward F.

    1975-01-01

    1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-α-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca2+, and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris–HCl buffer (pH8.2) activation by Ca2+ did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca2+ no longer exerts an activating effect and may become inhibitory. Ca2+ is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca2+ on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca2+. Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca2+. 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-α-benzoyl-dl-arginine 2-naphthylamide. 8. Km values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and kcat. values are slightly higher than those for trypsin. Considerations

  5. 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.

  6. 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…

  7. 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…

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.…

  16. 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.

  17. 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

  18. Protection against acute lung injury by intravenous or intratracheal pretreatment with EPI-HNE-4, a new potent neutrophil elastase inhibitor.

    PubMed

    Delacourt, Christophe; Hérigault, Sabine; Delclaux, Christophe; Poncin, Alain; Levame, Micheline; Harf, Alain; Saudubray, François; Lafuma, Chantal

    2002-03-01

    Excessive accumulation of active neutrophil elastase (NE) in pulmonary fluids and tissues of patients with cystic fibrosis (CF) is thought to act on the lungs, compromising their structure and function. The aim of this study was to investigate the in vitro and in vivo protective effect of a new, rapidly acting, potent (Ki = 5.45 x 10(-12) M and Kon = 8 x 10(6) M(-1) s(-1)) and specific human NE inhibitor, EPI-HNE-4, engineered from the Kunitz domain. The results demonstrated that this inhibitor was able to (i) effectively inhibit in vitro the high levels of active NE present in a medium as complex as sputum from children with CF, with a measured IC(50) equal or close to the calculated IC(50) in 60% of cases, and (ii) almost completely block (91%) the N-formyl-methionine-leucine-phenylalanine-induced migration of purified human neutrophils across a Matrigel basement membrane. Intratracheal administration (250, 175, or 100 microg per rat) of the inhibitor 5 min before instillation of pure human NE (HNE) (150 microg per rat) to rats induced effective, dose-dependent protection of the lungs, 4 h later, from hemorrhage, serum albumin leakage, residual active NE, and discrete neutrophil influx in air spaces induced by instillation of pure HNE. Intravenous administration (3 mg per rat) of EPI-HNE-4, 15 min before instillation of the soluble fraction of pooled sputum (delivering 120 microg of active NE per rat) from children with CF, effectively reduced (64%), 4 h later, the massive neutrophil influx induced by sputum instillation. Overall, these data strongly suggest that associated aerosol and systemic administration of EPI-HNE-4 would be beneficial in the treatment of CF.

  19. 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.

  20. Synthesis and characterization of Sant-75 derivatives as Hedgehog-pathway inhibitors.

    PubMed

    Che, Chao; Li, Song; Yang, Bo; Xin, Shengchang; Yu, Zhixiong; Shao, Taofeng; Tao, Chuanye; Lin, Shuo; Yang, Zhen

    2012-01-01

    Sant-75 is a newly identified potent inhibitor of the hedgehog pathway. We designed a diversity-oriented synthesis program, and synthesized a series of Sant-75 analogues, which lays the foundation for further investigation of the structure-activity relationship of this important class of hedgehog-pathway inhibitors.

  1. 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.

  2. Natural inhibitors of thrombin.

    PubMed

    Huntington, James A

    2014-04-01

    The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.

  3. [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.

  4. 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.

  5. Investigational cell cycle inhibitors in clinical trials for bladder cancer.

    PubMed

    Yun, Seok Joong; Moon, Sung-Kwon; Kim, Wun-Jae

    2013-03-01

    Cancer-related cell cycle defects are often mediated by alterations in activity of diverse cell cycle regulators. The development of cell cycle inhibitors has undergone a gradual evolution, and new investigational drugs have been extensively tested as a single agent or combination with conventional chemotherapeutic drugs. This review covers a broad perspective of how the cell cycle is deregulated in bladder cancer and discusses the clinical trials of cell cycle inhibitors. Although diverse cell cycle inhibitors have been considered as relevant drug candidates for cancer therapy owing to their potential role in restoring control of the cell cycle, these inhibitors have not been yet widely tested in human bladder cancer. Numerous studies already reported that deregulation of cell cycle controls has been commonly observed in bladder cancer cells, thus warranting clinical trials of these inhibitors in advanced bladder cancer patients. In addition, nonmuscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC) show different clinical and molecular biological characteristics, although ∼ 10 - 20% of NMIBC will progress to MIBC. Therefore, adequate cell cycle inhibitors have to be chosen for bladder cancer treatment based on the different genetic features between NMIBC and MIBC related to cell cycle regulators.

