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Sample records for bowman-birk protease inhibitor

  1. Bowman-Birk protease inhibitor from the seeds of Vigna unguiculata forms a highly stable dimeric structure.

    PubMed

    Rao, K N; Suresh, C G

    2007-10-01

    Different protease inhibitors including Bowman-Birk type (BBI) have been reported from the seeds of Vigna unguiculata. Protease isoinhibitors of double-headed Bowman-Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI from Vigna unguiculata (Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to form homodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 A resolution. Although, the inhibitor has a monomer fold similar to that found in other known structures of Bowman-Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structural elements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higher quaternary association to maintain the physiological state and to execute its biological function.

  2. A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.

    PubMed

    Vogtentanz, Gudrun; Collier, Katherine D; Bodo, Michael; Chang, Judy H; Day, Anthony G; Estell, David A; Falcon, Brandy C; Ganshaw, Grant; Jarnagin, Alisha S; Kellis, James T; Kolkman, Marc A B; Lai, Cindy S; Meneses, Renato; Miller, Jeffrey V; de Nobel, Hans; Power, Scott; Weyler, Walter; Wong, David L; Schmidt, Brian F

    2007-09-01

    A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.

  3. 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...

  4. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean

    USDA-ARS?s Scientific Manuscript database

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327bp encoding 109 amino acids w...

  5. Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

    PubMed Central

    Souza, Larissa da Costa; Camargo, Ricardo; Demasi, Marilene; Santana, Jaime Martins; de Freitas, Sonia Maria

    2014-01-01

    Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis. PMID:24475156

  6. Bowman-Birk inhibitors from legumes as colorectal chemopreventive agents

    PubMed Central

    Clemente, Alfonso; Arques, Maria del Carmen

    2014-01-01

    Aberrant functioning of serine proteases in inflammatory and carcinogenic processes within the gastrointestinal tract (GIT) has prompted scientists to investigate the potential of serine protease inhibitors, both natural and synthetic, as modulators of their proteolytic activities. Protease inhibitors of the Bowman-Birk type, a major protease inhibitor family in legume seeds, which inhibit potently and specifically trypsin- and chymotrypsin-like proteases, are currently being investigated as colorectal chemopreventive agents. Physiologically relevant amounts of Bowman-Birk inhibitors (BBI) can reach the large intestine in active form due to their extraordinary resistance to extreme conditions within the GIT. Studies in animal models have proven that dietary BBI from several legume sources, including soybean, pea, lentil and chickpea, can prevent or suppress carcinogenic and inflammatory processes within the GIT. Although the therapeutic targets and the action mechanism of BBI have not yet been elucidated, the emerging evidence suggests that BBI exert their preventive properties via protease inhibition; in this sense, serine proteases should be considered as primary targets in early stages of carcinogenesis. The validation of candidate serine proteases as therapeutic targets together with the identification, within the wide array of natural BBI variants, of the most potent and specific protease inhibitors, are necessary to better understand the potential of this protein family as colorectal chemopreventive agents. PMID:25132747

  7. A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

    PubMed

    Indarte, Martín; Lazza, Cristian M; Assis, Diego; Caffini, Néstor O; Juliano, María A; Avilés, Francesc X; Daura, Xavier; López, Laura M I; Trejo, Sebastián A

    2017-02-01

    A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M(-1) cm(-1). MpBBI inhibits strongly trypsin with K i in the 10(-10) M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

  8. Lunasin and Bowman-Birk protease inhibitor concentrations of protein extracts from enzyme-assisted aqueous extraction of soybeans.

    PubMed

    Leite Nobrega de Moura, Juliana Maria; Hernandez-Ledesma, Blanca; de Almeida, Neiva Maria; Hsieh, Chia-Chien; de Lumen, Ben O; Johnson, Lawrence A

    2011-07-13

    Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.

  9. 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.

  10. 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

  11. Effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid Macrosiphum euphorbiae (Homoptera, Aphididae) and its parasitoid Aphelinus abdominalis (Hymenoptera, Aphelinidae).

    PubMed

    Azzouz, H; Cherqui, A; Campan, E D M; Rahbé, Y; Duport, G; Jouanin, L; Kaiser, L; Giordanengo, P

    2005-01-01

    Transgenic plants expressing protease inhibitors (PIs) have emerged in recent years as an alternative strategy for pest control. Beneficial insects such as parasitoids may therefore be exposed to these entomotoxins either via the host or by direct exposure to the plant itself. With the objective of assessing the effects of PIs towards aphid parasitoids, bioassays using soybean Bowman-Birk inhibitor (SbBBI) or oryzacystatin I (OCI) on artificial diet were performed on Macrosiphum euphorbiae-Aphelinus abdominalis system. OCI significantly reduced nymphal survival of the potato aphid M. euphorbiae and prevented aphids from reproducing. This negative effect was much more pronounced than with other aphid species. On the contrary, SbBBI did not affect nymphal viability but significantly altered adult demographic parameters. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of Aphelinus abdominalis predominantly relies on serine proteases and especially on chymotrypsin-like activity. Immunoassays suggested that OCI bound to aphid proteins and accumulated in aphid tissues, whereas SbBBI remained unbound in the gut. Bioassays using M. euphorbiae reared on artificial diets supplemented with both OCI and SbBBI showed a fitness impairment of Aphelinus abdominalis that developed on intoxicated aphids. However, only SbBBI was detected in parasitoid larvae, while no PI could be detected in adult parasitoids that emerged from PI-intoxicated aphids. The potential impact of PI-expressing plants on aphid parasitoids and their combined efficiency for aphid control are discussed.

  12. Bowman-Birk inhibitor attenuates dystrophic pathology in mdx mice.

    PubMed

    Morris, C A; Selsby, J T; Morris, L D; Pendrak, K; Sweeney, H L

    2010-11-01

    Bowman-Birk inhibitor concentrate (BBIC), a serine protease inhibitor, has been shown to diminish disuse atrophy of skeletal muscle. Duchenne muscular dystrophy (DMD) results from a loss of dystrophin protein and involves an ongoing inflammatory response, with matrix remodeling and activation of transforming growth factor (TGF)-β(1) leading to tissue fibrosis. Inflammatory-mediated increases in extracellular protease activity may drive much of this pathological tissue remodeling. Hence, we evaluated the ability of BBIC, an extracellular serine protease inhibitor, to impact pathology in the mouse model of DMD (mdx mouse). Mdx mice fed 1% BBIC in their diet had increased skeletal muscle mass and tetanic force and improved muscle integrity (less Evans blue dye uptake). Importantly, mdx mice treated with BBIC were less susceptible to contraction-induced injury. Changes consistent with decreased degeneration/regeneration, as well as reduced TGF-β(1) and fibrosis, were observed in the BBIC-treated mdx mice. While Akt signaling was unchanged, myostatin activitation and Smad signaling were reduced. Given that BBIC treatment increases mass and strength, while decreasing fibrosis in skeletal muscles of the mdx mouse, it should be evaluated as a possible therapeutic to slow the progression of disease in human DMD patients.

  13. 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...

  14. Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and Bowman-Birk protease inhibitor in seeds.

    PubMed

    Kim, Won-Seok; Chronis, Demosthenis; Juergens, Matthew; Schroeder, Amy C; Hyun, Seung Won; Jez, Joseph M; Krishnan, Hari B

    2012-01-01

    Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur-containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins have met with limited success. Here, we report a successful strategy to increase the cysteine content of soybean seed through the overexpression of a key sulfur assimilatory enzyme. We have generated several transgenic soybean plants that overexpress a cytosolic isoform of O-acetylserine sulfhydrylase (OASS). These transgenic soybean plants exhibit a four- to tenfold increase in OASS activity when compared with non-transformed wild-type. The OASS activity in the transgenic soybeans was significantly higher at all the stages of seed development. Unlike the non-transformed soybean plants, there was no marked decrease in the OASS activity even at later stages of seed development. Overexpression of cytosolic OASS resulted in a 58-74% increase in protein-bound cysteine levels compared with non-transformed wild-type soybean seeds. A 22-32% increase in the free cysteine levels was also observed in transgenic soybeans overexpressing OASS. Furthermore, these transgenic soybean plants showed a marked increase in the accumulation of Bowman-Birk protease inhibitor, a cysteine-rich protein. The overall increase in soybean total cysteine content (both free and protein-bound) satisfies the recommended levels required for the optimal growth of monogastric animals.

  15. Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.

    PubMed

    Katoch, Rajan; Singh, Sunil Kumar; Thakur, Neelam; Dutt, Som; Yadav, Sudesh Kumar; Shukle, Rich

    2014-08-10

    This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Potential effects of plant protease inhibitors, oryzacystatin I and soybean Bowman-Birk inhibitor, on the aphid parasitoid Aphidius ervi Haliday (Hymenoptera, Braconidae).

    PubMed

    Azzouz, H; Campan, E D M; Cherqui, A; Saguez, J; Couty, A; Jouanin, L; Giordanengo, P; Kaiser, L

    2005-08-01

    Protease inhibitors (PIs) have been shown to cause lethal and sublethal effects on aphids depending on the kind of PI and aphid species. Therefore, these proteins might affect aphid parasitoids directly by inhibiting their digestive proteolysis or indirectly via their development in a less suitable host. In our study, the risk of exposure and the potential effects of soybean Bowman-Birk inhibitor (SbBBI) and oryzacystatin I (OCI) on the aphid endoparasitoid Aphidius ervi were investigated using artificial diet to deliver PIs. Immunoassays showed that both SbBBI and OCI were detected in the honeydew of aphids reared on artificial diet containing these recombinant proteins at 100 microg/mL. However, only SbBBI was detected in parasitoid larvae, while this PI could not be detected in adult parasitoids emerged from PI-intoxicated aphids. Enzymatic inhibition assays showed that digestive proteolytic activity of larvae and adults of A. ervi predominantly relies on serine proteases and especially on chymotrypsin-like activity. Bioassays using SbBBI and OCI on artificial diet were performed. A. ervi that developed on intoxicated aphids had impaired fitness. Thus development and parasitism success of parasitoids exposed to OCI were severely affected. On the contrary, SbBBI only altered significantly female size and sex ratio. Direct exposure to PIs through adult food intake did not affect female's longevity, while SbBBI and OCI (100 microg/mL) induced 69% and 30% inhibition of digestive protease activity, respectively. These studies made it possible to estimate the risk of exposure to plant PIs and the sensitivity of the aphid parasitoid A. ervi to these entomotoxins, by combining immunological, biochemical and biological approaches. First it pointed out that only immature stages are affected by PIs. Secondly, it documented two different modes of effect, according to the nature of the PIs and both host and parasitoid susceptibility. OCI prevented the development of A. ervi

  17. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

    PubMed

    da Cunha Morales Álvares, Alice; Schwartz, Elisabeth Ferroni; Amaral, Nathalia Oda; Trindade, Neidiane Rosa; Pedrino, Gustavo Rodrigues; Silva, Luciano Paulino; de Freitas, Sonia Maria

    2014-10-30

    The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

  18. Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases.

    PubMed

    Brand, G D; Pires, D A T; Furtado, J R; Cooper, A; Freitas, S M; Bloch, C

    2017-03-15

    The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. The biochemical and functional food properties of the bowman-birk inhibitor.

    PubMed

    Losso, Jack N

    2008-01-01

    The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. In 1999, FDA allowed a health claim on food labels stating that a daily diet containing 25 grams of soy protein, also low in saturated fat and cholesterol, may reduce the risk of heart disease [corrected] This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor.

  20. Bowman-Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster.

    PubMed

    Li, H-M; Sun, L; Mittapalli, O; Muir, W M; Xie, J; Wu, J; Schemerhorn, B J; Jannasch, A; Chen, J Y; Zhang, F; Adamec, J; Murdock, L L; Pittendrigh, B R

    2010-06-01

    Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy-associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI-fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.

  1. Bowman-Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster

    USDA-ARS?s Scientific Manuscript database

    Bowman-Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acid...

  2. Chemoprevention of rat prostate carcinogenesis by soy isoflavones and by Bowman-Birk inhibitor.

    PubMed

    McCormick, David L; Johnson, William D; Bosland, Maarten C; Lubet, Ronald A; Steele, Vernon E

    2007-01-01

    Epidemiology studies suggest that soy consumption confers protection against human prostate cancer. To identify the soy component(s) that may be responsible for this chemopreventive activity, studies were conducted to determine the influence of a soy isoflavone mixture (PTI G-2535; 45% genistein, 22% daidzein, 2% glycitein) and a soy-derived protease inhibitor (Bowman-Birk Inhibitor Concentrate; BBIC) on prostate carcinogenesis in rats. Prostate cancers were induced in male Wistar-Unilever rats by a sequential regimen of cyproterone acetate and testosterone propionate, followed by a single intravenous injection of N-methyl-N-nitrosourea (MNU) and chronic androgen stimulation. In separate studies, PTI G-2535 and BBIC were administered continuously at 0 (control), 200, or 2000 mg/kg diet, beginning 1 wk post-MNU. PTI G-2535 and BBIC both conferred modest, but statistically significant and dose-related protection against carcinogenesis in the dorsolateral+anterior prostate. These data demonstrate that both the isoflavone and protein (protease inhibitor) components of soy can inhibit prostate carcinogenesis in the rat. However, the modest individual activities of soy isoflavones and BBIC suggest that while both components may contribute to the chemopreventive activity of soy, combination administration (or exposure to whole soy) may be more effective in prostate cancer prevention than is administration of either component alone.

  3. Soybean-derived Bowman-Birk Inhibitor (BBI) Inhibits HIV Replication in Macrophages

    PubMed Central

    Ma, Tong-Cui; Zhou, Run-Hong; Wang, Xu; Li, Jie-Liang; Sang, Ming; Zhou, Li; Zhuang, Ke; Hou, Wei; Guo, De-Yin; Ho, Wen-Zhe

    2016-01-01

    The Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, is known to have anti-inflammatory effect in both in vitro and in vivo systems. Macrophages play a key role in inflammation and immune activation, which is implicated in HIV disease progression. Here, we investigated the effect of BBI on HIV infection of peripheral blood monocyte-derived macrophages. We demonstrated that BBI could potently inhibit HIV replication in macrophages without cytotoxicity. Investigation of the mechanism(s) of BBI action on HIV showed that BBI induced the expression of IFN-β and multiple IFN stimulated genes (ISGs), including Myxovirus resistance protein 2 (Mx2), 2′,5′-oligoadenylate synthetase (OAS-1), Virus inhibitory protein (viperin), ISG15 and ISG56. BBI treatment of macrophages also increased the expression of several known HIV restriction factors, including APOBEC3F, APOBEC3G and tetherin. Furthermore, BBI enhanced the phosphorylation of IRF3, a key regulator of IFN-β. The inhibition of IFN-β pathway by the neutralization antibody to type I IFN receptor (Anti-IFNAR) abolished BBI-mediated induction of the anti-HIV factors and inhibition of HIV in macrophages. These findings that BBI could activate IFN-β-mediated signaling pathway, initialize the intracellular innate immunity in macrophages and potently inhibit HIV at multiple steps of viral replication cycle indicate the necessity to further investigate BBI as an alternative and cost-effective anti-HIV natural product. PMID:27734899

  4. A Bowman-Birk inhibitor with anti-elastase activity from Lathyrus sativus L. seeds.

    PubMed

    Rocco, Micaela; Malorni, Livia; Chambery, Angela; Poerio, Elia; Parente, Augusto; Di Maro, Antimo

    2011-08-01

    Four Bowman-Birk inhibitors, named LSI-1/4, were isolated and purified from Lathyrus sativus L. seeds. The purification procedure consisted of two cation-exchange chromatography steps, followed by gel-filtration and RP-HPLC. Mass spectrometry analysis of LSI-1/4 inhibitors yielded relative molecular masses of 7914.41 for LSI-1, 6867.67 for LSI-2, 7341.24 for LSI-3 and 7460.01 for LSI-4. N-terminal sequences (up to 30 residues) of LSI-1/4 inhibitors were identical with the exception of sequence positions 21, 27 and 28 and highly similar to those of other Bowman-Birk inhibitors isolated from Leguminosae plants. Inhibitors LSI-1/4 were active towards trypsin and α-chymotrypsin, with IC(50) values for 12.6 nM of trypsin ranging from 4.9 to 24.3 nM. A lower activity was observed against bovine α-chymotrypsin (IC(50) values ranging from 0.5 to 3.4 μM for 15.0 nM of α-chymotrypsin). Peptide mapping of the LSI-1 sequence showed the presence of an Ala residue in the second reactive site, thus explaining the low anti-chymotrypsin activity of this inhibitor. In addition, LSI-1 was endowed with anti-elastase activity, being able to inhibit human leukocyte elastase.

  5. 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...

  6. Crystal structure of the anticarcinogenic Bowman-Birk inhibitor from snail medic (Medicago scutellata) seeds complexed with bovine trypsin.

    PubMed

    Capaldi, Stefano; Perduca, Massimiliano; Faggion, Beniamino; Carrizo, Maria E; Tava, Aldo; Ragona, Laura; Monaco, Hugo L

    2007-04-01

    The structure of the ternary complex of the anticarcinogenic Bowman-Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 A. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2' residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.

  7. Probing the soybean Bowman-Birk inhibitor using recombinant antibody fragments.

    PubMed

    Muzard, Julien; Fields, Conor; O'Mahony, James John; Lee, Gil U

    2012-06-20

    The nutritional and health benefits of soy protein have been extensively studied over recent decades. The Bowman-Birk inhibitor (BBI), derived from soybeans, is a double-headed inhibitor of chymotrypsin and trypsin with anticarcinogenic and anti-inflammatory properties, which have been demonstrated in vitro and in vivo. However, the lack of analytical and purification methodologies complicates its potential for further functional and clinical investigations. This paper reports the construction of anti-BBI antibody fragments based on the principle of protein design. Recombinant antibody (scFv and diabody) molecules targeting soybean BBI were produced and characterized in vitro (K(D)~1.10(-9) M), and the antibody-binding site (epitope) was identified as part of the trypsin-specific reactive loop. Finally, an extremely fast purification strategy for BBI from soybean extracts, based on superparamagnetic particles coated with antibody fragments, was developed. To the best of the authors' knowledge, this is the first report on the design and characterization of recombinant anti-BBI antibodies and their potential application in soybean processing.

  8. Heat-induced inactivation mechanism of soybean Bowman-Birk inhibitors.

    PubMed

    He, Hui; Li, Xingfei; Kong, Xiangzhen; Hua, Yufei; Chen, Yeming

    2017-10-01

    Due to the complications of the soymilk system, the heat-induced Bowman-Birk inhibitor (BBI) inactivation mechanism is not well known. In this study, two BBI samples with low and high purities were prepared from soymilk. It was confirmed that three groups (A, C, and D) of BBI, which are contained in soybean seeds, were transferred into soymilk during processing. On heating, it was found that 1) the two subdomains of BBI were not equally heat stable, 2) the conformation of BBI gradually changed, 3) some amino acid residues (namely, cystine, serine and lysine) in BBI were degraded, 4) BBI did not tend to form intermolecular cross-links with another BBI, but did slightly with non-BBI proteins. Based on some previous studies, the conformational change of BBI was attributed to β-elimination reactions on the amino acid residues of BBI and the subsequent intramolecular reactions induced by the products yielded by the β-elimination reactions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Fabrication of a Soybean Bowman-Birk Inhibitor (BBI) Nanodelivery Carrier To Improve Bioavailability of Curcumin.

    PubMed

    Liu, Chun; Cheng, Fenfen; Yang, Xiaoquan

    2017-03-22

    Curcumin is a poorly water-soluble drug, and its oral bioavailability is very low. Here, a novel self-assembly nanoparticle delivery carrier has been successfully developed by using soybean Bowman-Birk inhibitor (BBI) to improve the solubility, bioaccessibility, and oral absorption of curcumin. BBI is a unique protein, which can be resistant to the pH range and proteolytic enzymes in the gastrointestinal tract (GIT), bioavailable, and not allergenic. The encapsulation efficiencies (EE) and the loading capacities (LC) of curcumin in the curcumin-loaded BBI nanoparticles (Cur-BBI-NPs, size = 90.09 nm, PDI = 0.103) were 86.17 and 10.31%, respectively. The in vitro bioaccessibility of Cur-BBI-NPs was superior to that of curcumin-loaded sodium caseinate (SC) nanoparticles (Cur-SC-NPs) (as control). Moreover, Cur-BBI-NPs significantly enhanced the bioavailability of curcumin in rats compared with Cur-SC-NPs, and the clathrin-mediated endocytosis pathway probably contributed to the favorable bioavailability of Cur-BBI-NPs, as revealed by the cellular uptake inhibition study.

  10. Bowman-Birk inhibitor concentrate prevents atrophy, weakness, and oxidative stress in soleus muscle of hindlimb-unloaded mice.

    PubMed

    Arbogast, Sandrine; Smith, Jacqueline; Matuszczak, Yves; Hardin, Brian J; Moylan, Jennifer S; Smith, Jeffrey D; Ware, Jeffrey; Kennedy, Ann R; Reid, Michael B

    2007-03-01

    Antigravity muscles atrophy and weaken during prolonged mechanical unloading caused by bed rest or spaceflight. Unloading also induces oxidative stress in muscle, a putative cause of weakness. We tested the hypothesis that dietary supplementation with Bowman-Birk inhibitor concentrate (BBIC), a soy protein extract, would oppose these changes. Adult mice were fed a diet supplemented with 1% BBIC during hindlimb unloading for up to 12 days. Soleus muscles of mice fed the BBIC-supplemented diet weighed less, developed less force per cross-sectional area, and developed less total force after unloading than controls. BBIC supplementation was protective, blunting decrements in soleus muscle weight and force. Cytosolic oxidant activity was assessed using 2',7'-dichlorofluorescin diacetate. Oxidant activity increased in unloaded muscle, peaking at 3 days and remaining elevated through 12 days of unloading. Increases in oxidant activity correlated directly with loss of muscle mass and were abolished by BBIC supplementation. In vitro assays established that BBIC directly buffers reactive oxygen species and also inhibits serine protease activity. We conclude that dietary supplementation with BBIC protects skeletal muscle during prolonged unloading, promoting redox homeostasis in muscle fibers and blunting atrophy-induced weakness.

  11. Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential.

    PubMed

    Dantzger, Miriam; Vasconcelos, Ilka Maria; Scorsato, Valéria; Aparicio, Ricardo; Marangoni, Sergio; Macedo, Maria Lígia Rodrigues

    2015-10-01

    Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 × 10(-10) and 1.5 × 10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973 kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development.

  12. A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor.

    PubMed

    Kumar, Vinod; Murugeson, Saravanan; Vithani, Neha; Prakash, Balaji; Gowda, Lalitha R

    2015-01-15

    Legume Bowman-Birk inhibitors (BBIs) that inhibit mammalian proteases exist as dimers in solution. The structural basis governing dimerization of HGI-III (horsegram seed BBI) was investigated. An intra-monomer salt bridge (D76-K71) stabilizes an atypical hook-like conformation at the C-terminus. We postulate that this hook, positions D75 to enable an inter-monomer salt-bridge D75(a)-K24(b), which results in dimerization. We verify this by K71A and D76A mutations of HGI-III. The mutants were both monomers, likely due to destabilization of the C-terminal hook. Dimerization was sustained in a double mutant K71D/D76K that was anticipated to form a similar hook critical for dimerization. Conversely, K24(b) that interacts with D75(a) of the loop is the specificity determining residue that interacts with trypsin to inhibit its activity. The inter-monomer salt bridge D75(a)-K24(b) must be disrupted for the inhibition of trypsin, requiring HGI-III to transition into a monomer. Size exclusion studies and a model of HGI-III-trypsin complex support this notion. Interestingly, isoforms of the inhibitor present in germinated seeds (HGGIs) are monomers; and most strikingly, the C-termini of these inhibitors are truncated with the loss the C-terminal hook critical for dimerization. The tendency of HGI-III to self-associate seems to relate to its physiological function of a storage protein.

  13. Anticarcinogenic Bowman Birk inhibitor isolated from snail medic seeds (Medicago scutellata): solution structure and analysis of self-association behavior.

    PubMed

    Catalano, M; Ragona, L; Molinari, H; Tava, A; Zetta, L

    2003-03-18

    The high-resolution three-dimensional structure of a Bowman Birk inhibitor, purified from snail medic seeds (Medicago scutellata) (MSTI), has been determined in solution by 1H NMR spectroscopy at pH 5.6 and 27 degrees C. The structure of MSTI comprises two distinct symmetric domains each composed of a three-stranded beta-sheet containing a VIb type loop, where the active sites are located. A characteristic geometry of three aromatic residues confers stability to this protein, and we observe that this feature is conserved in all the Bowman Birk inhibitors of known structure. The two active domains exhibit different conformational features: the second domain displays higher flexibility and hydrophobicity with respect to the first one, and these properties have been correlated to a lower trypsin inhibitory specificity, in agreement with titration studies that have shown a stoichiometric ratio MSTI:trypsin of 1:1.5. NMR analysis indicated that MSTI undergoes self-association at concentrations higher than 2 mM, and the residues involved in this mechanism are localized at opposite faces of the molecule, having the highest positive and negative potential, respectively, thus indicating that electrostatic intermolecular interactions are the driving forces for MSTI association. Most of the residues affected by self-association are highly conserved in BBIs from different seeds, suggesting a functional relevance for these charged superficial patches, possibly involved in the interaction with other enzymes or macromolecules, thus triggering anti-carcinogenic activity.

  14. Effect of a Bowman-Birk proteinase inhibitor from Phaseolus coccineus on Hypothenemus hampei gut proteinases in vitro.

    PubMed

    de Azevedo Pereira, Railene; Valencia-Jiménez, Arnubio; Magalhães, Cláudio Picanço; Prates, Maura Vianna; Melo, Jorge Alex Taquita; de Lima, Liziane Maria; de Sales, Maurício Pereira; Tempel Nakasu, Erich Yukio; da Silva, Maria Cristina Mattar; Grossi-de-Sá, Maria Fátima

    2007-12-26

    The coffee berry borer, Hypothenemus hampei (Ferrari), is an important devastating coffee pest worldwide. Both trypsin and chymotrypsin enzyme activities from H. hampei larval midgut can be inactivated by proteinaceous enzyme-inhibitors. A serine proteinase inhibitor belonging to the Bowman-Birk class was purified from a wild accession of Phaseolus coccineus L. seeds. The inhibitor (PcBBI1) is a cysteine-rich protein that is heat-stable at alkaline pH. MALDI-TOF/MS analysis showed that PcBBI1 occurs in seeds as a monomer (8689 Da) or dimer (17,378 Da). Using in vitro inhibition assays, it was found that PcBBI1 has a high inhibitory activity against H. hampei trypsin-like enzymes, bovine pancreatic chymotrypsin, and trypsin. According to this, PcBBI1 could be a promising tool to make genetically modified coffee with resistance to coffee berry borer.

  15. Brown Kidney Bean Bowman-Birk Trypsin Inhibitor is Heat and pH Stable and Exhibits Anti-proliferative Activity.

    PubMed

    Chan, Yau Sang; Zhang, Yanbo; Ng, Tzi Bun

    2013-02-01

    A trypsin inhibitor with a molecular mass around 17 kDa was purified from the seeds of Phaseolus vulgaris cv. 'brown kidney bean'. The purification protocol involved, in sequence, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Q-Sepharose and Mono Q, and gel filtration on Superdex 75. The molecular size and N-terminal amino acid sequence of the trypsin inhibitor resembled leguminous Bowman-Birk protease inhibitors (BBIs), signifying that brown kidney bean trypsin inhibitor is a BBI. Brown kidney bean trypsin inhibitor exhibited pronounced thermostability and pH stability. Its trypsin inhibitory activity was retained at all pH values (0-14) and up to 90 °C. The trypsin inhibitor also inhibited the proliferation of human breast cancer MCF7 cells with an IC(50) of 71.52 μM. On the other hand, it only slightly inhibited proliferation of hepatoma HepG2 and embryonic liver WRL68 cells at a concentration over 110 μM.

  16. 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

  17. De novo sequencing and characterization of a novel Bowman-Birk inhibitor from Lathyrus sativus L. seeds by electrospray mass spectrometry.

    PubMed

    Tamburino, Rachele; Severino, Valeria; Sandomenico, Annamaria; Ruvo, Menotti; Parente, Augusto; Chambery, Angela; Di Maro, Antimo

    2012-10-30

    Bowman-Birk serine protease inhibitors (BBIs) from legume seeds are small proteins showing a two-head structure with distinct reactive site loops, which inhibit two molecules of the same enzyme or two different proteases. Purification and characterization of new BBIs is of broad interest for understanding the basic molecular mechanisms underlying natural defence against the action of proteolytic enzymes. In this study, two novel acidic BBIs (LSI-1a and LSI-2a) were isolated from L. sativus seeds using classical biochemical techniques and characterized for their inhibitory activity. In addition, the N-terminal sequencing of LSI-1a was performed by Edman degradation up to residue 10 and the complete primary structure of the most abundant form (LSI-2a) was determined by using a combination of mass spectrometry approaches, including MALDI-TOF MS, tandem MS and Electron Transfer Dissociation coupled with Proton Transfer Reaction (ETD/PTR) top-down sequencing of N- and C-termini. Furthermore, the LSI-2a dimerization surface has also been investigated by a combination of gel filtration, electrophoretic techniques and homology modelling. Knowing the structure of small proteins inhibiting proteolytic enzymes is of general importance for understanding the defence mechanisms against degradation for their use in biological applications as well as for designing artificial inhibitors.

  18. Cloning, characterization and differential expression of a Bowman-Birk inhibitor during progressive water deficit and subsequent recovery in peanut (Arachis hypogaea) leaves.

    PubMed

    Dramé, Khady Nani; Passaquet, Chantal; Repellin, Anne; Zuily-Fodil, Yasmine

    2013-01-15

    Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors well known for their anticarcinogenic properties and roles in plant defense against insects and pathogens. Here we investigated the expression of a BBI gene in response to water deficit, recovery and phytohormones. A full length cDNA encoding a novel BBI (AhBBI) was isolated from peanut (Arachis hypogaea L.) leaves. The deduced protein is a polypeptide of 11.5kDa containing a signal peptide of 20 amino acids which is missing from peanut seed full-length BBI. Sequence analysis showed that AhBBI presents the characteristic features of BBIs but its first inhibitory loop is unique among the Fabaceae species. Real-time PCR analyses indicated that in peanut leaves, AhBBI is upregulated by water deficit and exogenous jasmonic acid (JA) but repressed by abscissic acid (ABA) after 24h of treatment. The transcripts accumulation patterns during water deficit differed between two cultivars studied in relation to their tolerance levels to drought. AhBBI transcripts accumulated earlier and stronger in the tolerant cultivar (cv. Fleur11) compared to the susceptible one (cv. 73-30) suggesting that BBI genes are involved in drought stress tolerance. Subsequent rehydration reversed the accumulation of AhBBI transcripts in both cultivars but at different levels. The overall role of BBI in abiotic stress tolerance and the possible mechanisms of action are discussed. Copyright © 2012 Elsevier GmbH. All rights reserved.

  19. Phase I randomized double-blind placebo-controlled single-dose safety studies of Bowman-Birk inhibitor concentrate

    PubMed Central

    LIN, LILIE L.; MICK, ROSEMARIE; WARE, JEFFREY; METZ, JAMES; LUSTIG, ROBERT; VAPIWALA, NEHA; RENGAN, RAMESH; KENNEDY, ANN R.

    2014-01-01

    In previously performed animal studies and Phase I–II human trials, Bowman-Birk inhibitor concentrate (BBIC) appeared to be a promising cancer chemopreventive agent. The present study describes the results of two phase I randomized double-blind placebo-controlled trials performed in male subjects to assess the safety and toxicity of the original and new formulations of BBIC administered in a single dose as a suspension in orange juice. The dose of BBIC varied from 800–2,000 chymotrypsin inhibitor (CI) units. The BBI concentration in the serum samples collected from the subjects was analyzed by a dot-blot analysis procedure using the 5G2 monoclonal antibody, which is specific for reduced BBI. A total of 41 subjects were enrolled, 20 in the initial BBIC study and 21 in the second BBIC study. In these human trials, no clinically relevant changes in hematological or biochemical parameters were observed. Overall, BBIC was found to be well-tolerated. For these BBIC single-dose phase I trials, there was no dose-limiting toxicity for BBIC, even at the highest dose evaluated, and there were no apparent differences between the clinical trial results for the two formulations of BBIC. The bioavailability of BBI in the second clinical trial, which used the new BBIC formulation, was approximately 40 to 43% of the BBI bioavailability reached in the first clinical trial, which used the original BBIC formulation. The observed bioavailability difference was attributed to the different BBIC formulations used in these two clinical trials. These trials demonstrated that BBIC is safe when administered in a single dose of up to 2,000 CI units. Therefore, the results from the two trials indicate that a multi-dose trial of BBIC may be safely performed with doses of up to 2,000 CI units per day. PMID:24944684

  20. pLR-HL: A Novel Amphibian Bowman-Birk-type Trypsin Inhibitor from the Skin Secretion of the Broad-folded Frog, Hylarana latouchii.

    PubMed

    Lin, Yan; Hang, Haiying; Chen, Tianbao; Zhou, Mei; Wang, Lei; Shaw, Chris

    2016-01-01

    In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a 'shotgun' cloning technique. It contains a disulphide loop between Cys(5) and Cys(15) which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse-phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesized by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nm. The substitution of Lys-8 by Phe (Phe(8) -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μm. Additionally, both the disulphide loops of pLR-HL and Phe(8) -pLR-HL were synthesized and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically active moiety.

  1. Oligomerization states of Bowman-Birk inhibitor by atomic force microscopy and computational approaches.

    PubMed

    Silva, Luciano P; Azevedo, Ricardo B; Morais, Paulo C; Ventura, Manuel M; Freitas, Sonia M

    2005-11-15

    Several methods have been applied to study protein-protein interaction from structural and thermodynamic point of view. The present study reveals that atomic force microscopy (AFM), molecular modeling, and docking approaches represent alternative methods offering new strategy to investigate structural aspects in oligomerization process of proteinase inhibitors. The topography of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) was recorded by AFM and compared with computational rigid-bodies docking approaches. Multimeric states of BTCI identified from AFM analysis showed globular-ellipsoidal shapes. Monomers, dimers, trimers, and hexamers were the most prominent molecular arrays observed in AFM images as evaluated by molecular volume calculations and corroborated by in silico docking and theoretical approaches. We therefore propose that BTCI adopts stable and well-packed self-assembled states in monomer-dimer-trimer-hexamer equilibrium. Although there are no correlation between specificity and packing efficiency among proteinases and proteinase inhibitors, the AFM and docked BTCI analyses suggest that these assemblies may exist in situ to play their potential function in oligomerization process.

  2. The cytotoxic effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases.

    PubMed

    Clemente, Alfonso; Moreno, Francisco Javier; Marín-Manzano, Maria del Carmen; Jiménez, Elisabeth; Domoney, Claire

    2010-03-01

    Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.

  3. Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman-Birk inhibitor.

    PubMed

    Tang, MingXi; Asamoto, Makoto; Ogawa, Kumiko; Naiki-Ito, Aya; Sato, Shinya; Takahashi, Satoru; Shirai, Tomoyuki

    2009-11-01

    The soybean-derived serine protease inhibitor, Bowman-Birk inhibitor (BBI), has been reported as a potent chemoprevention agent against several types of tumors. The present study was undertaken to evaluate the effects of BBI on androgen-sensitive/dependent prostate cancers using a human prostate cancer cell (LNCaP) and the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. Treatment of LNCaP prostate cancer cells with 500 microg/mL BBI resulted in inhibition of viability measured on WST-1 assays, with induction of connexin 43 (Cx43) and cleaved caspase-3 protein expression. Feeding of 3% roughly prepared BBI (BBIC) to TRAP from the age 3 weeks to 13 weeks resulted in significant reduction of the relative epithelial areas within the acinus and multiplicity of the adenocarcinomas in the lateral prostate lobes. Cx43- and terminal deoxynucleotidyl transferase mediated dUTP-biotin end labeling of fragmented DNA (TUNEL)-positive apoptotic cancer cells were more frequently observed in the lateral prostates treated with BBIC than in the controls. These in vivo and in vitro results suggest that BBI possesses chemopreventive activity associated with induction of Cx43 expression and apoptosis.

  4. Simultaneous and rapid determination of the anticarcinogenic proteins Bowman-Birk inhibitor and lectin in soybean crops by perfusion RP-HPLC.

    PubMed

    Anta, Lucía; Luisa Marina, M; García, M Concepción

    2010-11-05

    There are numerous studies demonstrating a direct association between the ingestion of soybean and low cancer incidence. This fact has been related to the presence of Bowman-Birk inhibitor (BBI) and lectin in soybean. The simultaneous and fast determination of BBI and lectin in soybean is proposed, for the first time, in this work. Two different strategies were designed for the extraction of BBI and lectin: extraction of soybean proteins using a Tris-HCl buffer followed by isolation of BBI and lectin by the isoelectric precipitation of other soybean proteins (method I) or by the direct extraction of BBI and lectin using an acetate buffer (method II). The effect of the previous soybean defating on the extraction of BBI and lectin was also studied. Moreover, the possibility of using a high-intensity focalized ultrasonic probe for accelerating the extraction was explored and an optimization of the extraction time and ultrasound amplitude was performed. The extracts obtained were analysed by RP-HPLC-ESI-MS for the correct identification of BBI and lectin in soybean. Moreover, a fast chromatographic methodology using a perfusion column and UV detection was optimized for the rapid determination of BBI and lectin in soybean. After evaluating its analytical characteristics (linearity, precision, and recovery), the method was applied to the quantitation of BBI and lectin in different soybean varieties. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    DTIC Science & Technology

    1988-01-31

    PROTEASES; INSECT TRYPSINS and CHYMOTRYPSINS; BOWMAN-BIRK TRYPSIN-CHYMOTRYPSIN INHIBITOR (SOYBEANS); CHICKPEAS TRYPSIN-CHYMOTRYPSIN INHIBLTOR; SOYBEAN...inhibitors from legume .seeds, such as the Bowman-Birk inhibitor (BBI) from soybeans and CI from chickpeas . The purified and partially-characterized insect DD...inhibited by the trypsin - chymotrypsin inhibitors BBI from soybeans and CI from chickpeas . Separation and purification of these enzymes by gel

  6. Crystal structure of the Bowman-Birk Inhibitor from Vigna unguiculata seeds in complex with beta-trypsin at 1.55 A resolution and its structural properties in association with proteinases.

    PubMed

    Barbosa, João Alexandre R G; Silva, Luciano P; Teles, Rozeni C L; Esteves, Gisele F; Azevedo, Ricardo B; Ventura, Manuel M; de Freitas, Sonia M

    2007-03-01

    The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with beta-trypsin was solved and refined at 1.55 A to a crystallographic R(factor) of 0.154 and R(free) of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.

  7. 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

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

    DTIC Science & Technology

    1989-01-31

    CHYMOTRYPSINS; BOWMAN-BIRK TRYPSIN-CHYMOTRYPSIN INHIBITOR (SOYBEANS); CHICKPEAS TRYPSIN-CHYMOTRYPSIN INHIBITOR; SOYBEAN PROTEASE INHIBITORS 20. ABSTRACT...could be fully inhibited at a 1:1 molar ratio by the naturally-occuring proteinaceous trypsin inhibitors BBI from soybeans and CI from chickpeas ...substrates. These activities were fully inhibited by the proteinaceous trypsin-chymotrypsin inhibitors BBI from soybeans and CI from chickpeas when assayed

  9. 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.

  10. Transgenic soybean plants overexpressing O-acetylserine sulfhydrylase accumulate enhanced levels of cysteine and bowman-birk protease inhibitor in seeds

    USDA-ARS?s Scientific Manuscript database

    Soybeans provide an excellent source of protein in animal feed. Soybean protein quality can be enhanced by increasing the concentration of sulfur containing amino acids. Previous attempts to increase the concentration of sulfur-containing amino acids through the expression of heterologous proteins...

  11. Effects of Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and Semipure Lectin Fractions on Cancer Cells

    PubMed Central

    García-Gasca, Teresa; García-Cruz, Marlen; Hernandez-Rivera, Elisa; López-Matínez, Josue; Castañeda-Cuevas, Ana L.; Yllescas-Gasca, Lorena; Rodríguez-Méndez, Adriana J.; Mendiola-Olaya, Elizabeth; Castro-Guillén, José L.; Blanco-Labra, Alejandro

    2012-01-01

    Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes’ extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells. PMID:23163855

  12. 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.

  13. 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

  14. Plant Protease Inhibitors in Therapeutics-Focus on Cancer Therapy.

    PubMed

    Srikanth, Sandhya; Chen, Zhong

    2016-01-01

    Plants are known to have many secondary metabolites and phytochemical compounds which are highly explored at biochemical and molecular genetics level and exploited enormously in the human health care sector. However, there are other less explored small molecular weight proteins, which inhibit proteases/proteinases. Plants are good sources of protease inhibitors (PIs) which protect them against diseases, insects, pests, and herbivores. In the past, proteinaceous PIs were considered primarily as protein-degrading enzymes. Nevertheless, this view has significantly changed and PIs are now treated as very important signaling molecules in many biological activities such as inflammation, apoptosis, blood clotting and hormone processing. In recent years, PIs have been examined extensively as therapeutic agents, primarily to deal with various human cancers. Interestingly, many plant-based PIs are also found to be effective against cardiovascular diseases, osteoporosis, inflammatory diseases and neurological disorders. Several plant PIs are under further evaluation in in vitro clinical trials. Among all types of PIs, Bowman-Birk inhibitors (BBI) have been studied extensively in the treatment of many diseases, especially in the field of cancer prevention. So far, crops such as beans, potatoes, barley, squash, millet, wheat, buckwheat, groundnut, chickpea, pigeonpea, corn, and pineapple have been identified as good sources of PIs. The PI content of such foods has a significant influence on human health disorders, particularly in the regions where people mostly depend on these kind of foods. These natural PIs vary in concentration, protease specificity, heat stability, and sometimes several PIs may be present in the same species or tissue. However, it is important to carry out individual studies to identify the potential effects of each PI on human health. PIs in plants make them incredible sources to determine novel PIs with specific pharmacological and therapeutic effects due

  15. Plant Protease Inhibitors in Therapeutics-Focus on Cancer Therapy

    PubMed Central

    Srikanth, Sandhya; Chen, Zhong

    2016-01-01

    Plants are known to have many secondary metabolites and phytochemical compounds which are highly explored at biochemical and molecular genetics level and exploited enormously in the human health care sector. However, there are other less explored small molecular weight proteins, which inhibit proteases/proteinases. Plants are good sources of protease inhibitors (PIs) which protect them against diseases, insects, pests, and herbivores. In the past, proteinaceous PIs were considered primarily as protein-degrading enzymes. Nevertheless, this view has significantly changed and PIs are now treated as very important signaling molecules in many biological activities such as inflammation, apoptosis, blood clotting and hormone processing. In recent years, PIs have been examined extensively as therapeutic agents, primarily to deal with various human cancers. Interestingly, many plant-based PIs are also found to be effective against cardiovascular diseases, osteoporosis, inflammatory diseases and neurological disorders. Several plant PIs are under further evaluation in in vitro clinical trials. Among all types of PIs, Bowman-Birk inhibitors (BBI) have been studied extensively in the treatment of many diseases, especially in the field of cancer prevention. So far, crops such as beans, potatoes, barley, squash, millet, wheat, buckwheat, groundnut, chickpea, pigeonpea, corn, and pineapple have been identified as good sources of PIs. The PI content of such foods has a significant influence on human health disorders, particularly in the regions where people mostly depend on these kind of foods. These natural PIs vary in concentration, protease specificity, heat stability, and sometimes several PIs may be present in the same species or tissue. However, it is important to carry out individual studies to identify the potential effects of each PI on human health. PIs in plants make them incredible sources to determine novel PIs with specific pharmacological and therapeutic effects due

  16. Identification of a bioactive Bowman-Birk inhibitor from an insect-resistant early maize inbred

    USDA-ARS?s Scientific Manuscript database

    Breeding of maize, Zea mays, has improved insect resistance, but the genetic and biochemical basis of many of these improvements is unknown. Maize oligonucleotide microarrays were utilized to identify differentially expressed genes in leaves of three maize inbreds, parents Oh40B and W8 and progeny O...