  6. 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

  7. 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

  8. 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…

  9. 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…

  10. Workforce diversity in hospitals.

    PubMed

    Mateo, Magdalena A; Smith, Suzanne P

    2003-01-01

    With an increasingly diverse population and staff, leaders of organizations must play an active role in diversity management. Nurse leaders (n = 231) responded to a survey concerning diversity initiatives, outcomes, and issues related to staff and patients. The findings suggest that most respondents have not made diversity management an important issue. Few have goals related to diversity with indices to measure outcomes. The majority of the respondents did not report that diversity in their staffs or patients resulted in management or patient care issues. The authors suggest that managers revisit the policies, procedures, and practices related to their diversity management initiatives.

  11. 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.

  12. 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.

  13. LpxC inhibitors: a patent review (2010-2016).

    PubMed

    Kalinin, Dmitrii V; Holl, Ralph

    2017-08-04

    The Zn(2+)-dependent deacetylase LpxC is an essential enzyme of lipid A biosynthesis in Gram-negative bacteria and a promising target for the development of antibiotics selectively combating Gram-negative pathogens. Researchers from industry and academia have synthesized structurally diverse LpxC inhibitors, exhibiting different LpxC inhibitory and antibacterial activities. Areas covered: A brief introduction into the structure and function of LpxC, showing its suitability as antibacterial target, along with the structures of several reported LpxC inhibitors, is given. The article reviews patents (reported between 2010 and 2016) and related research publications on novel small-molecule LpxC inhibitors. Emphasis is placed on structure-activity relationships within the reported series of LpxC inhibitors. Expert opinion: The performed analysis of patents revealed that the current search for novel LpxC inhibitors is focused on small molecules, sharing common structural features like a Zn(2+)-chelating group as well as a highly lipophilic side-chain. However, despite the promising preclinical data of many of the reported compounds, besides the recently withdrawn clinical candidate ACHN-975, no other LpxC inhibitor has entered clinical trials. The lack of clinical candidates might be related with undesired effects caused by the common structural elements of the LpxC inhibitors.

  14. 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…

  15. 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…

  16. 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…

  17. 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…

  18. 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…

  19. 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…

  20. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. 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.

  2. 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.

  3. [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.

  4. 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.

  5. Protease inhibitor studies enrolling.

    PubMed

    1995-01-01

    The protease enzyme is essential for HIV to make copies of itself. So far, research has failed to find a protease inhibitor that works against HIV. It is believed that, regardless of what type of protease inhibitor someone takes, it will need to be supplemented with other anti-HIV drugs. Three protease inhibitors have thus far been found to be safe, although long-term effects are unknown. These drugs are saquinavir, ABT-538, and L-735,524 produced by Hoffman-LaRoche, Abbott, and Merck respectively. Clinical trials of saquinavir are promising but it has not been shown to be the knock-out drug needed. ABT-538 has high bioavailability, but studies are showing it can cause liver and eye damage. L-735,524 studies are showing that resistance develops quite quickly. Future studies at higher doses are expected. To obtain information on protease studies currently looking for participants, contact The Network. Information on other approved, alternative, and experimental drugs is also available.

  6. 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.

  7. Cox-2 inhibitors.

    PubMed

    Brown, E

    1999-01-01

    Increasing pharmacy costs are among the fastest growing segments of the health care budget. Health plans are focusing on appropriately managing pharmaceutical costs, both from a long-term global perspective and a short-term approach emphasizing newly marketed products. Over the next six months, cox-2 inhibitors are expected to be approved by the FDA. This new class of drugs, investigated as a safer alternative to non-steroidal anti-inflammatory drugs (NSAIDs), is among the most highly anticipated medications to hit the marketplace. How health plans react to the launch of cox-2 inhibitors may serve as an example for future pharmacy management efforts. A proactive policy regarding the use of cox-2 inhibitors may be challenging, but should include: Reviewing clinical information; evaluating the cost of the new drug; and identifying appropriate patient selection criteria. The available management strategies include precertification, a tiered co-payment system, restricting prescriptions to a provider specialty, retrospective physician profiling, and physician education.

  8. 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.