  17. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding

    PubMed Central

    Medeiros, Ane H.; Mingossi, Fabiana B.; Dias, Renata O.; Franco, Flávia P.; Vicentini, Renato; Mello, Marcia O.; Moura, Daniel S.; Silva-Filho, Marcio C.

    2016-01-01

    Sugarcane’s (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory. PMID:27598134

  18. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

    PubMed

    Medeiros, Ane H; Mingossi, Fabiana B; Dias, Renata O; Franco, Flávia P; Vicentini, Renato; Mello, Marcia O; Moura, Daniel S; Silva-Filho, Marcio C

    2016-09-01

    Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.

  19. 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.

  20. The proteolysis of trypsin inhibitors in legume seeds.

    PubMed

    Wilson, K A

    1988-01-01

    The seeds of plants often contain large amounts of proteins, which are subjected to extensive proteolytic processing during seed development and subsequent germination. One class of legume seed proteins, the Bowman-Birk-type trypsin inhibitors, has proved especially useful as a subject in studying these events. Sequence studies of the trypsin inhibitors from a number of legume species suggest that many of the inhibitors undergo a limited shortening at the amino terminus during seed development. However, during germination, the inhibitors appear to function as storage proteins. As such, they are subjected to extensive proteolysis, ultimately leading to their destruction. This degradative process has been studied extensively in the mung bean (Vigna radiata [L.] Wilczek). Proteolysis of the mung bean trypsin inhibitor involves, at least initially, an ordered sequence of limited proteolytic cleavages. The two proteases involved in the initial phases of this degradation have been identified and partially characterized.

  1. Eliminating anti-nutritional plant food proteins: the case of seed protease inhibitors in pea.

    PubMed

    Clemente, Alfonso; Arques, Maria C; Dalmais, Marion; Le Signor, Christine; Chinoy, Catherine; Olias, Raquel; Rayner, Tracey; Isaac, Peter G; Lawson, David M; Bendahmane, Abdelhafid; Domoney, Claire

    2015-01-01

    Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving

  2. Eliminating Anti-Nutritional Plant Food Proteins: The Case of Seed Protease Inhibitors in Pea

    PubMed Central

    Clemente, Alfonso; Arques, Maria C.; Dalmais, Marion; Le Signor, Christine; Chinoy, Catherine; Olias, Raquel; Rayner, Tracey; Isaac, Peter G.; Lawson, David M.; Bendahmane, Abdelhafid; Domoney, Claire

    2015-01-01

    Several classes of seed proteins limit the utilisation of plant proteins in human and farm animal diets, while plant foods have much to offer to the sustainable intensification of food/feed production and to human health. Reduction or removal of these proteins could greatly enhance seed protein quality and various strategies have been used to try to achieve this with limited success. We investigated whether seed protease inhibitor mutations could be exploited to enhance seed quality, availing of induced mutant and natural Pisum germplasm collections to identify mutants, whilst acquiring an understanding of the impact of mutations on activity. A mutant (TILLING) resource developed in Pisum sativum L. (pea) and a large germplasm collection representing Pisum diversity were investigated as sources of mutations that reduce or abolish the activity of the major protease inhibitor (Bowman-Birk) class of seed protein. Of three missense mutations, predicted to affect activity of the mature trypsin / chymotrypsin inhibitor TI1 protein, a C77Y substitution in the mature mutant inhibitor abolished inhibitor activity, consistent with an absolute requirement for the disulphide bond C77-C92 for function in the native inhibitor. Two further classes of mutation (S85F, E109K) resulted in less dramatic changes to isoform or overall inhibitory activity. The alternative strategy to reduce anti-nutrients, by targeted screening of Pisum germplasm, successfully identified a single accession (Pisum elatius) as a double null mutant for the two closely linked genes encoding the TI1 and TI2 seed protease inhibitors. The P. elatius mutant has extremely low seed protease inhibitory activity and introgression of the mutation into cultivated germplasm has been achieved. The study provides new insights into structure-function relationships for protease inhibitors which impact on pea seed quality. The induced and natural germplasm variants identified provide immediate potential for either halving

  3. The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm.

    PubMed

    Poerio, E; Caporale, C; Carrano, L; Caruso, C; Vacca, F; Buonocore, V

    1994-02-01

    The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion with Staphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.

  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. A trypsin inhibitor from snail medic seeds active against pest proteases.

    PubMed

    Ceciliani, F; Tava, A; Iori, R; Mortarino, M; Odoardi, M; Ronchi, S

    1997-02-01

    A protein trypsin inhibitor from seeds of snail medic (Medicago scutellata), MsTI, has been purified by ion-exchange chromatography, gel-filtration chromatography and reverse-phase HPLC. The protein inhibits the catalytic activity of bovine beta-trypsin, with an apparent Kd of 1.8 x 10(-9), but exhibits no activity towards bovine alpha-chymotrypsin. Moreover, MsTI inhibits the trypsin-like proteinase activity present in larvae of the crop pests Adoxophyes orana, Hyphantria cunea, Lobesia botrana and Ostrinia nubilalis. The complete amino acid sequence of MsTI consists of 62 residues corresponding to a M(r) of 6925. Sequence comparison shows that MsTI exhibits significant similarity to other proteins belonging to the Bowman-Birk trypsin inhibitor family, and the closest identity (81%) with the wound-induced trypsin inhibitor from Medicago sativa leaves.

  7. 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.

  8. Bowman-Birk inhibitor and genistein among soy compounds that synergistically inhibit nitric oxide and prostaglandin E2 pathways in lipopolysaccharide-induced macrophages

    USDA-ARS?s Scientific Manuscript database

    Inflammation has an important role in the development of chronic diseases. In this study, we evaluated the anti-inflammatory properties of eight soybean bioactive compounds using lipopolysaccharide-induced RAW 264.7 macrophages. Genistein, daidzein, mix isoflavone glucosides, saponin A group glyco...

  9. Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria.

    PubMed

    Spit, Jornt; Zels, Sven; Dillen, Senne; Holtof, Michiel; Wynant, Niels; Vanden Broeck, Jozef

    2014-05-01

    While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized. Phylogenetic analysis revealed that these are distributed among 3 evolutionary conserved clusters. In addition, we have determined the relative gene expression levels of representative members in the gut under different feeding conditions. This study demonstrated that the transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, larvae of L. migratoria displayed compensatory effects to the presence of Soybean Bowman Birk (SBBI) and Soybean Trypsin (SBTI) inhibitors in their diet by differential upregulation of multiple proteases. A rapid initial upregulation was observed for all tested serine protease transcripts, while only for members belonging to class I, the transcript levels remained elevated after prolonged exposure. In full agreement with these results, we also observed an increase in proteolytic activity in midgut secretions of locusts that were accustomed to the presence of protease inhibitors in their diet, while no change in sensitivity to these inhibitors was observed. Taken together, this paper is the first comprehensive study on dietary dependent transcript levels of proteolytic enzymes in Orthoptera. Our data suggest that compensatory response mechanisms to protease inhibitor ingestion may have appeared early in insect evolution.

  10. Purification, amino acid sequence, and cDNA cloning of trypsin inhibitors from onion (Allium cepa L.) bulbs.

    PubMed

    Deshimaru, Masanobu; Watanabe, Akira; Suematsu, Keiko; Hatano, Maki; Terada, Shigeyuki

    2003-08-01

    Three protease inhibitors (OTI-1-3) have been purified from onion (Allium cepa L.) bulbs. Molecular masses of these inhibitors were found to be 7,370.2, 7,472.2, and 7,642.6 Da by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. Based on amino acid composition and N-terminal sequence, OTI-1 and -2 are the N-terminal truncated proteins of OTI-3. All the inhibitors are stable to heat and extreme pH. OTI-3 inhibited trypsin, chymotrypsin, and plasmin with dissociation constants of 1.3 x 10(-9) M, 2.3 x 10(-7) M, and 3.1 x 10(-7) M, respectively. The complete amino acid sequence of OTI-3 showed a significant homology to Bowman-Birk family inhibitors, and the first reactive site (P1) was found to be Arg17 by limited proteolysis by trypsin. The second reactive site (P1) was estimated to be Leu46, that may inhibit chymotrypsin. OTI-3 lacks an S-S bond near the second reactive site, resulting in a low affinity for the enzyme. The sequence of OTI-3 was also ascertained by the nucleotide sequence of a cDNA clone encoding a 101-residue precursor of the onion inhibitor.

  11. Crystallization and preliminary X-ray diffraction studies on a trypsin/chymotrypsin double-headed inhibitor from horse gram.

    PubMed

    Prakash, B; Murthy, M R; Sreerama, Y N; Sarma, P R; Rao, D R

    1994-01-07

    The Bowman-Birk family of proteinase inhibitors from seeds of leguminous plants usually have a molecular mass of 8000 to 10,000 Da. Horse gram (Dolichos bifloros or Macrotyloma uniflorum) seeds contain an unusual Bowman-Birk inhibitor of molecular mass 15,500 Da active against both trypsin and chymotrypsin. In order to elucidate its three-dimensional structure, its evolutionary relationship with the more usual Bowman-Birk inhibitors and to study the structure-function properties, this inhibitor has been purified and crystallized. The purified protein crystallizes easily under a variety of conditions in different crystal forms. Crystals obtained by precipitating the protein (3 to 5 mg/ml in 50mM Tris.HCl (pH 8.0)) with 5% ammonium sulphate and 2 to 3% PEG 4000 appear to be suitable for structure determination by X-ray diffraction. The crystals belong to cubic space group P2(1)3 (a = 110.81 A) and diffract X-rays to beyond 3.0 A resolution.

  12. Rational tailoring of substrate and inhibitor affinity via ATRP polymer-based protein engineering.

    PubMed

    Murata, Hironobu; Cummings, Chad S; Koepsel, Richard R; Russell, Alan J

    2014-07-14

    Atom transfer radical polymerization (ATRP)-based protein engineering of chymotrypsin with a cationic polymer was used to tune the substrate specificity and inhibitor binding. Poly(quaternary ammonium) was grown from the surface of the enzyme using ATRP after covalent attachment of a protein reactive, water-soluble ATRP-initiator. This "grafting from" conjugation approach generated a high density of cationic ammonium ions around the biocatalytic core. Modification increased the surface area of the protein over 40-fold, and the density of modification on the protein surface was approximately one chain per 4 nm(2). After modification, bioactivity was increased at low pH relative to the activity of the native enzyme. In addition, the affinity of the enzyme for a peptide substrate was increased over a wide pH range. The massively cationic chymotrypsin, which included up to 2000 additional positive charges per molecule of enzyme, was also more stable at extremes of temperature and pH. Most interestingly, we were able to rationally control the binding of two oppositely charged polypeptide protease inhibitors, aprotinin and the Bowman-Birk trypsin-chymotrypsin inhibitor from Glycine max, to the cationic derivative of chymotrypsin. This study expands upon our efforts to use polymer-based protein engineering to predictably engineer enzyme properties without the need for molecular biology.

  13. 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

  14. Nelfinavir: fourth protease inhibitor approved.

    PubMed

    1997-01-01

    The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.

  15. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  16. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors.

    PubMed

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; Roush, William R

    2008-11-15

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

  17. 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.

  18. Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

    PubMed

    Nduaguibe, Chikodili C; Bentsi-Barnes, Kwamina; Mullen, Yoko; Kandeel, Fouad; Al-Abdullah, Ismail

    2010-01-01

    Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

  19. Intervention for hyperlipidemia associated with protease inhibitors.

    PubMed

    Melroe, N H; Kopaczewski, J; Henry, K; Huebsch, J

    1999-01-01

    In the past 3 years, treatment for HIV infection has significantly improved the prognosis for HIV-infected persons. The administration of protease inhibitors for the treatment of HIV infection has had a significant role in the reduction of AIDS-related complications. Recent findings have indicated that protease inhibitors may significantly increase lipids to levels that pose a health risk that may be greater than the illness itself. This article reviews the initial findings of a study that investigated the impact of interventions for the treatment of protease inhibitor-related hyperlipidemia. The purpose of the study was to determine if initiation of interventions based on the National Cholesterol Education Program Guidelines would be effective in lowering protease inhibitor-related hyperlipidemia without disrupting the effectiveness of the HIV therapy. A total of 45 HIV-infected individuals who were taking a protease inhibitor and had abnormally elevated lipids were enrolled into this study. Mean serum cholesterol level prior to initiation of a protease inhibitor regimen was 170 mg/dl as compared to a mean cholesterol at time of enrollment of 289 mg/dl and triglycerides of 879 mg/dl. Interventions included diet and exercise and the prescription of gemfibrozil alone or in combination with atorvatstatin. During the course of the study, overall intervention significantly reduced serum cholesterol level to 201 mg/dl (p. 01) over a study period of ten months. Case studies of five medical events related to hyperlipidemia are included. Currently, 26 participants continue in the study. Sixteen participants discontinued protease inhibitor therapy during the course of the study and thus ended their participation.

  20. Seminal and colostral protease inhibitors on leukocytes.

    PubMed

    Veselský, L; Cechová, D; Hruban, V; Klaudy, J

    1982-01-01

    For detection of protease inhibitors from cow colostrum (CTI) and bull seminal plasma (BUSI I and BUSI II) on the surface of leukocytes, immunological methods were used. An agglutination and an immunofluorescence test demonstrated components on the surface of bovine, porcine and ovine granulocytes and lymphocytes which were immunologically identical with the protease inhibitors isolated from cow colostrum and bull seminal plasma. When antisera against (CTI, BUSI and BUSI II were absorbed by bovine and porcine liver, kidney and spleen homogenate or by bovine and porcine granulocytes or lymphocytes, the immunological tests were negative.

  1. Saquinavir, the pioneer antiretroviral protease inhibitor.

    PubMed

    la Porte, Charles J L

    2009-10-01

    The treatment of HIV infection underwent a major change in 1995 when saquinavir was the first protease inhibitor introduced into the market. This drug made the use of combination therapy in the treatment of HIV possible and increased the success rate of treatment. This article will review recent literature on saquinavir to define its current role in HIV treatment, among the newer antiretroviral drugs. Scientific literature and conference presentations were evaluated for relevant information pertaining to saquinavir. Although underused, saquinavir has good efficacy and tolerability when compared to other protease inhibitors. The film-coated tablet formulation improved pill burden. Saquinavir still has potential in the treatment of adults, children and pregnant women.

  2. Current and Novel Inhibitors of HIV Protease

    PubMed Central

    Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. PMID:21994591

  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. Investigational protease inhibitors as antiretroviral therapies

    PubMed Central

    Midde, Narasimha M.; Patters, Benjamin J.; Rao, PSS; Cory, Theodore J.; Kumar, Santosh

    2017-01-01

    Introduction Highly Active Antiretroviral Therapy (HAART) has tremendously improved the life expectancy of the HIV-infected population over the past three decades. Protease inhibitors have been one of the major classes of drugs in HAART regimens that are effective in treating HIV. However, the emergence of resistance and cross-resistance against protease inhibitors encourages researchers to develop new PIs with broad-spectrum activity, as well as novel means of enhancing the efficacy of existing PIs. Areas covered In this article we discuss recent advances in HIV protease inhibitor (PI) development, focusing on both investigational and experimental agents. We also include a section on pharmacokinetic booster drugs for improved bioavailability of protease inhibitors. Further, we discuss novel drug delivery systems using a variety of nanocarriers for the delivery of PIs across the blood-brain barrier to treat the HIV in the brain. Expert opinion We discuss our opinion on the promises and challenges on the development of novel investigational and experimental PIs that are less toxic and more effective in combating drug-resistance. Further, we discuss the future of novel nanocarriers that have been developed to deliver PIs to the brain cells. Although these are promising findings, many challenges need to be overcome prior to making them a viable option. PMID:27415449

  5. Management of protease inhibitor-associated hyperlipidemia.

    PubMed

    Penzak, Scott R; Chuck, Susan K

    2002-01-01

    Dyslipidemia, characterized by elevated serum levels of triglycerides and reduced levels of total cholesterol, low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol, has been recognized in patients with human immunodeficiency virus (HIV) infection. It is thought that elevated levels of circulating cytokines, such as tumor necrosis factor-alpha and interferon-alpha, may alter lipid metabolism in patients with HIV infection. Protease inhibitors, such as saquinavir, indinavir and ritonavir, have been found to decrease mortality and improve quality of life in patients with HIV infection. However, these drugs have been associated with a syndrome of fat redistribution, insulin resistance, and hyperlipidemia. Elevations in serum total cholesterol and triglyceride levels, along with dyslipidemia that typically occurs in patients with HIV infection, may predispose patients to complications such as premature atherosclerosis and pancreatitis. It has been estimated that hypercholesterolemia and hypertriglyceridemia occur in greater than 50% of protease inhibitor recipients after 2 years of therapy, and that the risk of developing hyperlipidemia increases with the duration of treatment with protease inhibitors. In general, treatment of hyperlipidemia should follow National Cholesterol Education Program guidelines; efforts should be made to modify/control coronary heart disease risk factors (i.e. smoking; hypertension; diabetes mellitus) and maximize lifestyle modifications, primarily dietary intervention and exercise, in these patients. Where indicated, treatment usually consists of either pravastatin or atorvastatin for patients with elevated serum levels of LDL-C and/or total cholesterol. Atorvastatin is more potent in lowering serum total cholesterol and triglycerides compared with other hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, but it is also associated with more drug interactions compared with pravastatin. Simvastatin

  6. Amino Acid Sequence of Mung Bean Trypsin Inhibitor and Its Modified Forms Appearing during Germination.

    PubMed

    Wilson, K A; Chen, J C

    1983-02-01

    The amino acid sequence of the major trypsin inhibitor, F, of ungerminated mung beans (Vigna radiata [L.] Wilczek) was determined by a combination of automatic solid phase and manual sequencing techniques. F is a typical Bowman-Birk-type proteinase inhibitor with 80 amino acid residues and exhibits a high degree of identity with the other sequenced members of the Bowman-Birk family of inhibitors. Thin layer peptide maps of mung bean inhibitors E and C (which appear during germination) indicate that both are derived from inhibitor F by limited specific proteolysis. Loss of the carboxyl-terminal residues 77 to 80 from F produces inhibitor E, while the loss of an additional two carboxyl-terminal residues, the loss of the amino-terminal residues 1 to 8, and an internal cleavage at Ala(35)-Asp(36) produces inhibitor C from E. Another inhibitor species, E', was isolated from ungerminated seeds. It differs from F in the loss of residues 1 to 6. The majority of the proteolytic cleavages noted in the F-E-C-E' system are at peptide bonds involving aspartyl residues.

  7. Comparative study on the protease inhibitors from fish eggs

    NASA Astrophysics Data System (ADS)

    Ustadi; Kim, K. Y.; Kim, S. M.

    2005-07-01

    The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and l6.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 Umg-1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50-65°C and pH 8, which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant ( K i) of 4.44 nmolL-1.

  8. Insect response to plant defensive protease inhibitors.

    PubMed

    Zhu-Salzman, Keyan; Zeng, Rensen

    2015-01-07

    Plant protease inhibitors (PIs) are natural plant defense proteins that inhibit proteases of invading insect herbivores. However, their anti-insect efficacy is determined not only by their potency toward a vulnerable insect system but also by the response of the insect to such a challenge. Through the long history of coevolution with their host plants, insects have developed sophisticated mechanisms to circumvent antinutritional effects of dietary challenges. Their response takes the form of changes in gene expression and the protein repertoire in cells lining the alimentary tract, the first line of defense. Research in insect digestive proteases has revealed the crucial roles they play in insect adaptation to plant PIs and has brought about a new appreciation of how phytophagous insects employ this group of molecules in both protein digestion and counterdefense. This review provides researchers in related fields an up-to-date summary of recent advances.

  9. Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118).

    PubMed

    Swathi, Marri; Lokya, Vadthya; Swaroop, Vanka; Mallikarjuna, Nalini; Kannan, Monica; Dutta-Gupta, Aparna; Padmasree, Kollipara

    2014-10-01

    Proteinase inhibitors (C11PI) from mature dry seeds of Cajanus cajan (cv. ICP 7118) were purified by chromatography which resulted in 87-fold purification and 7.9% yield. SDS-PAGE, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrum and two-dimensional (2-D) gel electrophoresis together resolved that C11PI possessed molecular mass of 8385.682 Da and existed as isoinhibitors. However, several of these isoinhibitors exhibited self association tendency to form small oligomers. All the isoinhibitors resolved in Native-PAGE and 2-D gel electrophoresis showed inhibitory activity against bovine pancreatic trypsin and chymotrypsin as well as Achaea janata midgut trypsin-like proteases (AjPs), a devastating pest of castor plant. Partial sequences of isoinhibitor (pI 6.0) obtained from MALDI-TOF/TOF analysis and N-terminal sequencing showed 100% homology to Bowman-Birk Inhibitors (BBIs) of leguminous plants. C11PI showed non-competitive inhibition against trypsin and chymotrypsin. A marginal loss (<15%) in C11PI activity against trypsin at 80 (°)C and basic pH (12.0) was associated with concurrent changes in its far-UV CD spectra. Further, in vitro assays demonstrated that C11PI possessed significant inhibitory potential (IC50 of 78 ng) against AjPs. On the other hand, in vivo leaf coating assays demonstrated that C11PI caused significant mortality rate with concomitant reduction in body weight of both larvae and pupae, prolonged the duration of transition from larva to pupa along with formation of abnormal larval-pupal and pupal-adult intermediates. Being smaller peptides, it is possible to express C11PI in castor to protect them against its devastating pest A. janata.

  10. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    PubMed

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

  11. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior

    PubMed Central

    Almonte, Antoine G.; Sweatt, J. David

    2011-01-01

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes—coagulation, inflammation, and digestion, for example—have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation. PMID:21782155

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

    DTIC Science & Technology

    1989-12-15

    PROTEASES; PROTEASE INHIBITORS; STORED-PRODUCT INISECTS; TRIBOLIUM CASIANEUH; MIDGUT PROTEASES; TENEBRIO MOLITOR MIDGUT-PROTEASES; LOCUST CAECAL...separation and identification of numerous midgut proteases in Tenebrio and Tribolium . The PAGE-gelatin matrix revealed the inhibitory effect of BBI...the proteinaceous trypsin-chymotrypsin inhibitor from soybeans) on several Tribolium proteases - an effect which was not detectable in inhibition

  13. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  14. Novel pseudosymmetric inhibitors of HIV-1 protease

    SciTech Connect

    Faessler, A.; Roesel, J.; Gruetter, M.; Tintelnot-Blomley, M.; Alteri, E.; Bold, G.; Lang, M.

    1993-12-31

    Taking into account the unique C-2 symmetric nature of the HIV-1 protease homodimer, the authors have designed and synthesized novel inhibitors featuring an almost symmetric structure. Compounds containing the easily accessible Phe[CH(OH)CH{sub 2}N(NH)]Cha dipeptide isostere as a nonhydrolyzable replacement of the scissile amide bond of the natural substrate are potent inhibitors in vitro with IC{sub 50} values of 9 to 50 nM. The antiviral activity depends mainly on the nature of the anylated valine residues linked to the dipeptide mimic. In this series, CGP 53820 combines both high potency and excellent specificity. Its predicted symmetric binding pattern is illustrated by the X-ray structure analysis performed with the corresponding enzyme-inhibitor complex.

  15. A Multifunctional Protease Inhibitor To Regulate Endolysosomal Function

    PubMed Central

    2011-01-01

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin–pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses. PMID:21910425

  16. A multifunctional protease inhibitor to regulate endolysosomal function.

    PubMed

    van Kasteren, Sander I; Berlin, Ilana; Colbert, Jeff D; Keane, Doreen; Ovaa, Huib; Watts, Colin

    2011-11-18

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin-pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses.

  17. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    PubMed Central

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  18. HIV-1 protease inhibitors in development.

    PubMed

    Rusconi, Stefano; La Seta Catamancio, Simona

    2002-03-01

    Several pharmaceutical companies have developed an increasing number of second generation protease inhibitors (PI) during the last few years. Many of these compounds have been in preclinical trials and some are now in clinical use. All drugs in this category have been designed to be well absorbed and overcome the crucial problem of cross-resistance within this class of compounds. Taking into account the rapid occurrence of PI cross-resistance, clinicians who are treating patients with the HIV-1 infection will need new active PIs in the near future. The clinical and antiviral efficacy of the new molecules versus the older PIs will be investigated through comparative trials that are likely to be completed over the next 12 months. These third-generation PIs currently in development will be the subject of our review.

  19. Evaluation of proteases and protease inhibitors in Heterodera glycines cysts obtained from laboratory and field populations

    USDA-ARS?s Scientific Manuscript database

    Proteases and proteases inhibitors were evaluated in a number of preparations of Heterodera glycines cysts obtained from glasshouse cultures (GH) and field (LR) populations. Using a FRET-peptide library comprising 512 peptide substrate pools that detect 4 endoprotease types (aspartic, cysteine, meta...

  20. Protease inhibitors and proteolytic signalling cascades in insects.

    PubMed

    Gubb, David; Sanz-Parra, Arantza; Barcena, Laura; Troxler, Laurent; Fullaondo, Ane

    2010-12-01

    Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.

  1. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    PubMed Central

    Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes. PMID:25874235

  2. Improving Viral Protease Inhibitors to Counter Drug Resistance

    PubMed Central

    Yilmaz, Nese Kurt; Swanstrom, Ronald; Schiffer, Celia A.

    2016-01-01

    Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design. PMID:27090931

  3. Botulinum neurotoxin A protease: discovery of natural product exosite inhibitors.

    PubMed

    Silhár, Peter; Capková, Katerina; Salzameda, Nicholas T; Barbieri, Joseph T; Hixon, Mark S; Janda, Kim D

    2010-03-10

    A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product d-chicoric acid (I1) is disclosed. A detailed evaluation of chicoric acid's mechanism of inhibition reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and is synergistic with a competitive active site inhibitor when used in combination. Other components found in Echinacea, I3 and I4, were also inhibitors of the protease.

  4. Phage display as a powerful tool to engineer protease inhibitors.

    PubMed

    Zani, Marie-Louise; Moreau, Thierry

    2010-11-01

    Since its introduction by Georges Smith some 25 years ago, phage display has proved to be a powerful molecular technique for selecting proteins with desired biological properties from huge libraries. Early on, various protease inhibitor scaffolds were displayed at the surface of filamentous phages to select new inhibitors with shifted specificities and enhanced affinities towards one or more target protease(s). The past two decades have seen a number of natural protease inhibitors subjected to phage display, mostly to shift and increase their inhibitory specificity, but also to explore the molecular mechanisms by which they interact with their cognate enzymes with low or very high selectivity. This review focuses on the major uses of phage display in the field of protein protease inhibitors. The exquisite molecular mechanisms by which natural protease inhibitors prevent unwanted or excessive proteolysis in cells and tissues are also examined along with some of the general principles underlying the way phage display is applied to these molecules. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  5. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  6. 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.

  7. 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.

  8. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    PubMed Central

    Zhang, Huan; Fei, Rui; Xue, Baigong; Yu, Shanshan; Zhang, Zuoming; Zhong, Sheng; Gao, Yuanqi; Zhou, Xiaoli

    2017-01-01

    Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles. PMID:28067849

  9. New therapeutic strategies in HCV: second-generation protease inhibitors.

    PubMed

    Clark, Virginia C; Peter, Joy A; Nelson, David R

    2013-02-01

    Telaprevir and boceprevir are the first direct-acting antiviral agents approved for use in HCV treatment and represent a significant advance in HCV therapy. However, these first-generation drugs also have significant limitations related to thrice-daily dosing, clinically challenging side-effect profiles, low barriers to resistance and a lack of pan-genotype activity. A second wave of protease inhibitors are in phase II and III trials and promise to provide a drug regimen with a better dosing schedule and improved tolerance. These second-wave protease inhibitors will probably be approved in combination with PEG-IFN and Ribavirin (RBV), as well as future all-oral regimens. The true second-generation protease inhibitors are in earlier stages of development and efficacy data are anxiously awaited as they may provide pan-genotypic antiviral activity and a high genetic barrier to resistance. © 2012 John Wiley & Sons A/S.

  10. Protease inhibitor therapy in children with perinatally acquired HIV infection.

    PubMed

    Rutstein, R M; Feingold, A; Meislich, D; Word, B; Rudy, B

    1997-10-01

    To review the short-term response and safety of protease inhibitor therapy in HIV-infected children. Retrospective chart review of open-label protease inhibitor-containing combination therapy. Two urban pediatric HIV centers. Twenty-eight HIV-infected children were prescribed 30 protease inhibitor-containing antiretroviral therapy combinations. The median age at initiation of protease inhibitor antiretroviral therapy was 79 months. Patients had been on previous antiretroviral therapy for a mean of 45.5 months. Of the 28 children who completed at least 1 month of therapy, 26 experienced marked virologic and immunologic improvement (mean maximal decrease in viral load 1.90 log10 copies/ml; SD, 0.8; mean maximal rise in CD4+ lymphocytes of 279 x 10(6)/l; SD, 300 x 10(6)/l). Eleven patients achieved a viral nadir of < 400 copies/ml, and seven sustained this level of viral suppression for a mean of 6 months. Indinavir use was associated with a high incidence of renal side-effects, including two patients who developed interstitial nephritis. Two patients on ritonavir experienced a significant elevation of liver enzymes. Protease inhibitor therapy was associated with substantial short-term virologic and immunologic improvement in this primarily heavily pretreated cohort, with 25% maintaining a viral load of < 400 copies/ml after 6 months of therapy. There was a significant rate of adverse events. Pharmacokinetic and safety data are needed to guide aggressive antiretroviral therapy in HIV-infected children, and further treatment options are required for those failing or intolerant to the available protease inhibitors.

  11. Evolution of Primary Protease Inhibitor Resistance Mutations during Protease Inhibitor Salvage Therapy

    PubMed Central

    Kantor, Rami; Fessel, W. Jeffrey; Zolopa, Andrew R.; Israelski, Dennis; Shulman, Nancy; Montoya, Jose G.; Harbour, Michael; Schapiro, Jonathan M.; Shafer, Robert W.

    2002-01-01

    In order to track the evolution of primary protease inhibitor (PI) resistance mutations in human immunodeficiency virus type 1 (HIV-1) isolates, baseline and follow-up protease sequences were obtained from patients undergoing salvage PI therapy who presented initially with isolates containing a single primary PI resistance mutation. Among 78 patients meeting study selection criteria, baseline primary PI resistance mutations included L90M (42% of patients), V82A/F/T (27%), D30N (21%), G48V (6%), and I84V (4%). Despite the switching of treatment to a new PI, primary PI resistance mutations present at the baseline persisted in 66 of 78 (85%) patients. D30N persisted less frequently than L90M (50% versus 100%, respectively; P < 0.001) and V82A/F/T (50% versus 81%, respectively; P = 0.05). HIV-1 isolates from 38 (49%) patients failing PI salvage therapy developed new primary PI resistance mutations including L90M, I84V, V82A, and G48V. Common combinations of primary and secondary PI resistance mutations after salvage therapy included mutations at amino acid positions 10, 82, and 46 and/or 54 in 16 patients; 10, 90, and 71 and/or 73 in 14 patients; 10, 73, 84, 90, and 46 and/or 54 in 5 patients; 10, 48, and 82 in 5 patients; and 30, 88 and 90 in 5 patients. In summary, during salvage PI therapy, most HIV-1 isolates with a single primary PI resistance mutation maintained their original mutations, and 49% developed additional primary PI resistance mutations. The persistence of L90M, V82A/F/T, G48V, and I84V during salvage therapy suggests that these mutations play a role in clinical resistance to multiple PIs. PMID:11897594

  12. From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

    PubMed Central

    Cígler, Petr; Kožíšek, Milan; Řezáčová, Pavlína; Brynda, Jíří; Otwinowski, Zbyszek; Pokorná, Jana; Plešek, Jaromír; Grüner, Bohumír; Dolečková-Marešová, Lucie; Máša, Martin; Sedláček, Juraj; Bodem, Jochen; Kräusslich, Hans-Georg; Král, Vladimír; Konvalinka, Jan

    2005-01-01

    HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. PMID:16227435

  13. 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

  14. Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

    PubMed Central

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

  15. Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

    ERIC Educational Resources Information Center

    Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

    2002-01-01

    Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

  16. Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

    ERIC Educational Resources Information Center

    Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

    2002-01-01

    Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

  17. Proteases.

    PubMed

    Barrett, A J

    2001-05-01

    The processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly-synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein--e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

  18. Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors.

    PubMed

    Jones, Kristen L G; Holloway, M Katharine; Su, Hua-Poo; Carroll, Steven S; Burlein, Christine; Touch, Sinoeun; DiStefano, Daniel J; Sanchez, Rosa I; Williams, Theresa M; Vacca, Joseph P; Coburn, Craig A

    2010-07-15

    A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

  19. Protease Inhibitors Do Not Affect Antibody Responses to Pneumococcal Vaccination.

    PubMed

    De La Rosa, Indhira; Munjal, Iona M; Rodriguez-Barradas, Maria; Yu, Xiaoying; Pirofski, Liise-Anne; Mendoza, Daniel

    2016-06-01

    HIV(+) subjects on optimal antiretroviral therapy have persistently impaired antibody responses to pneumococcal vaccination. We explored the possibility that this effect may be due to HIV protease inhibitors (PIs). We found that in humans and mice, PIs do not affect antibody production in response to pneumococcal vaccination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. Protease inhibitors interfere with the necessary factors of carcinogenesis.

    PubMed

    Troll, W

    1989-05-01

    Many tumor promoters are inflammatory agents that stimulate the formation of oxygen radicals (.O2-) and hydrogen peroxide (H2O2) in phagocytic neutrophils. The neutrophils use the oxygen radicals to kill bacteria, which are recognized by the cell membrane of phagocytic cells causing a signal to mount the oxygen response. The tumor promoter isolated from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimics the signal, causing an oxygen radical release that is intended to kill bacteria; instead, it injures cells in the host. Oxygen radicals cause single strand breaks in DNA and modify DNA bases. These damaging reactions appear to be related to tumor promotion, as three types of chemopreventive agents, retinoids, onion oil, and protease inhibitors, suppress the induction of oxygen radicals in phagocytic neutrophils and suppress tumor promotion in skin cancer in mice. Protease inhibitors also suppress breast and colon cancers in mice. Protease inhibitors capable of inhibiting chymotrypsin show a greater suppression of the oxygen effect and are better suppressors of tumor promotion. In addition, oxygen radicals may be one of the many agents that cause activation of oncogenes. Since retinoids and protease inhibitors suppress the expression of the ras oncogene in NIH 3T3 cells, NIH 3T3 cells may serve as a relatively facile model for finding and measuring chemopreventive agents that interfere with the carcinogenic process.

  1. 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.

  2. Mutation Patterns and Structural Correlates in Human Immunodeficiency Virus Type 1 Protease following Different Protease Inhibitor Treatments

    PubMed Central

    Wu, Thomas D.; Schiffer, Celia A.; Gonzales, Matthew J.; Taylor, Jonathan; Kantor, Rami; Chou, Sunwen; Israelski, Dennis; Zolopa, Andrew R.; Fessel, W. Jeffrey; Shafer, Robert W.

    2003-01-01

    Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease—including 22 not previously associated with drug resistance—were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 Å of each other—many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance. PMID:12663790

  3. Boosted protease inhibitors and the electrocardiographic measures of QT and PR durations

    PubMed Central

    Soliman, Elsayed Z.; Lundgren, Jens D.; Roediger, Mollie P.; Duprez, Daniel A.; Temesgen, Zelalem; Bickel, Markus; Shlay, Judith C.; Somboonwit, Charurut; Reiss, Peter; Stein, James H.; Neaton, James D.

    2011-01-01

    Background There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown. Methods This analysis included 3719 participants from the Strategies for Management of Antiretroviral Therapy (SMART) study, of whom 1879 were randomized to receive intermittent antiretroviral therapy (ART) (drug conservation group), whereas the rest received these drugs continuously (viral suppression group). Linear regression analysis was used to compare four ritonavir-boosted protease inhibitor (protease inhibitor/r) regimens [saquinavir (SQV/r), lopinavir (LPV/r), atazanavir (ATV/r), and other protease inhibitor/r], and nonboosted protease inhibitor regimens with nonnucleoside reverse transcriptase inhibitor (NNRTI) regimens for Bazett’s (QTcB) and Fredericia’s (QTcF) heart rate corrected QT and PR. Changes in QTcB, QTcF, and PR after 12 and 24 months of randomization were compared in the drug conservation group and viral suppression group. Results Average levels of QTcB, QTcF, and PR duration at entry were 415, 406, and 158 ms. At study entry, 49% of participants were taking an NNRTI (no protease inhibitor)-based regimen and 31% were prescribed a boosted protease inhibitor, the most common being LPV/r. After adjustment for baseline factors, QTcB and QTcF levels did not vary by boosted protease inhibitor group (P = 0.26 and P = 0.34, respectively). For those given any of the boosted protease inhibitors, QTcB was 1.5 ms lower than the NNRTI group (P = 0.04). Both boosted and nonboosted protease inhibitor-containing regimens were significantly associated (P <0.01 for each) with longer PR intervals compared to the NNRTI group. After adjustment, the difference between boosted protease inhibitors and the NNRTI group was 5.11 ms (P <0.01); for nonboosted protease inhibitors, this difference was 3.00 ms (P <0.01). Following ART

  4. Identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library.

    PubMed

    Shah, Falgun; Mukherjee, Prasenjit; Gut, Jiri; Legac, Jennifer; Rosenthal, Philip J; Tekwani, Babu L; Avery, Mitchell A

    2011-04-25

    Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

  5. Purification and identification of a protease inhibitor from glassfish (Liparis tanakai) eggs.

    PubMed

    Ustadi, Ustadi; Kim, K Y; Kim, S M

    2005-10-05

    Two protease inhibitors of 67 and 18 kDa, respectively, were purified from glassfish, Liparis tanakai, eggs by affinity chromatography. The smaller protein was purified with a yield and purity of 0.25% and 49.69-fold, respectively, and was characterized for further study. The glassfish egg protease inhibitor exhibited stability between 50 and 65 degrees C in an alkaline environment (pH 8). It was shown to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 4.44 nM. Potent glassfish protease inhibitor with N-Val-Gly Ser-Met-Thr-Gly-Gly-Phe-Thr-Asp-C amino acid residues was synthesized and its inhibitory activity was compared. Moreover, the 18-kDa protein inhibited cathepsin, a cysteine protease, more effectively than did egg white protease inhibitor, whereas the reverse was true for papain. Glassfish egg protease inhibitor is classified as a member of the family I cystatins.

  6. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. A Culture-Based Method for Determining the Production of Secreted Protease Inhibitors

    PubMed Central

    Quintero, David; Bermudes, David

    2014-01-01

    We have developed a culture-based method for determining the production of secreted protease inhibitors. The assay utilizes standard proteolysis detection plates to support microbial growth followed by infiltrating the plate with a protease and subsequently detecting the remaining protein by trichloroacetic acid (TCA) precipitation, or by bromocreosol green (BCG) or Ponseau S (PS) staining. The presence of a protease inhibitor can be observed in the form of a protected zone of protein around the protease inhibitor-producing strain. Using the protease inhibitors α-2-macroglobulin, aprotinin, leupeptin, and bestatin and the primary and secondary forms of Photorhabdus luminescens in combination with the protease trypsin, we were able to demonstrate that the assay is specific for the cognate inhibitor of the protease and for bacteria secreting protease inhibitors. In addition, when casein-containing plates were used, the size of the diffusion zone was inversely correlated with the molecular weight of the inhibitor allowing a relative estimation of the protease inhibitor molecular weight. This assay is useful for detecting the presence of microbial secreted protease inhibitors and may reveal their production by microorganisms that were not previously recognized to produce them. PMID:24632514

  8. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    SciTech Connect

    Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M. )

    1989-12-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.

  9. Design and Validation of Novel Chikungunya Virus Protease Inhibitors.

    PubMed

    Das, Pratyush Kumar; Puusepp, Laura; Varghese, Finny S; Utt, Age; Ahola, Tero; Kananovich, Dzmitry G; Lopp, Margus; Merits, Andres; Karelson, Mati

    2016-12-01

    Chikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome. Active forms of replicase enzymes are generated using the autoproteolytic activity of nsP2. The available three-dimensional (3D) structure of nsP2 protease has made it a target for in silico drug design; however, there is thus far little evidence that the designed compounds indeed inhibit the protease activity of nsP2 and/or suppress CHIKV replication. In this study, a set of 12 compounds, predicted to interact with the active center of nsP2 protease, was designed using target-based modeling. The majority of these compounds were shown to inhibit the ability of nsP2 to process recombinant protein and synthetic peptide substrates. Furthermore, all compounds found to be active in these cell-free assays also suppressed CHIKV replication in cell culture, the 50% effective concentration (EC50) of the most potent inhibitor being ∼1.5 μM. Analysis of stereoisomers of one compound revealed that inhibition of both the nsP2 protease activity and CHIKV replication depended on the conformation of the inhibitor. Combining the data obtained from different assays also indicates that some of the analyzed compounds may suppress CHIKV replication using more than one mechanism.

  10. 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

  11. A specific inhibitor of Colletotrichum lindemuthianum protease from kidney bean (Phaseolus vulgaris) seeds.

    PubMed

    Mosolov, V V; Loginova, M D; Malova, E L; Benken, I I

    1979-01-01

    A specific protein-an inhibitor of Colletotrichum lindemuthianum protease-was isolated from kidney bean seeds in a homogeneous form. The purification procedure included gel filtration, isoelectric focusing and affinity chromatography on trypsin-Sepharose column. The latter was introduced to separate the fungal protease inhibitor from closely related trypsin and chymotrypsin inhibitors present in kidney bean seeds.

  12. 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.

  13. Design of HIV Protease Inhibitors Based on Inorganic Polyhedral Metallacarboranes

    SciTech Connect

    Rezacova, Pavlina; Pokorna, Jana; Brynda, Ji; Kozisek, Milan; Cigler, Petr; Lesik, Martin; Fanfrlik, Jindrich; Rezac, Jan; Saskova, Klara Grantz; Sieglova, Irena; Plesek, Jaromir; Sicha, Vaclav; Gruner, Bohumir; Oberwinkler, Heike; Sedlacek, Juraj; Krausslich, Hans-Georg; Hobza, Pavel; Kral, Vladimir; Konvalinka, Jan

    2010-04-19

    HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H{sub 2}N-(8-(C{sub 2}H{sub 4}O){sub 2}-1,2-C{sub 2}B{sub 9}H{sub 10})(1',2'-C{sub 2}B{sub 9}H{sub 11})-3,3'-Co){sub 2}]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.

  14. A New Class of Serine and Cysteine Protease Inhibitor with Chemotherapeutic Potential

    DTIC Science & Technology

    1999-06-01

    also be used to produce a serine protease inhibitor. Similar to the cysteine inhibitors, a dipeptide side chain is attached to the ring which is...which relieves the 7 strain (Figure 3). Serine and cysteine proteases use a mechanism to cleave peptide bonds which involves addition of a catalytic...serine and cysteine proteases share a similar mechanism for hydrolyzing amide bonds , we expect that 4-heterocyclohexanones should be good inhibitors

  15. Ergotism associated with HIV antiviral protease inhibitor therapy.

    PubMed

    Baldwin, Zachary K; Ceraldi, Chris C

    2003-03-01

    Ergotism is a rare condition of acute vasospasm found classically in young and middle-aged women taking ergot alkaloid agents to treat migraine headache. We report the case of a young man with human immunodeficiency virus (HIV) positivity and describe the drug interaction between protease inhibitors and ergot alkaloid agents, which most likely predisposed to development of ergot toxicity. The HIV-positive population receiving antiviral therapy may be an under-recognized group at risk for ergotism through decreased hepatic metabolism of ergot preparations.

  16. Zebra chip disease decreases tuber (Solanum tuberosum L.) protein content by attenuating protease inhibitor levels and increasing protease activities.

    PubMed

    Kumar, G N Mohan; Knowles, Lisa O; Knowles, N Richard

    2015-11-01

    Zebra chip disease of potato decreases protease inhibitor levels resulting in enhanced serine-type protease activity, decreased protein content and altered protein profiles of fully mature tubers. Zebra-chip (ZC), caused by Candidatus Liberibacter solanacearum (CLso), is a relatively new disease of potato that negatively affects growth, yield, propagation potential, and fresh and process qualities of tubers. Diseased plants produce tubers with characteristic brown discoloration of vascular tissue accompanied by elevated levels of free amino acids and reducing sugars. Here we demonstrate that ZC disease induces selective protein catabolism in tubers through modulating protease inhibitor levels. Soluble protein content of tubers from CLso-infected plants was 33% lower than from non-infected plants and electrophoretic analyses revealed substantial reductions in major tuber proteins. Patatin (~40 kDa) and ser-, asp- (22 kDa) and cys-type (85 kDa) protease inhibitors were either absent or greatly reduced in ZC-afflicted tubers. In contrast to healthy (non-infected) tubers, the proteolytic activity in CLso infected tubers was high and the ability of extracts from infected tubers to inhibit trypsin (ser-type) and papain (cys-type) proteases greatly attenuated. Moreover, extracts from CLso-infected tubers rapidly catabolized proteins purified from healthy tubers (40 kDa patatin, 22 kDa protease inhibitors, 85 kDa potato multicystatin) when subjected to proteolysis individually. In contrast, crude extracts from non-infected tubers effectively inhibited the proteolytic activity from ZC-afflicted tubers. These results suggest that the altered protein profile of ZC afflicted tubers is largely due to loss of ser- and cys-type protease inhibitors. Further analysis revealed a novel PMSF-sensitive (ser) protease (ca. 80-120 kDa) in CLso infected tubers. PMSF abolished the proteolytic activities responsible for degrading patatin, the 22 kDa protease inhibitor(s) and potato

  17. Protease inhibitor monotherapy is not associated with increased viral replication in lymph nodes.

    PubMed

    Vinuesa, David; Parra-Ruiz, Jorge; Chueca, Natalia; Alvarez, Marta; Muñoz-Medina, Leopoldo; Garcia, Federico; Hernandez-Quero, Jose

    2014-07-31

    There are concerns about residual viremia in sanctuary sites among patients on protease inhibitor monotherapy, so we aimed to study viro-immunological parameters in tonsil's lymphoid tissue of patients on highly active antiretroviral therapy (HAART) and on protease inhibitor monotherapy. Despite fully suppressed serum HIV viral load, we found viral replication in both groups; in addition, more patients had detectable proviral DNA among those on HAART, compared to those on protease inhibitor monotherapy (P = 0.08), supporting the absence of a deleterious effect of protease inhibitor monotherapy.

  18. Conformational study of a putative HLTV-1 retroviral protease inhibitor.

    PubMed

    Llido, S; d'Estaintot, B L; Dautant, A; Geoffre, S; Picard, P; Precigoux, G

    1993-05-01

    The crystal structure of prolyl-glutaminyl-valyl-statyl-alanyl-leucine (Pro-Gln-Val-Sta-Ala-Leu, C(32)H(57)N(7)0(9).5H(2)0, M(r) = 683.9 + 90.1), a putative HTLV-1 protease inhibitor based on one of the consensus retroviral protease cleavage sequences, and containing the statine residue [(4S,3S)-4-amino-3-hydroxy-6-methylheptanoic acid], has been determined by X-ray diffraction. The same molecule has been modelled in the active site of the HTLV-1 protease and both conformations have been compared. The peptide crystallizes as a pentahydrate in space group P2(1) with a = 10.874(2), b = 9.501(2), c = 21.062(5) A, beta = 103.68 (1) degrees, Z = 2, V= 2114.3 A(3), D(x) = 1.21 g cm(-3), micro = 8.02 cm(-1), T= 293 K, lambda(Cu Kalpha) = 1.5418 A. The structure has been refined to an R value of 0.070 for 2152 observed reflections. The peptide main chain can be described as extended and adopts the usual zigzag conformation from the prolyl to the statyl residue. The main difference in conformation between the individual observed and modelled molecules is located on the Sta, Ala and Leu residues with the main chain of the modelled molecule rotated by about 180 degrees as compared to the observed conformation in the crystal state.

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

    DTIC Science & Technology

    1987-06-30

    CHMOTRYPSIN INHIBITOR (SOYBEANS) CHICKPEAS TRYPSIN-CHYMOTRYPSIN INHIBITOR; SOYBEAN PROTEASE INHIBITORS 20. ABSTRACT (Coninue, on reverse aide It necessary...CI from chickpeas . Attempts are now in progress to separate and isolate these trypsin-and chymotrypsin-like enzymes. (3) Locust proteinases...and from chickpeas (CI). In addition, a specific Tribolium proteinase inhibitor from soybeans was separated. SIGNIFICANT FINDINGS A. The detection of

  20. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  1. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  2. Identification of Genotypic Changes in Human Immunodeficiency Virus Protease That Correlate with Reduced Susceptibility to the Protease Inhibitor Lopinavir among Viral Isolates from Protease Inhibitor-Experienced Patients

    PubMed Central

    Kempf, Dale J.; Isaacson, Jeffrey D.; King, Martin S.; Brun, Scott C.; Xu, Yi; Real, Kathryn; Bernstein, Barry M.; Japour, Anthony J.; Sun, Eugene; Rode, Richard A.

    2001-01-01

    The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with reduced susceptibility. Mutations at positions 82, 54, 10, 63, 71, and 84 were most closely associated with relatively modest (4- and 10-fold) changes in phenotype, while the K20M/R and F53L mutations, in conjunction with multiple other mutations, were associated with >20- and >40-fold-reduced susceptibility, respectively. The median 50% inhibitory concentrations (IC50) of lopinavir against isolates with 0 to 3, 4 or 5, 6 or 7, and 8 to 10 of the above 11 mutations were 0.8-, 2.7-, 13.5-, and 44.0-fold higher, respectively, than the IC50 against wild-type HIV. On average, the IC50 of lopinavir increased by 1.74-fold per mutation in isolates containing three or more mutations. Each of the 16 viruses that displayed a >20-fold change in susceptibility contained mutations at residues 10, 54, 63, and 82 and/or 84, along with a median of three mutations at residues 20, 24, 46, 53, 71, and 90. The number of protease mutations from the 11 identified in these analyses (the lopinavir mutation score) may be useful for the interpretation of HIV genotypic resistance testing with respect to lopinavir-ritonavir (Kaletra) regimens and may provide insight into the genetic barrier to resistance to lopinavir-ritonavir in both antiretroviral therapy-naive and protease inhibitor-experienced patients. PMID:11462018

  3. Elevated concentration of secretory leukocyte protease inhibitor in the cervical mucus before delivery.

    PubMed

    Samejima, Taiki; Nagamatsu, Takeshi; Schust, Danny J; Itaoka, Nao; Iriyama, Takayuki; Nakayama, Toshio; Komatsu, Atsushi; Kawana, Kei; Osuga, Yutaka; Fujii, Tomoyuki

    2016-06-01

    Cervical remodeling during parturition progresses under exquisite regulation by immunologic mediators and proteases. Secretory leukocyte protease inhibitor is a secretory protein that can function as an antimicrobial peptide, an antiinflammatory molecule, and a protease inhibitor. The involvement of secretory leukocyte protease inhibitor in cervical remodeling before and during parturition is understood poorly. We aimed to reveal the role of secretory leukocyte protease inhibitor in the cervical remodeling process before normal term delivery and to evaluate its utility as a predictive biomarker for timing of delivery. Cervical mucus samples were collected prospectively at weekly prenatal visits from a cohort of pregnant women at term. The secretory leukocyte protease inhibitor concentrations in 95 mucus samples that were obtained from 49 women with uncomplicated pregnancy who subsequently underwent normal vaginal delivery were assessed. Alterations in secretory leukocyte protease inhibitor concentrations at term and the association of secretory leukocyte protease inhibitor levels with the time to delivery were analyzed. A moderate positive correlation with significance was detected between cervical mucus secretory leukocyte protease inhibitor concentrations and days to delivery (r = 0.38; P = .0001). The secretory leukocyte protease inhibitor concentration was significantly higher in samples that were collected within 7 days of delivery when compared with samples that were collected >7 days before delivery (P = .001). Secretory leukocyte protease inhibitor concentrations were also significantly higher in samples from women with premature rupture of membranes when compared with those without premature rupture of membranes (P = .01), all of whom delivered within 7 days. A logistic regression analysis revealed that the cervical secretory leukocyte protease inhibitor level was a significant parameter for the prediction of the onset of delivery. (P = .017; unit

  4. Proteinaceous protease inhibitor from Lawsonia inermis: purification, characterization and antibacterial activity.

    PubMed

    Dabhade, Arvind; Patel, Priti; Pati, Ulhas

    2013-10-01

    A thermo-stable, proteinaceous protease inhibitor (LPI) from Lawsonia inermis is reported. The LPI was purified from Lawsonia inermis seeds by subsequent ammonium sulfate precipitation, ion exchange chromatography (DEAE-Cellulose) and gel permeation chromatography (Sephadex-50). The purified protease inhibitor is effective against a wide range of proteases viz. papain, trypsin, pepsin and metallo-protease. The apparent molecular weight of the protease inhibitor is 19 kDa, determined by SDS-PAGE electrophoresis. The protease inhibitor was found to be stable at 70 degrees C for 30 min. It was also examined for antibacterial activity against Pseudomonas aeruginosa MTCC 7926 and Staphylococcus aureus NCIM 2079; the IC50 values of the purified LPI were 11.4 microg/mL and 16.6 microg/mL respectively.

  5. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Ballard, Jeanine E; Bennett, David J; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S; Chang, Ronald K; Dubost, David C; Fay, John F; Diamond, Tracy L; Greshock, Thomas J; Hao, Li; Holloway, M Katharine; Felock, Peter J; Gesell, Jennifer J; Su, Hua-Poo; Manikowski, Jesse J; McKay, Daniel J; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M; Nantermet, Philippe G; Nadeau, Christian; Sanchez, Rosa I; Satyanarayana, Tummanapalli; Shipe, William D; Singh, Sanjay K; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M; Vacca, Joseph P; Crane, Sheldon N; McCauley, John A

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  6. Autoantibodies directed against the protease inhibitor calpastatin in psoriasis

    PubMed Central

    Matsushita, Y; Shimada, Y; Kawara, S; Takehara, K; Sato, S

    2005-01-01

    Psoriasis is believed to be a T cell-mediated autoimmune disease, but also exhibits autoantibody production. Calpastatin is an endogenous inhibitor of calpain, a ubiquitous protease that regulates inflammatory processes. Anti-calpastatin autoantibody was first identified as an autoantibody specific to rheumatoid arthritis, but has been also detected in other autoimmune diseases. In this study, we examined the presence and levels of anti-calpastatin antibody in 77 psoriasis patients by enzyme-linked immunosorbent assay. Compared with normal controls, psoriasis patients exhibited significantly elevated IgG anti-calpastatin antibody levels that were similar to those found in rheumatoid arthritis patients. Remarkably, IgG anti-calpastatin autoantibody in sera from psoriasis patients inhibited calpastatin activity. Calpain II expression was up-regulated in psoriasis skin lesions compared with normal skin while calpastatin expression was normal. The results of this study reveal the presence of anti-calpastatin autoantibody in psoriasis. PMID:15654835

  7. Cardiovascular considerations in patients treated with HIV protease inhibitors.

    PubMed

    Colagreco, Joseph P

    2004-01-01

    Highly active antiretroviral therapy (HAART) has dramatically reduced mortality from HIV infection, transforming it in many cases to a chronic condition. However, protease inhibitors (PIs), which are integral components of most HAART regimens, are commonly associated with a host of metabolic disturbances that may increase the risk of cardiovascular disease in patients with HIV infection, potentially counteracting some of the positive health effects of PIs. Dyslipidemia is of particular concern. The Adult AIDS Clinical Trials Group has established preliminary guidelines to evaluate and treat PI-associated dyslipidemia. A number of strategies exist for the management of PI-based dyslipidemia in HAART recipients; their advantages and disadvantages should be considered when treating patients with HIV infection.

  8. Development of potent inhibitors of the coxsackievirus 3C protease

    SciTech Connect

    Lee, Eui Seung; Lee, Won Gil; Yun, Soo-Hyeon; Rho, Seong Hwan; Im, Isak; Yang, Sung Tae; Sellamuthu, Saravanan; Lee, Yong Jae; Kwon, Sun Jae; Park, Ohkmae K.; Jeon, Eun-Seok; Park, Woo Jin . E-mail: wjpark@gist.ac.kr; Kim, Yong-Chul . E-mail: yongchul@gist.ac.kr

    2007-06-22

    Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.

  9. Rhodanine derivatives as selective protease inhibitors against bacterial toxins.

    PubMed

    Johnson, Sherida L; Chen, Li-Hsing; Harbach, Rebecca; Sabet, Mojgan; Savinov, Alexei; Cotton, Naomi J H; Strongin, Alex; Guiney, Donald; Pellecchia, Maurizio

    2008-02-01

    In this study, we analyzed a series of rhodanine derivatives, as potential inhibitors of bacterial toxins, namely the proteases anthrax lethal factor and the botulinum neurotoxin type A. Conducting an extensive structure-activity relationship study on rhodanine derivatives, we profiled their selectivity against the two bacterial toxins and two related human metalloproteases using in vitro assays. In addition, we examined initial in vitro ADME-Tox properties of selected compounds and their ability to protect lethal factor-induced cell death of macrophages. These data allowed the selection of one additional drug candidate for which preliminary in vivo efficacy studies against anthrax spores were conducted. Integration of these results with our structure-activity relationship studies provides a framework for the development of potential drug candidates against anthrax and botulinum.

  10. 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.

  11. Mechanism-Based Inhibitors of Serine Proteases with High Selectivity Through Optimization of S’ Subsite Binding

    PubMed Central

    Li, Yi; Dou, Dengfeng; He, Guijia; Lushington, Gerald H.; Groutas, William C.

    2009-01-01

    A series of mechanism-based inhibitors designed to interact with the S’ subsites of serine proteases was synthesized and their inhibitory activity toward the closely-related serine proteases human neutrophil elastase (HNE) and proteinase 3 (PR 3) was investigated. The compounds were found to be time-dependent inhibitors of HNE and were devoid of any inhibitory activity toward PR 3. The results suggest that highly selective inhibitors of serine proteases whose primary substrate specificity and active sites are similar can be identified by exploiting differences in their S’ subsites. The best inhibitor (compound 16) had a kinact/KI value of 4580 M−1 s−1. PMID:19394830

  12. High-throughput screening of improved protease inhibitors using a yeast cell surface display system and a yeast cell chip.

    PubMed

    Aoki, Wataru; Yoshino, Yuichi; Morisaka, Hironobu; Tsunetomo, Keiji; Koyo, Hirotaka; Kamiya, Shinji; Kawata, Noriyuki; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2011-01-01

    Protease-targeted inhibitors have been promising pharmaceuticals. Here, we combined a yeast cell surface display system with a yeast cell chip for the high-throughput screening of protease inhibitors, and succeeded in improving the activity of a protease inhibitor.

  13. Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).

    PubMed

    Sharma, Ranu; Suresh, C G

    2015-01-01

    Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein-ligand and inter and intramolecular protein-protein hydrogen bonds. The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Amelioration of immune complex-mediated glomerulonephritis by synthetic protease inhibitors.

    PubMed

    Jennette, J C; Tidwell, R R; Geratz, J D; Bing, D H; Falk, R J

    1987-06-01

    Proteases are involved in the pathogenesis of inflammatory diseases by participating in the activation of mediator systems and by producing proteolytic tissue injury. Homeostatic control of inflammation is accomplished in part by physiologic protease inhibitors. The authors investigated the effectiveness of a number of synthetic protease inhibitors in ameliorating the glomerular injury induced by immune complex-mediated glomerulonephritis in mice. Two amidine-type protease inhibitors, bis (5-amidino-2-benzimidazolyl)methane and 1,2-bis (5-amidino-2-benzimidazolyl)ethane, had the greatest effects. They caused a marked reduction in glomerular necrosis (P less than 0.001) but did not affect the amount or site of immune complex localization or leukocyte influx. The inhibition constants of the protease inhibitors against nine purified physiologic proteases were determined. These results were discussed in relation to the effectiveness of the protease inhibitors in reducing glomerular injury. This investigation indicates that the administration of synthetic protease inhibitors can have a beneficial effect on immune-mediated inflammatory injury.

  15. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  16. PlantPIs--an interactive web resource on plant protease inhibitors.

    PubMed

    Consiglio, Arianna; Grillo, Giorgio; Licciulli, Flavio; Ceci, Luigi R; Liuni, Sabino; Losito, Nicola; Volpicella, Mariateresa; Gallerani, Raffaele; De Leo, Francesca

    2011-08-01

    PlantPIs is a web querying system for a database collection of plant protease inhibitors data. Protease inhibitors in plants are naturally occurring proteins that inhibit the function of endogenous and exogenous proteases. In this paper the design and development of a web framework providing a clear and very flexible way of querying plant protease inhibitors data is reported. The web resource is based on a relational database, containing data of plants protease inhibitors publicly accessible, and a graphical user interface providing all the necessary browsing tools, including a data exporting function. PlantPIs contains information extracted principally from MEROPS database, filtered, annotated and compared with data stored in other protein and gene public databases, using both automated techniques and domain expert evaluations. The data are organized to allow a flexible and easy way to access stored information. The database is accessible at http://www.plantpis.ba.itb.cnr.it/.

  17. [Advances in researches on epididymal WFDC-type serine protease inhibitors].

    PubMed

    Liu, Juan; Wang, Hai-Yan; Li, Jian-Yuan

    2008-11-01

    Sperm maturation in the epididymis is regulated by changes of luminal ion concentration and processing of sperm surface membrane by several glycosidases and proteases, and the actions of the proteases are controlled by protease inhibitors present in specific areas of the epididymis. WFDC-type serine protease inhibitors that are highly expressed in the epididymis play an important role in natural immunity and male reproduction. This paper gives an overview of the structure and function of the protein and its application prospects in the development of drugs for male reproductive tract infection and immunocontraception.

  18. Resistance mechanism of human immunodeficiency virus type-1 protease to inhibitors: A molecular dynamic approach

    PubMed Central

    Dayer, Mohammad Reza; Dayer, Mohammad Saaid

    2014-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease inhibitors comprise an important class of drugs used in HIV treatments. However, mutations of protease genes accelerated by low fidelity of reverse transcriptase yield drug resistant mutants of reduced affinities for the inhibitors. This problem is considered to be a serious barrier against HIV treatment for the foreseeable future. In this study, molecular dynamic simulation method was used to examine the combinational and additive effects of all known mutations involved in drug resistance against FDA approved inhibitors. Results showed that drug resistant mutations are not randomly distributed along the protease sequence; instead, they are localized on flexible or hot points of the protein chain. Substitution of more hydrophobic residues in flexible points of protease chains tends to increase the folding, lower the flexibility and decrease the active site area of the protease. The reduced affinities of HIV-1 protease for inhibitors seemed to be due to substantial decrease in the size of the active site and flap mobility. A correlation was found between the binding energy of inhibitors and their affinities for each mutant suggesting the distortion of the active site geometry in drug resistance by preventing effective fitting of inhibitors into the enzymes' active site. To overcome the problem of drug resistance of HIV-1 protease, designing inhibitors of variable functional groups and configurations is proposed. PMID:27843989

  19. Triple Therapy with First Generation Protease Inhibitors for Hepatitis C Markedly Impairs Function of Neutrophil Granulocytes.

    PubMed

    Spindelboeck, Walter; Horvath, Angela; Tawdrous, Monika; Schmerböck, Bianca; Zettel, Gabriele; Posch, Andreas; Streit, Andrea; Jurse, Petra; Lemesch, Sandra; Horn, Martin; Wuensch, Gerit; Stiegler, Philipp; Stauber, Rudolf E; Leber, Bettina; Stadlbauer, Vanessa

    2016-01-01

    First-generation HCV protease inhibitors represent a milestone in antiviral therapy for chronic hepatitis C infection (CHC), but substantially increased rates of viral clearance are offset by increased rates of infection and infection-associated deaths, especially of patients with advanced liver disease. We aimed to assess whether first generation protease inhibitors interfere with neutrophil function. We included 108 consecutive, retrospective CHC patients and 44 consecutive, prospective CHC patients who were treated with peginterferon and ribavirin with or without protease inhibitors according to the guidelines in the period of November 2012 to June 2015. 33 healthy volunteers served as controls. Infection data were evaluated in all patients. Neutrophil phagocytosis, oxidative burst, elastase and diamine oxidase levels during 12 weeks of triple (n = 23) or dual therapy (n = 21) were studied in the prospective part. In the retro- and prospective cohorts patients experiencing clinically relevant infections were significantly more frequent during protease inhibitor therapy (31% and 26%) than during therapy with peginterferon and ribavirin (13% and 0%). Neutrophil phagocytosis decreased to 40% of baseline with addition of protease inhibitors to P/R but recovered 6 months after end of treatment. Protease inhibitors also seemed to reduce serum elastase levels but did not impact on gut permeability. Impaired neutrophil function during triple therapy with first generation HCV protease inhibitors may explain the high infection rate associated to these treatments and be of relevance for treatment success and patient survival.

  20. 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

  1. Prediction of HIV-1 protease inhibitor resistance using a protein-inhibitor flexible docking approach.

    PubMed

    Jenwitheesuk, Ekachai; Samudrala, Ram

    2005-01-01

    Emergence of drug resistance remains one of the most challenging issues in the treatment of HIV-1 infection. Here we focus on resistance to HIV-1 protease inhibitors (PIs) at a molecular level, which can be analysed genotypically or phenotypically. Genotypic assays are based on the analysis of mutations associated with reduced drug susceptibility, but are problematic because of the numerous mutations and mutational patterns that confer drug resistance. Phenotypic resistance or susceptibility can be experimentally evaluated by measuring the amount of free drug bound to HIV-1 protease molecules, but this procedure is expensive and time-consuming. To overcome these problems, we have developed a docking protocol that takes protein-inhibitor flexibility into account to predict phenotypic drug resistance. For six FDA-approved Pls and a total of 1792 HIV-1 protease sequence mutants, we used a combination of inhibitor flexible docking and molecular dynamics (MD) simulations to calculate protein-inhibitor binding energies. Prediction results were expressed as fold changes of the calculated inhibitory constant (Ki), and the samples predicted to have fold-increase in calculated Ki above the fixed cut-off were defined as drug resistant. Our combined docking and MD protocol achieved accuracies ranging from 72-83% in predicting resistance/susceptibility for five of the six drugs evaluated. Evaluating the method only on samples where our predictions concurred with established knowledge-based methods resulted in increased accuracies of 83-94% for the six drugs. The results suggest that a physics-based approach, which is readily applicable to any novel PI and/or mutant, can be used judiciously with knowledge-based approaches that require experimental training data to devise accurate models of HIV-1 Pl resistance prediction.

  2. Modulation of the epithelial sodium channel (ENaC) by bacterial metalloproteases and protease inhibitors.

    PubMed

    Butterworth, Michael B; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M; Thibodeau, Patrick H

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  3. Modulation of the Epithelial Sodium Channel (ENaC) by Bacterial Metalloproteases and Protease Inhibitors

    PubMed Central

    Butterworth, Michael B.; Zhang, Liang; Liu, Xiaoning; Shanks, Robert M.; Thibodeau, Patrick H.

    2014-01-01

    The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP) from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC), leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions. PMID:24963801

  4. Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

    PubMed Central

    Sutherland, Katherine A.; Parry, Chris M.; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Gilks, Charles F.; Goodall, Ruth; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Gupta, Ravindra K.

    2015-01-01

    Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic

  5. Calcineurin mediates pancreatic growth in protease inhibitor-treated mice.

    PubMed

    Tashiro, Mitsuo; Samuelson, Linda C; Liddle, Rodger A; Williams, John A

    2004-05-01

    CCK acts on pancreatic acinar cells to increase intracellular Ca(2+) leading to secretion of digestive enzymes and, in the long term, pancreatic growth. Calcineurin (CN) is a serine/threonine-specific protein phosphatase activated by Ca(2+) and calmodulin that recently has been shown to participate in the growth regulation of cardiac and skeletal myocytes. We therefore tested the effect of two different CN inhibitors, cyclosporine A (CsA) and FK506, on mouse pancreatic growth induced by oral administration of the synthetic protease inhibitor camostat, a known stimulator of endogenous CCK release. Mice were fed a powdered diet with or without 0.1% camostat. Pancreatic wet weight, protein, and DNA were increased in response to camostat in a time-dependent manner over 10 days in ICR mice but not in CCK-deficient mice. Both CsA (15 mg/kg) and FK506 (3 mg/kg) given twice daily blocked the increase in pancreatic wet weight and protein and DNA content induced by camostat. The increase in plasma CCK induced by camostat was not blocked by CsA or FK506. Camostat feeding also increased the relative amount of CN protein, whereas levels of MAPKs, ERKs, and p38 were not altered. In summary, 1) CCK released by chronic camostat feeding induces pancreatic growth in mice; 2) this growth is blocked by treatment with both CsA and FK506, indicating a role for CN; 3) CCK stimulation also increases CN protein. In conclusion, activation and possibly upregulation of CN may participate in regulation of pancreatic growth by CCK in mice.

  6. Protease-inhibitor interaction predictions: Lessons on the complexity of protein-protein interactions.

    PubMed

    Fortelny, Nikolaus; Butler, Georgina S; Overall, Christopher Mark; Pavlidis, Paul

    2017-04-06

    Protein interactions shape proteome function and thus biology. Identification of protein interactions is a major goal in molecular biology, but biochemical methods, although improving, remain limited in coverage and accuracy. Whereas computational predictions can guide biochemical experiments, low validation rates of predictions remain a major limitation. Here, we investigated computational methods in the prediction of a specific type of interaction, the inhibitory interactions between proteases and their inhibitors. Proteases generate thousands of proteoforms that dynamically shape the functional state of proteomes. Despite the important regulatory role of proteases, knowledge of their inhibitors remains largely incomplete with the vast majority of proteases lacking an annotated inhibitor. To link inhibitors to their target proteases on a large scale, we applied computational methods to predict inhibitory interactions between proteases and their inhibitors based on complementary data including coexpression, phylogenetic similarity, structural information, co-annotation, and colocalization, and also surveyed general protein interaction networks for potential inhibitory interactions. In testing nine predicted interactions biochemically, we validated the inhibition of kallikrein 5 by serpin B12. Despite the use of a wide array of complementary data, we found a high false positive rate of computational predictions in biochemical follow-up. Based on a protease-specific definition of true negatives derived from the biochemical classification of proteases and inhibitors, we analyzed prediction accuracy of individual features. Thereby we identified feature-specific limitations, which also affected general protein interaction prediction methods. Interestingly, proteases were often not coexpressed with most of their functional inhibitors, contrary to what is commonly assumed and extrapolated predominantly from cell culture experiments. Predictions of inhibitory interactions

  7. Protease Inhibitors of Parasitic Flukes: Emerging Roles in Parasite Survival and Immune Defence.

    PubMed

    Ranasinghe, Shiwanthi L; McManus, Donald P

    2017-05-01

    Protease inhibitors play crucial roles in parasite development and survival, counteracting the potentially damaging immune responses of their vertebrate hosts. However, limited information is currently available on protease inhibitors from schistosomes and food-borne trematodes. Future characterization of these molecules is important not only to expand knowledge on parasitic fluke biology but also to determine whether they represent novel vaccine and/or drug targets. Moreover, protease inhibitors from flukes may represent lead compounds for the development of a new range of therapeutic agents against inflammatory disorders and cancer. This review discusses already identified protease inhibitors of fluke origin, emphasizing their biological function and their possible future development as new intervention targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Interspecific Differences between D. pulex and D. magna in Tolerance to Cyanobacteria with Protease Inhibitors

    PubMed Central

    Kuster, Christian J.; Von Elert, Eric

    2013-01-01

    It is known that cyanobacteria negatively affect herbivores due to their production of toxins such as protease inhibitors. In the present study we investigated potential interspecific differences between two major herbivores, Daphnia magna and Daphnia pulex, in terms of their tolerance to cyanobacteria with protease inhibitors. Seven clones each of D. magna and of D. pulex were isolated from different habitats in Europe and North America. To test for interspecific differences in the daphnids’ tolerance to cyanobacteria, their somatic and population growth rates were determined for each D. magna and D. pulex clone after exposure to varying concentrations of two Microcystis aeruginosa strains. The M. aeruginosa strains NIVA and PCC− contained either chymotrypsin or trypsin inhibitors, but no microcystins. Mean somatic and population growth rates on a diet with 20% NIVA were significantly more reduced in D. pulex than in D. magna. On a diet with 10% PCC−, the population growth of D. pulex was significantly more reduced than that of D. magna. This indicates that D. magna is more tolerant to cyanobacteria with protease inhibitors than D. pulex. The reduction of growth rates was possibly caused by an interference of cyanobacterial inhibitors with proteases in the gut of Daphnia, as many other conceivable factors, which might have been able to explain the reduced growth, could be excluded as causal factors. Protease assays revealed that the sensitivities of chymotrypsins and trypsins to cyanobacterial protease inhibitors did not differ between D. magna and D. pulex. However, D. magna exhibited a 2.3-fold higher specific chymotrypsin activity than D. pulex, which explains the observed higher tolerance to cyanobacterial protease inhibitors of D. magna. The present study suggests that D. magna may control the development of cyanobacterial blooms more efficiently than D. pulex due to differences in their tolerance to cyanobacteria with protease inhibitors. PMID:23650523

  9. Assessment of the relative contribution of different protease inhibitors to the inhibition of plasmin in vivo.

    PubMed

    Levi, M; Roem, D; Kamp, A M; de Boer, J P; Hack, C E; ten Cate, J W

    1993-02-01

    It has been shown that the most important inhibitor of plasmin is alpha 2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed. To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin. It was confirmed that alpha 2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than alpha 2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-alpha 1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with alpha 2-macroglobulin and with antithrombin III were significantly elevated. In conclusion, we confirmed the important role of alpha 2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

    PubMed Central

    Tisdale, M; Myers, R E; Maschera, B; Parry, N R; Oliver, N M; Blair, E D

    1995-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy. PMID:7486905

  11. A novel class of cysteine protease inhibitors: solution structure of staphostatin A from Staphylococcus aureus.

    PubMed

    Dubin, Grzegorz; Krajewski, Marcin; Popowicz, Grzegorz; Stec-Niemczyk, Justyna; Bochtler, Matthias; Potempa, Jan; Dubin, Adam; Holak, Tad A

    2003-11-25

    A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of staphostatins, endogenous, highly specific, staphylococcal cysteine protease inhibitors, opened a possibility for structure-based design of low molecular weight analogues. Moreover, the crystal structure of staphostatin B revealed a distinct folding pattern and an unexpected, substrate-like binding mode. The solution structure of staphostatin A reported here confirms that staphostatins constitute a novel, distinct class of cysteine protease inhibitors. In addition, the structure knowledge-based mutagenesis studies shed light on individual structural features of staphostatin A, the inhibition mechanism, and the determinants of distinct specificity of staphostatins toward their target proteases.

  12. Metabolic Disorders in HIV-Infected Adolescents Receiving Protease Inhibitors

    PubMed Central

    Santiprabhob, Jeerunda; Tanchaweng, Surapong; Maturapat, Sirinoot; Lermankul, Watcharee; Sricharoenchai, Sirintip; Wittawatmongkol, Orasri; Lapphra, Keswadee; Phongsamart, Wanatpreeya

    2017-01-01

    Protease inhibitor (PI) may cause abnormal glucose metabolism, abnormal lipid metabolism, and metabolic syndrome in HIV-infected adults but less well studied in Asian adolescents. This cross-sectional study evaluated anthropometric factors, oral glucose tolerance test, and lipid profiles of perinatally HIV-infected Thai adolescents who had received PI-based antiretroviral therapy for at least 6 months. Eighty adolescents were enrolled [median (IQR) age 16.7 (14.6–18.0) years, 42 males]. Metabolic syndrome, prediabetes, and type 2 diabetes mellitus (T2DM) were found in 8 (10%), 17 (22.1%), and 3 (3.8%) adolescents, respectively. Dyslipidemia was found in 56 (70%) adolescents, with hypertriglyceridemia being the most common type. In multivariate analysis, presence of lipohypertrophy (OR: 25.7, 95% CI: 3.2–202.8; p = 0.002) and longer duration of PI use (OR: 1.04, 95% CI: 1.00–1.08; p = 0.023) were associated with metabolic syndrome. Obesity (OR: 7.71, 95% CI: 1.36–43.7; p = 0.021), presence of lipohypertrophy (OR: 62.9, 95% CI: 4.97–795.6; p = 0.001), and exposure to stavudine for ≥6 months (OR: 8.18, 95% CI: 1.37–48.7; p = 0.021) were associated with prediabetes/T2DM, while exposure to tenofovir for ≥6 months reduced the risk (OR: 0.17, 95% CI: 0.04–0.78; p = 0.022). Metabolic disorders were commonly found in adolescents receiving PI. Careful monitoring and early intervention to modify cardiovascular risk should be systematically implemented in this population particularly those with exposure to stavudine. PMID:28293638

  13. Haloperidol-based irreversible inhibitors of the HIV-1 and HIV-2 proteases.

    PubMed

    De Voss, J J; Sui, Z; DeCamp, D L; Salto, R; Babé, L M; Craik, C S; Ortiz de Montellano, P R

    1994-03-04

    The proteases expressed by the HIV-1 and HIV-2 viruses process the polyproteins encoded by the viral genomes into the mature proteins required for virion replication and assembly. Eight analogs of haloperidol have been synthesized that cause time-dependent inactivation of the HIV-1 protease and, in six cases, HIV-2 protease. The IC50 values for the analogues are comparable to that of haloperidol itself. Enzyme inactivation is due to the presence of an epoxide in two of the analogues and carbonyl-conjugated double or triple bonds in the others. Irreversible inactivation is confirmed by the failure to recover activity when one of the inhibitors is removed from the medium. At pH 8.0, the agents inactivate the HIV-1 protease 4-80 times more rapidly than the HIV-2 protease. Faster inactivation of the HIV-1 protease is consistent with alkylation of cysteine residues because the HIV-1 protease has four such residues whereas the HIV-2 protease has none. Inactivation of the HIV-2 protease requires modification of non-cysteine residues. The similarities in the rates of inactivation of the HIV-2 protease by six agents that have intrinsically different reactivities toward nucleophiles suggest that the rate-limiting step in the inactivation process is not the alkylation reaction itself. At least five of the agents inhibit polyprotein processing in an ex vivo cell assay system, but they are also toxic to the cells.

  14. Cysteine Proteases Inhibitors with immunoglobulin-like fold in protozoan parasites and their role in pathogenesis.

    PubMed

    Jimenez-Sandoval, Pedro; Lopez-Castillo, Laura Margarita; Trasviña-Arenas, Carlos H; Brieba, Luis G

    2016-08-13

    The number of protein folds in nature is limited, thus is not surprising that proteins with the same fold are able to exert different functions. The cysteine protease inhibitors that adopt an immunoglobulin-like fold (Ig-ICPs) are inhibitors encoded in bacteria and protozoan parasites. Structural studies indicate that these inhibitors resemble the structure of archetypical proteins with an Ig fold, like antibodies, cadherins or cell receptors. The structure of Ig-ICPs from four different protozoan parasites clearly shows the presence of three loops that form part of a protein-ligand interaction surface that resembles the antigen binding sites of antibodies. Thus, Ig-ICPs bind to different cysteine proteases using a tripartite mechanism in which their BC, DE and FG loops are responsible for the main interactions with the target cysteine protease. Ig-ICPs from different protozoan parasites regulate the enzymatic activity of host or parasite&#039;s proteases and thus regulate virulence and pathogenesis.

  15. Identification of inhibitors using a cell based assay for monitoring golgi-resident protease activity

    PubMed Central

    Coppola, Julia M.; Hamilton, Christin A.; Bhojani, Mahaveer S.; Larsen, Martha J.; Ross, Brian D.; Rehemtulla, Alnawaz

    2007-01-01

    Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE. PMID:17316541

  16. New protease inhibitors for the treatment of chronic hepatitis C: a cost-effectiveness analysis.

    PubMed

    Liu, Shan; Cipriano, Lauren E; Holodniy, Mark; Owens, Douglas K; Goldhaber-Fiebert, Jeremy D

    2012-02-21

    Chronic hepatitis C virus is difficult to treat and affects approximately 3 million Americans. Protease inhibitors increase the effectiveness of standard therapy, but they are costly. A genetic assay may identify patients most likely to benefit from this treatment advance. To assess the cost-effectiveness of new protease inhibitors and an interleukin (IL)-28B genotyping assay for treating chronic hepatitis C virus. Decision-analytic Markov model. Published literature and expert opinion. Treatment-naive patients with chronic, genotype 1 hepatitis C virus monoinfection. Lifetime. Societal. Strategies are defined by the use of IL-28B genotyping and type of treatment (standard therapy [pegylated interferon with ribavirin]; triple therapy [standard therapy and a protease inhibitor]). Interleukin-28B-guided triple therapy stratifies patients with CC genotypes to standard therapy and those with non-CC types to triple therapy. Discounted costs (in 2010 U.S. dollars) and quality-adjusted life-years (QALYs); incremental cost-effectiveness ratios. For patients with mild and advanced fibrosis, universal triple therapy reduced the lifetime risk for hepatocellular carcinoma by 38% and 28%, respectively, and increased quality-adjusted life expectancy by 3% and 8%, respectively, compared with standard therapy. Gains from IL-28B-guided triple therapy were smaller. If the protease inhibitor costs $1100 per week, universal triple therapy costs $102,600 per QALY (mild fibrosis) or $51,500 per QALY (advanced fibrosis) compared with IL-28B-guided triple therapy and $70,100 per QALY (mild fibrosis) and $36,300 per QALY (advanced fibrosis) compared with standard therapy. Results were sensitive to the cost of protease inhibitors and treatment adherence rates. Data on the long-term comparative effectiveness of the new protease inhibitors are lacking. Both universal triple therapy and IL-28B-guided triple therapy are cost-effective when the least-expensive protease inhibitor are used for

  17. Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease.

    PubMed

    Rosin, C D; Belew, R K; Morris, G M; Olson, A J; Goodsell, D S

    1999-02-16

    We have developed a coevolutionary method for the computational design of HIV-1 protease inhibitors selected for their ability to retain efficacy in the face of protease mutation. For HIV-1 protease, typical drug design techniques are shown to be ineffective for the design of resistance-evading inhibitors: An inhibitor that is a direct analogue of one of the natural substrates will be susceptible to resistance mutation, as will inhibitors designed to fill the active site of the wild-type or a mutant enzyme. Two design principles are demonstrated: (i) For enzymes with broad substrate specificity, such as HIV-1 protease, resistance-evading inhibitors are best designed against the immutable properties of the active site-the properties that must be conserved in any mutant protease to retain the ability to bind and cleave all of the native substrates. (ii) Robust resistance-evading inhibitors can be designed by optimizing activity simultaneously against a large set of mutant enzymes, incorporating as much of the mutational space as possible.

  18. Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease

    PubMed Central

    Rosin, Christopher D.; Belew, Richard K.; Morris, Garrett M.; Olson, Arthur J.; Goodsell, David S.

    1999-01-01

    We have developed a coevolutionary method for the computational design of HIV-1 protease inhibitors selected for their ability to retain efficacy in the face of protease mutation. For HIV-1 protease, typical drug design techniques are shown to be ineffective for the design of resistance-evading inhibitors: An inhibitor that is a direct analogue of one of the natural substrates will be susceptible to resistance mutation, as will inhibitors designed to fill the active site of the wild-type or a mutant enzyme. Two design principles are demonstrated: (i) For enzymes with broad substrate specificity, such as HIV-1 protease, resistance-evading inhibitors are best designed against the immutable properties of the active site—the properties that must be conserved in any mutant protease to retain the ability to bind and cleave all of the native substrates. (ii) Robust resistance-evading inhibitors can be designed by optimizing activity simultaneously against a large set of mutant enzymes, incorporating as much of the mutational space as possible. PMID:9990030

  19. Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors.

    PubMed

    Zeminian, Luciana Bonome; Padovani, Juliana Lara; Corvino, Sílvia Maria; Silva, Giovanni Faria; Pardini, Maria Inês de Moura Campos; Grotto, Rejane Maria Tommasini

    2013-02-01

    The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

  20. Peptide inhibitors of Dengue virus NS3 protease. Part 1: Warhead.

    PubMed

    Yin, Zheng; Patel, Sejal J; Wang, Wei-Ling; Wang, Gang; Chan, Wai-Ling; Rao, K R Ranga; Alam, Jenefer; Jeyaraj, Duraiswamy A; Ngew, Xinyi; Patel, Viral; Beer, David; Lim, Siew Pheng; Vasudevan, Subhash G; Keller, Thomas H

    2006-01-01

    Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a K(i) of 43 nM.

  1. Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain.

    PubMed

    McShan, Danielle; Kathman, Stefan; Lowe, Brittiney; Xu, Ziyang; Zhan, Jennifer; Statsyuk, Alexander; Ogungbe, Ifedayo Victor

    2015-10-15

    Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic assays will further expand the use of the covalent tethering method, a simple fragment-based drug discovery technique to discover covalent drug leads.

  2. Design of HIV Protease Inhibitors Targeting Protein Backbone: An Effective Strategy for Combating Drug Resistance

    SciTech Connect

    Ghosh, Arun K.; Chapsal, Bruno D.; Weber, Irene T.; Mitsuya, Hiroaki

    2008-06-03

    The discovery of human immunodeficiency virus (HIV) protease inhibitors (PIs) and their utilization in highly active antiretroviral therapy (HAART) have been a major turning point in the management of HIV/acquired immune-deficiency syndrome (AIDS). However, despite the successes in disease management and the decrease of HIV/AIDS-related mortality, several drawbacks continue to hamper first-generation protease inhibitor therapies. The rapid emergence of drug resistance has become the most urgent concern because it renders current treatments ineffective and therefore compels the scientific community to continue efforts in the design of inhibitors that can efficiently combat drug resistance.

  3. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases

    PubMed Central

    2015-01-01

    A novel fragment-based drug discovery approach is reported which irreversibly tethers drug-like fragments to catalytic cysteines. We attached an electrophile to 100 fragments without significant alterations in the reactivity of the electrophile. A mass spectrometry assay discovered three nonpeptidic inhibitors of the cysteine protease papain. The identified compounds display the characteristics of irreversible inhibitors. The irreversible tethering system also displays specificity: the three identified papain inhibitors did not covalently react with UbcH7, USP08, or GST-tagged human rhinovirus 3C protease. PMID:24870364

  4. Second locus involved in human immunodeficiency virus type 1 resistance to protease inhibitors.

    PubMed Central

    Doyon, L; Croteau, G; Thibeault, D; Poulin, F; Pilote, L; Lamarre, D

    1996-01-01

    Protease inhibitors are potent antiviral agents against human immunodeficiency virus type 1. As with reverse transcriptase inhibitors, however, resistance to protease inhibitors can develop and is attributed to the appearance of mutations in the protease gene. With the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS, 350- to 1,500-fold-resistant variants have been selected in vitro and were found not only to contain mutations in the protease gene but also to contain mutations in Gag precursor p1/p6 and/or NC (p7)/p1 cleavage sites. Mutations in cleavage sites give rise to better peptide substrates for the protease in vitro and to improved processing of p15 precursors in drug-resistant clones. Importantly, removal of cleavage site mutations in resistant clones leads to a decrease or even an absence of viral growth, confirming their role in viral fitness. Therefore, these second-locus mutations indicate that cleavage of p15 is a rate-limiting step in polyprotein processing in highly resistant viruses. The functional constraint of p15 processing also suggests that additional selective pressure could further compromise viral fitness and maintain the benefits of antiviral therapies. PMID:8648711

  5. Structures of HIV Protease Guide Inhibitor Design to Overcome Drug Resistance

    SciTech Connect

    Weber, Irene T.; Kovalevsky, Andrey Y.; Harrison, Robert W.