  9. Kinase inhibitor recognition by use of a multivariable QSAR model.

    PubMed

    Sprous, D G; Zhang, John; Zhang, Lei; Wang, Zhaolin; Tepper, M A

    2006-01-01

    We have applied a retrosynthetic program to determine the scaffold and R-group chemical space seen within a library of known kinase inhibitors and non-kinase drug-like molecules. Comparison of the differences quickly revealed that kinase inhibitors are distinct in several chemical fragment and physical properties. We then applied these descriptors in a multivariable quantitative structure-activity relationship (QSAR) model with the goal to distinguish kinase inhibitors from non-kinase drug-like molecules. This model is heuristic in that it was trained over a dataset of 258 known kinase inhibitors and 230 non-kinase drug molecules. The final model recognized 98% of the training set as being kinase inhibitors and had a false positive rate of 15%. This trait for false positives was accepted out of a desire to maintain diversity and not miss possible good kinase inhibitors for screening. The model was validated by reserving a portion of the datasets as test sets, which were not included in the QSAR model building stage. This was done repetitively for different percentiles of the total dataset population. It was seen that model recognition and false positive were only slightly damaged well down to a 70% reserve (30% dataset used for QSAR model training while 70% used for reserve test set). Beyond 70%, the QSAR models were inconsistent, signifying that the training sets were inadequately diverse to represent the greater reserve test sets. We applied this model to evaluate the commercial kinase libraries available from Asinex, BioFocus, ChemDiv and LifeChemicals to facilitate purchase decisions for compounds for HTS for lead compounds. We observed that there are significant differences in populations of recognizable kinase inhibitors across the vendors analyzed, with BioFocus showing the greatest population of kinase like molecules.

  10. 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…

  11. Managing Generational Diversity

    ERIC Educational Resources Information Center

    O'Donovan, Eamonn

    2009-01-01

    Many school leaders have explored the issue of diversity when it comes to students, teachers and staff. Their focus typically has been on gender and ethnicity. However, generational diversity, an area of diversity that warrants serious consideration, has received less attention. Generational intelligence is important today for two reasons. First…

  12. 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…

  13. 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…

  14. 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)…

  15. 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…

  16. 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…

  17. 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…

  18. Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration.

    PubMed

    Bilgin, Mehmet; Neuhof, Christiane; Doerr, Oliver; Benscheid, Utz; Andrade, Sheila S; Most, Astrid; Abdallah, Yaser; Parahuleva, Mariana; Guenduez, Dursun; Oliva, Maria L; Erdogan, Ali

    2010-12-01

    Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10-100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10-100 μM) on the membrane potential was measured with the fluorescence dye DiBAC4(3) and the effect on [Ca+2]i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10-100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1±1.8% as compared to control (p≤0.05; n=45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca2+ of 28.4±5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5±0.9% as compared to control (p≤0.05; n=80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.

  19. Phosphodiesterase Inhibitors as Therapeutics for Traumatic Brain Injury

    PubMed Central

    Titus, David J.; Oliva, Anthony A.; Wilson, Nicole M.; Atkins, Coleen M.

    2014-01-01

    Developing therapeutics for traumatic brain injury remains a challenge for all stages of recovery. The pathological features of traumatic brain injury are diverse, and it remains an obstacle to be able to target the wide range of pathologies that vary between traumatic brain injured patients and that evolve during recovery. One promising therapeutic avenue is to target the second messengers cAMP and cGMP with phosphodiesterase inhibitors due to their broad effects within the nervous system. Phosphodiesterase inhibitors have the capability to target different injury mechanisms throughout the time course of recovery after brain injury. Inflammation and neuronal death are early targets of phosphodiesterase inhibitors, and synaptic dysfunction and circuitry remodeling are late potential targets of phosphodiesterase inhibitors. This review will discuss how signaling through cyclic nucleotides contributes to the pathology of traumatic brain injury in the acute and chronic stages of recovery. We will review our current knowledge of the successes and challenges of using phosphodiesterase inhibitors for the treatment of traumatic brain injury and conclude with important considerations in developing phosphodiesterase inhibitors as therapeutics for brain trauma. PMID:25159077

  20. Work group diversity.

    PubMed

    van Knippenberg, Daan; Schippers, Michaéla C

    2007-01-01

    Work group diversity, the degree to which there are differences between group members, may affect group process and performance positively as well as negatively. Much is still unclear about the effects of diversity, however. We review the 1997-2005 literature on work group diversity to assess the state of the art and to identify key issues for future research. This review points to the need for more complex conceptualizations of diversity, as well as to the need for more empirical attention to the processes that are assumed to underlie the effects of diversity on group process and performance and to the contingency factors of these processes.

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

    PubMed Central

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

    2011-01-01

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

  2. 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.

  3. [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.

  4. 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

  5. Plant protein inhibitors of cell wall degrading enzymes.

    PubMed

    Juge, Nathalie

    2006-07-01

    Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.

  6. Marine-Derived Angiogenesis Inhibitors for Cancer Therapy

    PubMed Central

    Wang, Ying-Qing; Miao, Ze-Hong

    2013-01-01

    Angiogenesis inhibitors have been successfully used for cancer therapy in the clinic. Many marine-derived natural products and their analogues have been reported to show antiangiogenic activities. Compared with the drugs in the clinic, these agents display interesting characteristics, including diverse sources, unique chemical structures, special modes of action, and distinct activity and toxicity profiles. This review will first provide an overview of the current marine-derived angiogenesis inhibitors based on their primary targets and/or mechanisms of action. Then, the marine-derived antiangiogenic protein kinase inhibitors will be focused on. And finally, the clinical trials of the marine-derived antiangiogenic agents will be discussed, with special emphasis on their application potentials, problems and possible coping strategies in their future development as anticancer drugs. PMID:23502698

  7. 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.

  8. Discovery of potent wall teichoic acid early stage inhibitors.

    PubMed

    Labroli, Marc A; Caldwell, John P; Yang, Christine; Lee, Sang Ho; Wang, Hao; Koseoglu, Sandra; Mann, Paul; Yang, Shu-Wei; Xiao, Jing; Garlisi, Charles G; Tan, Christopher; Roemer, Terry; Su, Jing

    2016-08-15

    The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with β-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum β-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant Staphylococci. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Ethnic differences in acetylcholinesterase inhibitor use for Alzheimer disease.

    PubMed

    Mehta, Kala M; Yin, Maggie; Resendez, Cynthia; Yaffe, Kristine

    2005-07-12

    Acetylcholinesterase inhibitors (AChIs) have been demonstrated to improve Alzheimer disease symptoms. Whether the use of AChIs varies by ethnicity is unknown. More than 2500 ethnically diverse patients (6% African American, 14% Latino, and 7% Asian patients) from the Alzheimer's Disease Research Centers in California were studied. Compared with white patients with AD, minority patients had 40% lower odds of AChI use (odds ratio 0.6, 95% confidence interval: 0.5 to 0.7).

  10. 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.

  11. Managing diversity in hospitals.

    PubMed

    Schwartz, R H; Sullivan, D B

    1993-01-01

    Hospital work force diversity, although potentially a source of creativity and improved problem solving, is often a source of political strife and the mistreatment of people based on their identification with one or another of the diverse groups that are employed in hospitals. Factors linked to these phenomena are discussed and are the basis for suggestions about how administrators can deal with the organizational pathologies that are often associated with unmanaged work force diversity.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. Predictive QSAR modeling of phosphodiesterase 4 inhibitors.

    PubMed

    Kovalishyn, Vasyl; Tanchuk, Vsevolod; Charochkina, Larisa; Semenuta, Ivan; Prokopenko, Volodymyr

    2012-02-01

    A series of diverse organic compounds, phosphodiesterase type 4 (PDE-4) inhibitors, have been modeled using a QSAR-based approach. 48 QSAR models were compared by following the same procedure with different combinations of descriptors and machine learning methods. QSAR methodologies used random forests and associative neural networks. The predictive ability of the models was tested through leave-one-out cross-validation, giving a Q² = 0.66-0.78 for regression models and total accuracies Ac=0.85-0.91 for classification models. Predictions for the external evaluation sets obtained accuracies in the range of 0.82-0.88 (for active/inactive classifications) and Q² = 0.62-0.76 for regressions. The method showed itself to be a potential tool for estimation of IC₅₀ of new drug-like candidates at early stages of drug development. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. 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

  18. Biological abatement of cellulase inhibitors

    USDA-ARS?s Scientific Manuscript database

    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 ...

  19. Design of a series of bicyclic HIV-1 integrase inhibitors. Part 2: azoles: effective metal chelators.

    PubMed

    Le, Giang; Vandegraaff, Nick; Rhodes, David I; Jones, Eric D; Coates, Jonathan A V; Thienthong, Neeranat; Winfield, Lisa J; Lu, Long; Li, Xinming; Yu, Changjiang; Feng, Xiao; Deadman, John J

    2010-10-01

    Synthesis of a diverse set of azoles and their utilizations as an amide isostere in the design of HIV integrase inhibitors is described. The Letter identified thiazole, oxazole, and imidazole as the most promising heterocycles. Initial SAR studies indicated that these novel series of integrase inhibitors are amenable to lead optimization. Several compounds with low nanomolar inhibitory potency are reported. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. 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.

  1. 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

  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. 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

  4. 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.

  5. Chemosensitization potential of P-glycoprotein inhibitors in malaria parasites.

    PubMed

    Alcantara, Laura M; Kim, Junwon; Moraes, Carolina B; Franco, Caio H; Franzoi, Kathrin D; Lee, Sukjun; Freitas-Junior, Lucio H; Ayong, Lawrence S

    2013-06-01

    Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.

  6. 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…

  7. Racial Diversity Reconsidered.

    ERIC Educational Resources Information Center

    Rothman, Stanley; Lipset, Seymour Martin; Nevitte, Neil

    2003-01-01

    Surveyed college faculty, administrators, and students about their feelings on campus diversity programs and various aspects of the general educational experience and environment. Among faculty and administrators, diversity brought perceptions of better race relations, decreased educational quality, and decreased academic preparation. As black…

  8. 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,…

  9. 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…

  10. Diversity at Work.

    ERIC Educational Resources Information Center

    Sabo, Sandra R.