    2008-06-03

    The HIV/AIDS infection continues to be a major epidemic worldwide despite the initial promise of antiviral drugs. Current therapy includes a combination of drugs that inhibit two of the virally-encoded enzymes, the reverse transcriptase and the protease. The first generation of HIV protease inhibitors that have been in clinical use for treatment of AIDS since 1995 was developed with the aid of structural analysis of protease-inhibitor complexes. These drugs were successful in improving the life span of HIV-infected people. Subsequently, the rapid emergence of drug resistance has necessitated the design of new inhibitors that target mutant proteases. This second generation of antiviral protease inhibitors has been developed with the aid of data from medicinal chemistry, kinetics, and X-ray crystallographic analysis. Traditional computational methods such as molecular mechanics and dynamics can be supplemented with intelligent data mining approaches. One approach, based on similarities to the protease interactions with substrates, is to incorporate additional interactions with main chain atoms that cannot easily be eliminated by mutations. Our structural and inhibition data for darunavir have helped to understand its antiviral activity and effectiveness on drug resistant HIV and demonstrate the success of this approach.

  6. Functional proteomics-aided selection of protease inhibitors for herbivore insect control

    PubMed Central

    Rasoolizadeh, Asieh; Munger, Aurélie; Goulet, Marie-Claire; Sainsbury, Frank; Cloutier, Conrad; Michaud, Dominique

    2016-01-01

    Studies have reported the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains to be established. Insects have evolved strategies to cope with dietary protease inhibitors, such as the use of proteases recalcitrant to inhibition, that often make the selection of effective inhibitors very challenging. Here, we used a functional proteomics approach for the ‘capture’ of Cys protease targets in crude protein extracts as a tool to identify promising cystatins for plant improvement. Two cystatins found to differ in their efficiency to capture Cys proteases of the coleopteran pest Leptinotarsa decemlineata also differed in their usefulness to produce transgenic potato lines resistant to this insect. Plants expressing the most potent cystatin at high level had a strong repressing effect on larval growth and leaf intake, while plants expressing the weakest cystatin showed no effect on both two parameters compared to untransformed parental line used for genetic transformation. Our data underline the relevance of considering the whole range of possible protease targets when selecting an inhibitor for plant pest control. They also confirm the feasibility of developing cystatin-expressing transgenics resistant to a major pest of potato. PMID:27958307

  7. Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System

    PubMed Central

    Mahdi, Mohamed; Szojka, Zsófia; Mótyán, János András; Tőzsér, József

    2015-01-01

    Retroviral protease inhibitors (PIs) are fundamental pillars in the treatment of HIV infection and acquired immunodeficiency syndrome (AIDS). Currently used PIs are designed against HIV-1, and their effect on HIV-2 is understudied. Using a modular HIV-2 protease cassette system, inhibition profiling assays were carried out for protease inhibitors both in enzymatic and cell culture assays. Moreover, the treatment-associated resistance mutations (I54M, L90M) were introduced into the modular system, and comparative inhibition assays were performed to determine their effect on the susceptibility of the protease. Our results indicate that darunavir, saquinavir, indinavir and lopinavir were very effective HIV-2 protease inhibitors, while tipranavir, nelfinavir and amprenavir showed a decreased efficacy. I54M, L90M double mutation resulted in a significant reduction in the susceptibility to most of the inhibitors with the exception of tipranavir. To our knowledge, this modular system constitutes a novel approach in the field of HIV-2 protease characterization and susceptibility testing. PMID:26633459

  8. Degradation of immunoglobulins, protease inhibitors, and interleukin-1 by a secretory proteinase of Acanthamoeba castellanii

    PubMed Central

    Na, Byoung-Kuk; Cho, Jong-Hwa; Song, Chul-Yong; Kim, Tong-Soo

    2002-01-01

    The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (sIgA), IgG, and IgM. It also degraded interleukin-1α (IL-1α) and IL-1β. Its activity was not inhibited by endogenous protease inhibitors, such as α2-macroglobulin, α1-trypsin inhibitor, and α2-antiplasmin. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthamoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection. PMID:12073735

  9. Decreased expression of protease inhibitor 9, a granzyme B inhibitor, in celiac disease: a potential mechanism in enterocyte destruction and villous atrophy.

    PubMed

    Pohjanen, V-M; Kokkonen, T S; Arvonen, M; Augustin, M A; Patankar, M; Turunen, S; Vähäsalo, P; Karttunen, T J

    2013-01-01

    The objective of this study was to assess the expression of protease inhibitor 9, a granzyme B inhibitor, in human small intestine, and to evaluate its cytoprotective role in the celiac disease of children. Twelve subjects with untreated celiac disease and thirteen healthy controls were examined by endoscopy. The expression of protease inhibitor 9 was analyzed immunohistochemically from duodenal biopsies and compared to granzyme B expression, apoptosis rate, number of intraepithelial lymphocytes and villus and crypt height data from the biopsies. We discovered that protease inhibitor 9 is expressed in the cytoplasm of the duodenal epithelial cells in the majority of cases. The enterocyte expression of protease inhibitor 9 was lower in celiac disease patients than in controls. Protease inhibitor 9 expression also showed a negative correlation with the number of apoptotic cells, overall density of granzyme B expressing intraepithelial lymphocytes, the height of the crypts and the severity of villous atrophy in duodenum. Therefore, we conclude that the protease inhibitor 9 is constantly expressed in the enterocytes of normal duodenum and the expression is decreased in celiac disease. These findings suggest that protease inhibitor 9 has a role in duodenal homeostasis and in the protection of enterocytes from misdirected granzyme B. Indeed, observed associations of lowered protease inhibitor 9 expression together with increased granzyme B expression, apoptosis rate and severity of villous atrophy suggest that impaired balance between granzyme B mediated cytotoxicity and its inhibition by protease inhibitor 9 forms an important factor in the pathogenesis of villous atrophy in celiac disease.

  10. Rosuvastatin vs. protease inhibitor switching for hypercholesterolaemia: a randomized trial.

    PubMed

    Lee, F J; Monteiro, P; Baker, D; Bloch, M; Roth, N; Finlayson, R; Moore, R; Hoy, J; Martinez, E; Carr, A

    2016-09-01

    The aim of the study was to compare the efficacy and safety of rosuvastatin initiation with those of switching of ritonavir-boosted protease inhibitors (PI/rs) in HIV-1-infected adults with hypercholesterolaemia and increased cardiovascular risk scores. In this open-label, multicentre study, HIV-1-infected adults on PI/r-based therapy with viral load < 50 HIV-1 RNA copies/mL, fasting total cholesterol ≥ 5.5 mmol/L (both for ≥ 6 months) and elevated cardiovascular risk (Framingham score ≥ 8% or diabetes or family history), and not on lipid-lowering therapy, were randomized to open-label rosuvastatin 10 mg/day or to PI/r switching, both with standardized diet/exercise advice. The primary endpoint was change in total cholesterol at week 12 (intention to treat). There were 43 participants (23 on rosuvastatin). Baseline characteristics were: mean [± standard deviation (SD)] age 55 (8.5) years, 42 (98%) male, 41 (95%) white race, and mean (± SD) total cholesterol 6.2 (1.2) mmol/L. At enrolment, PI/rs were lopinavir/ritonavir (n = 22; 51%), atazanavir/ritonavir (n = 12; 28%) and darunavir/ritonavir (n = 9; 21%). The commonest PI/r substitutes were raltegravir (n = 9; 45%) and rilpivirine (n = 4; 20%). All participants were adherent through to week 12. Rosuvastatin yielded greater declines than PI/r switching in total (- 21.4% vs. - 8.7%, respectively; P = 0.003) and low-density lipoprotein (- 29.9% vs. - 1.0%, respectively; P < 0.001) cholesterol, but smaller declines in very low-density lipoprotein cholesterol and triglycerides (P < 0.01). Cholesterol lowering was greater in participants on atazanavir/ritonavir or once-daily darunavir/ritonavir (vs. lopinavir/ritonavir). More study drug-related adverse events (mostly grade 1 nausea/diarrhoea; 10 vs. one, respectively; P = 0.001) occurred with PI/r switching than with rosuvastatin. In adults receiving a PI/r, rosuvastatin 10 mg/day for 12 weeks yielded larger decreases in total and low-density lipoprotein

  11. Impact of protease inhibitors on the evolution of urinary markers

    PubMed Central

    Bonjoch, Anna; Puig, Jordi; Pérez-Alvarez, Nuria; Juega, Javier; Echeverría, Patricia; Clotet, Bonaventura; Romero, Ramón; Bonet, J.; Negredo, E.

    2016-01-01

    Abstract Kidney injury (defined as the presence of albuminuria, proteinuria, glycosuria [without hyperglycemia], hematuria, and/or renal hypophosphatemia) is an emerging problem in human immunodeficiency virus (HIV)-infected patients, although few data are available on the role of protease inhibitors (PIs) in this condition. To determine the time to kidney injury in a cohort of HIV-infected patients receiving a PI-containing regimen. We report the results of a subanalysis of a published cross-sectional study. The subanalysis included only patients receiving PI-containing regimens for more than 6 months (377 of the overall 970 patients). We determined associated factors and constructed receiver operating characteristic curves to estimate time to kidney injury depending on the PI used. The percentage of patients with kidney injury was 27.7% for darunavir, 27.9% for lopinavir, and 30% for atazanavir. Time to kidney injury was as follows: 229 days for atazanavir/ritonavir (area under the curve [AUC], 0.639; sensitivity, 0.89; specificity, 0.41); 332 days for atazanavir/ritonavir plus tenofovir (AUC, 0.603; sensitivity, 0.75; and specificity, 0.29); 318 days for nonboosted atazanavir (AUC, 0.581; sensitivity, 0.89; and specificity, 0.29); 478 days for lopinavir/ritonavir (AUC, 0.566; sensitivity, 0.864; and specificity, 0.44); 1339 days for lopinavir/ritonavir plus tenofovir (AUC, 0.667; sensitivity, 0.86; and specificity, 0.77); 283 days for darunavir/ritonavir (AUC, 0.523; sensitivity, 0.80; and specificity, 0.261); and 286 days for darunavir/ritonavir plus tenofovir (AUC, 0.446; sensitivity, 0.789; and specificity, 0.245). The use of lopinavir/ritonavir without tenofovir was a protective factor (odds ratio = 1.772; 95%CI, 1.070–2.93; P = 0.026). For all PIs, the percentage of patients with kidney injury exceeded 27%, irrespective of tenofovir use. The longest time to kidney injury was recorded with lopinavir/ritonavir. These results demonstrate the need for

  12. Protease inhibitors, part 13: Specific, weakly basic thrombin inhibitors incorporating sulfonyl dicyandiamide moieties in their structure.

    PubMed

    Clare, B W; Scozzafava, A; Supuran, C T

    2001-01-01

    A series of compounds has been prepared by reaction of dicyandiamide with alkyl/arylsulfonyl halides as well as arylsulfonylisocyanates to locate a lead for obtaining weakly basic thrombin inhibitors with sulfonyldicyandiamide moieties as the S1 anchoring group. The detected lead was sulfanilyl-dicyandiamide (K1 of 3 microM against thrombin, and 15 microM against trypsin), which has been further derivatized at the 4-amino group by incorporating arylsulfonylureido as well as amino acyl/dipeptidyl groups protected at the amino terminal moiety with benzyloxycarbonyl or tosylureido moieties. The best compound obtained (ts-D-Phe-Pro-sulfanilyl-dicyandiamide) showed inhibition constants of 9 nM against thrombin and 1400 nM against trypsin. pKa measurements showed that the new derivatives reported here do indeed possess a reduced basicity, with the pKa of the modified guanidine moieties in the range 7.9-8.3 pKa units. Molecular mechanics calculations showed that the preferred tautomeric form of these compounds is of the type ArSO2N=C(NH2) NH-CN, probably allowing for the formation of favorable interaction between this new anchoring group and the active site amino acid residue Asp 189, critical for substrate/inhibitor binding to this type of serine protease. Thus, the main finding of the present paper is that the sulfonyldicyandiamide group may constitute an interesting alternative for obtaining weakly basic, potent thrombin inhibitors, which bind with less affinity to trypsin.

  13. Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease.

    PubMed

    Konno, Hiroyuki; Wakabayashi, Masaki; Takanuma, Daiki; Saito, Yota; Akaji, Kenichi

    2016-03-15

    Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Potent inhibitors of HCV-NS3 protease derived from boronic acids

    SciTech Connect

    Venkatraman, Srikanth; Wu, Wanli; Prongay, Andrew; Girijavallabhan, Viyyoor; Njoroge, F. George

    2009-07-23

    Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200 pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.

  15. Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

    PubMed Central

    Walsh, P N; Sinha, D; Kueppers, F; Seaman, F S; Blankstein, K B

    1987-01-01

    We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug. Images PMID:3500185

  16. 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.

  17. Drug-drug interactions between HMG-CoA reductase inhibitors (statins) and antiviral protease inhibitors.

    PubMed

    Chauvin, Benoit; Drouot, Sylvain; Barrail-Tran, Aurélie; Taburet, Anne-Marie

    2013-10-01

    The HMG-CoA reductase inhibitors are a class of drugs also known as statins. These drugs are effective and widely prescribed for the treatment of hypercholesterolemia and prevention of cardiovascular morbidity and mortality. Seven statins are currently available: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. Although these drugs are generally well tolerated, skeletal muscle abnormalities from myalgia to severe lethal rhabdomyolysis can occur. Factors that increase statin concentrations such as drug-drug interactions can increase the risk of these adverse events. Drug-drug interactions are dependent on statins' pharmacokinetic profile: simvastatin, lovastatin and atorvastatin are metabolized through cytochrome P450 (CYP) 3A, while the metabolism of the other statins is independent of this CYP. All statins are substrate of organic anion transporter polypeptide 1B1, an uptake transporter expressed in hepatocyte membrane that may also explain some drug-drug interactions. Many HIV-infected patients have dyslipidemia and comorbidities that may require statin treatment. HIV-protease inhibitors (HIV PIs) are part of recommended antiretroviral treatment in combination with two reverse transcriptase inhibitors. All HIV PIs except nelfinavir are coadministered with a low dose of ritonavir, a potent CYP3A inhibitor to improve their pharmacokinetic properties. Cobicistat is a new potent CYP3A inhibitor that is combined with elvitegravir and will be combined with HIV-PIs in the future. The HCV-PIs boceprevir and telaprevir are both, to different extents, inhibitors of CYP3A. This review summarizes the pharmacokinetic properties of statins and PIs with emphasis on their metabolic pathways explaining clinically important drug-drug interactions. Simvastatin and lovastatin metabolized through CYP3A have the highest potency for drug-drug interaction with potent CYP3A inhibitors such as ritonavir- or cobicistat-boosted HIV-PI or the

  18. Design of new potent HTLV-1 protease inhibitors: in silico study.

    PubMed

    Kheirabadi, Mitra; Maleki, Javad; Soufian, Safieh; Hosseini, Samaneh

    2016-03-01

    HTLV-1 and HIV-1 are two major causes for severe T-cell leukemia disease and acquired immune deficiency syndrome (AIDS). HTLV-1 protease, a member of aspartic acid protease family, plays important roles in maturation during virus replication cycle. The impairment of these proteases results in uninfectious HTLV-1virions.Similar to HIV-1protease deliberate mutations that confer drug resistance on HTLV-1 are frequently seen in this protease. Therefore, inhibition of HTLV-1 protease activity is expected to disrupt HTLV-1's ability to replicate and infect additional cells. In this study, we initially designed fifteen inhibitory compounds based on the conformations of a class of HIV-1 aspartyl protease inhibitors, sulfonamid-peptoid. Five compounds were chosen based on the goodness of their Drug-Likeness scoreusing "Lipinsk's rule of five". Here, using protein-ligand docking approach we compared the inhibitory constants of these compounds to those available in literatures and observed significantly higher inhibition for two compounds, SP-4 and SP-5. Our data suggest that the addition of two cyclic hydrocarbons to both ends of sulfonamide peptoids leads to the formation of new hydrophobic interactions due to the semi-circular form of these compounds, connecting the first chain of protease to the two ends of tested ligands via Hydrophobic interactions. We conclude that hydrophobic force plays an important role in suppressing protease activity especially for HTLV-1 protease, which in turn prevents the virus maturity. Therefore, designing and development of new ligands based on aromatic hydrocarbons in both ends of inhibitors is very promising for efficient treatment.

  19. Design of new potent HTLV-1 protease inhibitors: in silico study

    PubMed Central

    Kheirabadi, Mitra; Maleki, Javad; Soufian, Safieh; Hosseini, Samaneh

    2016-01-01

    HTLV-1 and HIV-1 are two major causes for severe T-cell leukemia disease and acquired immune deficiency syndrome (AIDS). HTLV-1 protease, a member of aspartic acid protease family, plays important roles in maturation during virus replication cycle. The impairment of these proteases results in uninfectious HTLV-1virions.Similar to HIV-1protease deliberate mutations that confer drug resistance on HTLV-1 are frequently seen in this protease. Therefore, inhibition of HTLV-1 protease activity is expected to disrupt HTLV-1’s ability to replicate and infect additional cells. In this study, we initially designed fifteen inhibitory compounds based on the conformations of a class of HIV-1 aspartyl protease inhibitors, sulfonamid-peptoid. Five compounds were chosen based on the goodness of their Drug-Likeness scoreusing “Lipinsk’s rule of five”. Here, using protein-ligand docking approach we compared the inhibitory constants of these compounds to those available in literatures and observed significantly higher inhibition for two compounds, SP-4 and SP-5. Our data suggest that the addition of two cyclic hydrocarbons to both ends of sulfonamide peptoids leads to the formation of new hydrophobic interactions due to the semi-circular form of these compounds, connecting the first chain of protease to the two ends of tested ligands via Hydrophobic interactions. We conclude that hydrophobic force plays an important role in suppressing protease activity especially for HTLV-1 protease, which in turn prevents the virus maturity. Therefore, designing and development of new ligands based on aromatic hydrocarbons in both ends of inhibitors is very promising for efficient treatment. PMID:27844017

  20. Michael Acceptor-Based Peptidomimetic Inhibitor of Main Protease from Porcine Epidemic Diarrhea Virus.

    PubMed

    Wang, Fenghua; Chen, Cheng; Yang, Kailin; Xu, Yang; Liu, Xiaomei; Gao, Fan; Liu, He; Chen, Xia; Zhao, Qi; Liu, Xiang; Cai, Yan; Yang, Haitao

    2017-03-13

    Porcine epidemic diarrhea virus (PEDV) causes high mortality in pigs. PEDV main protease (Mpro) plays an essential role in viral replication. We solved the structure of PEDV Mpro complexed with peptidomimetic inhibitor N3 carrying a Michael acceptor warhead, revealing atomic level interactions. We further designed a series of 17 inhibitors with altered side groups. Inhibitors M2 and M17 demonstrated enhanced specificity against PEDV Mpro. These compounds have potential as future therapeutics to combat PEDV infection.

  1. Oxadiazole-Based Cell Permeable Macrocyclic Transition State Inhibitors of Norovirus 3CL Protease.

    PubMed

    Damalanka, Vishnu C; Kim, Yunjeong; Alliston, Kevin R; Weerawarna, Pathum M; Galasiti Kankanamalage, Anushka C; Lushington, Gerald H; Mehzabeen, Nurjahan; Battaile, Kevin P; Lovell, Scott; Chang, Kyeong-Ok; Groutas, William C

    2016-03-10

    Human noroviruses are the primary causative agents of acute gastroenteritis and a pressing public health burden worldwide. There are currently no vaccines or small molecule therapeutics available for the treatment or prophylaxis of norovirus infections. Norovirus 3CL protease plays a vital role in viral replication by generating structural and nonstructural proteins via the cleavage of the viral polyprotein. Thus, molecules that inhibit the viral protease may have potential therapeutic value. We describe herein the structure-based design, synthesis, and in vitro and cell-based evaluation of the first class of oxadiazole-based, permeable macrocyclic inhibitors of norovirus 3CL protease.

  2. Isolation of a trypsin-chymotrypsin inhibitor and its functional properties.

    PubMed

    Wang, Shaoyun; Shao, Biao; Lu, Wei; Hong, Jing; Rao, Pingfan

    2014-01-01

    A novel trypsin inhibitor with thermal and pH stability, designated Merrtine, was isolated from Glycine max L. merr. The procedure involved ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, and affinity chromatography on Affi-gel blue gel. The 20 N-terminal amino acid sequences were determined to be DEYSKPCCDLCMCTRRCPPQ, demonstrating high homology with the sequence of Bowman-Birk type trypsin inhibitors. The molecular mass and isoelectric point of the inhibitor were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to be 20.0 kD and 5.8, respectively. Trypsin could be completely inhibited by Merrtine when the molar ratio was 8.1. The inhibitory activity of Merrtine was unaffected after exposure to temperatures up to 85 °C, as well as within the pH range 2-12. Besides inhibiting trypsin-chymotrypsin, the inhibitor demonstrated additional antifungal activity against the species of Alternaria alternate, Fusarium oxysporum, Pythium aphanidermatum, Physalospora piricola, Botrytis cinerea, and Fusarium solani. We herein report not only the trypsin inhibitor's extraction and isolation for the first time, but also its physiochemical and antifungal properties.

  3. Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L.

    PubMed

    Devaraj, V R; Manjunatha, N H

    1999-01-01

    A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS-PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an Mr of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI-trypsin/chymotrypsin complexes by difference spectral studies. Apparent Ka values of complexes of inhibitor with trypsin and chymotrypsin were 2.1x10(7) M(-1) and 3.1x10(7) M(-1), respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.

  4. Complete amino acid sequences of three proteinase inhibitors from white sword bean (Canavalia gladiata).

    PubMed

    Park, S S; Sumi, T; Ohba, H; Nakamura, O; Kimura, M

    2000-10-01

    Three major serine proteinase inhibitors (SBI-1, -2, and -3) were purified from the seeds of white sword bean (Canavalia gladiata) by FPLC and reversed-phase HPLC. The sequences of these inhibitors were established by automatic Edman degradation and TOF-mass spectrometry. SBI-1, -2, and -3 consisted of 72, 73, and 75 amino acid residues, with molecular masses of 7806.5, 7919.8, and 8163.4, respectively. The sequences of SBI-1 and -2 coincided with those of CLT I and II [Terada et al. (1994) Biosci. Biotech. Biochem., 58, 376-379] except only N- or C-terminal amino acid residues. Analysis of the amino acid sequences showed that the active sites of the inhibitors contained a Lys21-Ser22 against trypsin and Leu48-Ser49 against chymotrypsin, respectively. Further, it became apparent that about seven disulfide bonds were present. These results suggest that sword bean inhibitors are members of the Bowman-Birk proteinase inhibitor family.

  5. Factors associated with virological rebound in HIV-infected patients receiving protease inhibitor monotherapy.

    PubMed

    Stöhr, Wolfgang; Dunn, David T; Arenas-Pinto, Alejandro; Orkin, Chloe; Clarke, Amanda; Williams, Ian; Johnson, Margaret; Beeching, Nicholas J; Wilkins, Edmund; Sanders, Karen; Paton, Nicholas I

    2016-11-13

    The Protease Inhibitor Monotherapy Versus Ongoing Triple Therapy (PIVOT) trial found that protease inhibitor monotherapy as a simplification strategy is well tolerated in terms of drug resistance but less effective than combination therapy in suppressing HIV viral load. We sought to identify factors associated with the risk of viral load rebound in this trial. PIVOT was a randomized controlled trial in HIV-positive adults with suppressed viral load for at least 24 weeks on combination therapy comparing a strategy of physician-selected ritonavir-boosted protease inhibitor monotherapy versus ongoing triple therapy. In participants receiving monotherapy, we analysed time to confirmed viral load rebound and its predictors using flexible parametric survival models. Of 290 participants initiating protease inhibitor monotherapy (80% darunavir, 14% lopinavir, and 6% other), 93 developed viral load rebound on monotherapy. The risk of viral load rebound peaked at 9 months after starting monotherapy and then declined to approximately 5 per 100 person-years from 18 months onwards. Independent predictors of viral load rebound were duration of viral load suppression before starting monotherapy (hazard ratio 0.81 per additional year <50 copies/ml; P < 0.001), CD4 cell count (hazard ratio 0.73 per additional 100 cells/μl for CD4 nadir; P = 0.008); ethnicity (hazard ratio 1.87 for nonwhite versus white, P = 0.025) but not the protease inhibitor agent used (P = 0.27). Patients whose viral load was analysed with the Roche TaqMan-2 assay had a 1.87-fold risk for viral load rebound compared with Abbott RealTime assay (P = 0.012). A number of factors can identify patients at low risk of rebound with protease inhibitor monotherapy, and this may help to better target those who may benefit from this management strategy.

  6. Structure-guided fragment-based in silico drug design of dengue protease inhibitors.

    PubMed

    Knehans, Tim; Schüller, Andreas; Doan, Danny N; Nacro, Kassoum; Hill, Jeffrey; Güntert, Peter; Madhusudhan, M S; Weil, Tanja; Vasudevan, Subhash G

    2011-03-01

    An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC(50) = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC(50) = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.

  7. Structure-guided fragment-based in silico drug design of dengue protease inhibitors

    NASA Astrophysics Data System (ADS)

    Knehans, Tim; Schüller, Andreas; Doan, Danny N.; Nacro, Kassoum; Hill, Jeffrey; Güntert, Peter; Madhusudhan, M. S.; Weil, Tanja; Vasudevan, Subhash G.

    2011-03-01

    An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC50 = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC50 = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.

  8. Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors.

    PubMed

    Qasmi, D; de Rosny, E; René, L; Badet, B; Vergely, I; Boggetto, N; Reboud-Ravaux, M

    1997-04-01

    A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

  9. Chymotrypsin protease inhibitor gene family in rice: Genomic organization and evidence for the presence of a bidirectional promoter shared between two chymotrypsin protease inhibitor genes.

    PubMed

    Singh, Amanjot; Sahi, Chandan; Grover, Anil

    2009-01-01

    Protease inhibitors play important roles in stress and developmental responses of plants. Rice genome contains 17 putative members in chymotrypsin protease inhibitor (ranging in size from 7.21 to 11.9 kDa) gene family with different predicted localization sites. Full-length cDNA encoding for a putative subtilisin-chymotrypsin protease inhibitor (OCPI2) was obtained from Pusa basmati 1 (indica) rice seedlings. 620 bp-long OCPI2 cDNA contained 219 bp-long ORF, coding for 72 amino acid-long 7.7 kDa subtilisin-chymotrypsin protease inhibitor (CPI) cytoplasmic protein. Expression analysis by semi-quantitative RT-PCR analysis showed that OCPI2 transcript is induced by varied stresses including salt, ABA, low temperature and mechanical injury in both root and shoot tissues of the seedlings. Transgenic rice plants produced with OCPI2 promoter-gus reporter gene showed that this promoter directs high salt- and ABA-regulated expression of the GUS gene. Another CPI gene (OCPI1) upstream to OCPI2 (with 1126 bp distance between the transcription initiation sites of the two genes; transcription in the reverse orientation) was noted in genome sequence of rice genome. A vector that had GFP and GUS reporter genes in opposite orientations driven by 1881 bp intergenic sequence between the OCPI2 and OCPI1 (encompassing the region between the translation initiation sites of the two genes) was constructed and shot in onion epidermal cells by particle bombardment. Expression of both GFP and GUS from the same epidermal cell showed that this sequence represents a bidirectional promoter. Examples illustrating gene pairs showing co-expression of two divergent neighboring genes sharing a bidirectional promoter have recently been extensively worked out in yeast and human systems. We provide an example of a gene pair constituted of two homologous genes showing co-expression governed by a bidirectional promoter in rice.

  10. Detection of alpha 2-macroglobulin, alpha 1-protease inhibitor, and neutral protease-antiprotease complexes within liver granulomas of Schistosoma mansoni-infected mice.

    PubMed

    Truden, J L; Boros, D L

    1988-02-01

    In schistosomiasis mansoni the parasite egg-induced granulomatous tissue inflammations resolve by fibrosis. Intralesional collagen synthesis and deposition are influenced by collagenase, elastase activity that is diminished at the chronic stage of the disease. To determine the cause of diminished neutral protease activity, the authors determined levels of the antiprotease/alpha 2-macroglobulin (alpha 2M) and alpha 1-protease inhibitor (alpha 1Pi) in extracts or secretions of liver granulomas of infected mice. By ELISA, both antiproteases were detected in granuloma-derived substances, as well as supernatants of cultured, adherent granuloma macrophages. In all samples, alpha 2M was the predominant inhibitor. Antiprotease levels were similar in granuloma-derived samples obtained from acutely and chronically infected mice. However, supernatants of cultured adherent macrophages isolated from granulomas of mice with acute infection contained levels of protease inhibitors several times higher than those of similar preparations obtained from chronically infected animals. Gel filtration of samples on Sephacryl S-200 columns did not separate collagenase and elastase from protease inhibitors. By chromatofocusing, a few inhibitor-free collagenase as well as enzyme-free alpha 2M and alpha 1Pi-active peaks were eluted. The bulk of the material that eluted at the acidic region contained protease-antiprotease activity indicating the presence of enzyme-inhibitor complexes. The intragranulomatous presence of antiproteases complexed with protease enzymes emphasizes their importance in the possible enhancement of fibrosis.

  11. Detection of alpha 2-macroglobulin, alpha 1-protease inhibitor, and neutral protease-antiprotease complexes within liver granulomas of Schistosoma mansoni-infected mice.

    PubMed Central

    Truden, J. L.; Boros, D. L.

    1988-01-01

    In schistosomiasis mansoni the parasite egg-induced granulomatous tissue inflammations resolve by fibrosis. Intralesional collagen synthesis and deposition are influenced by collagenase, elastase activity that is diminished at the chronic stage of the disease. To determine the cause of diminished neutral protease activity, the authors determined levels of the antiprotease/alpha 2-macroglobulin (alpha 2M) and alpha 1-protease inhibitor (alpha 1Pi) in extracts or secretions of liver granulomas of infected mice. By ELISA, both antiproteases were detected in granuloma-derived substances, as well as supernatants of cultured, adherent granuloma macrophages. In all samples, alpha 2M was the predominant inhibitor. Antiprotease levels were similar in granuloma-derived samples obtained from acutely and chronically infected mice. However, supernatants of cultured adherent macrophages isolated from granulomas of mice with acute infection contained levels of protease inhibitors several times higher than those of similar preparations obtained from chronically infected animals. Gel filtration of samples on Sephacryl S-200 columns did not separate collagenase and elastase from protease inhibitors. By chromatofocusing, a few inhibitor-free collagenase as well as enzyme-free alpha 2M and alpha 1Pi-active peaks were eluted. The bulk of the material that eluted at the acidic region contained protease-antiprotease activity indicating the presence of enzyme-inhibitor complexes. The intragranulomatous presence of antiproteases complexed with protease enzymes emphasizes their importance in the possible enhancement of fibrosis. PMID:2449083

  12. Prevalence, mutation patterns, and effects on protease inhibitor susceptibility of the L76V mutation in HIV-1 protease.

    PubMed

    Young, Thomas P; Parkin, Neil T; Stawiski, Eric; Pilot-Matias, Tami; Trinh, Roger; Kempf, Dale J; Norton, Michael

    2010-11-01

    Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir.

  13. Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris.

    PubMed

    Luniak, Nora; Meiser, Peter; Burkart, Sonja; Müller, Rolf

    2017-01-01

    Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi-component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54-65, 2017. © 2016 American Institute of Chemical Engineers.

  14. Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

    PubMed Central

    Burchacka, Ewa; Zdzalik, Michal; Niemczyk, Justyna-Stec; Pustelny, Katarzyna; Popowicz, Grzegorz; Wladyka, Benedykt; Dubin, Adam; Potempa, Jan; Sienczyk, Marcin; Dubin, Grzegorz; Oleksyszyn, Jozef

    2014-01-01

    Staphylococcus aureus is responsible for a variety of human infections, including life-threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of α-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-PheP-(OC6H4−4-SO2CH3)2 and Suc-Val-Pro-PheP-(OC6H5)2 are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of α-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases. PMID:24375505

  15. 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.

  16. Hepatitis C virus NS3-4A serine protease inhibitors: SAR of P'2 moiety with improved potency.

    PubMed

    Arasappan, A; Njoroge, F G; Chan, T-Y; Bennett, F; Bogen, S L; Chen, K; Gu, H; Hong, L; Jao, E; Liu, Y-T; Lovey, R G; Parekh, T; Pike, R E; Pinto, P; Santhanam, B; Venkatraman, S; Vaccaro, H; Wang, H; Yang, X; Zhu, Z; Mckittrick, B; Saksena, A K; Girijavallabhan, V; Pichardo, J; Butkiewicz, N; Ingram, R; Malcolm, B; Prongay, A; Yao, N; Marten, B; Madison, V; Kemp, S; Levy, O; Lim-Wilby, M; Tamura, S; Ganguly, A K

    2005-10-01

    We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.

  17. Molecular mechanisms of tolerance to cyanobacterial protease inhibitors revealed by clonal differences in Daphnia magna.

    PubMed

    Schwarzenberger, Anke; Kuster, Christian J; Von Elert, Eric

    2012-10-01

    Protease inhibitors of primary producers are a major food quality constraint for herbivores. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is mainly represented by Daphnia and cyanobacteria. Protease inhibitors have been found in many cyanobacterial blooms. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the daphnid's gut, that is, trypsins and chymotrypsins. In this study, we fed four different Daphnia magna genotypes with the trypsin-inhibitor-containing cyanobacterial strain Microcystis aeruginosa PCC 7806 Mut. Upon exposure to dietary trypsin inhibitors, all D. magna genotypes showed increased gene expression of digestive trypsins and chymotrypsins. Exposure to dietary trypsin inhibitors resulted in increased activity of chymotrypsins and reduced activity of trypsin. Strong intraspecific differences in tolerance of the four D. magna genotypes to the dietary trypsin inhibitors were found. The degree of tolerance depended on the D. magna genotype. The genotypes' tolerance was positively correlated with the residual trypsin activity and the different IC(50) values of the trypsins. On the genetic level, the different trypsin loci varied between the D. magna genotypes. The two tolerant Daphnia genotypes that both originate from the same lake, which frequently produces cyanobacterial blooms, clustered in a neighbour-joining phylogenetic tree based on the three trypsin loci. This suggests that the genetic variability of trypsin loci was an important cause for the observed intraspecific variability in tolerance to cyanobacterial trypsin inhibitors. Based on these findings, it is reasonable to assume that such genetic variability can also be found in natural populations and thus constitutes the basis for local adaptation of natural populations to dietary protease inhibitors.

  18. Purification and some characteristics of the human epidermal SH-protease inhibitor.

    PubMed

    Järvinen, M

    1978-08-01

    An inhibitor of papain and other SH-proteases was purified 520-fold from human epidermis extracts by acetone fractionation, heat treatment, papain-Sepharose affinity chromatography, and Sephadex G-50 chromatography. The purified inhibitor had a molecular weight of 12,600 and contained no hexose, as tested by the anthrone reaction. The inhibitor survived in a boiling water bath, in 5% trichloroacetic acid, 20 mM Na3PO4 (pH 12.1) and 4 M NH4OH (pH 11.9). By isoelectric focusing 2 major activity peaks with pI's of 4.6 and 4.8, and a minor peak with a pI of 4.9 was fractioned, and 3 corresponding protein bands were seen after analytical isoelectric focusing. Immunization of rabbits with the purified inhibitor yielded a highly specific anti-inhibitor serum. The purified inhibitor inhibited papain, ficin, human cathepsins B and C, and slightly inhibited bromelain. No inhibition of serine proteases (bovine trypsin and chymotrypsin A, porcine elastase) or an acid protease (human cathepsin D) was observed. Evidence was obtained that the inhibitor formed a complex with both dithiothreitol-activated papain and enzymatically inactive mercuripapain.

  19. 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

  20. Conformer and pharmacophore based identification of peptidomimetic inhibitors of chikungunya virus nsP2 protease.

    PubMed

    Dhindwal, Sonali; Kesari, Pooja; Singh, Harvijay; Kumar, Pravindra; Tomar, Shailly

    2016-12-02

    Chikungunya virus nsP2 replication protein is a cysteine protease, which cleaves the nonstructural nsP1234 polyprotein into functional replication components. The cleavage and processing of nsP1234 by nsP2 protease is essential for the replication and proliferation of the virus. Thus, ChikV nsP2 protease is a promising target for antiviral drug discovery. In this study, the crystal structure of the C-terminal domain of ChikV nsP2 protease (PDB ID: 4ZTB) was used for structure based identification and rational designing of peptidomimetic inhibitors against nsP2 protease. The interactions of the junction residues of nsP3/4 polyprotein in the active site of nsP2 protease have been mimicked to identify and design potential inhibitory molecules. Molecular docking of the nsP3/4 junction peptide in the active site of ChikV nsP2 protease provided the structural insight of the probable binding mode of nsP3/4 peptide and pigeonholed the molecular interactions critical for the substrate binding. Further, the shape and pharmacophoric properties of the viral nsP3/4 substrate peptide were taken into consideration and the mimetic molecules were identified and designed. The designed mimetic compounds were then analyzed by docking and their binding affinity was assessed by molecular dynamics simulations.

  1. Cysteine protease inhibitor (AcStefin) is required for complete cyst formation of Acanthamoeba.

    PubMed

    Lee, Jung-Yub; Song, Su-Min; Moon, Eun-Kyung; Lee, Yu-Ran; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Cha, Hee-Jae; Yu, Hak Sun; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-04-01

    The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba.

  2. Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

    PubMed Central

    Lee, Jung-Yub; Song, Su-Min; Moon, Eun-Kyung; Lee, Yu-Ran; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Cha, Hee-Jae; Yu, Hak Sun; Kong, Hyun-Hee; Chung, Dong-Il

    2013-01-01

    The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba. PMID:23397569

  3. A novel protease inhibitor in Bombyx mori is involved in defense against Beauveria bassiana.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Shiping; Dong, Zhaoming; Chen, Jianping; Xiang, Zhonghuai; Xia, Qingyou

    2012-10-01

    Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle using a plethora of hydrolytic enzymes including cuticle-degrading proteases and chitinases, which are important virulence factors. The insect integument and hemolymph contains a relatively high concentration of protease inhibitors, which are closely involved with defense against pathogenic microorganisms. To elucidate the molecular mechanism underlying resistance against entomopathogenic fungi and to identify a new molecular target for improving fungal resistance in the silkworm, Bombyx mori, we cloned and expressed a novel silkworm TIL-type protease inhibitor BmSPI38, which was very stable over a wide range of temperatures and pH values. An activity assay suggested that BmSPI38 potently inactivated the insecticidal cuticle-degrading enzyme (CDEP-1) produced by B. bassiana and subtilisin A produced by Bacillus licheniformis. The melanization of silkworm induced by CDEP-1 protease could also be blocked by BmSPI38. These results provided new insights into the molecular mechanisms whereby insect protease inhibitors provide resistance against entomopathogenic fungi, suggesting the possibility of using fungal biopesticides in sericulture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Isosorbide-based peptidomimetics as inhibitors of hepatitis C virus serine protease.

    PubMed

    Portela, Aline C; Barros, Thalita G; Lima, Camilo H da S; Dias, Luiza R S; Azevedo, Pedro H R de A; Dantas, Anna Sophia C L; Mohana-Borges, Ronaldo; Ventura, Gustavo T; Pinheiro, Sergio; Muri, Estela M F

    2017-08-15

    Hepatitis C infection is a cause of chronic liver diseases such as cirrhosis and carcinoma. The current therapy for hepatitis C has limited efficacy and low tolerance. The HCV encodes a serine protease which is critical for viral replication, and few protease inhibitors are currently on the market. In this paper, we describe the synthesis and screening of novel isosorbide-based peptidomimetic inhibitors, in which the compounds 1d, 1e, and 1i showed significant inhibition of the protease activity in vitro at 100µM. The compound 1e also showed dose-response (IC50=36±3µM) and inhibited the protease mutants D168A and V170A at 100µM, indicating it as a promising inhibitor of the HCV NS3/4A protease. Our molecular modeling studies suggest that the activity of 1e is associated with a change in the interactions of S2 and S4 subsites, since that the increased flexibility favors a decrease in activity against D168A, whereas the appearance of a hydrophobic cavity in the S4 subsite increase the inhibition against V170A strain. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Fluorogenic Assay for Inhibitors of HIV-1 Protease with Sub-picomolar Affinity

    NASA Astrophysics Data System (ADS)

    Windsor, Ian W.; Raines, Ronald T.

    2015-08-01

    A fluorogenic substrate for HIV-1 protease was designed and used as the basis for a hypersensitive assay. The substrate exhibits a kcat of 7.4 s-1, KM of 15 μM, and an increase in fluorescence intensity of 104-fold upon cleavage, thus providing sensitivity that is unmatched in a continuous assay of HIV-1 protease. These properties enabled the enzyme concentration in an activity assay to be reduced to 25 pM, which is close to the Kd value of the protease dimer. By fitting inhibition data to Morrison’s equation, Ki values of amprenavir, darunavir, and tipranavir were determined to be 135, 10, and 82 pM, respectively. This assay, which is capable of measuring Ki values as low as 0.25 pM, is well-suited for characterizing the next generation of HIV-1 protease inhibitors.

  6. Three-dimensional structure of a simian immunodeficiency virus protease/inhibitor complex. Implications for the design of human immunodeficiency virus type 1 and 2 protease inhibitors.

    PubMed

    Zhao, B; Winborne, E; Minnich, M D; Culp, J S; Debouck, C; Abdel-Meguid, S S

    1993-12-07

    Simian immunodeficiency virus (SIV) proteins have considerable amino acid sequence homology to those from human immunodeficiency virus (HIV); thus monkeys are considered useful models for the preclinical evaluation of acquired immune deficiency syndrome (AIDS) therapeutics. We have crystallized and determined the three-dimensional structure of SIV protease bound to the hydroxyethylene isostere inhibitor SKF107457. Crystals of the complex were grown from 25-32% saturated sodium chloride, by the hanging drop method of vapor diffusion. They belong to the orthorhombic space group I222, with a = 46.3 A, b = 101.5 A, and c = 118.8 A. The structure has been determined at 2.5-A resolution by molecular replacement and refined to a crystallographic discrepancy factor, R (= sigma parallel Fo magnitude of - magnitude of Fc parallel/sigma magnitude of Fo magnitude of), of 0.189. The overall structure of the complex is very similar to previously reported structures of HIV-1 protease bound to inhibitors. The inhibitor is bound in a conformation that is almost identical to that found for the same inhibitor bound to HIV-1 protease, except for an overall translation of the inhibitor, varying along the backbone atoms from about 1.0 A at the termini to about 0.5 A around the scissile bond surrogate. The structures of the SIV and HIV-1 proteins vary significantly only in three surface loops composed of amino acids 15-20, 34-45, and 65-70. Superposition of the 1188 protein backbone atoms from the two structures gives an rms deviation of 1.0 A; this number is reduced to 0.6 A when atoms from the three surface loops are eliminated from the rms calculation.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Protonation state and free energy calculation of HIV-1 protease-inhibitor complex based on electrostatic polarisation effect

    NASA Astrophysics Data System (ADS)

    Yang, Maoyou; Jiang, Xiaonan; Jiang, Ning

    2014-06-01

    The protonation states of catalytic Asp25/25‧ residues remarkably affect the binding mechanism of the HIV-1 protease-inhibitor complex. Here we report a molecular dynamics simulation study, which includes electrostatic polarisation effect, to investigate the influence of Asp25/25‧ protonation states upon the binding free energy of the HIV-1 protease and a C2-symmetric inhibitor. Good agreements are obtained on inhibitor structure, hydrogen bond network, and binding free energy between our theoretical calculations and the experimental data. The calculations show that the Asp25 residue is deprotonated, and the Asp25‧ residue is protonated. Our results reveal that the Asp25/25‧ residues can have different protonation states when binding to different inhibitors although the protease and the inhibitors have the same symmetry. This study offers some insights into understanding the protonation state of HIV-1 protease-inhibitor complex, which could be helpful in designing new inhibitor molecules.

  8. Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors.