    2000-01-01

    Diversity in the workplace goes beyond racial, ethnic, and cultural backgrounds. It extends to those with disabilities of all types and older workers. Students must be able to acknowledge and appreciate peoples' differences and educators must integrate diversity into the classroom. (JOW)

  11. Chapter 14: Genetic diversity

    Treesearch

    C. I. Millar

    1999-01-01

    Genetic diversity rarely makes headline news. Whereas species extinctions, loss of old-growth forests, and catastrophic forest fires are readily grasped public issues, genetic diversity is often perceived as arcane and academic. Yet genes are the fundamental unit of biodiversity, the raw material for evolution, and the ultimate source of all variation among plants and...

  12. 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)

  13. Diversity in the Workplace.

    ERIC Educational Resources Information Center

    1996

    This document contains three papers presented at a symposium on diversity in the workplace moderated by Sandra Johnson at the 1996 conference of the Academy of Human Resource Development (AHRD). "Diversity and Development: An Assessment of Equal Opportunities and the Role of HRD in the Police Service" (Rashmi Biswas, Penny Dick) examines…

  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. 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…

  16. The Diversity Machine.

    ERIC Educational Resources Information Center

    Lynch, Frederick R.

    1997-01-01

    Discusses social policy influences on the workplace and how the linkage of demographic change with multiculturalism has led to changes in organizational policies. It examines the emergence of diversity management practice, the influence of globalizing markets in driving corporate diversity policies, and the roles of corporate and government…

  17. 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…

  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. 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.

  20. 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)

  1. 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…

  2. 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…

  3. Indonesia's Unity through Diversity.

    ERIC Educational Resources Information Center

    Sa'ud, Udin

    1988-01-01

    Discusses cultural diversity and national unity in Indonesia, a country with a population of 165 million people from over 300 ethnic groups. Examines the philosophical basis of the Indonesian way of life and the country's national symbol of unity, "Bhineka Tunggal Ika," which means "unity in diversity." (GEA)

  4. 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…

  5. 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…

  6. 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…

  7. 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…

  8. 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…

  9. 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)

  10. Embracing cultural diversity.

    PubMed

    Casady, W M

    2001-01-01

    Healthcare providers from all backgrounds are taught the Western medicine approach with little consideration given to cultural-specific care. Yet, today it is difficult to ignore that approximately 33 percent of Americans originate from ethnically diverse groups. As our population continues to become more diversified, it is imperative that healthcare professionals become more sensitive to cultural differences. Effectively managing cultural diversity in the workplace requires a complex set of skills as well as an understanding of the concept. Communication skills will be challenged in a complex and diverse work environment. Managers must learn to listen. Embracing cultural diversity is a two-step process. The first step begins with personal self-interest and self-examination. The second step in the process is the "awakening." Tomorrow's successful managers will take an active role today in creating an environment that views diversity as an asset to the work force.

  11. 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…

  12. Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins.

    PubMed

    van Ree, R; Hoffman, D R; van Dijk, W; Brodard, V; Mahieu, K; Koeleman, C A; Grande, M; van Leeuwen, W A; Aalberse, R C

    1995-05-01

    Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. We sought to characterize the allergen biochemically and immunologically. The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

  13. 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.

  14. 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

  15. Tryptase inhibitors: a patent review.

    PubMed

    Ni, Wei-Wei; Cao, Meng-Da; Huang, Wen; Meng, Ling; Wei, Ji-Fu

    2017-08-01

    Tryptase is one of the main serine-proteinases located in the secretory granules of mast cells, and is released through degranulation, which is involved in the pathogenesis of allergic inflammatory disease, cardiovascular diseases, lung fibrosis and tumor. Therefore, inhibitors targeting tryptase may represent a new direction for the treatment of allergic inflammatory disease and other diseases. Areas covered: In this article, we discussed the history and development of tryptase inhibitors and described a variety of tryptase inhibitors via their structures and biological importance in clinical studies and drug development for tryptase-related diseases. Expert opinion: Initial tryptase inhibitors based on indole structure as the hydrophobic substituent on a benzylamine-piperidine template have low specificity and poor bioavailability. Therefore, designing new and specific inhibitors targeting tryptase should be involved in future clinical studies. Modifications toward indoles with varying N-substitution, introducing an amide bond, and growing the chain length contribute to an increase in the specific selectivity and potency of tryptase inhibitors. Tryptase has become the research hotspot to explore many related diseases. Therefore, there has been growing appreciation for the potential importance of the tryptase inhibitors as a target for treating these diseases.

  16. Diacylglycerol Metabolism and Signaling is a Driving Force Underlying FASN Inhibitor Sensitivity in Cancer Cells

    PubMed Central

    Benjamin, Daniel I.; Li, Daniel S.; Lowe, Wallace; Heuer, Timothy; Kemble, George; Nomura, Daniel K.

    2015-01-01

    Fatty acid synthase (FASN) generates the de novo source of lipids for cell proliferation and is a promising cancer therapy target. Development of FASN inhibitors, however, necessitates a better understanding of sensitive and resistant cancer types to optimize patient treatment. Indeed, testing the cytotoxic effects of FASN inhibition across human cancer cells revealed diverse sensitivities. We show here that metabolic incorporation of glucose into specific complex lipid species strongly predicts FASN inhibitor sensitivity. We also show that the levels of one of these lipid classes, protein kinase C (PKC) stimulators diacylglycerols, are lowered upon FASN inhibitor treatment in sensitive compared to resistant cells and that PKC activators and inhibitors rescue cell death in sensitive cells and sensitize resistant cells, respectively. Our findings not only reveal a biomarker for predicting FASN sensitivity in cancer cells, but also a put forth a heretofore unrecognized mechanism underlying the anti-cancer effects of FASN inhibitors. PMID:25871544

  17. 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

  18. Inhibitors of plant hormone transport.

    PubMed

    Klíma, Petr; Laňková, Martina; Zažímalová, Eva

    2016-11-01

    Here we present an overview of what is known about endogenous plant compounds that act as inhibitors of hormonal transport processes in plants, about their identity and mechanism of action. We have also summarized commonly and less commonly used compounds of non-plant origin and synthetic drugs that show at least partial 'specificity' to transport or transporters of particular phytohormones. Our main attention is focused on the inhibitors of auxin transport. The urgent need to understand precisely the molecular mechanism of action of these inhibitors is highlighted.

  19. 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

  20. 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

  1. "No. 190. Grand Valley Diversion Dam. Diversion gates, water flowing ...

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

    "No. 190. Grand Valley Diversion Dam. Diversion gates, water flowing into high line. June, 1917. R.B.D." - Grand Valley Diversion Dam, Half a mile north of intersection of I-70 & Colorado State Route 65, Cameo, Mesa County, CO

  2. NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes1[W][OA

    PubMed Central

    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. PMID:23150644

  3. 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

  4. Cholinesterase inhibitors and beyond.

    PubMed

    Pepeu, Giancarlo; Giovannini, Maria Grazia

    2009-04-01

    Cholinesterase inhibitors (ChEIs) were introduced in the therapy of Alzheimer Disease (AD) in the nineteen nineties with great expectations. The hopes and large interest raised by these drugs are well demonstrated by 12,000 references listed by PubMed under 'ChEI' for 1995-2007. The list is reduced to 2500 if we confine ourselves to 'ChEIs and dementia'. Of them, about 500 were published in the last two years. Whereas an increase in brain acetylcholine and an improvement of cognitive deficits have been consistently demonstrated in animal models of AD, from aging rats to transgenic mice, the clinical effectiveness of ChEIs has been and is still a matter of contrasting opinions. These range from the negative conclusions of the AD2000 trial on donepezil, claiming that it is not cost effective, with benefits below a minimally relevant threshold, to the NICE appraisal of 2007 declaring that donepezil, rivastigmine, galantamine are efficacious for mild to moderate AD, irrespective of their different selectivity for acetyl- (AChE) and butyrylcholinesterase (BuChE). The possibility that ChEIs may exert their effects through mechanisms beyond cholinesterase inhibition has been envisaged. However, according to the information presented in this review, the "classical" ChEIs, donepezil, rivastigmine and galantamine, show no pharmacological actions beyond cholinesterase inhibition which may play an important role in their therapeutic efficacy. The diverging opinions on clinical efficacy do not discourage from developing new ChEIs, and particularly the so called multifunctional ChEIs. They represent the future of the cholinergic therapy for AD but other indications for these drugs may be considered, including vascular dementia, mild cognitive impairment, and the ethically sensitive improvement of memory and learning in healthy subjects.

  5. [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.

  6. Thrombin Inhibitors from Different Animals

    PubMed Central

    Tanaka-Azevedo, A. M.; Morais-Zani, K.; Torquato, R. J. S.; Tanaka, A. S.

    2010-01-01

    Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor Xa. Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals. PMID:20976270

  7. 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

  8. 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)

  9. 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)

  10. 2012 Diversity Day

    NASA Image and Video Library

    2012-10-31

    John C. Stennis Space Center employees enjoyed 2012 Diversity Day activities Oct. 31. During the day, Stennis employees were able to visit exhibits highlighting different cultures and participate in a range of activities.

  11. 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)

  12. 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

  13. 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.

  14. River Diversions and Shoaling

    DTIC Science & Technology

    2008-11-01

    ERDC/CHL CHETN-VII-9 November 2008 River Diversions and Shoaling by Joseph V. Letter, Jr., C. Fred Pinkard , Jr., and Nolan K. Raphelt...V. Letter, Jr. (772-342- 1295), email: Joseph.V.Letter@usace.army.mil, or C. Fred Pinkard , Jr. (601-634-3086), email: Fred.Pinkard@usace.army.mil...note should be cited as follows: Letter, J. V., Jr., C. F. Pinkard , Jr., and N. K. Raphelt. 2008. River diversions and shoaling. Coastal and

  15. 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)

  16. 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.