    PubMed

    Mandadapu, Sivakoteswara Rao; Gunnam, Mallikarjuna Reddy; Tiew, Kok-Chuan; Uy, Roxanne Adeline Z; Prior, Allan M; Alliston, Kevin R; Hua, Duy H; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C

    2013-01-01

    Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.

  9. Potent and Selective Peptidyl Boronic Acid Inhibitors of the Serine Protease Prostate-Specific Antigen

    PubMed Central

    LeBeau, Aaron M.; Singh, Pratap; Isaacs, John T.; Denmeade, Samuel R.

    2012-01-01

    SUMMARY Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a Ki for PSA of 65 nM. The inhibitor had a 60-fold higher Ki for chymotrypsin. A validated model of PSA’s catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme. PMID:18635003

  10. Validation of phage display method for protease inhibitor selection using synthetic hybrid peptides.

    PubMed

    de Marco, Renato; Azzolini, Simone S; Lovato, Diogo V; Torquato, Ricardo J S; Amino, Rogerio; de Miranda, Antonio; Tanaka, Aparecida S

    2010-11-01

    A recombinant Haematobia irritans irritans trypsin inhibitor (HiTI - Mw 7030 kDa)) phagemid library was constructed and displayed functionally on the tip of the filamentous M13 phage. A combinatorial library of 7.2 x 10(6) mutants was created with HiTI mutations restricted to the P1'-P3' and P5' positions of the reactive site. This combinatorial library was selected for trypsin-like Pr2 proteases of Metarhizium anisopliae fungus, and 11 HiTI mutants containing the following substitutions: K17G, S18R, D19G, S21A, among 60 sequenced clones, were obtained. In order to confirm the inhibitory activity of the selected sequences, we transferred the selected sequence to the shortest protease inhibitor, the sunflower trypsin inhibitor (SFTI), for inhibitory activity analysis. The hybrid peptide containing the mutated sequence (SFTI-Mut, GRCTRGRGLACFPD-NH2; Ki = 14 µM) presented an apparent inhibition constant (Ki(app)) for Pr2 proteases ≈20-fold lower than the control peptide containing the original HiTI sequence (SFTI-HiTI, GRCTRKSDLSCFPD-NH2; Ki = 259 µM). In conclusion, the present work enabled the selection of a specific HiTI mutant for Pr2 proteases of M. anisopliae fungus using a HiTI combinatorial library on M13 phage surface. Selection of strong binders by phage display and their validation as inhibitors using synthetic hybrid peptides proved to be a powerful technique to generate specific serine protease inhibitors suitable for studies of drug design and enzyme-inhibitor interaction.

  11. Targeting dynamic pockets of HIV-1 protease by structure-based computational screening for allosteric inhibitors.

    PubMed

    Kunze, Jens; Todoroff, Nickolay; Schneider, Petra; Rodrigues, Tiago; Geppert, Tim; Reisen, Felix; Schreuder, Herman; Saas, Joachim; Hessler, Gerhard; Baringhaus, Karl-Heinz; Schneider, Gisbert

    2014-03-24

    We present the discovery of low molecular weight inhibitors of human immunodeficiency virus 1 (HIV-1) protease subtype B that were identified by structure-based virtual screening as ligands of an allosteric surface cavity. For pocket identification and prioritization, we performed a molecular dynamics simulation and observed several flexible, partially transient surface cavities. For one of these presumable ligand-binding pockets that are located in the so-called "hinge region" of the identical protease chains, we computed a receptor-derived pharmacophore model, with which we retrieved fragment-like inhibitors from a screening compound pool. The most potent hit inhibited protease activity in vitro in a noncompetitive mode of action. Although attempts failed to crystallize this ligand bound to the enzyme, the study provides proof-of-concept for identifying innovative tool compounds for chemical biology by addressing flexible protein models with receptor pocket-derived pharmacophore screening.

  12. Effects of tobacco genetically modified to express protease inhibitor bovine spleen trypsin inhibitor on non-target soil organisms.

    PubMed

    O'Callaghan, Maureen; Brownbridge, Michael; Stilwell, Wendy B; Gerard, Emily M; Burgess, Elisabeth P J; Barraclough, Emma I; Christeller, John T

    2007-01-01

    Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil.

  13. New cyanopeptide-derived low molecular weight inhibitors of trypsin-like serine proteases.

    PubMed

    Radau, Gregor; Schermuly, Sonja; Fritsche, Alexandra

    2003-08-01

    This paper deals with the design, syntheses, and inhibition tests of new low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of cyanobacteria with interesting biological activities, as new lead structures. Starting with aeruginosin 98-B (1) as a lead structure, we have developed and synthesised new, selective acting inhibitors of serine proteases (RA-1005 and RA-1009, which are suitable targets for further structure-activity studies.

  14. 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.

  15. Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

    PubMed

    Šmid, Ida; Rotter, Ana; Gruden, Kristina; Brzin, Jože; Buh Gašparič, Meti; Kos, Janko; Žel, Jana; Sabotič, Jerica

    2015-07-01

    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

  16. Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound.

    PubMed

    Karthik, L; Kumar, Gaurav; Keswani, Tarun; Bhattacharyya, Arindam; Chandar, S Sarath; Bhaskara Rao, K V

    2014-01-01

    The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608). The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml). In in vivo model, the highest level of parasitemia suppression (≈ 45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development.

  17. Lazarus and Group Psychotherapy: AIDS in the Era of Protease Inhibitors

    ERIC Educational Resources Information Center

    Gushue, George V.; Brazaitis, Sarah J.

    2003-01-01

    A new class of medications, protease inhibitors, has dramatically improved the health of many people with Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS). This development has had a major impact on the lives of those affected by HIV/AIDS. This article considers how a group is affected by the larger systems of…

  18. Inhibitors of hepatitis C virus NS3.4A protease 2. Warhead SAR and optimization.

    PubMed

    Perni, Robert B; Pitlik, Janos; Britt, Shawn D; Court, John J; Courtney, Lawrence F; Deininger, David D; Farmer, Luc J; Gates, Cynthia A; Harbeson, Scott L; Levin, Rhonda B; Lin, Chao; Lin, Kai; Moon, Young-Choon; Luong, Yu-Ping; O'Malley, Ethan T; Rao, B Govinda; Thomson, John A; Tung, Roger D; Van Drie, John H; Wei, Yunyi

    2004-03-22

    The alpha-ketoamide warhead (e.g., 15) was found to be a practical replacement for aliphatic aldehydes in a series of HCV NS3.4A protease inhibitors. Structure-activity relationships and prime side optimization are discussed.

  19. Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

    PubMed

    Black, P L; Downs, M B; Lewis, M G; Ussery, M A; Dreyer, G B; Petteway, S R; Lambert, D M

    1993-01-01

    Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors.

  20. Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

    PubMed Central

    Black, P L; Downs, M B; Lewis, M G; Ussery, M A; Dreyer, G B; Petteway, S R; Lambert, D M

    1993-01-01

    Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors. Images PMID:8381640

  1. Lazarus and Group Psychotherapy: AIDS in the Era of Protease Inhibitors

    ERIC Educational Resources Information Center

    Gushue, George V.; Brazaitis, Sarah J.

    2003-01-01

    A new class of medications, protease inhibitors, has dramatically improved the health of many people with Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS). This development has had a major impact on the lives of those affected by HIV/AIDS. This article considers how a group is affected by the larger systems of…

  2. Protease Inhibitors from Marine Actinobacteria as a Potential Source for Antimalarial Compound

    PubMed Central

    Karthik, L.; Kumar, Gaurav; Keswani, Tarun; Bhattacharyya, Arindam; Chandar, S. Sarath; Bhaskara Rao, K. V.

    2014-01-01

    The study was planned to screen the marine actinobacterial extract for the protease inhibitor activity and its anti- Pf activity under in vitro and in vivo conditions. Out of 100 isolates, only 3 isolates exhibited moderate to high protease inhibitor activities on trypsin, chymotrypsin and proteinase K. Based on protease inhibitor activity 3 isolates were chosen for further studies. The potential isolate was characterized by polyphasic approach and identified as Streptomyces sp LK3 (JF710608). The lead compound was identified as peptide from Streptomyces sp LK3. The double-reciprocal plot displayed inhibition mode is non-competitive and it confirms the irreversible nature of protease inhibitor. The peptide from Streptomyces sp LK3 extract showed significant anti plasmodial activity (IC50: 25.78 µg/ml). In in vivo model, the highest level of parasitemia suppression (≈45%) was observed in 600 mg/kg of the peptide. These analyses revealed no significant changes were observed in the spleen and liver tissue during 8 dpi. The results confirmed up-regulation of TGF-β and down regulation of TNF-α in tissue and serum level in PbA infected peptide treated mice compared to PbA infection. The results obtained infer that the peptide possesses anti- Pf activity activity. It suggests that the extracts have novel metabolites and could be considered as a potential source for drug development. PMID:24618707

  3. 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.

  4. Protease inhibitors activity in lepromatous leprosy and lepra reaction.

    PubMed

    Yemul, V L; Sengupta, S R; Dhole, T N

    1983-01-01

    Serum alpha one antitrypsin levels were measured in 50 healthy age and sex matched controls with 45 lepromatous leprosy cases and 5 cases of lepra reaction. It was noted that the mean level in healthy controls was 281.00 mg%, while the mean levels in LL patients was 421.00 mg% and in LR 570.00 mg%. The elevation of Alpha one antitrypsin was statistically significant in LL patients. It is possible that the rise is a reaction to release of proteases and or higher complement activity, which are the results of a high bacillary loading to formation of immune complexes.

  5. Protease Inhibitors Extracted from Caesalpinia echinata Lam. Affect Kinin Release during Lung Inflammation

    PubMed Central

    Cruz-Silva, Ilana; Praxedes-Garcia, Priscila; Tanaka, Aparecida Sadae; Shimamoto, Kazuaki

    2016-01-01

    Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI (C. echinata kallikrein inhibitor), a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor), which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control) or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymes; however, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases. PMID:28044105

  6. Contribution of Aspartic Proteases in Candida Virulence. Protease Inhibitors against Candida Infections.

    PubMed

    Staniszewska, Monika; Małgorzata, Bondaryk; Zbigniew, Ochal

    2016-08-09

    Candida species are the major opportunistic human pathogens accounting for 70-90% of all invasive fungal infections. Candida spp, especially C. albicans, are able to produce and secrete hydrolytic enzymes, particularly aspartic proteases (Saps). These enzymes production is an evolutionary adaptation of pathogens to utilize nutrients and survive in host. Sap1-10 are believed to contribute to the adhesion and invasion of host tissues through the degradation of cell surface structures. Aspartic proteases control several steps in innate immune evasion and they degrade proteins related to immunological defense (antibodies, complement and cytokines), allowing the fungus to escape from the first line of host defense. The existing ways to identify potential drug targets rely on specific subset like virulence genes, transcriptional and stress response factors. Candida virulence factors like Sap isoenzymes can be pivotal targets for drug development. The identification of mechanism of a non-canonical inflammasome exerted by Saps could open novel therapeutic strategies to dampen hyperinflammatory response in candidiasis.

  7. Biopotency of serine protease inhibitors from cowpea (Vigna unguiculata) seeds on digestive proteases and the development of Spodoptera littoralis (Boisduval).

    PubMed

    Abd El-latif, Ashraf Oukasha

    2015-05-01

    Serine protease inhibitors (PIs) have been described in many plant species and are universal throughout the plant kingdom, where trypsin inhibitors is the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of 13 selected cultivars/accessions of cowpea. Two cowpea cultivars, Cream7 and Buff, were found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested cultivars for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Cream7-purified proteins showed two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) corresponding to molecular mass of 17.10 and 14.90 kDa, while the purified protein from Buff cultivar showed a single band corresponding mass of 16.50 kDa. The purified inhibitors were stable at temperature below 60°C and were active at wide range of pH from 2 to 12. The kinetic analysis revealed noncompetitive type of inhibition for both inhibitors against both enzymes. The inhibitor constant (Ki ) values suggested high affinity between inhibitors and enzymes. Purified inhibitors were found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis, where Buff PI was more effective than Cream7 PI. It may be concluded that cowpea PI gene(s) could be potential insect control protein for future studies in developing insect-resistant transgenic plants. © 2014 Wiley Periodicals, Inc.

  8. Natural cysteine protease inhibitors in protozoa: Fifteen years of the chagasin family.

    PubMed

    Costa, Tatiana F R; Lima, Ana Paula C A

    2016-03-01

    Chagasin-type inhibitors comprise natural inhibitors of papain-like cysteine proteases that are distributed among Protist, Bacteria and Archaea. Chagasin was identified in the pathogenic protozoa Trypanosoma cruzi as an approximately 11 kDa protein that is a tight-binding and highly thermostable inhibitor of papain, cysteine cathepsins and endogenous parasite cysteine proteases. It displays an Imunoglobulin-like fold with three exposed loops to one side of the molecule, where amino acid residues present in conserved motifs at the tips of each loop contact target proteases. Differently from cystatins, the loop 2 of chagasin enters the active-site cleft, making direct contact with the catalytic residues, while loops 4 and 6 embrace the enzyme from the sides. Orthologues of chagasin are named Inhibitors of Cysteine Peptidases (ICP), and share conserved overall tri-dimensional structure and mode of binding to proteases. ICPs are tentatively distributed in three families: in family I42 are grouped chagasin-type inhibitors that share conserved residues at the exposed loops; family I71 contains Plasmodium ICPs, which are large proteins having a chagasin-like domain at the C-terminus, with lower similarity to chagasin in the conserved motif at loop 2; family I81 contains Toxoplasma ICP. Recombinant ICPs tested so far can inactivate protozoa cathepsin-like proteases and their mammalian counterparts. Studies on their biological roles were carried out in a few species, mainly using transgenic protozoa, and the conclusions vary. However, in all cases, alterations in the levels of expression of chagasin/ICPs led to substantial changes in one or more steps of parasite biology, with higher incidence in influencing their interaction with the hosts. We will cover most of the findings on chagasin/ICP structural and functional properties and overview the current knowledge on their roles in protozoa.

  9. 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.

  10. Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter.

    PubMed

    Gaffaney, Jon D; Vaughan, Roxanne A

    2004-03-01

    Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.

  11. 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

  12. 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.

  13. Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB.

    PubMed

    Schröder, Jörg; Noack, Sandra; Marhöfer, Richard J; Mottram, Jeremy C; Coombs, Graham H; Selzer, Paul M

    2013-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

  14. Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

    PubMed Central

    Schröder, Jörg; Noack, Sandra; Marhöfer, Richard J.; Mottram, Jeremy C.; Coombs, Graham H.; Selzer, Paul M.

    2013-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases. PMID:24146999

  15. ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease

    PubMed Central

    Sham, Hing L.; Kempf, Dale J.; Molla, Akhteruzammen; Marsh, Kennan C.; Kumar, Gondi N.; Chen, Chih-Ming; Kati, Warren; Stewart, Kent; Lal, Ritu; Hsu, Ann; Betebenner, David; Korneyeva, Marina; Vasavanonda, Sudthida; McDonald, Edith; Saldivar, Ayda; Wideburg, Norm; Chen, Xiaoqi; Niu, Ping; Park, Chang; Jayanti, Venkata; Grabowski, Brian; Granneman, G. Richard; Sun, Eugene; Japour, Anthony J.; Leonard, John M.; Plattner, Jacob J.; Norbeck, Daniel W.

    1998-01-01

    The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS. PMID:9835517

  16. Acipimox, an inhibitor of lipolysis, attenuates atherogenesis in LDLR-null mice treated with HIV protease inhibitor ritonavir.

    PubMed

    Guo, Wen; Wong, Siu; Pudney, Jeffrey; Jasuja, Ravi; Hua, Ning; Jiang, Lan; Miller, Andrew; Hruz, Paul W; Hamilton, James A; Bhasin, Shalender

    2009-12-01

    The advent of HIV protease inhibitors has greatly extended the life span of AIDS patients. With an aging HIV(+) population, the cardiometabolic side effects of these drugs are becoming increasingly important clinical concerns. The purpose of this study was to test the hypothesis that inhibition of adipose lipolysis will retard atherogenic lesion development induced by the antiviral protease inhibitors. LDLR-null mice receiving ritonavir were compared with those receiving ritonavir plus lipolysis inhibitor acipimox or vehicle alone to determine how acipimox would affect ritonavir-induced atherogenesis. Intermittent high-fat high-cholesterol diet was used to facilitate optimal atheromatous lesion development. Drug effects were assessed as changes in aortic lesion score, plasma lipid and lipoprotein profile, body fat mass, and insulin-induced suppression of plasma fatty acid concentrations. Ritonavir increased aortic lesions, in association with decreased body fat mass, impaired antilipolysis action of insulin, and increased proatherogenic plasma lipoproteins. All these adverse effects were attenuated by cotreatment with acipimox. Our results provide the first direct evidence that supports the hypothesis that dysregulation of adipose lipolysis is an important contributor to the proatherogenic role of selected HIV protease inhibitors.

  17. Relation between flexibility and positively selected HIV-1 protease mutants against inhibitors.

    PubMed

    Braz, Antônio S K; Tufanetto, Patrícia; Perahia, David; Scott, Luis P B

    2012-12-01

    The antiretroviral chemotherapy helps to reduce the mortality of HIVs infected patients. However, RNA dependant virus replication has a high mutation rate. Human immunodeficiency virus Type 1 protease plays an essential role in viral replication cycle. This protein is an important target for therapy with viral protein inhibitors. There are few works using normal mode analysis to investigate this problem from the structural changes viewpoint. The investigation of protein flexibility may be important for the study of processes associated with conformational changes and state transitions. The normal mode analysis allowed us to investigate structural changes in the protease (such as flexibility) in a straightforward way and try to associate these changes with the increase of fitness for each positively selected HIV-1 mutant protease of patients treated with several protease inhibitors (saquinavir, indinavir, ritonavir, nelfinavir, lopinavir, fosamprenavir, atazanavir, darunavir, and tripanavir) in combination or separately. These positively selected mutations introduce significant flexibility in important regions such as the active site cavity and flaps. These mutations were also able to cause changes in accessible solvent area. This study showed that the majority of HIV-1 protease mutants can be grouped into two main classes of protein flexibility behavior. We presented a new approach to study structural changes caused by positively selected mutations in a pathogen protein, for instance the HIV-1 protease and their relationship with their resistance mechanism against known inhibitors. The method can be applied to any pharmaceutically relevant pathogen proteins and could be very useful to understand the effects of positively selected mutations in the context of structural changes.

  18. Cardioprotection by a novel recombinant serine protease inhibitor in myocardial ischemia and reperfusion injury.

    PubMed

    Murohara, T; Guo, J P; Lefer, A M

    1995-09-01

    Polymorphonuclear neutrophils (PMN) play an important role in myocardial ischemia/reperfusion (MI/R) injury; however, the role of neutrophilic proteases is less understood. The effects of a novel serine protease inhibitor (serpin), LEX032, were investigated in a murine model of MI (20 min) and R (24 hr) injury in vivo. LEX032 is a recombinant human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of alpha-1 protease inhibitor. LEX032 has the ability to inhibit both neutrophil elastase and cathepsin G, two major neutral serine proteases in neutrophils, as well as superoxide generation. LEX032 (25 or 50 mg/kg) administered i.v. 1 min before reperfusion significantly attenuated myocardial necrotic injury evaluated by cardiac creatine kinase loss compared to MI/R rats receiving only vehicle (P < .001). Moreover, cardiac myeloperoxidase activity, an index of PMN accumulation, in the ischemic myocardium was significantly attenuated by LEX032 as compared with rats receiving vehicle (P < .001). LEX032 also moderately attenuated leukotriene B4-stimulated PMN adherence to rat superior mesenteric artery endothelium and markedly diminished superoxide radical release from LTB4-stimulated PMN in vitro. In a glycogen-induced rat peritonitis model, LEX032 (50 mg/kg) significantly attenuated PMN transmigration into the peritoneal cavity in vivo. In conclusion, the recombinant serine protease inhibitor, LEX032, appears to be an effective agent for attenuating MI/R injury by inhibiting neutrophil-accumulation into the ischemic-reperfused myocardium and by inactivating cytotoxic metabolites (proteases and superoxide radical) released from neutrophils.

  19. A potential Kazal-type serine protease inhibitor involves in kinetics of protease inhibition and bacteriostatic activity.

    PubMed

    Kumaresan, Venkatesh; Harikrishnan, Ramaswamy; Arockiaraj, Jesu

    2015-02-01

    Kazal-type serine protease inhibitor (KSPI) is a pancreatic secretary trypsin inhibitor which involves in various cellular component regulations including development and defense process. In this study, we have characterized a KSPI cDNA sequence of freshwater striped murrel fish Channa striatus (Cs) at molecular level. Cellular location analysis predicted that the CsKSPI was an extracellular protein. The domain analysis showed that the CsKSPI contains a Kazal domain at 47-103 along with its family signature between 61 and 83. Phylogenetically, CsKSPI is closely related to KSPI from Maylandia zebra and formed a sister group with mammals. The 2D structure of CsKSPI showed three α-helical regions which are connected with random coils, one helix at signal sequence and two at the Kazal domain region. The relative gene expression showed that the CsKSPI was highly expressed in gills and its expression was induced upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and poly I:C (a viral analogue) challenge. The CsKSPI recombinant protein was produced to characterize and study the CsKSPI gene specific functions. The recombinant CsKSPI strongly inhibited trypsin compared to other tested proteases. The results of the kinetic activity of CsKSPI against trypsin was V(max)s = 1.62 nmol/min, K(M)s = 0.21 mM and K(i)s = 15.37 nM. Moreover, the recombinant CsKSPI inhibited the growth of Gram-negative bacteria A. hydrophila at 20 μM and Gram-positive bacteria Bacillus subtilis at the MIC50 of 15 μM. Overall, the study indicated that the CsKSPI was a potential trypsin inhibitor which involves in antimicrobial activity.

  20. Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease

    SciTech Connect

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri; Kiso, Yoshiaki; Gustchina, Alla; Wlodawer, Alexander

    2010-09-28

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.

  1. Non-ribosomal halogenated protease inhibitors from cyanobacterial isolates as attractive drug targets.

    PubMed

    Silva-Stenico, M E; Rigonato, J; Leal, M G; Vaz, M G M V; Andreote, A P D; Fiore, M F

    2012-01-01

    Cyanobacteria possess the ability to produce compounds with remarkable biological activity, and have thus attracted the attention of the pharmaceutical industry. Cyanopeptides acting as protease inhibitors have shown potential in the field of pharmacotherapy through regulation of abnormal physiological processes in the human body. Despite the already described cyanopeptide protease inhibitors, the search for new congeners is of considerable interest which may pave the way for more efficient molecules. In this study, the presence of the protease inhibitors aeruginosin and cyanopeptolin with non-, mono- and dichlorination and also genes coding for their synthetases was investigated in 90 cyanobacterial strains. Mass spectrometry analyses highlighted production of 91, 19 and 3 non-, mono- and dichlorinated congeners, respectively. The purified extract of Microcystis botrys SPC759 inhibited 61% of pepsin protease. PCR amplifications of aeruginosin and cyanopeptolin synthetase gene regions were observed in 41 and 28% of evaluated strains, respectively. The sequences obtained for the aerA-aerB (aeruginosin) and mcnC-mcnE (cyanopeptolin) gene regions grouped together with their homologues found in other cyanobacterial strains in the phylogenetic analyses with high bootstrap support. Antimicrobial activity assays performed using all intracellular extracts inhibited 31 and 26% of Gram-negative and Gram-positive pathogenic bacterial growth, respectively. The results of this study showed the production of aeruginosin and cyanopeptolin and the presence of their genes in several cyanobacterial genera for the first time besides the discovery of novel congeners.

  2. Evaluation of trypanocidal activity of combinations of anti-sleeping sickness drugs with cysteine protease inhibitors.

    PubMed

    Steverding, Dietmar

    2015-01-01

    Chemotherapy of human African trypanosomiasis (HAT) is unsatisfactory because only a few drugs, with serious side effects and poor efficacy, are available. As drug combination regimes often achieve greater therapeutic efficacy than monotherapies, here the trypanocidal activity of the cysteine protease inhibitor K11777 in combination with current anti-HAT drugs using bloodstream forms of Trypanosoma brucei was investigated. Isobolographic analysis was used to determine the interaction between cysteine protease inhibitors (K11777, CA-074Me and CAA0225) and anti-HAT drugs (suramin, pentamidine, melarsoprol and eflornithine). Bloodstream forms of T. brucei were incubated in culture medium containing cysteine protease inhibitors or anti-HAT drugs alone or in combination at a 1:1 fixed-dose ratio. After 48 h incubation, live cells were counted, the 50% growth inhibition values determined and combination indices calculated. The general cytotoxicity of drug combinations was evaluated with human leukaemia HL-60 cells. Combinations of K11777 with suramin, pentamidine and melarsoprol showed antagonistic effects while with eflornithine a synergistic effect was observed. Whereas eflornithine antagonises with CA-074Me, an inhibitor inactivating the targeted TbCATL only under reducing conditions, it synergises with CAA0255, an inhibitor structurally related to CA-074Me which inactivates TbCATL independently of thiols. These findings indicate an essential role of thiols for the synergistic interaction between K11777 and eflornithine. Encouragingly, the K11777/eflornithine combination displayed higher trypanocidal than cytotoxic activity. The results of this study suggest that the combination of the cysteine protease inhibitor K11777 and eflornithine display promising synergistic trypanocidal activity that warrants further investigation of the drug combination as possible alternative treatment of HAT.

  3. Deep sequencing of protease inhibitor resistant HIV patient isolates reveals patterns of correlated mutations in Gag and protease.

    PubMed

    Flynn, William F; Chang, Max W; Tan, Zhiqiang; Oliveira, Glenn; Yuan, Jinyun; Okulicz, Jason F; Torbett, Bruce E; Levy, Ronald M

    2015-04-01

    While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle.

  4. Lead expansion and virtual screening of Indinavir derivate HIV-1 protease inhibitors using pharmacophoric - shape similarity scoring function

    PubMed Central

    Shityakov, Sergey; Dandekar, Thomas

    2010-01-01

    Indinavir (Crivaxan®) is a potent inhibitor of the HIV (human immunodeficiency virus) protease. This enzyme has an important role in viral replication and is considered to be very attractive target for new antiretroviral drugs. However, it becomes less effective due to highly resistant new viral strains of HIV, which have multiple mutations in their proteases. For this reason, we used a lead expansion method to create a new set of compounds with a new mode of action to protease binding site. 1300 compounds chemically diverse from the initial hit were generated and screened to determine their ability to interact with protease and establish their QSAR properties. Further computational analyses revealed one unique compound with different protease binding ability from the initial hit and its role for possible new class of protease inhibitors is discussed in this report. PMID:20978602

  5. Structural basis for the immunomodulatory function of cysteine protease inhibitor from human roundworm Ascaris lumbricoides.

    PubMed

    Mei, Guoqiang; Dong, Jianmei; Li, Zhaotao; Liu, Sanling; Liu, Yunfeng; Sun, Mingze; Liu, Guiyun; Su, Zhong; Liu, Jinsong

    2014-01-01

    Immunosuppression associated with infections of nematode parasites has been documented. Cysteine protease inhibitor (CPI) released by the nematode parasites is identified as one of the major modulators of host immune response. In this report, we demonstrated that the recombinant CPI protein of Ascaris lumbricoides (Al-CPI) strongly inhibited the activities of cathepsin L, C, S, and showed weaker effect to cathepsin B. Crystal structure of Al-CPI was determined to 2.1 Å resolution. Two segments of Al-CPI, loop 1 and loop 2, were proposed as the key structure motifs responsible for Al-CPI binding with proteases and its inhibitory activity. Mutations at loop 1 and loop 2 abrogated the protease inhibition activity to various extents. These results provide the molecular insight into the interaction between the nematode parasite and its host and will facilitate the development of anthelmintic agents or design of anti-autoimmune disease drugs.

  6. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

    PubMed

    Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R

    2017-02-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

  7. Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors

    PubMed Central

    Ponder, Elizabeth L.; Albrow, Victoria E.; Leader, Brittany A.; Békés, Miklós; Mikolajczyk, Jowita; Fonović, Urša Pečar; Shen, Aimee; Drag, Marcin; Xiao, Junpeng; Deu, Edgar; Campbell, Amy J.; Powers, James C.; Salvesen, Guy S.; Bogyo, Matthew

    2011-01-01

    SUMMARY Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite lifecycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a novel class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum. PMID:21700207

  8. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors

    PubMed Central

    Burtoloso, Antonio C. B.; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C.; Gonçalez, Cristiana; Kenny, Peter W.; Leitão, Andrei; Quilles, José Carlos; Ribeiro, Jean F. R.; Rocha, Josmar R.

    2017-01-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases. PMID:28222138

  9. Functional characterization of a SUMO deconjugating protease of Plasmodium falciparum using newly identified small molecule inhibitors.

    PubMed

    Ponder, Elizabeth L; Albrow, Victoria E; Leader, Brittany A; Békés, Miklós; Mikolajczyk, Jowita; Fonović, Urša Pečar; Shen, Aimee; Drag, Marcin; Xiao, Junpeng; Deu, Edgar; Campbell, Amy J; Powers, James C; Salvesen, Guy S; Bogyo, Matthew

    2011-06-24

    Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Recent patents and emerging therapeutics for HIV infections: a focus on protease inhibitors.

    PubMed

    Patel, Mitesh; Mandava, Nanda K; Vadlapatla, Ramya Krishna; Mitra, Ashim K

    2013-07-01

    The inclusion of protease inhibitors (PIs) in highly active antiretroviral therapy has significantly improved clinical outcomes in HIV-1-infected patients. To date, PIs are considered to be the most important therapeutic agents for the treatment of HIV infections. Despite high anti-HIV-1 potency, poor oral bioavailability of PIs has been a major concern. For achieving therapeutic concentrations, large doses of PIs are administered, which results in unacceptable systemic toxicities. Such severe and long-term toxicities necessitate the development of safer and potentially promising PIs. Recently, considerable attention has been paid to the development of newer compounds capable of inhibiting wild-type and resistant HIV-1 protease. Some of these PIs have displayed potent HIV-1 protease inhibitory activity. In this review, we have made an attempt to provide an overview on clinically approved and newly developing PIs, and related recent patents in the development of novel PIs.

  11. Functional diversification of a protease inhibitor gene in the genus Drosophila and its molecular basis.

    PubMed

    Börner, Stefan; Ragg, Hermann

    2008-05-31

    The mutually exclusive use of alternative reactive site loop (RSL) cassettes due to alternative splicing of serpin (serine protease inhibitor) gene transcripts is a widespread strategy to create target-selective protease inhibitors in the animal kingdom. Since molecular basis and evolution of serpin RSL cassette exon amplification and diversification are unexplored, the exon-intron organization of the serpin gene spn4 from 12 species of the genus Drosophila was studied. The analysis of the gene structures shows that both number and target enzyme specificities of Spn4 RSL cassettes are highly variable in fruit flies and includes inhibitor variants with novel antiproteolytic activities in some species, indicating that RSL diversity is the result of adaptive evolution. Comparative genomics suggests that interallelic gene conversion and/or recombination events contribute to RSL cassette exon amplification. Due to an intron that is located at the most suitable position within the RSL region, multiple inhibitors can be formed in an economic manner that are both efficient and target-selective, allowing fruit flies to control an astonishing variety of proteases with different cleavage chemistry and evolutionary ancestry.

  12. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

    SciTech Connect

    Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D.

    2008-10-27

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

  13. Protease inhibitor homologues from mamba venoms: facilitation of acetylcholine release and interactions with prejunctional blocking toxins.

    PubMed

    Harvey, A L; Karlsson, E

    1982-09-01

    1 Five polypeptides, which were isolated from elapid snake venoms and which are structurally related to protease inhibitors, were tested for action on isolated biventer cervicis nerve-muscle preparations of the chick. 2 Dendrotoxin from the Eastern green mamba (Dendroaspis angusticeps) and toxins K and I from the black mamba (Dendroaspis polylepis polylepis) increased to indirect stimulation without affecting responses to exogenous acetylcholine, carbachol of KCl. 3 The two other protease inhibitor homologues, HHV-II from Ringhals cobra (Hemachatus haemachatus) and NNV-II from Cape cobra (Naja nivea) did not increase responses to nerve stimulation. Trypsin inhibitor from bovine pancreas also had no facilitatory effects on neuromuscular transmission. 4 The facilitatory toxins from mamba venoms interacted with the prejunctional blocking toxins, beta-bungarotoxin, crotoxin and notexin, but not with taipoxin. The blocking effects of beta-bungarotoxin were reduced by pretreatment with the mamba toxins, whereas the blocking actions of crotoxin and notexin were enhanced. 5 The results indicate that protease inhibitor homologues from mamba venoms form a new class of neurotoxin, which acts to increase the release of acetylcholine in response to motor nerve stimulation. 6 From the interaction studies it is concluded that the facilitatory toxins bind to motor nerve terminals at sites related to those occupied by the prejunctional blocking toxins. However, differences in interactions with individual toxins suggest that there must be several related binding sites on the nerve terminals.

  14. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

    PubMed Central

    Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D.

    2008-01-01

    We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC50 value of 20 μM, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC50 of 15 μM and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs. PMID:18852458

  15. In vitro ANTIGIARDIAL ACTIVITY OF THE CYSTEINE PROTEASE INHIBITOR E-64

    PubMed Central

    de Carvalho, Thaís Batista; Oliveira-Sequeira, Teresa Cristina Goulart; Guimarães, Semíramis

    2014-01-01

    The quest for new antiparasitic alternatives has led researchers to base their studies on insights into biology, host-parasite interactions and pathogenesis. In this context, proteases and their inhibitors are focused, respectively, as druggable targets and new therapy alternatives. Herein, we proposed to evaluate the in vitro effect of the cysteine protease inhibitor E-64 on Giardia trophozoites growth, adherence and viability. Trophozoites (105) were exposed to E-64 at different final concentrations, for 24, 48 and 72 h at 37 °C. In the growth and adherence assays, the number of trophozoites was estimated microscopically in a haemocytometer, whereas cell viability was evaluated by a dye-reduction assay using MTT. The E-64 inhibitor showed effect on growth, adherence and viability of trophozoites, however, its better performance was detected in the 100 µM-treated cultures. Although metronidazole was more effective, the E-64 was shown to be able to inhibit growth, adherence and viability rates by ≥ 50%. These results reveal that E-64 can interfere in some crucial processes to the parasite survival and they open perspectives for future investigations in order to confirm the real antigiardial potential of the protease inhibitors. PMID:24553607

  16. 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.

  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. Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens

    PubMed Central

    López-Cortés, Luis F.; Castaño, Manuel A.; López-Ruz, Miguel A.; Rios-Villegas, María J.; Hernández-Quero, José; Merino, Dolores; Jiménez-Aguilar, Patricia; Marquez-Solero, Manuel; Terrón-Pernía, Alberto; Tellez-Pérez, Francisco; Viciana, Pompeyo; Orihuela-Cañadas, Francisco; Palacios-Baena, Zaira; Vinuesa-Garcia, David; Fajardo-Pico, Jose M.; Romero-Palacios, Alberto; Ojeda-Burgos, Guillermo; Pasquau-Liaño, Juan

    2016-01-01

    Background and Objective Significant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv) as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF) while on protease inhibitor (PI) -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens. Methods This retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure). Results A total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis) and virological efficacy (on-treatment analysis) at week 96 were 79.3% (CI95, 76.8−81.8) and 91.5% (CI95, 89.6–93.4), respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations. Conclusion Switching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for

  19. Inhibitory Activity of Human Immunodeficiency Virus Aspartyl Protease Inhibitors against Encephalitozoon intestinalis Evaluated by Cell Culture-Quantitative PCR Assay

    PubMed Central

    Menotti, Jean; Santillana-Hayat, Maud; Cassinat, Bruno; Sarfati, Claudine; Derouin, Francis; Molina, Jean-Michel

    2005-01-01

    Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis. PMID:15917534

  20. Characterization of human immunodeficiency virus type 1 variants with increased resistance to a C2-symmetric protease inhibitor.

    PubMed Central

    Ho, D D; Toyoshima, T; Mo, H; Kempf, D J; Norbeck, D; Chen, C M; Wideburg, N E; Burt, S K; Erickson, J W; Singh, M K

    1994-01-01

    Inhibitors of the human immunodeficiency virus type 1 protease represent a promising class of antiviral drugs for the treatment of AIDS, and several are now in clinical trials. Here, we report the in vitro selection of viral variants with decreased sensitivity to a C2-symmetric protease inhibitor (A-77003). We show that a single amino acid substitution (Arg to Gln or Lys) at position 8 of the protease results in a substantial decrease in the inhibitory activity of the drug on the enzyme and a comparable increase in viral resistance. These findings, when analyzed by using the three-dimensional structure of the protease-drug complex, provide a strategic guide for the future development of inhibitors of the human immunodeficiency virus type 1 protease. Images PMID:8107264

  1. Design, Synthesis and Biological Evaluation of a Library of Thiocarbazates and their Activity as Cysteine Protease Inhibitors

    PubMed Central

    Liu, Zhuqing; Myers, Michael C.; Shah, Parag P.; Beavers, Mary Pat; Benedetti, Phillip A.; Diamond, Scott L.

    2010-01-01

    Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead. Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity against a panel proteases indicated a significant preference for members of the papain family of cysteine proteases over serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several very potent inhibitors of Cathepsin L and S were identified. The SAR data was employed in docking studies in an effort to understand the structural elements required for Cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of the papain family of cysteine proteases. PMID:20438448

  2. Gene expression of proteases and protease inhibitors in the human ciliary epithelium and ODM-2 cells.

    PubMed

    Ortego, J; Escribano, J; Coca-Prados, M

    1997-08-01

    Complementary DNAs (cDNAs), corresponding to the human proteinases cathepsins D and O and proteinase inhibitors alpha2-macroglobulin and PP5/TFPI-2, have recently been isolated and identified from a subtractive human ciliary body library. In the present study we determined: (i) their pattern of expression in the human eye; (ii) the ability of the ciliary body and/or ciliary epithelial cells to synthesize and secrete cathepsin D and alpha1-antitrypsin in vitro; and (iii) whether alpha1-antitrypsin expression in cultured ciliary epithelial cells is modulated by protein kinase C activation. Northern analysis demonstrated that the ciliary body expresses high levels of cathepsins D and O, alpha2-macroglobulin, alpha1-antitrypsin and PP5/TFPI-2 transcripts. Western blot analysis and immunoprecipitation experiments with cathepsin D and alpha1-antitrypsin antibodies indicated that metabolically labeled ciliary body explants and/or ciliary epithelial cells in vitro with 35S-methionine, synthesize and secrete these proteins. Cultured nonpigmented ciliary epithelial ODM-2 cells, in response to phorbol-12-myristate 13-acetate (PMA), but not to the non-protein kinase C binding phorbol ester 4 alpha-phorbol didecanoate (PDBu), elicited up-regulation (up to 5-fold) of transcription, synthesis and secretion of alpha1-antitrypsin. These results provide in vitro evidence that the ciliary epithelium synthesizes and secretes a selective group of proteinases and proteinase inhibitors detected also in aqueous humor. The expression of at least of one of the proteinase inhibitors, alpha1-antitrypsin, can be modulated in response to phorbol ester.

  3. Novel inhibitors of human leukocyte elastase and cathepsin G. Sequence variants of squash seed protease inhibitor with altered protease selectivity

    SciTech Connect

    McWherter, C.A.; Walkenhorst, W.F.; Glover, G.I. ); Campbell, E.J. )

    1989-07-11

    Novel peptide inhibitors of human leukocyte elastase (HLE) and cathepsin G (CG) were prepared by solid-phase peptide synthesis of P1 amino acid sequence variants of Curcurbita maxima trypsin inhibitor III (CMTI-III), a 29-residue peptide found in squash seed. A systematic study of P1 variants indicated that P1, Arg, Lys, Leu, Ala, Phe, and Met inhibit trypsin; P1, Val, Ile, Gly, Leu, Ala, Phe, and Met inhibit HLE; P1 Leu, Ala, Phe, and Met inhibit CG and chymotrypsin. Variants with P1, Val, Ile, or Gly were selective inhibitors of HLE, while inhibition of trypsin required P1 amino acids with an unbranched {beta} carbon. Studies of Val-5-CMTI-III (P1 Val) inhibition of HLE demonstrated a 1:1 binding stoichiometry with a (K{sub i}){sub app} of 8.7 nM. Inhibition of HLE by Gly-5-CMTI-III indicated a significant role for reactive-site structural moieties other than the P1 side chain. Val-5-CMTI-III inhibited both HLE and human polymorphonuclear leukocyte (PMN) proteolysis of surface-bound {sup 125}I-labeled fibronectin. Val-5-CMTI-III was more effective at preventing turnover of a peptide p-nitroanilide substrate than halting dissolution of {sup 125}I-labeled fibronectin. It was about as effective as human serum {alpha}{sub 1}-proteinase inhibitor in preventing PMN degradation of the connective tissue substrate. In addition to providing interesting candidates for controlling inflammatory cell proteolytic injury, the CMTI-based inhibitors are ideal for studying molecular recognition because of their small size, their ease of preparation, and the availability of sensitive and quantitative assays for intermolecular interactions.

  4. Identification of chikungunya virus nsP2 protease inhibitors using structure-base approaches.

    PubMed

    Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

    2015-04-01

    The nsP2 protease of chikungunya virus (CHIKV) is one of the essential components of viral replication and it plays a crucial role in the cleavage of polyprotein precursors for the viral replication process. Therefore, it is gaining attention as a potential drug design target against CHIKV. Based on the recently determined crystal structure of the nsP2 protease of CHIKV, this study identified potential inhibitors of the virus using structure-based approaches with a combination of molecular docking, virtual screening and molecular dynamics (MD) simulations. The top hit compounds from database searching, using the NCI Diversity Set II, with targeting at five potential binding sites of the nsP2 protease, were identified by blind dockings and focused dockings. These complexes were then subjected to MD simulations to investigate the stability and flexibility of the complexes and to gain a more detailed insight into the interactions between the compounds and the enzyme. The hydrogen bonds and hydrophobic contacts were characterized for the complexes. Through structural alignment, the catalytic residues Cys1013 and His1083 were identified in the N-terminal region of the nsP2 protease. The absolute binding free energies were estimated by the linear interaction energy approach and compared with the binding affinities predicted with docking. The results provide valuable information for the development of inhibitors for CHIKV.

  5. Low molecular weight serine protease inhibitors from insects are proteins with highly conserved sequences.

    PubMed

    Boigegrain, R A; Pugnière, M; Paroutaud, P; Castro, B; Brehélin, M

    2000-02-01

    A low molecular weight protease inhibitor peptide found in ovaries of the desert locust Schistocerca gregaria (SGPI-2), was purified from plasma of the same locust and sequenced. It was named SGCI. It was found active towards chymotrypsin and human leukocyte elastase. SGCI was synthesized using a solid-phase procedure and the sequence of its reactive site for chymotrypsin was determined. Compared with an inhibitor purified earlier from another locust species, the total sequence of SGCI showed 88% identity. In particular, the sequence of the reactive site of these inhibitors was identical. Our search for a closely related peptide in an insect species far removed from locusts, the lepidopteran Spodoptera littoralis, was unfruitful but a different chymotrypsin inhibitor, belonging to the Kazal family, was found whose mass is greater than that of SGCI (20 vs 3.6 kDa). Its N-terminal sequence shares 80% identity with that of a chymotrypsin inhibitor purified earlier from the haemolymph of another lepidopteran. Conservation of the amino acid sequence in the reactive site seems to be an exception among protease inhibitors.