  17. 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.

  18. 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

  19. Heat shock protein 90 inhibitors repurposed against Entamoeba histolytica

    PubMed Central

    Shahinas, Dea; Debnath, Anjan; Benedict, Christan; McKerrow, James H.; Pillai, Dylan R.

    2015-01-01

    Hsp90 is an essential chaperone responsible for trafficking a vast array of client proteins, which are substrates that Hsp90 regulates in eukaryotic cells under stress conditions. The ATP-binding N-terminal domain of Hsp90 (also known as a GHKL type ATPase domain) can serve as a specific drug target, because sufficient structural diversity in the ATP-binding pocket of Hsp90 allows for ortholog selectivity of Hsp90 inhibitors. The primary objective of this study is to identify inhibitors specific for the ATP-binding domain of Entamoeba histolytica Hsp90 (EhHsp90). An additional aim, using a combination of site-directed mutagenesis and a protein in vitro assay, is to show that the antiparasitic activity of Hsp90 inhibitors is dependent on specific residues within the ATP-binding domain. Here, we tested the activity of 43 inhibitors of Hsp90 that we previously identified using a high-throughput screen. Of the 43 compounds tested, 19 competed for binding of the EhHsp90 ATP-binding domain. Five out of the 19 EhHsp90 protein hits demonstrated activity against E. histolytica in vitro culture: rifabutin, rutilantin, cetylpyridinium chloride, pararosaniline pamoate and gentian violet. These five top E. histolytica Hsp90 inhibitors showed 30–100% inhibition of E. histolytica in culture in the micromolar range. These data suggest that E. histolytica-specific Hsp90 inhibitors are possible to identify and provide important lead compounds for the development of novel antiamebic drugs. PMID:26029171

  20. Natural and synthetic inhibitors of kallikrein-related peptidases (KLKs)

    PubMed Central

    Goettig, Peter; Magdolen, Viktor; Brandstetter, Hans

    2010-01-01

    Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn2+ ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α2-macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches. PMID:20615447

  1. 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.

  2. 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

  3. 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.

  4. Navigating the Differences [and] Diversity Grows More Diverse.

    ERIC Educational Resources Information Center

    Galagan, Patricia A.; Allerton, Haidee

    1993-01-01

    This report of an American Society for Training and Development symposium discusses how important it is for companies to deal with diversity issues. It offers guidelines for making sure business communications reflect the diversity of the work force and customer base, and it compares affirmative action, valuing diversity, and managing diversity.…

  5. 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…

  6. 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…

  7. 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…

  8. Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis.

    PubMed

    Kawashima, Shigehiro A; Chen, Zhen; Aoi, Yuki; Patgiri, Anupam; Kobayashi, Yuki; Nurse, Paul; Kapoor, Tarun M

    2016-10-06

    All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins' physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin's role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Discovery of novel human acrosin inhibitors by virtual screening

    NASA Astrophysics Data System (ADS)

    Liu, Xuefei; Dong, Guoqiang; Zhang, Jue; Qi, Jingjing; Zheng, Canhui; Zhou, Youjun; Zhu, Ju; Sheng, Chunquan; Lü, Jiaguo

    2011-10-01

    Human acrosin is an attractive target for the discovery of male contraceptive drugs. For the first time, structure-based drug design was applied to discover structurally diverse human acrosin inhibitors. A parallel virtual screening strategy in combination with pharmacophore-based and docking-based techniques was used to screen the SPECS database. From 16 compounds selected by virtual screening, a total of 10 compounds were found to be human acrosin inhibitors. Compound 2 was found to be the most potent hit (IC50 = 14 μM) and its binding mode was investigated by molecular dynamics simulations. The hit interacted with human acrosin mainly through hydrophobic and hydrogen-bonding interactions, which provided a good starting structure for further optimization studies.

  3. 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

  4. 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

  5. [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.

  6. 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.

  7. Continent cutaneous diversion.

    PubMed

    Skinner, Eila C

    2015-11-01

    This article updates the recently reported intermediate to long-term results with the most commonly used forms of continent cutaneous urinary diversion, and to discuss approaches to early and late complications. Many variations on construction of a continent cutaneous diversion have been described. Results with large series of patients demonstrate acceptable results with all of them, but with a significant revision rate. Long-term complication rates and adaptation to robotic approaches have recently been described. Continent cutaneous diversion is rarely offered in the USA to patients undergoing cystectomy except in a few centers. Most studies have found a high complication rate and need for revision surgery in 10-20% of patients. However, functional results are acceptable and many patients are willing to accept the complications in exchange for avoiding an external appliance.

  8. Interaction of ABC multidrug transporters with anticancer protein kinase inhibitors: substrates and/or inhibitors?

    PubMed

    Hegedus, Csilla; Ozvegy-Laczka, Csilla; Szakács, Gergely; Sarkadi, Balázs

    2009-05-01

    Protein kinase inhibitors (PKI) are becoming key agents in modern cancer chemotherapy, and combination of PKIs with classical chemotherapeutic drugs may help to overcome currently untreatable metastatic cancers. Since chemotherapy resistance is a recurrent problem, mechanisms of resistance should be clarified in order to help further drug development. Here we suggest that in addition to PKI resistance based on altered target structures, the active removal of these therapeutic agents by the MDR-ABC transporters should also be considered as a major cause of clinical resistance. We discuss the occurring systemic and cellular mechanisms, which may hamper PKI efficiency, and document the role of selected MDR-ABC transporters in these phenomena through their interactions with these anticancer agents. Moreover, we suggest that PKI interactions with ABC transporters may modulate overall drug metabolism, including the fate of diverse, chemically or target-wise unrelated drugs. These effects are based on multiple forms of MDR-ABC transporter interaction with PKIs, as these compounds may be both substrates and/or inhibitors of an ABC transporter. We propose that these interactions should be carefully considered in clinical application, and a combined MDR-ABC transporter and PKI effect may bring a major advantage in future drug development.

  9. 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.

  10. 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. (c) 2015 APA, all rights reserved).

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Coking products as corrosion inhibitors

    SciTech Connect

    Antonov, A.V.; Petrenko, V.G.; Frolova, R.P.; Kurinnaya, S.N.

    1982-11-06

    Activated sludge and froth from the biological treatment of coke plant waste waters has been determined to be a corrosion inhibitor in both neutral and acidic media, due to the presence of unreacted coking derived inhibitors, bacteriological formation of inhibitors, bacterial organisms, humic-type organics and traces of germanium, zinc, mercury and manganese. The corrosive liquids tested were, river water, technical system water, gas cooler aqueous condensate, gas collector condensate and coking waste water before and after treatment, the substrate being St 3 steel plates (45 X 45 X 5 M) (time 24-30 hr (acid media) and 934 hr (neutral media)). The activated sludge (25 g/l) reduced acid media corrosion rate by 10/sup 3/, the protective effect being 99% for the test liquids: Sludge is more effective than the froth.

  19. 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%.

  20. 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

  1. Algorithm Diversity for Resilent Systems

    DTIC Science & Technology

    2016-06-27

    data structures. 15. SUBJECT TERMS computer security, software diversity, program transformation 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18...shared vulnerabilities. A promising way to use diversity to increase the resilience of a software application is to run multiple diverse versions of the...Review. We surveyed over 20 papers related to automated software diversity and summarized each paper’s diversity generation technique and evaluation

  2. Increasing diversity in radiologic technology.

    PubMed

    Carwile, Laura

    2003-01-01

    Diversity is increasingly important in the radiologic technology workplace. For significant changes to occur in work force diversity, educators must first recruit and retain students from a wide variety of backgrounds. This article examines personality, race and gender as factors affecting career choice and how educators can use these factors to increase diversity in their programs. An overview of the ASRT's efforts to improve diversity within the profession is presented, along with suggestions for developing effective recruitment and retention plans to increase diversity.

  3. 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

  4. [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.

  5. 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.

  6. 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

  7. Lipases and their inhibitors in health and disease.

    PubMed

    Nomura, Daniel K; Casida, John E

    2016-11-25

    Lipids play diverse and important biological roles including maintaining cellular integrity, storing fat for energy, acting as signaling molecules, and forming microdomains to support membrane protein signaling. Altering the levels of specific lipid species through activating or inactivating their biosynthetic or degradative pathways has been shown to provide either therapeutic benefit or cause disease. This review focuses on the functional, therapeutic, and (patho)physiological roles of lipases within the serine hydrolase superfamily and their inhibitors, with particular emphasis on the pharmacological tools, drugs, and environmental chemicals that inhibit these lipases. Copyright © 2016. Published by Elsevier Ireland Ltd.

  8. 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…

  9. "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…

  10. 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…

  11. 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…

  12. 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…

  13. 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.

  14. 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,…

  15. "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…

  16. Diversity on the Docket

    ERIC Educational Resources Information Center

    Trotter, Andrew

    2006-01-01

    School leaders attest to educational and social benefits from diversity. They argue that local housing patterns historically tend to separate families of different races and may lead to schools that are racially homogeneous if the districts do not counter them with assignment policies that consider race. This article discusses race in education…

  17. 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…

  18. 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)…

  19. 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…

  20. 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…