  6. Efficacy of Bacillus thuringiensis Cry3Aa protoxin and protease inhibitors against coleopteran storage pests.

    PubMed

    Oppert, Brenda; Morgan, Tom D; Kramer, Karl J

    2011-05-01

    Environmental impacts and resistance to insecticides pose serious challenges to stored-product insect and other types of pest control. Insect-resistant transgenic grain is a potential alternative to fumigants, but candidate control proteins are needed, especially for coleopterans. Therefore, we evaluated the efficacy of a coleopteran-active toxin, Bacillus thuringiensis Cry3Aa, with or without protease inhibitors, in laboratory feeding assays against coleopteran storage pests. In a comparison of the toxicity of Cry3Aa protoxin towards three species of coleopteran storage pests, Tenebrio molitor L. was found to be most sensitive, Tribolium castaneum (Herbst.) was most refractory and Rhyzopertha dominica F. displayed an intermediate response. For R. dominica, Cry3Aa combined with 3500 mg potato carboxypeptidase inhibitor or 5000 mg aprotinin kg(-1) diet resulted in both delayed development and increased mortality. Potato carboxypeptidase inhibitor and bovine aprotinin reduced the LC(50) of Cry3Aa for R. dominica two- and threefold respectively. Cry3Aa treatment resulted in fewer progeny from R. dominica, and progeny was further reduced when the protoxin was combined with potato carboxypeptidase inhibitor. These data support the hypothesis that a combination of Cry3Aa protoxin and protease inhibitors, particularly a potato carboxypeptidase inhibitor, may have applications in control strategies for preventing damage to stored products and grains by coleopteran pests. This article is a US Government work and is in the public domain in the USA. Published 2011 by John Wiley & Sons, Ltd.

  7. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields.

  8. Protease inhibitors decrease rabbit cartilage degradation after meniscectomy

    SciTech Connect

    Caputo, C.B.; Sygowski, L.A.; Patton, S.P.; Wolanin, D.J.; Shaw, A.; Roberts, R.A.; DiPasquale, G.

    1988-01-01

    In vitro proteoglycan (PG) synthesis and release were measured on cartilage removed from rabbit knees within 1 week of meniscectomy. Three days following partial lateral meniscectomy, 72% of the femurs and 82% of the tibias had visible ulcers. Cartilage from the weight-bearing areas incorporated 2.0-2.9 times more /sup 35/S-sulfate in vitro than cartilage from the opposite, unoperated knees. /sup 3/H-thymidine incorporation was 2.5-3.4 times higher for surgical than control groups. /sup 35/S-sulfate incorporation by the surgical group was inhibited by 22% in the presence of 10(-4) M U24522, an inhibitor of rabbit chondrocyte metalloprotease (CMP). /sup 3/H-thymidine incorporation by the surgical group was inhibited by 28% by 10(-4) M U24522. In vitro PG release from cartilage removed 2 days after surgery was 1.6-3.7 times higher for the surgical than the control group. PG release by the surgical group after 22 h of incubation was reduced to the control level by three CMP inhibitors, U24278, U24279, and U24522. PG release by cartilage from the nonsurgical group was also reduced by these compounds at 22 h. These results suggest that both the anabolic and catabolic processes that are stimulated by surgery can be isolated in vitro and that CMP may be involved in the catabolic process.

  9. Reaching the melting point: Degradative enzymes and protease inhibitors involved in baculovirus infection and dissemination.

    PubMed

    Ishimwe, Egide; Hodgson, Jeffrey J; Clem, Rollie J; Passarelli, A Lorena

    2015-05-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in "melting" or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Computational mutation scanning and drug resistance mechanisms of HIV-1 protease inhibitors.

    PubMed

    Hao, Ge-Fei; Yang, Guang-Fu; Zhan, Chang-Guo

    2010-07-29

    The drug resistance of various clinically available HIV-1 protease inhibitors has been studied using a new computational protocol, that is, computational mutation scanning (CMS), leading to valuable insights into the resistance mechanisms and structure-resistance correction of the HIV-1 protease inhibitors associated with a variety of active site and nonactive site mutations. By using the CMS method, the calculated mutation-caused shifts of the binding free energies linearly correlate very well with those derived from the corresponding experimental data, suggesting that the CMS protocol may be used as a generalized approach to predict drug resistance associated with amino acid mutations. Because it is essentially important for understanding the structure-resistance correlation and for structure-based drug design to develop an effective computational protocol for drug resistance prediction, the reasonable and computationally efficient CMS protocol for drug resistance prediction should be valuable for future structure-based design and discovery of antiresistance drugs in various therapeutic areas.

  11. Reversible detection of proteases and their inhibitors by a pulsed chronopotentiometric polyion-sensitive electrode.

    PubMed

    Xu, Yida; Shvarev, Alexey; Makarychev-Mikhailov, Sergey; Bakker, Eric

    2008-03-15

    Polymer membrane electrodes operated by pulsed chronopotentiometry have recently been introduced to replace traditional ion-selective electrodes for a number of applications. While ion-selective electrodes for the polycation protamine have been reported, for instance, a pulsed chronopotentiometric readout mode (called here pulstrode) provides improved stability and reproducibility while exhibiting sufficient selectivity for the direct detection of protamine in undiluted whole blood samples. Here, such protamine-sensitive pulstrodes are applied for the real-time detection of the activity of the protease trypsin and its soybean inhibitor. This is possible because small fragments produced by the trypsin digestion are not detectable by the protamine-sensing membrane. The real-time response to the proteolytic reaction is shown to exhibit good reproducibility and reversibility, and the initial reaction rate is dependent on the concentration of the protease and its inhibitor.

  12. Tetrahydrofuran, tetrahydropyran, triazoles and related heterocyclic derivatives as HIV protease inhibitors

    PubMed Central

    Ghosh, Arun K; Anderson, David D

    2011-01-01

    HIV/AIDS remains a formidable disease with millions of individuals inflicted worldwide. Although treatment regimens have improved considerably, drug resistance brought on by viral mutation continues to erode their effectiveness. Intense research efforts are currently underway in search of new and improved therapies. This review is concerned with the design of novel HIV-1 protease inhibitors that incorporate heterocyclic scaffolds and which have been reported within the recent literature (2005–2010). Various examples in this review showcase the essential role heterocycles play as scaffolds and bioisosteres in HIV-1 protease inhibitor drug development. This review will hopefully stimulate the widespread application of these heterocycles in the design of other therapeutic agents. PMID:21806380

  13. Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies.

    PubMed

    Sabotič, Jerica; Ohm, Robin A; Künzler, Markus

    2016-01-01

    Fruiting bodies or sporocarps of dikaryotic (ascomycetous and basidiomycetous) fungi, commonly referred to as mushrooms, are often rich in entomotoxic and nematotoxic proteins that include lectins and protease inhibitors. These protein toxins are thought to act as effectors of an innate defense system of mushrooms against animal predators including fungivorous insects and nematodes. In this review, we summarize current knowledge about the structures, target molecules, and regulation of the biosynthesis of the best characterized representatives of these fungal defense proteins, including galectins, beta-trefoil-type lectins, actinoporin-type lectins, beta-propeller-type lectins and beta-trefoil-type chimerolectins, as well as mycospin and mycocypin families of protease inhibitors. We also present an overview of the phylogenetic distribution of these proteins among a selection of fungal genomes and draw some conclusions about their evolution and physiological function. Finally, we present an outlook for future research directions in this field and their potential applications in medicine and crop protection.

  14. Crystallization and preliminary X-ray crystallographic analysis of the cysteine protease inhibitor clitocypin

    SciTech Connect

    Galeša, Katja; Brzin, Jože; Sabotič, Jerica; Turk, Dušan

    2006-01-01

    Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. Clitocypin is a cysteine protease inhibitor from the mushroom Clitocybe nebularis. The protein has been purified from natural sources and crystallized in a variety of non-isomorphous forms belonging to monoclinic and triclinic space groups. A diffraction data set to 1.55 Å resolution was obtained from a crystal belonging to space group P2, with unit-cell parameters a = 38.326, b = 33.597, c = 55.568 Å, β = 104°. An inability to achieve isomorphism forced the use of MAD and SAD phasing methods. Phasing is in progress.

  15. Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

    PubMed Central

    Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

    2015-01-01

    Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

  16. Design, Synthesis, and Evaluation of Novel Prodrugs of Transition State Inhibitors of Norovirus 3CL Protease.

    PubMed

    Galasiti Kankanamalage, Anushka C; Kim, Yunjeong; Rathnayake, Athri D; Alliston, Kevin R; Butler, Michelle M; Cardinale, Steven C; Bowlin, Terry L; Groutas, William C; Chang, Kyeong-Ok

    2017-07-27

    Ester and carbamate prodrugs of aldehyde bisulfite adduct inhibitors were synthesized in order to improve their pharmacokinetic and pharmacodynamic properties. The inhibitory activity of the compounds against norovirus 3C-like protease in enzyme and cell-based assays was determined. The ester and carbamate prodrugs displayed equivalent potency to those of the precursor aldehyde bisulfite adducts and precursor aldehydes. Furthermore, the rate of ester cleavage was found to be dependent on alkyl chain length. The generated prodrugs exhibited low cytotoxicity and satisfactory liver microsomes stability and plasma protein binding. The methodology described herein has wide applicability and can be extended to the bisulfite adducts of common warheads employed in the design of transition state inhibitors of serine and cysteine proteases of medical relevance.

  17. Quinolylhydrazones as novel inhibitors of Plasmodium falciparum serine protease PfSUB1.

    PubMed

    Gemma, Sandra; Giovani, Simone; Brindisi, Margherita; Tripaldi, Pierangela; Brogi, Simone; Savini, Luisa; Fiorini, Isabella; Novellino, Ettore; Butini, Stefania; Campiani, Giuseppe; Penzo, Maria; Blackman, Michael J

    2012-08-15

    Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.

  18. Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamide-substituted cyclooctylpyramones.

    PubMed

    Skulnick, H I; Johnson, P D; Aristoff, P A; Morris, J K; Lovasz, K D; Howe, W J; Watenpaugh, K D; Janakiraman, M N; Anderson, D J; Reischer, R J; Schwartz, T M; Banitt, L S; Tomich, P K; Lynn, J C; Horng, M M; Chong, K T; Hinshaw, R R; Dolak, L A; Seest, E P; Schwende, F J; Rush, B D; Howard, G M; Toth, L N; Wilkinson, K R; Romines, K R

    1997-03-28

    Recently, cyclooctylpyranone derivatives with m-carboxamide substituents (e.g. 2c) were identified as potent, nonpeptidic HIV protease inhibitors, but these compounds lacked significant antiviral activity in cell culture. Substitution of a sulfonamide group at the meta position, however, produces compounds with excellent HIV protease binding affinity and antiviral activity. Guided by an iterative structure-based drug design process, we have prepared and evaluated a number of these derivatives, which are readily available via a seven-step synthesis. A few of the most potent compounds were further evaluated for such characteristics as pharmacokinetics and toxicity in rats and dogs. From this work, the p-cyanophenyl sulfonamide derivative 35k emerged as a promising inhibitor, was selected for further development, and entered phase I clinical trials.

  19. Enhanced bioavailability of subcutaneously injected insulin by pretreatment with ointment containing protease inhibitors

    SciTech Connect

    Takeyama, M.; Ishida, T.; Kokubu, N.; Komada, F.; Iwakawa, S.; Okumura, K.; Hori, R. )

    1991-01-01

    The present study was undertaken to develop an ointment preparation containing a protease inhibitor for stabilizing subcutaneously injected insulin. The ointment containing the protease inhibitor, gabexate mesilate or nafamostat mesilate, was applied to the skin around the insulin injection site. Three results were obtained. First, gabexate and nafamostat inhibited insulin degradation in subcutaneous tissue homogenates in vitro. Second, after application of gabexate or nafamostat ointment, an appreciable amount of gabexate or nafamostat appeared in the subcutaneous tissue of rats or hairless mice and their concentrations were comparable to those seen in the in vitro experiment. Third, insulin degradation at the subcutaneous injection site in the rat was depressed after pretreatment with gabexate or nafamostat ointment. Pretreatment with gabexate or nafamostat ointment increased the plasma immunoreactive insulin (IRI) levels and the hypoglycermic effect of insulin in healthy volunteers. These results indicate that gabexate or nafamostat ointments stabilize subcutaneously injected insulin.

  20. Dipeptide-derived nitriles containing additional electrophilic sites: potentially irreversible inhibitors of cysteine proteases.

    PubMed

    Löser, Reik; Gütschow, Michael

    2009-12-01

    Heterocyclic and open-chain dipeptide-derived nitriles have been synthesized, containing an additional electrophilic center enabling the subsequent covalent modification of the thioimidate nitrogen formed in situ at the active site of the enzyme. The inhibitory potential of these nitriles against the cysteine proteases papain and cathepsins L, S, and K was determined. The open-chain dipeptide nitriles 8 and 10 acted as moderate reversible inhibitors, but no evidence for an irreversible inhibition of these enzymes was discernable.

  1. Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors

    PubMed Central

    Bayés, Alex; Comellas-Bigler, Mireia; de la Vega, Monica Rodríguez; Maskos, Klaus; Bode, Wolfram; Aviles, Francesc X.; Jongsma, Maarten A.; Beekwilder, Jules; Vendrell, Josep

    2005-01-01

    Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A/B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase α/β-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1′ substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase–PCI complexes, the β8-α9 loop, and α7 together with the α7-α8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors. PMID:16260742

  2. Structural basis of the resistance of an insect carboxypeptidase to plant protease inhibitors.

    PubMed

    Bayés, Alex; Comellas-Bigler, Mireia; Rodríguez de la Vega, Monica; Maskos, Klaus; Bode, Wolfram; Aviles, Francesc X; Jongsma, Maarten A; Beekwilder, Jules; Vendrell, Josep

    2005-11-15

    Corn earworm (Helicoverpa zea), also called tomato fruitworm, is a common pest of many Solanaceous plants. This insect is known to adapt to the ingestion of plant serine protease inhibitors by using digestive proteases that are insensitive to inhibition. We have now identified a B-type carboxypeptidase of H. zea (CPBHz) insensitive to potato carboxypeptidase inhibitor (PCI) in corn earworm. To elucidate the structural features leading to the adaptation of the insect enzyme, the crystal structure of the recombinant CPBHz protein was determined by x-ray diffraction. CPBHz is a member of the A/B subfamily of metallocarboxypeptidases, which displays the characteristic metallocarboxypeptidase alpha/beta-hydrolase fold, and does not differ essentially from the previously described Helicoverpa armigera CPA, which is very sensitive to PCI. The data provide structural insight into several functional properties of CPBHz. The high selectivity shown by CPBHz for C-terminal lysine residues is due to residue changes in the S1' substrate specificity pocket that render it unable to accommodate the side chain of an arginine. The insensitivity of CPBHz to plant inhibitors is explained by the exceptional positioning of two of the main regions that stabilize other carboxypeptidase-PCI complexes, the beta8-alpha9 loop, and alpha7 together with the alpha7-alpha8 loop. The rearrangement of these two regions leads to a displacement of the active-site entrance that impairs the proper interaction with PCI. This report explains a crystal structure of an insect protease and its adaptation to defensive plant protease inhibitors.

  3. Macrocyclic Hepatitis C Virus NS3/4A Protease Inhibitors: An Overview of Medicinal Chemistry.

    PubMed

    Pillaiyar, Thanigaimalai; Namasivayam, Vigneshwaran; Manickam, Manoj

    2016-01-01

    Hepatitis C virus (HCV) is a causative agent of hepatitis C infectious disease that primarily affects the liver, ranging in severity from a mild illness lasting a few weeks to a lifelong illness. The 9.6 kb RNA genome of HCV encodes approximately 3000 amino acid polyprotein that must be processed by host and viral proteases into both structural (S) and non-structural (NS) proteins, respectively. Targeting the serine protease NS3 with an activating factor NS4A, i.e., NS3/4A has been considered as one of the most attractive targets for the development of anti-HCV therapy. Although there is no vaccine available, antiviral medicines cure approximately 90% of the persons with hepatitis C infection. On the other hand, efficacy of these medications can be hampered due to the rapid drug and cross resistances. To date, all developed HCV NS3/4A inhibitors are mainly peptide-based compounds derived from the cleavage products of substrate. Specifically macrocyclic peptidomimetics have rapidly emerged as a classical NS3/4A protease inhibitors for treating the HCV infection. This review highlights the development of macrocyclic anti-HCV NS3/4A protease, as well as clinically important inhibitors developed from linear peptides, discovered during the last 12 years (2003-2015) from all sources, including laboratory synthetic methods, virtual screening and structure-based molecular docking studies. We emphasize the rationale behind the design, study of structure-activity relationships, and mechanism of inhibitions and cellular effect of the macrocyclic inhibitors.

  4. Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

    PubMed Central

    Sheaffer, Amy K.; Friborg, Jacques; Hernandez, Dennis; Falk, Paul; Zhai, Guangzhi; Levine, Steven; Chaniewski, Susan; Yu, Fei; Barry, Diana; Chen, Chaoqun; Lee, Min S.; Mosure, Kathy; Sun, Li-Qiang; Sinz, Michael; Meanwell, Nicholas A.; Colonno, Richard J.; Knipe, Jay; Scola, Paul

    2012-01-01

    Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with Ki values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC50s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC50, 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC50, >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC50s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients. PMID:22869577

  5. Nagashima-type palmoplantar keratosis: a common Asian type caused by SERPINB7 protease inhibitor deficiency.

    PubMed

    Kubo, Akiharu

    2014-08-01

    Nagashima-type palmoplantar keratosis (NPPK) is an autosomal recessive diffuse non-epidermolytic palmoplantar keratosis caused by mutations in SERPINB7, a member of the serine protease inhibitor superfamily. Genetic studies suggest that NPPK is the most common palmoplantar keratosis in Japan, and probably Asia, but one that is extremely rare in Western countries. In this issue, Yin et al. report a founder effect of a SERPINB7 mutation in Chinese populations.

  6. Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease.

    PubMed

    Bodvard, Kristofer; Mohlin, Johanna; Knecht, Wolfgang

    2007-02-01

    Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.

  7. Toxic uveitis caused by pharmacodynamic interactions of Rifabutin and protease inhibitors: a case report.

    PubMed

    Ebraert, H; Salu, P

    2007-01-01

    Toxic reactions (uveitis, arthritis and leucopenia) of Rifabutin at normal doses should always be considered because of pharmacokinetic interactions with other drugs (e.g. the protease inhibitors). This case demonstrates that this kind of uveitis is clinically significant as the diagnosis of uveitis, particularly hypopyon uveitis, in an immunocompromised patient generally mandates extensive systemic diagnostic evaluation, as well as intensive ophthalmic and systemic monitoring and therapy.

  8. Virtual screening of novel reversible inhibitors for marine alkaline protease MP.

    PubMed

    Ji, Xiaofeng; Zheng, Yuan; Wang, Wei; Sheng, Jun; Hao, Jianhua; Sun, Mi

    2013-11-01

    Marine alkaline protease (MP,(2) accession no. ACY25898) is produced by a marine bacterium strain isolated from Yellow Sea sediment in China. Previous research has shown that this protease is a cold-adapted enzyme with antioxidant activity that could be used as a detergent additive. Owing to its instability in the liquid state, MP's application in liquid detergents was limited. Therefore, the discovery of reversible MP inhibitors to stabilize the protease was imperative. Here, we used the X-ray structure of MP and recompiled AutoDock 4.2 with refined Zn(2+) characters to screen the free chemical database ZINC. After completing the docking procedure, we applied strategies including the "initial filter", consensus scoring and pharmocophore model to accelerate the process and improve the virtual screening success rate. The "initial filter" was built based on the docking results of boronic acid derivatives validated as reversible inhibitors of MP by our previous studies. Finally, ten compounds were purchased or synthetized to test their binding affinity for MP. Three of the compounds could reversibly inhibit MP with apparent Ki values of 0.8-1.2 mmol. These active compounds and their binding modes provide useful information for understanding the molecular mechanism of reversible MP inhibition. The results may also serve as the foundation for further screening and design of reversible MP inhibitors.

  9. Multicenter Quality Control of Hepatitis C Virus Protease Inhibitor Resistance Genotyping

    PubMed Central

    Larrat, Sylvie; Laperche, Syria; Le Guillou-Guillemette, Hélène; Legrand-Abravanel, Florence; Bouchardeau, Françoise; Pivert, Adeline; Henquell, Cécile; Mirand, Audrey; André-Garnier, Elisabeth; Giordanengo, Valérie; Lagathu, Gisèle; Thibault, Vincent; Scholtes, Caroline; Schvoerer, Evelyne; Gaudy-Graffin, Catherine; Maylin, Sarah; Trimoulet, Pascale; Brochot, Etienne; Hantz, Sébastien; Gozlan, Joël; Roque-Afonso, Anne-Marie; Soussan, Patrick; Plantier, Jean-Christophe; Charpentier, Charlotte; Chevaliez, Stéphane; Colson, Philippe; Mackiewicz, Vincent; Aguilera, Lina; Rosec, Sylvain; Gouriou, Stéphanie; Magnat, Nelly; Lunel-Fabiani, Françoise; Izopet, Jacques; Morand, Patrice; Payan, Christopher; Pawlotsky, Jean-Michel

    2013-01-01

    Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents. PMID:23426922

  10. Multicenter quality control of hepatitis C virus protease inhibitor resistance genotyping.

    PubMed

    Vallet, Sophie; Larrat, Sylvie; Laperche, Syria; Le Guillou-Guillemette, Hélène; Legrand-Abravanel, Florence; Bouchardeau, Françoise; Pivert, Adeline; Henquell, Cécile; Mirand, Audrey; André-Garnier, Elisabeth; Giordanengo, Valérie; Lagathu, Gisèle; Thibault, Vincent; Scholtes, Caroline; Schvoerer, Evelyne; Gaudy-Graffin, Catherine; Maylin, Sarah; Trimoulet, Pascale; Brochot, Etienne; Hantz, Sébastien; Gozlan, Joël; Roque-Afonso, Anne-Marie; Soussan, Patrick; Plantier, Jean-Christophe; Charpentier, Charlotte; Chevaliez, Stéphane; Colson, Philippe; Mackiewicz, Vincent; Aguilera, Lina; Rosec, Sylvain; Gouriou, Stéphanie; Magnat, Nelly; Lunel-Fabiani, Françoise; Izopet, Jacques; Morand, Patrice; Payan, Christopher; Pawlotsky, Jean-Michel

    2013-05-01

    Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.

  11. A structural and functional analogue of a Bowman–Birk-type protease inhibitor from Odorrana schmackeri

    PubMed Central

    Long, Qilin; Xu, Ying; Guo, Shaodong; Chen, Tianbao; Wang, Lei; Zhou, Mei; Shaw, Chris; Walker, Brian

    2017-01-01

    Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the present study, by use of molecular ‘shotgun’ cloning and LC MS/MS fractionation sequencing, a novel Bowman–Birk-type heptadecapeptide (AALKGCWTKSIPPKPCF-amide), named Odorrana schmackeri Trypsin Inhibitor (OSTI), with a canonical Cys6–Cys16 disulfide bridge, was isolated and identified in piebald odorous frog (O. schmackeri) skin secretion. A synthetic replicate of OSTI-exhibited trypsin inhibitory activity with a Ki value of 0.3 ± 0.04 nM and also a tryptase inhibitory effect with a Ki of 2.5 ± 0.6 μM. This is the first time that this property has been reported for a peptide originating from amphibian sources. In addition, substituting lysine (K) with phenylalanine (F) at the presumed P1 position, completely abrogated the trypsin and tryptase inhibition, but produced a strong chymotrypsin inhibition with a Ki of 1.0 ± 0.1 μM. Thus, the specificity of this peptidic protease inhibitor could be optimized through modifying the amino acid residue at the presumed P1 position and this novel native OSTI, along with its analogue, [Phe9]-OSTI, have expanded the potential drug discovery and development pipeline directed towards alleviation of serine protease-mediated pathologies. PMID:28356487

  12. Design, Synthesis, Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    PubMed Central

    Parai, Maloy Kumar; Huggins, David J.; Cao, Hong; Nalam, Madhavi N. L.; Ali, Akbar; Schiffer, Celia A.; Tidor, Bruce; Rana, Tariq M.

    2012-01-01

    A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants, in particular inhibitors containing 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with Ki values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and C and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp-29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies. PMID:22708897

  13. Substitution of raltegravir for ritonavir-boosted protease inhibitors in HIV-infected patients: the SPIRAL study.

    PubMed

    Martínez, Esteban; Larrousse, María; Llibre, Josep M; Gutiérrez, Felix; Saumoy, Maria; Antela, Antonio; Knobel, Hernando; Murillas, Javier; Berenguer, Juan; Pich, Judit; Pérez, Ignacio; Gatell, José M

    2010-07-17

    Switching to raltegravir in selected patients treated with ritonavir-boosted protease inhibitors may result in similar efficacy and lower plasma lipids. SPIRAL is a 48-week multicentre, open-label trial in which HIV-infected adults with less than 50 copies/ml of plasma HIV RNA for at least the previous 6 months on ritonavir-boosted protease inhibitor-based therapy were randomized (1: 1) to switch from the ritonavir-boosted protease inhibitor to raltegravir or to continue on ritonavir-boosted protease inhibitor-based therapy. Primary endpoint was the proportion of patients free of treatment failure (noncompleter = failure) at 48 weeks. SPIRAL study was powered to show noninferior efficacy of raltegravir-based therapy with a margin of -12.5%. Two hundred and seventy-three patients assigned to switch to raltegravir (n = 139) or to continue ritonavir-boosted protease inhibitor (n = 134) were included in the efficacy analysis. At 48 weeks, 89.2% (raltegravir-based therapy) and 86.6% (ritonavir-boosted protease inhibitor-based therapy) of the patients remained free of treatment failure [difference 2.6%; 95% confidence interval (CI) -5.2 to 10.6]. A total of 96.9% (raltegravir-based therapy) and 95.1% (ritonavir-boosted protease inhibitor-based therapy) of the patients remained free of virological failure (difference 1.8%; 95% CI -3.5 to 7.5). Switching to raltegravir was associated with significant decreases in plasma lipids and total-to-HDL cholesterol ratio relative to continuing ritonavir-boosted protease inhibitor. Severe adverse events and study drug discontinuations due to any adverse event occurred in 4 and 2% of the patients in each group. In patients with sustained virological suppression on ritonavir-boosted protease inhibitor-based therapy, switching from ritonavir-boosted protease inhibitor to raltegravir demonstrated noninferior efficacy and resulted in a better lipid profile at 48 weeks than continuing ritonavir-boosted protease inhibitor.

  14. The design of potent, non-peptidic inhibitors of hepatitis C protease.

    PubMed

    Andrews, David M; Chaignot, Helene M; Coomber, Barry A; Dowle, Mike D; Hind, S Lucy; Johnson, Martin R; Jones, Paul S; Mills, Gail; Patikis, Angela; Pateman, Tony J; Robinson, J Ed; Slater, Martin J; Trivedi, Naimisha

    2003-04-01

    The pyrrolidine-5,5-trans-lactam template was used to design small, neutral, mechanism-based inhibitors of hepatitis C NS3/4A protease displaying potent activity in the replicon cell-based assay. The activity of this series is not dependent upon its chemical reactivity and molecules have been synthesised which combine enhanced biochemical potency with improved plasma stability. Promising initial pharmacokinetic data indicating the potential for further optimisation of this series into low molecular weight, drug-like inhibitors is presented.

  15. Extending the cellulosome paradigm: the modular Clostridium thermocellum cellulosomal serpin PinA is a broad-spectrum inhibitor of subtilisin-like proteases.

    PubMed

    O Cuív, Páraic; Gupta, Rajesh; Goswami, Hareshwar P; Morrison, Mark

    2013-10-01

    Clostridium thermocellum encodes a cellulosomal, modular, and thermostable serine protease inhibitor (serpin), PinA. PinA stability but not inhibitory activity is affected by the Fn(III) and Doc(I) domains, and PinA is a broad inhibitor of subtilisin-like proteases and may play a key role in protecting the cellulosome from protease attack.

  16. Contribution of Gag and protease to variation in susceptibility to protease inhibitors between different strains of subtype B human immunodeficiency virus type 1.

    PubMed

    Sutherland, Katherine A; Mbisa, Jean L; Cane, Patricia A; Pillay, Deenan; Parry, Chris M

    2014-01-01

    Recent reports have shown that human immunodeficiency virus type 1 (HIV-1) Gag can directly affect susceptibility to protease inhibitors (PIs) in the absence of known resistance mutations in protease. Inclusion of co-evolved Gag alongside protease in phenotypic drug susceptibility assays can alter PI susceptibility in comparison with protease with a WT Gag. Using a single-replication-cycle assay encompassing full-length Gag together with protease we demonstrated significant variation in PI susceptibility between a number of PI-naïve subtype B viruses. Six publicly available subtype B molecular clones, namely HXB2, NL4-3, SF2, YU2, JRFL and 89.6, displayed up to nine-fold reduced PI susceptibility in comparison with the assay reference strain. For two molecular clones, YU2 and JRFL, Gag contributed solely to the observed reduction in susceptibility, with the N-terminal region of Gag contributing significantly. Gag and protease from treatment-naïve, patient-derived viruses also demonstrated significant variation in susceptibility, with up to a 17-fold reduction to atazanavir in comparison with the assay reference strain. In contrast to the molecular clones, protease was the main determinant of the reduced susceptibility. Common polymorphisms in protease, including I13V, L63P and A71T, were shown to contribute to this reduction in PI susceptibility, in the absence of major resistance mutations. This study demonstrated significant variation in PI susceptibility of treatment-naïve patient viruses, and provided further evidence of the independent role of Gag, the protease substrate and in particular the N-terminus of Gag in PI susceptibility. It also highlighted the importance of considering co-evolved Gag and protease when assessing PI susceptibility.

  17. Basic Tetrapeptides as Potent Intracellular Inhibitors of Type A Botulinum Neurotoxin Protease Activity*

    PubMed Central

    Hale, Martha; Oyler, George; Swaminathan, Subramanyam; Ahmed, S. Ashraf

    2011-01-01

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development. PMID:20961849

  18. Basis Tetrapeptides as Potent Intracellular Inhibitors of type A Botulinum Neurotoxin Protease Activity

    SciTech Connect

    Hale, M.; Swaminathan, S.; Oyler, G.; Ahmed, S. A.

    2011-01-21

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

  19. Identification of novel parasitic cysteine protease inhibitors by use of virtual screening. 2. The available chemical directory.

    PubMed

    Desai, Prashant V; Patny, Akshay; Gut, Jiri; Rosenthal, Philip J; Tekwani, Babu; Srivastava, Anuradha; Avery, Mitchell

    2006-03-09

    The incidence of parasitic infections such as malaria, leishmaniasis, and trypanosomiasis has been steadily increasing. Since the existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance, there is an urgent need for development of potent, mechanism-based antiparasitic agents against these diseases. Cysteine proteases have been established as valid targets for this purpose. The Available Chemical Directory consisting of nearly 355,000 compounds was screened in silico against the homology models of plasmodial cysteine proteases, falcipain-2, and falcipain-3, to identify structurally diverse non-peptide inhibitors. The study led to identification of 22 inhibitors of parasitic cysteine proteases out of which 18 compounds were active against falcipain-2 and falcipain-3. Eight compounds exhibited dual activity against both enzymes. Additionally, four compounds were found to inhibit L. donovani cysteine protease. While one of the cysteine protease inhibitors also exhibited in vitro antiplasmodial activity with an IC50 value of 9.5 microM, others did not show noticeable antiplasmodial activity up to 20 microM. A model identifying important pharmacophoric features common to the structurally diverse falcipain-2 inhibitors has also been developed. Very few potent non-peptide inhibitors of the parasitic cysteine proteases have been reported so far, and identification of these novel and chemically diverse inhibitors should provide leads to be optimized into candidates to treat protozoal infections.

  20. Mung bean trypsin inhibitor is effective in suppressing the degradation of myofibrillar proteins in the skeletal muscle of blue scad (Decapterus maruadsi).

    PubMed

    Sun, Le-Chang; Yoshida, Asami; Cai, Qiu-Feng; Liu, Guang-Ming; Weng, Ling; Tachibana, Katsuyasu; Su, Wen-Jin; Cao, Min-Jie

    2010-12-22

    Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).

  1. Identification of potential transmembrane protease serine 4 inhibitors as anti-cancer agents by integrated computational approach.

    PubMed

    Ilamathi, M; Hemanth, R; Nishanth, S; Sivaramakrishnan, V

    2016-01-21

    Transmembrane protease serine 4 is a well known cell surface protease facilitating the extracellular matrix degradation and epithelial mesenchymal transition in hepatocellular carcinoma. Henceforth targeting transmembrane protease serine 4 is strongly believed to provide therapeutic intervention against hepatocellular carcinoma. Owing to lack of crystal structure for human transmembrane protease serine 4, we predicted its three dimensional structure for the first time in this study. Experimentally proven inhibitor-Tyroserleutide (TSL) against hepatocellular carcinoma via transmembrane protease serine 4 was used as a benchmark to identify structurally similar candidates from PubChem database to create the TSL library. Virtual screening of TSL library against modeled transmembrane protease serine 4 revealed the top four potential inhibitors. Further binding free energy (ΔGbind) analysis of the potential inhibitors revealed the best potential lead compound against transmembrane protease serine 4. Drug likeliness nature of the top four potential hits were additionally analyzed in comparison to TSL to confirm on the best potential lead compound with the highest % of human oral absorption. Consequently, e-pharmacophore mapping of the best potential lead compound yielded a six point feature. It was observed to contain four hydrogen bond donor sites (D), one positively ionizable site (P) and one aromatic ring (R). Such e-pharmacophore insight obtained from structural determinants by integrated computational analysis could serve as a framework for further advancement of drug discovery process of new anti-cancer agents with less toxicity and high specificity targeting transmembrane protease serine 4 and hepatocellular carcinoma.

  2. Crystal Structure of Feline Infectious Peritonitis Virus Main Protease in Complex with Synergetic Dual Inhibitors

    PubMed Central

    Wang, Fenghua; Chen, Cheng; Liu, Xuemeng; Yang, Kailin

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) can cause highly prevalent diseases in humans and animals. Feline infectious peritonitis virus (FIPV) belongs to the genus Alphacoronavirus, resulting in a lethal systemic granulomatous disease called feline infectious peritonitis (FIP), which is one of the most important fatal infectious diseases of cats worldwide. No specific vaccines or drugs have been approved to treat FIP. CoV main proteases (Mpros) play a pivotal role in viral transcription and replication, making them an ideal target for drug development. Here, we report the crystal structure of FIPV Mpro in complex with dual inhibitors, a zinc ion and a Michael acceptor. The complex structure elaborates a unique mechanism of two distinct inhibitors synergizing to inactivate the protease, providing a structural basis to design novel antivirals and suggesting the potential to take advantage of zinc as an adjunct therapy against CoV-associated diseases. IMPORTANCE Coronaviruses (CoVs) have the largest genome size among all RNA viruses. CoV infection causes various diseases in humans and animals, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). No approved specific drugs or vaccinations are available to treat their infections. Here, we report a novel dual inhibition mechanism targeting CoV main protease (Mpro) from feline infectious peritonitis virus (FIPV), which leads to lethal systemic granulomatous disease in cats. Mpro, conserved across all CoV genomes, is essential for viral replication and transcription. We demonstrated that zinc ion and a Michael acceptor-based peptidomimetic inhibitor synergistically inactivate FIPV Mpro. We also solved the structure of FIPV Mpro complexed with two inhibitors, delineating the structural view of a dual inhibition mechanism. Our study provides new insight into the pharmaceutical strategy against CoV Mpro through using zinc as an adjuvant therapy to enhance the efficacy of an irreversible

  3. Structure-based design of HIV protease inhibitors: 5,6-dihydro-4-hydroxy-2-pyrones as effective, nonpeptidic inhibitors.

    PubMed

    Thaisrivongs, S; Romero, D L; Tommasi, R A; Janakiraman, M N; Strohbach, J W; Turner, S R; Biles, C; Morge, R R; Johnson, P D; Aristoff, P A; Tomich, P K; Lynn, J C; Horng, M M; Chong, K T; Hinshaw, R R; Howe, W J; Finzel, B C; Watenpaugh, K D

    1996-11-08

    From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2-pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S1' and S2' subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (Ki = 15 nM) and showed antiviral activity (IC50 = 5 microM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.

  4. Developmentally linked changes in proteases and protease inhibitors suggest a role for potato multicystatin in regulating protein content of potato tubers.

    PubMed

    Weeda, Sarah M; Mohan Kumar, G N; Richard Knowles, N

    2009-06-01

    The soluble protein fraction of fully developed potato (Solanum tuberosum L.) tubers is dominated by patatin, a 40 kD storage glycoprotein, and protease inhibitors. Potato multicystatin (PMC) is a multidomain Cys-type protease inhibitor. PMC effectively inhibits degradation of patatin by tuber proteases in vitro. Herein we show that changes in PMC, patatin concentration, activities of various proteases, and their gene expression are temporally linked during tuber development, providing evidence that PMC has a role in regulating tuber protein content in vivo. PMC was barely detectable in non-tuberized stolons. PMC transcript levels increased progressively during tuberization, concomitant with a 40-fold increase in PMC concentration (protein basis) as tubers developed to 10 g fresh wt. Further increases in PMC were comparatively modest (3.7-fold) as tubers developed to full maturity (250 g). Protease activity declined precipitously as PMC levels increased during tuberization. Proteolytic activity was highest in non-tuberized stolons and fell substantially through the 10-g fresh wt stage. Cys-type proteases dominated the pre-tuberization and earliest stages of tuber development. Increases in patatin transcript levels during tuberization were accompanied by a notable lag in patatin accumulation. Patatin did not begin to accumulate substantially on a protein basis until tubers had reached the 10-g stage, wherein protease activity had been inhibited by approximately 60%. These results indicate that a threshold level of PMC (ca. 3 microg tuber(-1), 144 ng mg(-1) protein) is needed to favor patatin accumulation. Collectively, these results are consistent with a role for PMC in facilitating the accumulation of proteins in developing tubers by inhibiting Cys-type proteases.

  5. Structural Evidence for Effectiveness of Darunavir and Two Related Antiviral Inhibitors against HIV-2 Protease

    SciTech Connect

    Kovalevsky, Andrey Y.; Louis, John M.; Aniana, Annie; Ghosh, Arun K.; Weber, Irene T.

    2008-12-08

    No drug has been targeted specifically for HIV-2 (human immunodeficiency virus type 2) infection despite its increasing prevalence worldwide. The antiviral HIV-1 (human immunodeficiency virus type 1) protease (PR) inhibitor darunavir and the chemically related GRL98065 and GRL06579A were designed with the same chemical scaffold and different substituents at P2 and P2' to optimize polar interactions for HIV-1 PR (PR1). These inhibitors are also effective antiviral agents for HIV-2-infected cells. Therefore, crystal structures of HIV-2 PR (PR2) complexes with the three inhibitors have been solved at 1.2-{angstrom} resolution to analyze the molecular basis for their antiviral potency. Unusually, the crystals were grown in imidazole and zinc acetate buffer, which formed interactions with the PR2 and the inhibitors. Overall, the structures were very similar to the corresponding inhibitor complexes of PR1 with an RMSD of 1.1 {angstrom} on main-chain atoms. Most hydrogen-bond and weaker C-H...O interactions with inhibitors were conserved in the PR2 and PR1 complexes, except for small changes in interactions with water or disordered side chains. Small differences were observed in the hydrophobic contacts for the darunavir complexes, in agreement with relative inhibition of the two PRs. These near-atomic-resolution crystal structures verify the inhibitor potency for PR1 and PR2 and will provide the basis for the development of antiviral inhibitors targeting PR2.

  6. Exogenous and endogenous protease inhibitors in the gut of the fall armyworm larvae, Spodoptera frugiperda.

    PubMed

    Lwalaba, Digali; Weidlich, Sandy; Hoffmann, Klaus H; Woodring, Joseph

    2010-06-01

    A dose-dependent inhibition of endogenous trypsin and aminopeptidase occurs in the lumen of Spodoptera frugiperda after feeding L6 larvae exogenous inhibitors soybean trypsin inhibitor (SBTI), tosyl-L-lysine chloromethyl ketone-HCl (TLCK), or bestatin, respectively, for 3 days. TLCK inhibits trypsin in tissue extracts and in secretions more strongly than SBTI. The aminopeptidase released into the lumen (containing the peritrophic membrane) is strongly inhibited by bestatin, but the membrane-bound enzyme is not. A bound enzyme may be more resistant to an inhibitor than unbound. A cross-class elevation of aminopeptidase activity occurs in response to ingested trypsin inhibitor, but there was no cross-class effect of aminopeptidase inhibitor (bestatin) on trypsin activity. An endogenous trypsin and aminopeptidase inhibitor is present in the lumen and ventricular cells. The strength of the endogenous trypsin inhibition seems to be in the same range as that resulting from ingestion of the exogenous inhibitor SBTI. In some insect species, considerable trypsin secretion occurs in unfed as well as in fed animals, and endogenous protease inhibitors might function to protect the ventricular epithelium by inactivation of trypsin when less food is available.

  7. Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

    PubMed Central

    Pessoa, Luciana Santos; Vidal, Luãnna Liebscher; da Costa, Emmerson C.B.; Abreu, Celina Monteiro; da Cunha, Rodrigo Delvecchio; Valadão, Ana Luiza Chaves; dos Santos, André Felipe; Tanuri, Amilcar

    2016-01-01

    Abstract Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented. PMID:27575432

  8. Identification of inhibitors of the transmembrane protease FlaK of Methanococcus maripaludis.

    PubMed

    Coburger, Ina; Schaub, Yvonne; Roeser, Dirk; Hardes, Kornelia; Maeder, Patrick; Klee, Nina; Steinmetzer, Torsten; Imhof, Diana; Diederich, Wibke E; Than, Manuel E

    2016-08-01

    GxGD-type intramembrane cleaving proteases (I-CLiPs) form a family of proteolytic enzymes that feature an aspartate-based catalytic mechanism. Yet, they structurally and functionally largely differ from the classical pepsin-like aspartic proteases. Among them are the archaeal enzyme FlaK, processing its substrate FlaB2 during the formation of flagella and γ-secretase, which is centrally involved in the etiology of the neurodegenerative Alzheimer's disease. We developed an optimized activity assay for FlaK and based on screening of a small in-house library and chemical synthesis, we identified compound 9 as the first inhibitor of this enzyme. Our results show that this intramembrane protease differs from classical pepsin-like aspartic proteases and give insights into the substrate recognition of this enzyme. By providing the needed tools to further study the enzymatic cycle of FlaK, our results also enable further studies towards a functional understanding of other GxGD-type I-CLiPs. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  9. Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions.

    PubMed

    Pessoa, Luciana Santos; Vidal, Luãnna Liebscher; Costa, Emmerson C B da; Abreu, Celina Monteiro; Cunha, Rodrigo Delvecchio da; Valadão, Ana Luiza Chaves; Santos, André Felipe Dos; Tanuri, Amilcar

    2016-01-01

    Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented.

  10. A structural basis for the acute effects of HIV protease inhibitors on GLUT4 intrinsic activity.

    PubMed

    Hertel, Johann; Struthers, Heidi; Horj, Christal Baird; Hruz, Paul W

    2004-12-31

    Human immunodeficiency virus (HIV) protease inhibitors (PIs) act as reversible noncompetitive inhibitors of GLUT4 with binding affinities in the low micromolar range and are known to contribute to alterations in glucose homeostasis during treatment of HIV infection. As aspartyl protease inhibitors, these compounds all possess a core peptidomimetic structure together with flanking hydrophobic moieties. To determine the molecular basis for GLUT4 inhibition, a family of related oligopeptides containing structural elements found in PIs was screened for their ability to inhibit 2-deoxyglucose transport in primary rat adipocytes. The peptide oxybenzylcarbonyl-His-Phe-Phe-O-ethyl ester (zHFFe) was identified as a potent inhibitor of zero-trans glucose flux with a K(i) of 26 mum. Similar to PIs, transport inhibition by this peptide was acute, noncompetitive, and reversible. Within a Xenopus oocyte expression system, zHFFe acutely and reversibly inhibited GLUT4-mediated glucose uptake, whereas GLUT1 activity was unaffected at concentrations as high as 1 mm. The related photoactivatable peptide zHFF-p-benzoylphenylalanine-[(125)I]Tyr-O-ethyl ester selectively labeled GLUT4 in rat adipocytes and indinavir effectively protected against photolabeling. Furthermore, GLUT4 bound to a peptide affinity column containing the zHFF sequence and was eluted by indinavir. These data establish a structural basis for PI effects on GLUT4 activity and support the direct binding of PIs to the transport protein as the mechanism for acute inhibition of insulin-stimulated glucose uptake.

  11. Neutrophil serine proteases and their endogenous inhibitors in coronary artery ectasia patients

    PubMed Central

    Liu, Ruifeng; Chen, Lianfeng; Wu, Wei; Chen, Houzao; Zhang, Shuyang

    2016-01-01

    Objective: Proteolytic enzymes possibly contribute to coronary artery ectasia (CAE). This study aimed to determine whether neutrophils, neutrophil serine proteases (NSPs), and their endogenous inhibitors participated in the pathological process of CAE. Methods: The study consisted of 30 patients with CAE, 30 patients with coronary artery disease (CAD), and 29 subjects with normal coronary arteries (Control). The following circulating items were measured: the main NSPs, including human neutrophil elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3); soluble elastin (sElastin), which was a degradation product of elastin fibres; NSP inhibitors such as α1-protease inhibitor (α1-PI), α2-macroglobulin (α2-MG), secretory leucoprotease inhibitor (SLPI), and elafin; as well as two neutrophil activation markers (myeloperoxidase and lactoferrin) and three classic neutrophil activators [tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and bacterial endotoxin]. Results: The levels of HNE, CG, and sElastin were elevated in the CAE group. The levels of α1-PI and α2-MG were also significantly increased in the CAE group. The levels of myeloperoxidase and lactoferrin were higher in the CAE group. The levels of TNF-α, IL-8, and endotoxin were unchanged in the CAE group compared with those in the CAD group. Conclusion: Neutrophils may participate in the process of vessel extracellular matrix destruction and coronary ectasia by releasing NSPs in a non-classical manner. PMID:26467359

  12. Neutrophil serine proteases and their endogenous inhibitors in coronary artery ectasia patients.

    PubMed

    Liu, Ruifeng; Chen, Lianfeng; Wu, Wei; Chen, Houzao; Zhang, Shuyang

    2016-01-01

    Proteolytic enzymes possibly contribute to coronary artery ectasia (CAE). This study aimed to determine whether neutrophils, neutrophil serine proteases (NSPs), and their endogenous inhibitors participated in the pathological process of CAE. The study consisted of 30 patients with CAE, 30 patients with coronary artery disease (CAD), and 29 subjects with normal coronary arteries (Control). The following circulating items were measured: the main NSPs, including human neutrophil elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3); soluble elastin (sElastin), which was a degradation product of elastin fibres; NSP inhibitors such as α1-protease inhibitor (α1-PI), α2-macroglobulin (α2-MG), secretory leucoprotease inhibitor (SLPI), and elafin; as well as two neutrophil activation markers (myeloperoxidase and lactoferrin) and three classic neutrophil activators [tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and bacterial endotoxin]. The levels of HNE, CG, and sElastin were elevated in the CAE group. The levels of α1-PI and α2-MG were also significantly increased in the CAE group. The levels of myeloperoxidase and lactoferrin were higher in the CAE group. The levels of TNF-α, IL-8, and endotoxin were unchanged in the CAE group compared with those in the CAD group. Neutrophils may participate in the process of vessel extracellular matrix destruction and coronary ectasia by releasing NSPs in a non-classical manner.

  13. Effect of serine protease inhibitors on posttraumatic brain injury and neuronal apoptosis.

    PubMed

    Movsesyan, V A; Yakovlev, A G; Fan, L; Faden, A I

    2001-02-01

    N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K(+) deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 microM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death. Copyright 2001 Academic Press.

  14. Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor.

    PubMed Central

    Markowitz, M; Mo, H; Kempf, D J; Norbeck, D W; Bhat, T N; Erickson, J W; Ho, D D

    1995-01-01

    Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors. PMID:7815532

  15. Antimicrobial activity of protease inhibitor from leaves of Coccinia grandis (L.) Voigt.

    PubMed

    Satheesh, L Shilpa; Murugan, K

    2011-05-01

    Antimicrobial activity of protease inhibitor isolated from Coccinia grandis (L.) Voigt. has been reported. A 14.3 kDa protease inhibitor (PI) was isolated and purified to homogeneity by ammonium sulfate precipitation (20-85% saturation), sephadex G-75, DEAE sepharose column and trypsin-sepharose affinity chromatography from the leaves of C. grandis. The purity was checked by reverse phase high performance liquid chromatography. PI exhibited marked growth inhibitory effects on colon cell lines in a dose-dependent manner. PI was thermostable and showed antimicrobial activity without hemolytic activity. PI strongly inhibited pathogenic microbial strains, including Staphylococcus aureus, Klebsiella pneumoniae, Proteus vulgaris, Eschershia coli, Bacillus subtilis and pathogenic fungus Candida albicans, Mucor indicus, Penicillium notatum, Aspergillus flavus and Cryptococcus neoformans. Examination by bright field microscopy showed inhibition of mycelial growth and sporulation. Morphologically, PI treated fungus showed a significant shrinkage of hyphal tips. Reduced PI completely lost its activity indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. Results reported in this study suggested that PI may be an excellent candidate for development of novel oral or other anti-infective agents.

  16. Alphavirus protease inhibitors from natural sources: A homology modeling and molecular docking investigation.

    PubMed

    Byler, Kendall G; Collins, Jasmine T; Ogungbe, Ifedayo Victor; Setzer, William N

    2016-10-01

    Alphaviruses such as Chikungunya virus (CHIKV), O'Nyong-Nyong virus (ONNV), Ross River virus (RRV), Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV), and Western equine encephalitis virus (WEEV), are mosquito-transmitted viruses that can cause fevers, rash, and rheumatic diseases (CHIKV, ONNV, RRV) or potentially fatal encephalitis (EEEV, VEEV, WEEV) in humans. These diseases are considered neglected tropical diseases for which there are no current antiviral therapies or vaccines available. The alphavirus non-structural protein 2 (nsP2) contains a papain-like protease, which is considered to be a promising target for antiviral drug discovery. In this work, molecular docking analyses have been carried out on a library of 2174 plant-derived natural products (290 alkaloids, 664 terpenoids, 1060 polyphenolics, and 160 miscellaneous phytochemicals) with the nsP2 proteases of CHIKV, ONNV, RRV, EEEV, VEEV, WEEV, as well as Aura virus (AURV), Barmah Forest Virus (BFV), Semliki Forest virus (SFV), and Sindbis virus (SINV) in order to identity structural scaffolds for inhibitor design or discovery. Of the 2174 phytochemicals examined, a total of 127 showed promising docking affinities and poses to one or more of the nsP2 proteases, and this knowledge can be used to guide experimental investigation of potential inhibitors.

  17. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    PubMed

    Prohaska, Thomas A; Wahlmüller, Felix C; Furtmüller, Margareta; Geiger, Margarethe

    2012-01-01

    The serine protease inhibitor protein C inhibitor (PCI) is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP) is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4) M(-1) s(-1). Low molecular weight (LMWH) and unfractionated heparin (UFH) slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition) value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml). By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

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

    PubMed

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

    2016-07-15

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

  19. Novel Mycosin Protease MycP1 Inhibitors Identified by Virtual Screening and 4D Fingerprints

    PubMed Central

    2015-01-01

    The rise of drug-resistant Mycobacterium tuberculosis lends urgency to the need for new drugs for the treatment of tuberculosis (TB). The identification of a serine protease, mycosin protease-1 (MycP1), as the crucial agent in hydrolyzing the virulence factor, ESX-secretion-associated protein B (EspB), potentially opens the door to new tuberculosis treatment options. Using the crystal structure of mycobacterial MycP1 in the apo form, we performed an iterative ligand- and structure-based virtual screening (VS) strategy to identify novel, nonpeptide, small-molecule inhibitors against MycP1 protease. Screening of ∼485 000 ligands from databases at the Genomics Research Institute (GRI) at the University of Cincinnati and the National Cancer Institute (NCI) using our VS approach, which integrated a pharmacophore model and consensus molecular shape patterns of active ligands (4D fingerprints), identified 81 putative inhibitors, and in vitro testing subsequently confirmed two of them as active inhibitors. Thereafter, the lead structures of each VS round were used to generate a new 4D fingerprint that enabled virtual rescreening of the chemical libraries. Finally, the iterative process identified a number of diverse scaffolds as lead compounds that were tested and found to have micromolar IC50 values against the MycP1 target. This study validated the efficiency of the SABRE 4D fingerprints as a means of identifying novel lead compounds in each screening round of the databases. Together, these results underscored the value of using a combination of in silico iterative ligand- and structure-based virtual screening of chemical libraries with experimental validation for the identification of promising structural scaffolds, such as the MycP1 inhibitors. PMID:24628123

  20. Phylogenetic distribution of protease inhibitors of the Kazal-family within the Arthropoda.

    PubMed

    van Hoef, Vincent; Breugelmans, Bert; Spit, Jornt; Simonet, Gert; Zels, Sven; Vanden Broeck, Jozef

    2013-03-01

    In mammalian pancreatic cells, the pancreatic secretory trypsin inhibitor (PSTI) belonging to the Kazal-family prevents the premature activation of digestive enzymes and thus plays an important role in a protective mechanism against tissue destruction by autophagy. Although a similar protective mechanism exists in Arthropoda, the distribution of these inhibitors in this phylum remains obscure. A comprehensive in silico search of nucleotide databases, revealed the presence of members of the Kazal-family in the four major subphyla of the Arthropoda. Especially in the Hexapoda and the Crustacea these inhibitors are widespread, while in the Chelicerata and Myriapoda only a few Kazal-like protease inhibitors were found. A sequence alignment of inhibitors retrieved in the digestive system of insects revealed a conservation of the PSTI characteristics and strong resemblance to vertebrate PSTI. A phylogenetic analysis of these inhibitors showed that they generally cluster according to their order. The results of this data mining study provide new evidence for the existence of an ancient protective mechanism in metazoan digestive systems. Kazal-like inhibitors, which play an important protective role in the pancreas of vertebrates, also seem to be present in Arthropoda.

  1. Viral protease cleavage of inhibitor of κBα triggers host cell apoptosis

    PubMed Central

    Zaragoza, Carlos; Saura, Marta; Padalko, Elizaveta Y.; Lopez-Rivera, Ester; Lizarbe, Tania R.; Lamas, Santiago; Lowenstein, Charles J.

    2006-01-01

    Apoptosis is an innate immune response to viral infection that limits viral replication. However, the mechanisms by which cells detect viral infection and activate apoptosis are not completely understood. We now show that during Coxsackievirus infection, the viral protease 3Cpro cleaves inhibitor of κBα (IκBα). A proteolytic fragment of IκBα then forms a stable complex with NF-κB, translocates to the nucleus, and inhibits NF-κB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IκBα expression are more susceptible to viral infection, with less apoptosis and more viral replication. IκBα thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection. PMID:17138672

  2. In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors.

    PubMed

    Cassone, A; De Bernardis, F; Torosantucci, A; Tacconelli, E; Tumbarello, M; Cauda, R

    1999-08-01

    Highly active antiretroviral therapy that includes human immunodeficiency virus (HIV) aspartyl protease inhibitors (PIs) causes a decline in the incidence of some opportunistic infections in AIDS, and this decline is currently attributed to the restoration of specific immunity. The effect of two PIs (indinavir and ritonavir) on the enzymatic activity of a secretory aspartyl protease (Sap) of Candida albicans (a major agent of mucosal disease in HIV-infected subjects) and on growth and experimental pathogenicity of this fungus was evaluated. Both PIs strongly (>/=90%) and dose dependently (0.1-10 microM) inhibited Sap activity and production. They also significantly reduced Candida growth in a nitrogen-limited, Sap expression-dependent growth medium and exerted a therapeutic effect in an experimental model of vaginal candidiasis, with an efficacy comparable to that of fluconazole. Thus, besides the expected immunorestoration, patients receiving PI therapy may benefit from a direct anticandidal activity of these drugs.

  3. Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

    PubMed Central

    Kleina, L G; Grubman, M J

    1992-01-01

    The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed. Images PMID:1331517

  4. Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

    PubMed Central

    Yanuar, Arry; Suhartanto, Heru; Mun׳im, Abdul; Anugraha, Bram Hik; Syahdi, Rezi Riadhi

    2014-01-01

    HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4'-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside. PMID:24616554

  5. Quantitative structure activity relationship analysis of canonical inhibitors of serine proteases

    NASA Astrophysics Data System (ADS)

    Dell'Orco, Daniele; De Benedetti, Pier Giuseppe

    2008-06-01

    Correlation analysis was carried out between binding affinity data values from the literature and physicochemical molecular descriptors of two series of single point mutated canonical inhibitors of serine proteases, namely bovine pancreatic trypsin inhibitor (BPTI) and turkey ovomucoid third domain (OMTKY3), toward seven enzymes. Simple quantitative structure-activity relationship (QSAR) models based on either single or double linear regressions (SLR or DLR) were obtained, which highlight the role of hydrophobic and bulk/polarizability features of mutated amino acids of the inhibitors in modulating both affinity and specificity. The utility of the QSAR paradigm applied to the analysis of mutagenesis data was underlined, resulting in a simple tool to quantitatively help deciphering structure-function/activity relationships (SFAR) of different protein systems.

  6. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro

    PubMed Central

    Cao, Zeyu; Ding, Yue; Ke, Zhipeng; Cao, Liang; Li, Na; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

    2016-01-01

    Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro. PMID:26870944

  7. Isolation of a new trypsin inhibitor from the Faba bean (Vicia faba cv. Giza 843) with potential medicinal applications.

    PubMed

    Fang, Evandro Fei; Hassanien, Abdallah Abd Elazeem; Wong, Jack Ho; Bah, Clara Shui Fern; Soliman, Saeed Saad; Ng, Tzi Bun

    2011-01-01

    A new 15-kDa Bowman-Birk type trypsin inhibitor (termed VFTI-G1) was isolated from the seeds of Faba bean (Vicia faba cv. Giza 843) using cation exchange chromatography on an SP-Sepharose column, anion exchange chromatography on Q-Sepharose and Mono Q columns, and finally size exclusion chromatography on a Superdex 75 column. VFTI-G1 manifested significant antiproteolytic activity against trypsin (5761 BAEE units/mg, K(i) 20.4 × 10(-9) M), but only a slight chymotrypsin inhibitory activity (< 10 BTEE units/mg). The suitable environment to sustain its trypsin inhibitory activity was at temperatures below 60 °C and at pH 7. Its trypsin inhibitory activity was inhibited by the reducing agent dithiothreitol in a dose-dependent manner, indicating the significance of intact disulfide bonds to the trypsin inhibitory activity. It inhibited HIV-1 reverse transcriptase (RT) activity with an IC(50) of about 0.76 µM. Furthermore, VFTI-G1 showed specific antiproliferative activity toward HepG2 hepatoma cells by inducing chromatin condensation and cell apoptosis. The HIV-1 RT inhibitory activity of VFTI-G1 and its specific antiproliferative activity toward Hep G2 cells may find medical applications.

  8. Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

    PubMed Central

    Terras, FRG.; Schoofs, HME.; Thevissen, K.; Osborn, R. W.; Vanderleyden, J.; Cammue, BPA.; Broekaert, W. F.

    1993-01-01

    Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes. PMID:12232024

  9. An inhibitor from Lupinus bogotensis seeds effective against aspartic proteases from Hypothenemus hampei.

    PubMed

    Molina, Diana; Zamora, Humberto; Blanco-Labra, Alejandro

    2010-06-01

    The coffee berry borer, Hypothenemus hampei (Ferrari), is one of the most devastating coffee pests (Coffea arabica L.) worldwide. Digestion in the midgut of H. hampei is facilitated by aspartic proteases. This is the first report of an aspartic protease inhibitor from Lupinus bogotensis. The L. bogotensis aspartic protease inhibitor (LbAPI) exhibited a molecular mass of 12.84kDa, as determined by MALDI-TOF, and consists of a single polypeptide chain with an isoelectric point of 4.5. In thermal activity experiments, stability was retained at pH 2.5 after heating the protein at 70 degrees C for 30 min, but was unstable at 100 degrees C. The protein was also stable over a broad range of pH, from 2 to 11, at 30 degrees C. In in vitro assays, LbAPI was highly effective against aspartic proteases from H. hampei guts with a half maximal inhibitory concentration (IC(50)) of 2.9 microg. LbAPI inhibits pepsin in a stoichiometric ratio of 1:1. LbAPI inhibition of pepsin was competitive, with a K(i) of 3.1 microM, using hemoglobin as substrate. Its amino-terminal sequence had 76% homology with the seed storage proteins vicilin and beta-conglutin. The homology of LbAPI to vicilins from Lupinus albus L. suggests that they may also serve as storage proteins in the seed. LbAPI could be a promising tool to make genetically modified coffee with resistance to H. hampei. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes.

    PubMed

    Ahn, J-E; Lovingshimer, M R; Salzman, R A; Presnail, J K; Lu, A L; Koiwa, H; Zhu-Salzman, K

    2007-06-01

    Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

  11. HIV-1 Protease with 20 Mutations Exhibits Extreme Resistance to Clinical Inhibitors through Coordinated Structural Rearrangements

    SciTech Connect

    Agniswamy, Johnson; Shen, Chen-Hsiang; Aniana, Annie; Sayer, Jane M.; Louis, John M.; Weber, Irene T.

    2012-06-28

    The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35-37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2' pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.

  12. The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

    SciTech Connect

    Bandaranayake, Rajintha M.; Kolli, Madhavi; King, Nancy M.; Nalivaika, Ellen A.; Heroux, Annie; Kakizawa, Junko; Sugiura, Wataru; Schiffer, Celia A.

    2010-09-08

    The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01{_}AE (AE) strain is seen principally in Southeast Asia. AE protease differs by {approx}10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

  13. Production of a protease inhibitor from edible mushroom Agaricus bisporus and its statistical optimization by response surface methodology.

    PubMed

    Vishvakarma, Reena; Mishra, Abha

    2017-01-31

    The production of a protease inhibitor from Agaricus bisporus through solid state fermentation was studied. The purpose was to produce protease inhibitor from natural, cheap and readily available carbon and nitrogen sources. Solid state fermentation enhanced the mycelia growth and also gave a higher yield of the product. Further, fungal growth and other production parameters were statistically optimized. The specificity of the inhibitor was tested and was effective against trypsin. Screening of significant factors (wheat bran, cyanobacterial biomass, initial pH, temperature, incubation period, and moisture content and inoculum size) was done using Plackett-Burman Design. Central Composite Design was used to determine the optimized values of the significant variables which were found to be temperature (27.5 °C), incubation time (156 hrs.), cyanobacterial biomass (1 g) and moisture content (50%) and gave a statistical yield of 980 PIU/g which was 25.6% higher than experimental yield (780 PIU/g). The inhibitor was purified by ammonium sulphate precipitation and DEAE cellulose chromatography (yield 43.89% and 0.21% respectively) and subjected to Reversed-phase HPLC to validate its identity. Since protease inhibitors act against proteases, finding ample therapeutic roles; the isolated protease inhibitor from A. bisporus can also be a probable medicinal agent after its further characterization.

  14. Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B.

    PubMed

    Mahrus, Sami; Kisiel, Walter; Craik, Charles S

    2004-12-24

    Granzyme M is a trypsin-fold serine protease that is specifically found in the granules of natural killer cells. This enzyme has been implicated recently in the induction of target cell death by cytotoxic lymphocytes, but unlike granzymes A and B, the molecular mechanism of action of granzyme M is unknown. We have characterized the extended substrate specificity of human granzyme M by using purified recombinant enzyme, several positional scanning libraries of coumarin substrates, and a panel of individual p-nitroanilide and coumarin substrates. In contrast to previous studies conducted using thiobenzyl ester substrates (Smyth, M. J., O'Connor, M. D., Trapani, J. A., Kershaw, M. H., and Brinkworth, R. I. (1996) J. Immunol. 156, 4174-4181), a strong preference for leucine at P1 over methionine was demonstrated. The extended substrate specificity was determined to be lysine = norleucine at P4, broad at P3, proline > alanine at P2, and leucine > norleucine > methionine at P1. The enzyme activity was found to be highly dependent on the length and sequence of substrates, indicative of a regulatory function for human granzyme M. Finally, the interaction between granzyme M and the serpins alpha(1)-antichymotrypsin, alpha(1)-proteinase inhibitor, and proteinase inhibitor 9 was characterized by using a candidate-based approach to identify potential endogenous inhibitors. Proteinase inhibitor 9 was effectively hydrolyzed and inactivated by human granzyme M, raising the possibility that this orphan granzyme bypasses proteinase inhibitor 9 inhibition of granzyme B.

  15. Rapid emergence of hepatitis C virus protease inhibitor resistance is expected

    SciTech Connect

    Rong, Libin; Perelson, Alan S; Ribeiro, Ruy M

    2009-01-01

    Approximately 170 million people worldwide are infected with hepatitis C virus (HCV). Current therapy, consisting of pegylated interferon (PEG-IFN) and ribavirin (RBV), leads to sustained viral elimination in only about 45% of patients treated. Telaprevir (VX-950), a novel HCV NS3-4A serine protease inhibitor, has demonstrated substantial antiviral activity in patients with chronic hepatitis C genotype 1 infection. However, some patients experience viral breakthrough during dosing, with drug resistant variants being 5%-20% of the virus population as early as day 2 after treatment initiation. Why viral variants appear such a short time after the start of dosing is unclear, especially since this has not been seen with monotherapy for either human immunodeficiency virus or hepatitis B virus. Here, using a viral dynamic model, we explain why such rapid emergence of drug resistant variants is expected when potent HCV protease inhibitors are used as monotherapy. Surprisingly, our model also shows that such rapid emergence need not be the case with some potent HCV NS5B polymerase inhibitors. Examining the case of telaprevir therapy in detail, we show the model fits observed dynamics of both wild-type and drug-resistant variants during treatment, and supports combination therapy of direct antiviral drugs with PEG-IFN and/or RBV for hepatitis C.

  16. Determination of activable proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine protease inhibitor.

    PubMed

    Palencia, D D; Garner, D L; Hudig, D; Holcombe, D W; Burner, C A; Redelman, D; Fernandez, G C; Abuelyaman, A S; Kam, C M; Powers, J C

    1996-09-01

    The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumarin serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIC). The presence of bound inhibitor on sperm was then determined by secondary labeling with avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expose the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples from six bulls, the mean proportion of sperm with activable proacrosin/acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed acrosin after cryopreservation of these samples was 55.8 +/- 4.1%. Significant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryopreservation. Activable proacrosin/acrosin levels in samples of cryopreserved sperm from five bulls were not correlated with fertility. These results do indicate, however, that the irreversible isocoumarin serine protease inhibitor BIC can be used to determine the proportion of sperm cells that retain activable proacrosin/acrosin after cryopreservation and thawing.

  17. 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.

  18. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant–pathogen interactions

    PubMed Central

    Jashni, Mansoor Karimi; Mehrabi, Rahim; Collemare, Jérôme; Mesarich, Carl H.; de Wit, Pierre J. G. M.

    2015-01-01

    Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles. PMID:26284100

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

    PubMed

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

    2015-02-15

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

  20. A preference-based free-energy parameterization of enzyme-inhibitor binding. Applications to HIV-1-protease inhibitor design.

    PubMed Central

    Wallqvist, A.; Jernigan, R. L.; Covell, D. G.

    1995-01-01

    The interface between protein receptor-ligand complexes has been studied with respect to their binary interatomic interactions. Crystal structure data have been used to catalogue surfaces buried by atoms from each member of a bound complex and determine a statistical preference for pairs of amino-acid atoms. A simple free energy model of the receptor-ligand system is constructed from these atom-atom preferences and used to assess the energetic importance of interfacial interactions. The free energy approximation of binding strength in this model has a reliability of about +/- 1.5 kcal/mol, despite limited knowledge of the unbound states. The main utility of such a scheme lies in the identification of important stabilizing atomic interactions across the receptor-ligand interface. Thus, apart from an overall hydrophobic attraction (Young L, Jernigan RL, Covell DG, 1994, Protein Sci 3:717-729), a rich variety of specific interactions is observed. An analysis of 10 HIV-1 protease inhibitor complexes is presented that reveals a common binding motif comprised of energetically important contacts with a rather limited set of atoms. Design improvements to existing HIV-1 protease inhibitors are explored based on a detailed analysis of this binding motif. PMID:8528086

  1. HIV protease inhibitors disrupt astrocytic glutamate transporter function and neurobehavioral performance.

    PubMed

    Vivithanaporn, Pornpun; Asahchop, Eugene L; Acharjee, Shaona; Baker, Glen B; Power, Christopher

    2016-02-20

    The neurotoxic actions of the HIV protease inhibitors, amprenavir (APV) and lopinavir (LPV) were investigated. With combination antiretroviral therapy (cART), HIV-infected persons exhibit neurocognitive impairments, raising the possibility that cART might exert adverse central nervous system (CNS) effects. We examined the effects of LPV and APV using in-vitro and in-vivo assays of CNS function. Gene expression, cell viability and amino-acid levels were measured in human astrocytes, following exposure to APV or LPV. Neurobehavioral performance, amino-acid levels and neuropathology were examined in HIV-1 Vpr transgenic mice after treatment with APV or LPV. Excitatory amino-acid transporter-2 (EAAT2) expression was reduced in astrocytes treated with LPV or APV, especially LPV (P < 0.05), which was accompanied by reduced intracellular L-glutamate levels in LPV-treated cells (P < 0.05). Treatment of astrocytes with APV or LPV reduced the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 (P < 0.05) although cell survival was unaffected. Exposure of LPV to astrocytes augmented glutamate-evoked transient rises in [Cai] (P < 0.05). Vpr mice treated with LPV showed lower concentrations of L-glutamate, L-aspartate and L-serine in cortex compared with vehicle-treated mice (P < 0.05). Total errors in T-maze assessment were increased in LPV and APV-treated animals (P < 0.05). EAAT2 expression was reduced in the brains of protease inhibitor-treated animals, which was associated with gliosis (P < 0.05). These results indicated that contemporary protease inhibitors disrupt astrocyte functions at therapeutic concentrations with enhanced sensitivity to glutamate, which can lead to neurobehavioral impairments. ART neurotoxicity should be considered in future therapeutic regimens for HIV/AIDS.

  2. Purification and partial characterization of thiol protease inhibitors from embryos of the brine shrimp Artemia.

    PubMed

    Warner, A H; Sonnenfeld-Karcz, M J

    1992-01-01

    Thiol protease inhibitor (TPI) proteins in embryos of the brine shrimp Artemia were purified to apparent homogeneity and several of their properties were studied. Four protein fractions containing thiol protease inhibitor activity were obtained by high performance liquid chromatography of Artemia embryo proteins on a C-18 reverse-phase column and these were designated as TPI-1a, -1b, -2, and -3. Acrylamide gel electrophoresis showed that TPI-1a and TPI-1b each consisted of two bands of 11.8 and 13.6 kilodaltons (kDa), while TPI-2 and TPI-3 consisted of only one band of 12.5 kDa. Isoelectric focusing experiments demonstrated that TPI-3 contained one band at pH 5.3, while both TPI-1b and TPI-2 yielded bands at pH 5.2 and 5.3. TPI-1a did not yield any major bands. Amino acid composition analyses of the Artemia TPI proteins showed them to be remarkably similar to one another. All were rich in valine and aspartic and glutamic acids, and devoid of cysteine. Partial trypsin digestion of TPI-1b, TPI-2, and TPI-3 yielded several peptides with identical mobilities on a reverse-phase column and several other peptides with different mobilities, suggesting that the multiple forms of Artemia TPIs may have originated from the same parental protein. N-terminal amino acid sequence analyses of TPI-3 suggest that Artemia TPI proteins are members of the type I cystatin family of protease inhibitors.

  3. Neurocognitive Impairment in Patients Treated with Protease Inhibitor Monotherapy or Triple Drug Antiretroviral Therapy

    PubMed Central

    Pérez-Valero, Ignacio; González-Baeza, Alicia; Estébanez, Miriam; Montes-Ramírez, María L.; Bayón, Carmen; Pulido, Federico; Bernardino, José I.; Zamora, Francisco X.; Monge, Susana; Gaya, Francisco; Lagarde, María; Rubio, Rafael; Hernando, Asunción; Arnalich, Francisco; Arribas, José R.

    2013-01-01

    Background In patients who remain virologically suppressed in plasma with triple-drug ART a switch to protease inhibitor monotherapy maintains high rates of suppression; however it is unknown if protease inhibitor monotherapy is associated to a higher rate of neurocognitive impairment. Methods In this observational, cross-sectional study we included patients with plasma virological suppression (≥1 year) without concomitant major neurocognitive confounders, currently receiving for ≥1 year boosted lopinavir or darunavir as monotherapy or as triple ART. Neurocognitive impairment was defined as per the 2007 consensus of the American Association of Neurology. The association between neurocognitive impairment and protease inhibitor monotherapy, adjusted by significant confounders, was analysed. Results Of the 191 included patients - triple therapy: 96, 1–2 years of monotherapy: 40 and >2 years of monotherapy: 55 - proportions (95% CI) with neurocognitive impairment were: overall, 27.2% (20.9–33.6); triple therapy, 31.6% (22.1–41.0); short-term monotherapy, 25.0% (11.3–38.7); long-term monotherapy: 21.4% (10.5–32.3); p = 0.38. In all groups, neurocognitive impairment was mildly symptomatic or asymptomatic by self-report. There were not significant differences in Global Deficit Score by group. In the regression model confounding variables for neurocognitive impairment were years on ART, ethnicity, years of education, transmission category and the HOMA index. Adjusted by these variables the Odds Ratio (95% CI) for neurocognitive impairment of patients receiving short-term monotherapy was 0.85 (0.29–2.50) and for long-term monotherapy 0.40 (0.14–1.15). Conclusions Compared to triple drug antiretroviral therapy, monotherapy with lopinavir/ritonavir or darunavir/ritonavir in patients with adequate plasma suppression was not associated with a higher rate of asymptomatic neurocognitive impairment than triple drug ART. PMID:23936029

  4. The putative serine protease inhibitor Api m 6 from Apis mellifera venom: recombinant and structural evaluation.

    PubMed

    Michel, Y; McIntyre, M; Ginglinger, H; Ollert, M; Cifuentes, L; Blank, S; Spillner, E

    2012-01-01

    Immunoglobulin (Ig) E-mediated reactions to honeybee venom can cause severe anaphylaxis, sometimes with fatal consequences. Detailed knowledge of the allergic potential of all venom components is necessary to ensure proper diagnosis and treatment of allergy and to gain a better understanding of the allergological mechanisms of insect venoms. Our objective was to undertake an immunochemical and structural evaluation of the putative low-molecular-weight serine protease inhibitor Api m 6, a component of honeybee venom. We recombinantly produced Api m 6 as a soluble protein in Escherichia coli and in Spodoptera frugiperda (Sf9) insect cells.We also assessed specific IgE reactivity of venom-sensitized patients with 2 prokaryotically produced Api m 6 variants using enzyme-linked immunosorbent assay. Moreover, we built a structural model ofApi m 6 and compared it with other protease inhibitor structures to gain insights into the function of Api m 6. In a population of 31 honeybee venom-allergic patients, 26% showed specific IgE reactivity with prokaryotically produced Api m 6, showing it to be a minor but relevant allergen. Molecular modeling of Api m 6 revealed a typical fold of canonical protease inhibitors, supporting the putative function of this venom allergen. Although Api m 6 has a highly variant surface charge, its epitope distribution appears to be similar to that of related proteins. Api m 6 is a honeybee venom component with IgE-sensitizing potential in a fraction of venom-allergic patients. Recombinant Api m 6 can help elucidate individual component-resolved reactivity profiles and increase our understanding of immune responses to low-molecular-weight allergens

  5. HIV-1 evolution under pressure of protease inhibitors: climbing the stairs of viral fitness.

    PubMed

    Berkhout, B

    1999-01-01

    The human immunodeficiency virus (HIV-1) has evolved into a viral quasispecies with a high replication capacity or fitness. Antiretroviral drugs potently inhibit replication of the wild-type virus, but HIV-1 responds by selection of drug-resistant variants. Here we review, in brief, the evolution of resistance to protease inhibitors that is characterized by severe fitness losses and an abundance of subsequent repair strategies. The possibility to restrict HIV-1 fitness is discussed in relation to the control of HIV-1 pathogenesis.

  6. Inmate has no constitutional right to a protease inhibitor, court says.

    PubMed

    1999-02-19

    The 10th U.S. Circuit Court of Appeals has ruled that the Kansas Department of Corrections is not violating the rights of inmate [name removed] by refusing to give him protease inhibitors. [Name removed] charged this omission was cruel and unusual punishment even though he is being given an AZT-3TC combination. Although existing Federal guidelines state that effective antiviral therapy calls for the use of three drugs, Judge John Porfilio found that [name removed]' disagreement with the prison doctors about the course of his treatment did not constitute deliberate indifference.

  7. Design and synthesis of irreversible inhibitors of foot-and-mouth disease virus 3C protease.

    PubMed

    Roqué Rosell, Núria R; Mokhlesi, Ladan; Milton, Nicholas E; Sweeney, Trevor R; Zunszain, Patricia A; Curry, Stephen; Leatherbarrow, Robin J

    2014-01-15

    Foot-and-mouth disease virus (FMDV) causes a highly infectious and economically devastating disease of livestock. The FMDV genome is translated as a single polypeptide precursor that is cleaved into functional proteins predominantly by the highly conserved viral 3C protease, making this enzyme an attractive target for antiviral drugs. A peptide corresponding to an optimal substrate has been modified at the C-terminus, by the addition of a warhead, to produce irreversible inhibitors that react as Michael acceptors with the enzyme active site. Further investigation highlighted key structural determinants for inhibition, with a positively charged P2 being particularly important for potency.

  8. In Vitro Resistance Profile of the Hepatitis C Virus NS3 Protease Inhibitor BI 201335

    PubMed Central

    Lagacé, Lisette; White, Peter W.; Bousquet, Christiane; Dansereau, Nathalie; Dô, Florence; Llinas-Brunet, Montse; Marquis, Martin; Massariol, Marie-Josée; Maurice, Roger; Spickler, Catherine; Thibeault, Diane; Triki, Ibtissem; Zhao, Songping

    2012-01-01

    The in vitro resistance profile of BI 201335 was evaluated through selection and characterization of variants in genotype 1a (GT 1a) and genotype 1b (GT 1b) replicons. NS3 R155K and D168V were the most frequently observed resistant variants. Phenotypic characterization of the mutants revealed shifts in sensitivity specific to BI 201335 that did not alter susceptibility to alpha interferon. In contrast to macrocyclic and covalent protease inhibitors, changes at V36, T54, F43, and Q80 did not confer resistance to BI 201335. PMID:22024816

  9. Vascular Access System for Continuous Arterial Infusion of a Protease Inhibitor in Acute Necrotizing Pancreatitis

    SciTech Connect

    Ganaha, Fumikiyo; Yamada, Tetsuhisa; Yorozu, Naoya; Ujita, Masuo; Irie, Takeo; Fukuda, Yasushi; Fukuda, Kunihiko; Tada, Shimpei

    1999-09-15

    We used a vascular access system (VAS) for continuous arterial infusion (CAI) of a protease inhibitor in two patients with acute necrotizing pancreatitis. The infusion catheter was placed into the dorsal pancreatic artery in the first patient and into the gastroduodenal artery in the second, via a femoral artery approach. An implantable port was then connected to the catheter and was secured in a subcutaneous pocket prepared in the right lower abdomen. No complications related to the VAS were encountered. This system provided safe and uncontaminated vascular access for successful CAI for acute pancreatitis.

  10. Effect of nutrient limitation of cyanobacteria on protease inhibitor production and fitness of Daphnia magna.

    PubMed

    Schwarzenberger, Anke; Sadler, Thomas; Von Elert, Eric

    2013-10-01

    Herbivore-plant interactions have been well studied in both terrestrial and aquatic ecosystems as they are crucial for the trophic transfer of energy and matter. In nutrient-rich freshwater ecosystems, the interaction between primary producers and herbivores is to a large extent represented by Daphnia and cyanobacteria. The occurrence of cyanobacterial blooms in lakes and ponds has, at least partly, been attributed to cyanotoxins, which negatively affect the major grazer of planktonic cyanobacteria, i.e. Daphnia. Among these cyanotoxins are the widespread protease inhibitors. These inhibitors have been shown (both in vitro and in situ) to inhibit the most important group of digestive proteases in the gut of Daphnia, i.e. trypsins and chymotrypsins, and to reduce Daphnia growth. In this study we grew cultures of the cyanobacterium Microcystis sp. strain BM25 on nutrient-replete, N-depleted or P-depleted medium. We identified three different micropeptins to be the cause for the inhibitory activity of BM25 against chymotrypsins. The micropeptin content depended on nutrient availability: whereas N limitation led to a lower concentration of micropeptins per biomass, P limitation resulted in a higher production of these chymotrypsin inhibitors. The altered micropeptin content of BM25 was accompanied by changed effects on the fitness of Daphnia magna: a higher content of micropeptins led to lower IC50 values for D. magna gut proteases and vice versa. Following expectations, the lower micropeptin content in the N-depleted BM25 caused higher somatic growth of D. magna. Therefore, protease inhibitors can be regarded as a nutrient-dependent defence against grazers. Interestingly, although the P limitation of the cyanobacterium led to a higher micropeptin content, high growth of D. magna was observed when they were fed with P-depleted BM25. This might be due to reduced digestibility of P-depleted cells with putatively thick mucilaginous sheaths. These findings indicate that

  11. Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

    PubMed

    Schenk, Jörg A; Fettke, Joerg; Lenz, Christine; Albers, Katharina; Mallwitz, Frank; Gajovic-Eichelmann, Nenad; Ehrentreich-Förster, Eva; Kusch, Emely; Sellrie, Frank

    2012-03-31

    The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.

  12. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    SciTech Connect

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R.

    2014-07-25

    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

  13. Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

    PubMed

    Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

    2017-08-23

    Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Continuous Regional Arterial Infusion of Protease Inhibitors Has No Efficacy in the Treatment of Severe Acute Pancreatitis

    PubMed Central

    Horibe, Masayasu; Sasaki, Mitsuhito; Sanui, Masamitsu; Sugiyama, Daisuke; Iwasaki, Eisuke; Yamagishi, Yoshiyuki; Sawano, Hirotaka; Goto, Takashi; Ikeura, Tsukasa; Hamada, Tsuyoshi; Oda, Takuya; Yasuda, Hideto; Shinomiya, Wataru; Miyazaki, Dai; Hirose, Kaoru; Kitamura, Katsuya; Chiba, Nobutaka; Ozaki, Tetsu; Yamashita, Takahiro; Koinuma, Toshitaka; Oshima, Taku; Yamamoto, Tomonori; Hirota, Morihisa; Moriya, Takashi; Shirai, Kunihiro; Mayumi, Toshihiko; Kanai, Takanori

    2017-01-01

    Objective The aim of this study is to assess the effectiveness of continuous regional arterial infusion (CRAI) of protease inhibitors in patients with severe acute pancreatitis (SAP) including acute necrotizing pancreatitis. Methods This retrospective study was conducted among 44 institutions in Japan from 2009 to 2013. Patients 18 years or older diagnosed with SAP according to the criteria of the Japanese Ministry of Health, Labour and Welfare study group (2008) were consecutively enrolled. We evaluated the association between CRAI of protease inhibitors and mortality, incidence of infection, and the need for surgical intervention using multivariable logistic regression analysis. Results Of 1159 patients admitted, 1097 patients with all required data were included for analysis. Three hundred and seventy-four (34.1%) patients underwent CRAI of protease inhibitors and 723 (65.9%) did not. In multivariable analysis, CRAI of protease inhibitors was not associated with a reduction in mortality, infection rate, or need for surgical intervention (odds ratio [OR] 0.79, 95% confidence interval [CI] 0.47–1.32, P = 0.36; OR 0.97, 95% CI 0.61–1.54, P = 0.89; OR 0.76, 95% CI 0.50–1.15, P = 0.19; respectively). Conclusions Continuous regional arterial infusion of protease inhibitors was not efficacious in the treatment of patients with SAP. PMID:27977624

  15. Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors

    PubMed Central

    Stapels, Daphne A. C.; Ramyar, Kasra X.; Bischoff, Markus; von Köckritz-Blickwede, Maren; Milder, Fin J.; Ruyken, Maartje; Eisenbeis, Janina; McWhorter, William J.; Herrmann, Mathias; van Kessel, Kok P. M.; Geisbrecht, Brian V.; Rooijakkers, Suzan H. M.

    2014-01-01

    Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity. PMID:25161283

  16. Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease

    PubMed Central

    Tan, Yong Wah; Ang, Melgious Jin Yan; Lau, Qiu Ying; Poulsen, Anders; Ng, Fui Mee; Then, Siew Wen; Peng, Jianhe; Hill, Jeffrey; Hong, Wan Jin; Chia, Cheng San Brian; Chu, Justin Jang Hann

    2016-01-01

    Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery. PMID:27645381

  17. Protease inhibitor reduces airway response and underlying inflammation in cockroach allergen-induced murine model.

    PubMed

    Saw, Sanjay; Arora, Naveen

    2015-04-01

    Protease(s) enhances airway inflammation and allergic cascade. In the present study, effect of a serine protease inhibitor was evaluated in mouse model of airway disease. Mice were sensitized with cockroach extract (CE) or Per a 10 and treated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) 1 h before or after challenge to measure airway response. Mice were euthanized to collect bronchoalveolar lavage fluid (BALF), blood, and lung to evaluate inflammation. AEBSF treatment significantly reduced the AHR in allergen-challenged mice in dose-dependent manner (p≤ 0.01). IgE (p≤0.05) and Th2 cytokines (p≤0.05) were significantly reduced in treated mice. AEBSF treatment lowered total cell (p≤0.05), eosinophil (p≤0.05), and neutrophil (p≤0.05) in BALF and lung tissue. Oxidative stress parameters were impaired on treatment in allergen-challenged mice (p≤0.05). AEBSF had therapeutic effect in allergen-induced airway resistance and underling inflammation and had potential for combination or as add-on therapy for respiratory diseases.

  18. A designed P1 cysteine mimetic for covalent and non-covalent inhibitors of HCV NS3 protease.

    PubMed

    Narjes, Frank; Koehler, Konrad F; Koch, Uwe; Gerlach, Benjamin; Colarusso, Stefania; Steinkühler, Christian; Brunetti, Mirko; Altamura, Sergio; De Francesco, Raffaele; Matassa, Victor G

    2002-02-25

    The difluoromethyl group was designed by computational chemistry methods as a mimetic of the canonical P1 cysteine thiol for inhibitors of the hepatitis C virus NS3 protease. This modification led to the development of competitive, non-covalent inhibitor 4 (K(i) 30 nM) and reversible covalent inhibitors (6, K(i) 0.5 nM; and 8 K*(i) 10 pM).

  19. Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

    PubMed Central

    Weiss, André; Joerss, Hanna; Brockmeyer, Jens

    2014-01-01

    EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

  20. The Second-Generation Maturation Inhibitor GSK3532795 Maintains Potent Activity Toward HIV Protease Inhibitor-Resistant Clinical Isolates.

    PubMed

    Ray, Neelanjana; Li, Tianbo; Lin, Zeyu; Protack, Tricia; van Ham, Petronella Maria; Hwang, Carey; Krystal, Mark; Nijhuis, Monique; Lataillade, Max; Dicker, Ira

    2017-05-01

    Protease inhibitor (PI)-resistant HIV-1 isolates with primary substitutions in protease (PR) and secondary substitutions in Gag could potentially exhibit cross-resistance to maturation inhibitors. We evaluated the second-generation maturation inhibitor, GSK3532795, for activity toward clinical isolates with genotypic and phenotypic characteristics associated with PI resistance (longitudinal). Longitudinal clinical isolates from 15 PI-treated patients and 7 highly PI-resistant (nonlongitudinal) viruses containing major and minor PI resistance-associated mutations were evaluated for GSK3532795 sensitivity. Phenotypic sensitivity was determined using the PhenoSense Gag/PR assay (Monogram Biosciences) or in-house single- and multiple-cycle assays. Changes from baseline [CFB; ratio of post- to pre-treatment FC-IC50 (fold-change in IC50 versus wild-type virus)] <3 were considered to be within the no-effect level. All nonlongitudinal viruses tested were sensitive to GSK3532795 (FC-IC50 range 0.16-0.68). Among longitudinal isolates, all post-PI treatment samples had major PI resistance-associated mutations in PR and 17/21 had PI resistance-associated changes in Gag. Nineteen of the 21 post-PI treatment samples had GSK3532795 CFB <3. Median (range) CFB was 0.83 (0.05-27.4) [Monogram (11 patients)] and 1.5 (1.0-2.2) [single-cycle (4 patients)]. The 2 post-PI treatment samples showing GSK3532795 CFB >3 (Monogram) were retested using single- and multiple-cycle assays. Neither sample had meaningful sensitivity changes in the multiple-cycle assay. Gag changes were not associated with an increased GSK3532795 CFB. GSK3532795 maintained antiviral activity against PI-resistant isolates with emergent PR and/or Gag mutations. This finding supports continued development of GSK3532795 in treatment-experienced patients with or without previous PI therapy.

  1. 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.

  2. Quantitative analysis of protease recognition by inhibitors in plasma using microscale thermophoresis.

    PubMed

    Dau, T; Edeleva, E V; Seidel, S A I; Stockley, R A; Braun, D; Jenne, D E

    2016-10-14

    High abundance proteins like protease inhibitors of plasma display a multitude of interactions in natural environments. Quantitative analysis of such interactions in vivo is essential to study diseases, but have not been forthcoming, as most methods cannot be directly applied in a complex biological environment. Here, we report a quantitative microscale thermophoresis assay capable of deciphering functional deviations from in vitro inhibition data by combining concentration and affinity measurements. We obtained stable measurement signals for the substrate-like interaction of the disease relevant inhibitor α-1-antitrypsin (AAT) Z-variant with catalytically inactive elastase. The signal differentiates between healthy and sick AAT-deficient individuals suggesting that affinity between AAT and elastase is strongly modulated by so-far overlooked additional binding partners from the plasma.

  3. Quantitative analysis of protease recognition by inhibitors in plasma using microscale thermophoresis

    PubMed Central

    Dau, T.; Edeleva, E. V.; Seidel, S. A. I.; Stockley, R. A.; Braun, D.; Jenne, D. E.

    2016-01-01

    High abundance proteins like protease inhibitors of plasma display a multitude of interactions in natural environments. Quantitative analysis of such interactions in vivo is essential to study diseases, but have not been forthcoming, as most methods cannot be directly applied in a complex biological environment. Here, we report a quantitative microscale thermophoresis assay capable of deciphering functional deviations from in vitro inhibition data by combining concentration and affinity measurements. We obtained stable measurement signals for the substrate-like interaction of the disease relevant inhibitor α-1-antitrypsin (AAT) Z-variant with catalytically inactive elastase. The signal differentiates between healthy and sick AAT-deficient individuals suggesting that affinity between AAT and elastase is strongly modulated by so-far overlooked additional binding partners from the plasma. PMID:27739542

  4. Evaluation of dipeptide nitriles as inhibitors of rhodesain, a major cysteine protease of Trypanosoma brucei.

    PubMed

    Schirmeister, Tanja; Schmitz, Janina; Jung, Sascha; Schmenger, Torsten; Krauth-Siegel, R Luise; Gütschow, Michael

    2017-01-01

    A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8μM) the compounds represent promising starting points for new rhodesain inhibitors.

  5. A serine protease inhibitor from hemolymph of green mussel, Perna viridis.

    PubMed

    Khan, M S; Goswami, U; Rojatkar, S R; Khan, M I

    2008-07-15

    Bioactivity guided fractions of cell-free hemolymph of bacterially challenged marine mussel, Perna viridis led to the isolation of a novel quaternary alkaloid 1, which was identified by its spectral data. The isolated molecule 1 has been found to be a potent serine protease inhibitor (SPI) showing IC(50) and K(i) values of 102.5 and 97.1-104.68 microM, respectively. The E(t)/K(i) value of SPI is 6.3, whereas E(t)/K(m) value is 1.04. The Van't Hoff analysis showed that the value of K(i) decreases with increase in temperature, and the binding of the inhibitor is entropically driven.

  6. Development of a highly reliable assay for ubiquitin-specific protease 2 inhibitors.

    PubMed

    Wang, Zhongli; Xie, Wenjuan; Zhu, Mingyan; Zhou, Huchen

    2017-09-01

    The dynamic modification of proteins with ubiquitin plays crucial roles in major celluar functions, and is associated with a number of pathological conditions. Ubiquitin-specific proteases (USPs) cleave ubiquitin from substrate proteins, and rescue them from proteasomal degradation. Among them, USP2 is overexpressed and plays important roles in various cancers including prostate cancer. Thus, it represents an attractive target for drug discovery. In order to develop potent and selective USP2 inhibitors, a highly reliable assay is needed for in-depth structure-activity relationship study. We report the cloning, expression, and purification of USP2 and UBA52, and the development of a highly reliable assay based on readily available SDS-PAGE-Coomassie systeme using UBA52 as the substrate protein. A number of effective USP2 inhibitors were also identified using this assay. Copyright © 2017. Published by Elsevier Ltd.

  7. Engineering human immunodeficiency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation.

    PubMed Central

    McPhee, F; Good, A C; Kuntz, I D; Craik, C S

    1996-01-01

    Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876160

  8. Novel two-round phenotypic assay for protease inhibitor susceptibility testing of recombinant and primary HIV-1 isolates.

    PubMed

    Puertas, Maria C; Buzón, Maria J; Ballestero, Mònica; Van Den Eede, Peter; Clotet, Bonaventura; Prado, Julia G; Martinez-Picado, Javier

    2012-12-01

    Antiretroviral drug susceptibility tests facilitate therapeutic management of HIV-1-infected patients. Although genotyping systems are affordable, inaccuracy in the interpretation of complex mutational patterns may limit their usefulness. Currently available HIV-1 phenotypic assays are based on the generation of recombinant viruses in which the specific viral gene of interest, derived from a patient plasma sample, is cloned into a susceptible genetic viral backbone prior to in vitro drug susceptibility evaluation. Nevertheless, in the case of protease inhibitors, not only are mutations in the HIV-1 protease-coding region involved in resistance, but the role of Gag in drug susceptibility has also recently been reported. In order to avoid the inherent limitations resulting from partial cloning of the viral genome, we designed and evaluated a new experimental strategy to test the in vitro susceptibility of primary viral isolates to protease inhibitors. Our protocol, which is based on a two-round infection protocol using the reporter TZM-bl cell line, showed a good correlation with genotypic resistance prediction and with the Antivirogram phenotypic assay, in both protease-recombinant viruses and primary viral isolates. The protocol is suitable for any HIV-1 subtype and enables rapid in-house measurement of protease inhibitor susceptibility, thus making it possible to evaluate the concomitant effects of both patient-derived gag and protease-coding regions.

  9. Structure-based design of carbon nanotubes as HIV-1 protease inhibitors: atomistic and coarse-grained simulations.

    PubMed

    Cheng, Yuan; Li, Dechang; Ji, Baohua; Shi, Xinghua; Gao, Huajian

    2010-09-01

    Nanoparticles such as fullerenes and carbon nanotubes have been extensively studied for biomedical applications. In this paper, we report the design of carbon nanotubes as HIV-1 protease inhibitors. Docking and molecular dynamics calculations are performed using an atomistic model to explore the optimal interaction structure and free energy between the nanotube and HIV-1 protease. A coarse-grained model is then developed based on the atomistic model, allowing us to investigate the dynamic behaviors of the protease in the bound and unbound states. The dynamic process reveals that the carbon nanotube is able to bind to the active site of the protease and prevent the active flaps from opening up, thus blocking the function of the protease. This process is strongly influenced by the size of the nanotube. The binding of carbon nanotubes to an alternative binding site other than the active site is also explored. Therefore, carbon nanotube-based inhibitors have great potential for application as HIV-1 protease inhibitors.

  10. Heterodera glycines cysts contain an extensive array of endoproteases as well as inhibitors of proteases in H. glycines and Meloidogyne incognita infective juvenile stages

    USDA-ARS?s Scientific Manuscript database

    Heterodera glycines cysts contain proteases, and inhibitors of protease activities in various nematode species. In this investigation, proteases in H. glycines cysts were identified using a commercially available FRET-peptide library comprising 512 peptide pools qualified to detect up to 4 endoprot...

  11. Preclinical Characterization of the Novel Hepatitis C Virus NS3 Protease Inhibitor GS-9451

    PubMed Central

    Yang, Huiling; Robinson, Margaret; Corsa, Amoreena C.; Peng, Betty; Cheng, Guofeng; Tian, Yang; Wang, Yujin; Pakdaman, Rowchanak; Shen, Marian; Qi, Xiaoping; Mo, Hongmei; Tay, Chin; Krawczyk, Steve; Sheng, X. Christopher; Kim, Choung U.

    2014-01-01

    GS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitro antiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50 = 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection. PMID:23939899

  12. Preclinical characterization of the novel hepatitis C virus NS3 protease inhibitor GS-9451.

    PubMed

    Yang, Huiling; Robinson, Margaret; Corsa, Amoreena C; Peng, Betty; Cheng, Guofeng; Tian, Yang; Wang, Yujin; Pakdaman, Rowchanak; Shen, Marian; Qi, Xiaoping; Mo, Hongmei; Tay, Chin; Krawczyk, Steve; Sheng, X Christopher; Kim, Choung U; Yang, Chris; Delaney, William E

    2014-01-01

    GS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, including in vitro antiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50 = 316 nM). Additive to synergistic in vitro antiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results of in vitro cross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

  13. Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

    SciTech Connect

    Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.; Braz, Gloria R.C.; Tanaka, Aparecida S.

    2011-09-23

    Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

  14. Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1.

    PubMed

    Vago, Juliana P; Tavares, Luciana P; Sugimoto, Michelle A; Lima, Graziele Letícia N; Galvão, Izabela; de Caux, Thais R; Lima, Kátia M; Ribeiro, Ana Luíza C; Carneiro, Fernanda S; Nunes, Fernanda Freire C; Pinho, Vanessa; Perretti, Mauro; Teixeira, Mauro M; Sousa, Lirlândia P

    2016-02-15

    Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.

  15. A cysteine protease inhibitor rescues mice from a lethal Cryptosporidium parvum infection.

    PubMed

    Ndao, Momar; Nath-Chowdhury, Milli; Sajid, Mohammed; Marcus, Victoria; Mashiyama, Susan T; Sakanari, Judy; Chow, Eric; Mackey, Zachary; Land, Kirkwood M; Jacobson, Matthew P; Kalyanaraman, Chakrapani; McKerrow, James H; Arrowood, Michael J; Caffrey, Conor R

    2013-12-01

    Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

  16. A Cysteine Protease Inhibitor Rescues Mice from a Lethal Cryptosporidium parvum Infection

    PubMed Central

    Nath-Chowdhury, Milli; Sajid, Mohammed; Marcus, Victoria; Mashiyama, Susan T.; Sakanari, Judy; Chow, Eric; Mackey, Zachary; Land, Kirkwood M.; Jacobson, Matthew P.; Kalyanaraman, Chakrapani; McKerrow, James H.; Arrowood, Michael J.; Caffrey, Conor R.

    2013-01-01

    Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis. PMID:24060869

  17. Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19

    PubMed Central

    Kim, Tae K.; Radulovic, Zeljko; Mulenga, Albert

    2016-01-01

    Amblyomma americanum tick serine protease inhibitor (serpin, AAS) 19, is a highly conserved protein that is characterized by its functional domain being 100% conserved across tick species. We also reported that AAS19 was an immunogenic tick saliva protein with anti-haemostatic functions and an inhibitor of trypsin-like proteases including five of the eight serine protease factors in the blood clotting cascade. In this study the goal was to validate the importance of AAS19 in A. americanum tick physiology, assess immunogenicity and investigate tick vaccine efficacy of yeast-expressed recombinant (r) AAS19. We confirm that AAS19 is important to A. americanum fitness and blood meal feeding. AAS19 mRNA disruption by RNAi silencing caused ticks to obtain blood meals that were 50% smaller than controls, and treated ticks being morphologically deformed with 100% of the deformed ticks dying in incubation. We show that rAAS19 is highly immunogenic in that two 500 µg inoculations mixed with TiterMax Gold adjuvant provoked antibody titers of more than 1:320000 that specifically reacted with native AAS19 in unfed and partially fed tick tissue. Since AAS19 is injected into animals during tick feeding, we challenge infested immunized rabbits twice to test if tick infestations of immunized rabbits could act as booster. While in the first infestation significantly smaller tick blood meals were observed on one of the two immunized rabbits, smaller blood meals were observed on both rabbits, but 60% of ticks that engorged on immunized rabbits in the second infestation failed to lay eggs. It is notable that ticks fed faster on immunized animals despite obtaining smaller blood meals. We conclude that rAAS19 is a potential component of cocktail tick vaccine. PMID:26746129

  18. Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19.

    PubMed

    Kim, Tae K; Radulovic, Zeljko; Mulenga, Albert

    2016-04-01

    Amblyomma americanum tick serine protease inhibitor (serpin, AAS) 19, is a highly conserved protein that is characterized by its functional domain being 100% conserved across tick species. We also reported that AAS19 was an immunogenic tick saliva protein with anti-haemostatic functions and an inhibitor of trypsin-like proteases including five of the eight serine protease factors in the blood clotting cascade. In this study the goal was to validate the importance of AAS19 in A. americanum tick physiology, assess immunogenicity and investigate tick vaccine efficacy of yeast-expressed recombinant (r) AAS19. We confirm that AAS19 is important to A. americanum fitness and blood meal feeding. AAS19 mRNA disruption by RNAi silencing caused ticks to obtain blood meals that were 50% smaller than controls, and treated ticks being morphologically deformed with 100% of the deformed ticks dying in incubation. We show that rAAS19 is highly immunogenic in that two 500 μg inoculations mixed with TiterMax Gold adjuvant provoked antibody titers of more than 1:320,000 that specifically reacted with native AAS19 in unfed and partially fed tick tissue. Since AAS19 is injected into animals during tick feeding, we challenge infested immunized rabbits twice to test if tick infestations of immunized rabbits could act as booster. While in the first infestation significantly smaller tick blood meals were observed on one of the two immunized rabbits, smaller blood meals were observed on both rabbits, but 60% of ticks that engorged on immunized rabbits in the second infestation failed to lay eggs. It is notable that ticks fed faster on immunized animals despite obtaining smaller blood meals. We conclude that rAAS19 is a potential component of cocktail tick vaccine.

  19. Mapping inhibitor binding modes on an active cysteine protease via nuclear magnetic resonance spectroscopy.

    PubMed

    Lee, Gregory M; Balouch, Eaman; Goetz, David H; Lazic, Ana; McKerrow, James H; Craik, Charles S

    2012-12-18

    Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.

  20. Aberrant mucosal wound repair in the absence of secretory leukocyte protease inhibitor.

    PubMed

    Angelov, Nikola; Moutsopoulos, Niki; Jeong, Moon-Jin; Nares, Salvador; Ashcroft, Gillian; Wahl, Sharon M

    2004-08-01

    Secretory leukocyte protease inhibitor (SLPI) is a cationic serine protease inhibitor with anti-microbial and anti-inflammatory properties found in large quantities in mucosal fluids, including saliva. SLPI is expressed during cutaneous wound healing, however, its role in oral wound repair is unknown. We have used a novel approach involving a murine buccal mucosal acute wound model to investigate the role of SLPI in oral healing. In parallel to the observed cutaneous healing phenotype, an absence of SLPI results in markedly impaired oral wound healing associated with increased inflammation and raised elastase activity. Moreover, matrix deposition was decreased, while MMP activity was enhanced in the oral SLPI null wounds suggesting deregulated proteolysis. Intriguingly, regardless of genotype, reduced collagen deposition was observed in oral compared to dermal wounds, associated with reduced TGF-beta expression and decreased fibroblast collagen expression in vitro. We propose that SLPI is a pivotal endogenous factor necessary for optimal tissue repair including intra-oral wound healing. In addition, our model provides a unique opportunity to delineate the cellular and molecular mechanisms underlying the differences between dermal scarring and oral scar-free healing.

  1. Serine protease inhibitor attenuates intracerebral hemorrhage-induced brain injury and edema formation in rat.

    PubMed

    Nakamura, Takehiro; Kuroda, Yasuhiro; Hosomi, Naohisa; Okabe, Naohiko; Kawai, Nobuyuki; Tamiya, Takashi; Xi, Guohua; Keep, Richard F; Itano, Toshifumi

    2010-01-01

    Our previous studies have demonstrated that thrombin plays an important role in intracerebral hemorrhage (ICH)-induced brain injury and edema formation. We, therefore, examined whether nafamostat mesilate (FUT), a serine protease inhibitor, can reduce ICH-induced brain injury. Anesthetized male Sprague-Dawley rats received an infusion of autologous whole blood (100 microL), thrombin (5U/50 microL) or type VII collagenase (0.4 U/2 microL) into the right basal ganglia, the three ICH models used in the present study. FUT (10 mg/kg) or vehicle was administered intraperitoneally 6 h after ICH (or immediately after thrombin infusion) and then at 12-h intervals (six treatments in total, n = 5 in each group). All rats were sacrificed 72 h later. We also examined whether FUT promotes rebleeding in a model in which ICH was induced by intracerebral injection of collagenase. Systemic administration of FUT starting 6 h after ICH reduced brain water content in the ipsilateral basal ganglia 72 h after ICH compared with vehicle. FUT attenuated ICH-induced changes in 8-OHdG and thrombin-reduced brain edema. FUT did not increase collagenase-induced hematoma volume. FUT attenuates ICH-induced brain edema and DNA injury suggesting that serine protease inhibitor may be potential therapeutic agent for ICH.

  2. Characterization of a novel serine protease inhibitor gene from a marine metagenome.

    PubMed

    Jiang, Cheng-Jian; Hao, Zhen-Yu; Zeng, Rong; Shen, Pei-Hong; Li, Jun-Fang; Wu, Bo

    2011-01-01

    A novel serine protease inhibitor (serpin) gene designated as Spi1C was cloned via the sequenced-based screening of a metagenomic library from uncultured marine microorganisms. The gene had an open reading frame of 642 base pairs, and encoded a 214-amino acid polypeptide with a predicted molecular mass of about 28.7 kDa. The deduced amino acid sequence comparison and phylogenetic analysis indicated that Spi1C and some partial proteinase inhibitor I4 serpins were closely related. Functional characterization demonstrated that the recombinant Spi1C protein could inhibit a series of serine proteases. The Spi1C protein exhibited inhibitory activity against α-chymotrypsin and trypsin with K(i) values of around 1.79 × 10(-8) and 1.52 × 10(-8) M, respectively. No inhibition activity was exhibited against elastase. Using H-d-Phe-Pip-Arg-pNA as the chromogenic substrate, the optimum pH and temperature of the inhibition activity against trypsin were 7.0-8.0 and 25 °C, respectively. The identification of a novel serpin gene underscores the potential of marine metagenome screening for novel biomolecules.

  3. Resistance through inhibition: ectopic expression of serine protease inhibitor offers stress tolerance via delayed senescence in yeast cell.

    PubMed

    Joshi, Rakesh S; Tanpure, Rahul S; Singh, Rajan Kumar; Gupta, Vidya S; Giri, Ashok P

    2014-09-26

    Protease inhibitors have been known to confer multiple stress tolerance in transgenic plants. We have assessed growth of yeast (Pichia pastoris GS115) strains expressing inhibitory repeat domains (PpIRD(+)) of previously characterized Capsicum annuum protease inhibitors under high salt, heavy metal and oxidative stress. PpIRD(+) strains exhibited multiple stress tolerance and showed differential molecular responses at transcriptional and translational level on exposure to stress inducing agents like heavy metal, high salt and H2O2. PpIRD(+) strains display significant reduction in metacaspase (Yca1) activity, the key enzyme in apoptosis, indicates the possibility of cross reactivity of IRDs (serine protease inhibitor) with cysteine proteases. PpIRD(+) and Saccharomyces cerevisiae knockout with Yca1 (ΔYca1) strain showed similar growth characteristics under stress, which indicated the delayed senescence due to cellular metacaspase inhibition. Molecular docking study showed a close proximity of IRDs reactive site and the active site of metacaspase in the complex that signified their strong interactions. Maintenance of GAPDH activity, primary target of metacaspase, in PpIRD(+) strain evidenced the inhibition of metacaspase activity and survival of these cells under stress. This report demonstrates a potential molecular mechanism of protease inhibitor-based multiple stress tolerance in yeast strains.

  4. Novel Potent Hepatitis C Virus NS3 Serine Protease Inhibitors Derived from Proline-Based Macrocycles

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Arasappan, Ashok; Venkatraman, Srikanth; Vibulbhan, Bancha; Yang, Weiying; Parekh, Tejal N.; Pichardo, John; Prongay, Andrew; Cheng, Kuo-Chi; Butkiewicz, Nancy; Yao, Nanhua; Madison, Vincent; Girijavallabhan, Viyyoor

    2008-06-30

    The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic {alpha}-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K*{sub i}). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K*{sub i} = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K*{sub i} = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC{sub 50} of 130 nM in a cellular replicon assay, while IC{sub 50} for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.

  5. HIV-protease inhibitors for the treatment of cancer: Repositioning HIV protease inhibitors while developing more potent NO-hybridized derivatives?

    PubMed

    Maksimovic-Ivanic, Danijela; Fagone, Paolo; McCubrey, James; Bendtzen, Klaus; Mijatovic, Sanja; Nicoletti, Ferdinando

    2017-04-15

    The possible use of HIV protease inhibitors (HIV-PI) as new therapeutic option for the treatment of cancer primarily originated from their success in treating HIV-related Kaposi's sarcoma (KS). While these findings were initially attributed to immune reconstitution and better control of oncogenic viral infections, the number of reports on solid tumors, KS, lymphoma, fibrosarcoma, multiple myeloma and prostate cancer suggest other mechanisms for the anti-neoplastic activity of PIs. However, a major drawback for the possible adoption of HIV-PIs in the therapy of cancer relies on their relatively weak anticancer potency and important side effects. This has propelled several groups to generate derivatives of HIV-PIs for anticancer use, through modifications such as attachment of different moieties, ligands and transporters, including saquinavir-loaded folic acid conjugated nanoparticles and nitric oxide (NO) derivatives of HIV-PIs. In this article, we discuss the current preclinical and clinical evidences for the potential use of HIV-PIs, and of novel derivatives, such as saquinavir-NO in the treatment of cancer. © 2016 UICC.

  6. Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides.

    PubMed

    Liu, Sanling; Dong, Jianmei; Mei, Guoqiang; Liu, Guiyun; Xu, Wei; Su, Zhong; Liu, Jinsong

    2011-02-01

    The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, β = 118.26°. The crystal diffracted to 2.1 Å resolution and contained two molecules in the asymmetric unit.

  7. Expected response to protease inhibitors of HIV-1 non-B subtype viruses according to resistance algorithms.

    PubMed

    Champenois, Karen; Bocket, Laurence; Deuffic-Burban, Sylvie; Cotte, Laurent; André, Patrice; Choisy, Philippe; Yazdanpanah, Yazdan

    2008-05-31

    The expected effectiveness of protease inhibitors was assessed according to the Agence Nationale de Recherches sur le SIDA (ANRS), Rega and Stanford 2007 resistance algorithms in 93 and 87 antiretroviral therapy-naive patients, respectively, infected with B and non-B subtype viruses. Either B or non-B subtypes were considered fully susceptible to protease inhibitors, except to tipranavir/ritonavir, for which the 2007 ANRS algorithm scored non-B subtypes as naturally resistant when this algorithm was extended to these subtypes.

  8. Influence of the spacer on the inhibitory effect of different polycarbophil-protease inhibitor conjugates.

    PubMed

    Marschütz, M K; Veronese, F M; Bernkop-Schnürch, A

    2001-09-01

    Within the present study various polycarbophil (PCP)-serine protease inhibitor conjugates were synthesized and the influence of different spacers on their inhibitory efficacy was evaluated in vitro. Results demonstrated that 4.2+/-0.15 units (n=3; +/-SD) of alpha-chymotrypsin were inhibited by 50% utilizing 0.86% (w/v) of a PCP-tetramethylenediamine (TMDA)-chymostatin 20:1 conjugate. In contrast, only 0.6+/-0.05 units (n=3; +/-SD) of alpha-chymotrypsin were inhibited by a corresponding PCP-poly(ethylene glycol) (PEG)-chymostatin conjugate. Inhibitory effects of PCP-TMDA-antipain and -elastatinal conjugates towards trypsin and elastase, respectively, were also significantly higher (P<0.05) than those of corresponding PCP-PEG-inhibitor conjugates. Hence, the great impact of the molecular size as well as the structure of the spacer on resulting polymer-inhibitor conjugates could be demonstrated. The small and rigid C4-spacer TMDA (molecular weight (MW) 161.1) was thereby shown to be highly advantageous over a long, hydrophilic and flexible PEG-diamine spacer (MW 3400). Results obtained should provide helpful basic knowledge for the development of mucoadhesive polymer-inhibitor conjugates used as auxiliary agents for the oral administration of peptide drugs.

  9. Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir

    SciTech Connect

    Sasková, Klára Grantz; Koíek, Milan; Rezácová, Pavlína; Brynda, Jirí; Yashina, Tatyana; Kagan, Ron M.; Konvalinka, Jan

    2010-03-04

    Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2 binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients.

  10. The Molecular Basis of Drug Resistance against Hepatitis C Virus NS3/4A Protease Inhibitors

    PubMed Central

    Romano, Keith P.; Ali, Akbar; Aydin, Cihan; Soumana, Djade; Özen, Ayşegül; Deveau, Laura M.; Silver, Casey; Cao, Hong; Newton, Alicia; Petropoulos, Christos J.; Huang, Wei; Schiffer, Celia A.

    2012-01-01

    Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clinical development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the molecular basis of drug resistance. In this study, 16 high-resolution crystal structures of four representative protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique molecular underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymatic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the molecular basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus. PMID:22910833

  11. Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization.

    PubMed

    Hackl, Mathias W; Lakemeyer, Markus; Dahmen, Maria; Glaser, Manuel; Pahl, Axel; Lorenz-Baath, Katrin; Menzel, Thomas; Sievers, Sonja; Böttcher, Thomas; Antes, Iris; Waldmann, Herbert; Sieber, Stephan A

    2015-07-08

    Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by genetic knockouts and through the use of beta-lactones, which remain the only specific inhibitors of ClpP discovered to date. Beta-lactones have served as chemical tools to manipulate ClpP in several organisms; however, their potency, selectivity and stability is limited. Despite detailed structural insights into the composition and conformational flexibility of the ClpP active site, no rational efforts to design specific non-beta-lactone inhibitors have been reported to date. In this work, an unbiased screen of more than 137 000 compounds was used to identify five phenyl ester compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP. The potency of phenyl esters largely exceeded that of beta-lactones in ClpP peptidase and protease inhibition assays and displayed unique target selectivity in living S. aureus cells. Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates, they remain covalently trapped as acyl-enzyme intermediates in the active site. The synthesis of 36 derivatives and subsequent structure-activity relationship (SAR) studies provided insights into conserved structural elements that are important for inhibition potency and acylation reactivity. Moreover, the stereochemistry of a methyl-substituent at the alpha position to the ester, resembling amino acid side chains in peptide substrates, impacted ClpP complex stability, causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by molecular docking experiments.

  12. Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors

    PubMed Central

    Duan, Haichuan; Hu, Tao; Foti, Robert S.; Pan, Yongmei; Swaan, Peter W.

    2015-01-01

    Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1–3 (OCT1–3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1–3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC50 = 1.4 ± 0.2 µM) over OCT1 (IC50 = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1–3. PMID:26285765

  13. Drug-Drug Interactions with the NS3/4A Protease Inhibitor Simeprevir.

    PubMed

    Ouwerkerk-Mahadevan, Sivi; Snoeys, Jan; Peeters, Monika; Beumont-Mauviel, Maria; Simion, Alexandru

    2016-02-01

    Simeprevir is an NS3/4A protease inhibitor approved for the treatment of hepatitis C infection, as a component of combination therapy. Simeprevir is metabolized by the cytochrome P450 (CYP) system, primarily CYP3A, and is a substrate for several drug transporters, including the organic anion transporting polypeptides (OATPs). It is susceptible to metabolic drug-drug interactions with drugs that are moderate or strong CYP3A inhibitors (e.g. ritonavir and erythromycin) or CYP3A inducers (e.g. rifampin and efavirenz); coadministration of these drugs may increase or decrease plasma concentrations of simeprevir, respectively, and should be avoided. Clinical studies have shown that simeprevir is a mild inhibitor of CYP1A2 and intestinal CYP3A but does not inhibit hepatic CYP3A. The effects of simeprevir on these enzymes are of clinical relevance only for narrow-therapeutic-index drugs that are metabolized solely by these enzymes (e.g. oral midazolam). Simeprevir does not have a clinically relevant effect on the pharmacokinetics of rilpivirine, tacrolimus, oral contraceptives and several other drugs metabolized by CYP enzymes. Simeprevir is a substrate and inhibitor of the transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and OATP1B1/3. Cyclosporine is an inhibitor of OATP1B1/3, BCRP and P-gp, and a mild inhibitor of CYP3A; cyclosporine causes a significant increase in simeprevir plasma concentrations, and coadministration is not recommended. Clinical studies have demonstrated increases in coadministered drug concentrations for drugs that are substrates of the OATP1B1/3, BRCP (e.g. rosuvastatin) and P-gp (e.g. digoxin) transporters; these drugs should be administered with dose titration and or/close monitoring.

  14. Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

    PubMed

    Zhu-Salzman, K; Koiwa, H; Salzman, R A; Shade, R E; Ahn, J-E

    2003-04-01

    The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371-379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN-free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN-adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN-induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L-like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN-degrading protease activity.

  15. Antiviral and resistance studies of AG1343, an orally bioavailable inhibitor of human immunodeficiency virus protease.

    PubMed Central

    Patick, A K; Mo, H; Markowitz, M; Appelt, K; Wu, B; Musick, L; Kalish, V; Kaldor, S; Reich, S; Ho, D; Webber, S

    1996-01-01

    AG1343 ([3S-(3R*,4aR*,8aR*,2'S*,3'S*)]-2-[2' hydroxy-3'-phenylthiomethyl-4'-aza-5'-oxo-5'-(2''-methyl-3''-hydro xy-phenyl) pentyl]-decahydroiso-quinoline-3-N-t-butylcarboxamide methanesulfonic acid) is a selective, nonpeptidic inhibitor of human immunodeficiency virus (HIV) protease (Ki = 2 nM) that was discovered by protein structure-based drug design methodologies. AG1343 was effective against the replication of several laboratory and clinical HIV type 1 (HIV-1) or HIV-2 isolates including pyridinone- and zidovudine-resistant strains, with 50% effective concentrations ranging from 9 to 60 nM. In reversibility studies, inhibition of gag (p55) proteolytic processing in HIV-1 particles from cells treated with AG1343 was maintained for up to 36 h after drug removal. The ability of virus to develop resistance to AG1343 was studied by serial passage of HIV-1 NL4.3 in the presence of increasing concentrations of drug. After 28 passages, a variant with a 30-fold reduction in susceptibility to AG1343 was isolated. Molecular analysis of the protease from this variant indicated a double change from a Met to Ile at residue 46 and an Ile to Val or Ala at residue 84 (M46I+I84V, A). Consistent with these findings, reductions in susceptibility were observed for recombinant viruses constructed to contain the single I84V change or the double M46I+I84V substitutions. Resistance, however, was not detected for recombinant viruses containing other key mutations in HIV-1 protease, including a Val to Ile change at residue 32 or a Val to Ala or Phe at residue 82. The potent anti-HIV activity of AG1343 against several isolates suggests that AG1343 should perform well during ongoing human phase II clinical trials. PMID:8834868

  16. Prey-mediated effects of the protease inhibitor aprotinin on the predatory carabid beetle Nebria brevicollis.

    PubMed

    Burgess, E P.J.; Lövei, G L.; Malone, L A.; Nielsen, I W.; Gatehouse, H S.; Christeller, J T.

    2002-12-01

    To investigate the potential non-target impacts of transgenic pest-resistant plants, prey-mediated impacts of a protease inhibitor (PI) on the predatory carabid, Nebria brevicollis, were investigated. The PI used was aprotinin, a serine PI of mammalian origin with insecticidal properties when incorporated in artificial diet or expressed in transgenic plants. Field-collected N. brevicollis adults, kept at 23 degrees C, 16:8 L:D, were fed, over their pre-aestivation activity period of 24 days, with Helicoverpa armigera larvae reared on an artificial diet containing 0.5% (w:w, fresh mass) aprotinin. These larvae contained 22.62 &mgr;g aprotinin/g insect. Control prey was reared on diet without aprotinin. Beetle survival and body mass were unaffected by prey type. Beetles consuming PI-fed prey lost significantly more mass than the control beetles during two periods of mass loss, but gained significantly more mass during the final period of mass gain. This was not due to differences in amounts of prey supplied or consumed. The final mass gain coincided with increased consumption of PI-prey. Female beetles were significantly heavier than males, but we found no consistent gender-based differences in response to PI-prey. At the end of the experiment, body mass of all beetles was similar to field-collected ones (approximately 55 mg). All experimental beetles had significantly lower activities of digestive cysteine proteases and the serine proteases chymotrypsin and trypsin than field-collected ones. Beetles consuming PI-fed prey had significantly lower levels of trypsin and higher levels of chymotrypsin and elastase than the control beetles.

  17. Overview of transcriptomic analysis of all human proteases, non-proteolytic homologs and inhibitors: Organ, tissue and ovarian cancer cell line expression profiling of the human protease degradome by the CLIP-CHIP™ DNA microarray.

    PubMed

    Kappelhoff, Reinhild; Puente, Xose S; Wilson, Claire H; Seth, Arun; López-Otín, Carlos; Overall, Christopher M

    2017-08-07

    The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript. Using the CLIP-CHIP™ we mapped the expression profile of proteases and their inhibitors in 23 different human tissues and 6 ovarian cancer cell lines in 104 sample datasets. Hierarchical cluster analysis showed that expression profiles clustered according to their anatomic locations, cellular composition, physiologic functions, and the germ layer from which they are derived. The human ovarian cancer cell lines cluster according to malignant grade. 110 proteases and 42 inhibitors were tissue specific (1 to 3 tissues). Of these 110 proteases 69% (74) are mainly extracellular, 30% (34) intracellular and 1% intramembrane. Notably, 35% (197/565) of human proteases and 30% (47/156) of inhibitors were ubiquitously expressed in all 23 tissues; 27% (155) of proteases and 21% (32) of inhibitors were broadly expressed in 4-20 tissues. Our datasets provide a valuable resource for the community of baseline protease and inhibitor relative expression in normal human tissues and can be used for comparison with diseased tissue, e.g. ovarian cancer, to decipher pathogenesis, and to aid drug development. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  18. Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle.

    PubMed

    McGivern, David R; Masaki, Takahiro; Lovell, William; Hamlett, Chris; Saalau-Bethell, Susanne; Graham, Brent

    2015-05-01

    Hepatitis C virus (HCV) NS3 is a multifunctional protein composed of a protease domain and a helicase domain linked by a flexible linker. Protease activity is required to generate viral nonstructural (NS) proteins involved in RNA replication. Helicase activity is required for RNA replication, and genetic evidence implicates the helicase domain in virus assembly. Binding of protease inhibitors (PIs) to the protease active site blocks NS3-dependent polyprotein processing but might impact other steps of the virus life cycle. Kinetic analyses of antiviral suppression of cell culture-infectious genotype 1a strain H77S.3 were performed using assays that measure different readouts of the viral life cycle. In addition to the active-site PI telaprevir, we examined an allosteric protease-helicase inhibitor (APHI) that binds a site in the interdomain interface. By measuring nucleotide incorporation into HCV genomes, we found that telaprevir inhibits RNA synthesis as early as 12 h at high but clinically relevant concentrations. Immunoblot analyses showed that NS5B abundance was not reduced until after 12 h, suggesting that telaprevir exerts a direct effect on RNA synthesis. In contrast, the APHI could partially inhibit RNA synthesis, suggesting that the allosteric site is not always available during RNA synthesis. The APHI and active-site PI were both able to block virus assembly soon (<12 h) after drug treatment, suggesting that they rapidly engage with and block a pool of NS3 involved in assembly. In conclusion, PIs and APHIs can block NS3 functions in RNA synthesis and virus assembly, in addition to inhibiting polyprotein processing. The NS3/4A protease of hepatitis C virus (HCV) is an important antiviral target. Currently, three PIs have been approved for therapy of chronic hepatitis C, and several others are in development. NS3-dependent cleavage of the HCV polyprotein is required to generate the mature nonstructural proteins that form the viral replicase. Inhibition of

  19. Impact of Stereochemistry on Ligand Binding: X-ray Crystallographic Analysis of an Epoxide-Based HIV Protease Inhibitor.

    PubMed

    Benedetti, Fabio; Berti, Federico; Campaner, Pietro; Fanfoni, Lidia; Demitri, Nicola; Olajuyigbe, Folasade M; De March, Matteo; Geremia, Silvano

    2014-09-11

    A new pseudopeptide epoxide inhibitor, designed for irreversible binding to HIV protease (HIV-PR), has been synthesized and characterized in solution and in the solid state. However, the crystal structure of the complex obtained by inhibitor-enzyme cocrystallization revealed that a minor isomer, with inverted configuration of the epoxide carbons, has been selected by HIV-PR during crystallization. The structural characterization of the well-ordered pseudopeptide, inserted in the catalytic channel with its epoxide group intact, provides deeper insights into inhibitor binding and HIV-PR stereoselectivity, which aids development of future epoxide-based HIV inhibitors.

  20. 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.

  1. Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

    PubMed Central

    Mulenga, A.; Kim, T.; Ibelli, A. M. G.

    2013-01-01

    We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross-class inhibitor of both serine- and papain-like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics analyses revealed that Pichia pastoris-expressed recombinant (r) AamS6 reduced initial velocities of substrate hydrolysis (V0) and/or maximum enzyme velocity (Vmax) of trypsin, chymotrypsin, elastase, chymase, and papain in a dose–response manner. We speculate that rAamS6 inhibited plasmin in a temporary fashion in that while rAamS6 reduced V0 of plasmin by up to ~53%, it had no effect on Vmax. Our data also suggest that rAmS6 has minimal or no apparent effect on V0 or Vmax of thrombin, factor Xa, and kallikrein. We speculate that AamS6 is apparently involved in facilitating blood meal feeding in that various amounts of rAamS6 reduced platelet aggregation by up to ~47% and delayed plasma clotting time in the recalcification time assay by up to ~210 s. AamS6 is most likely not involved with the tick’s evasion of the host’s complement defense mechanism, in that rAamS6 did not interfere with the complement activation pathway. Findings in this study are discussed in the context of expanding our understanding of tick proteins that control bloodmeal feeding and hence tick-borne disease transmission by ticks. PMID:23521000

  2. The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosa infection.

    PubMed

    Benarafa, Charaf; Priebe, Gregory P; Remold-O'Donnell, Eileen

    2007-08-06

    Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1(-/-) mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein-D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance in serpinb1(-/-) mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.

  3. The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosa infection

    PubMed Central

    Benarafa, Charaf; Priebe, Gregory P.; Remold-O'Donnell, Eileen

    2007-01-01

    Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1−/− mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein–D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance in serpinb1−/− mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection. PMID:17664292

  4. Multi-step inhibition explains HIV-1 protease inhibitor pharmacodynamics and resistance.

    PubMed

    Rabi, S Alireza; Laird, Gregory M; Durand, Christine M; Laskey, Sarah; Shan, Liang; Bailey, Justin R; Chioma, Stanley; Moore, Richard D; Siliciano, Robert F

    2013-09-01

    HIV-1 protease inhibitors (PIs) are among the most effective antiretroviral drugs. They are characterized by highly cooperative dose-response curves that are not explained by current pharmacodynamic theory. An unresolved problem affecting the clinical use of PIs is that patients who fail PI-containing regimens often have virus that lacks protease mutations, in apparent violation of fundamental evolutionary theory. Here, we show that these unresolved issues can be explained through analysis of the effects of PIs on distinct steps in the viral life cycle. We found that PIs do not affect virion release from infected cells but block entry, reverse transcription, and post-reverse transcription steps. The overall dose-response curves could be reconstructed by combining the curves for each step using the Bliss independence principle, showing that independent inhibition of multiple distinct steps in the life cycle generates the highly cooperative dose-response curves that make these drugs uniquely effective. Approximately half of the inhibitory potential of PIs is manifest at the entry step, likely reflecting interactions between the uncleaved Gag and the cytoplasmic tail (CT) of the Env protein. Sequence changes in the CT alone, which are ignored in current clinical tests for PI resistance, conferred PI resistance, providing an explanation for PI failure without resistance.

  5. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection

    PubMed Central

    Lawrence, Amba; Fraser, Tamieka; Gillett, Amber; Tyndall, Joel D. A.; Timms, Peter; Polkinghorne, Adam; Huston, Wilhelmina M.

    2016-01-01

    The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas. PMID:27530689

  6. Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

    PubMed

    Kesteleyn, Bart; Amssoms, Katie; Schepens, Wim; Hache, Geerwin; Verschueren, Wim; Van De Vreken, Wim; Rombauts, Klara; Meurs, Greet; Sterkens, Patrick; Stoops, Bart; Baert, Lieven; Austin, Nigel; Wegner, Jörg; Masungi, Chantal; Dierynck, Inge; Lundgren, Stina; Jönsson, Daniel; Parkes, Kevin; Kalayanov, Genadiy; Wallberg, Hans; Rosenquist, Asa; Samuelsson, Bertil; Van Emelen, Kristof; Thuring, Jan Willem

    2013-01-01

    The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS.

  7. Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

    PubMed

    Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

    2014-01-01

    Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

  8. Positive and Negative Aspects of the Human Immunodeficiency Virus Protease: Development of Inhibitors versus Its Role in AIDS Pathogenesis

    PubMed Central

    Ikuta, Kazuyoshi; Suzuki, Satoko; Horikoshi, Haruko; Mukai, Tetsu; Luftig, Ronald B.

    2000-01-01

    In this review we summarize multiple aspects of the human immunodeficiency virus (HIV) protease from both structural and functional viewpoints. After an introductory overview, we provide an up-to-date status report on protease inhibitors (PI). This proceeds from a discussion of PI structural design, to how PI are optimally utilized in highly active antiretroviral triple therapy (one PI along with two reverse transcriptase inhibitors), the emergence of PI resistance, and the natural role of secretory leukocyte PI. Then we switch to another focus: the interaction of HIV protease with other genes in acute and persistent infection, which in turn may have an effect on AIDS pathogenesis. We conclude with a discussion on future directions in HIV treatment, involving multiple-target anti-HIV therapy, vaccine development, and novel reactivation-inhibitory reagents. PMID:11104817

  9. A bacterial protease inhibitor protects antigens delivered in oral vaccines from digestion while triggering specific mucosal immune responses.

    PubMed

    Ibañez, Andrés Esteban; Coria, Lorena Mirta; Carabajal, Marianela Verónica; Delpino, María Victoria; Risso, Gabriela Sofía; Cobiello, Paula Gonzalez; Rinaldi, Jimena; Barrionuevo, Paula; Bruno, Laura; Frank, Fernanda; Klinke, Sebastián; Goldbaum, Fernando Alberto; Briones, Gabriel; Giambartolomei, Guillermo Hernán; Pasquevich, Karina Alejandra; Cassataro, Juliana

    2015-12-28

    We report here that a bacterial protease inhibitor from Brucella spp. called U-Omp19 behaves as an ideal constituent for a vaccine formulation against infectious diseases. When co-administered orally with an antigen (Ag), U-Omp19: i) can bypass the harsh environment of the gastrointestinal tract by inhibiting stomach and intestine proteases and consequently increases the half-life of the co-administered Ag at immune inductive sites: Peyer's patches and mesenteric lymph nodes while ii) it induces the recruitment and activation of antigen presenting cells (APCs) and increases the amount of intracellular Ag inside APCs. Therefore, mucosal as well as systemic Ag-specific immune responses, antibodies, Th1, Th17 and CD8(+) T cells are enhanced when U-Omp19 is co-administered with the Ag orally. Finally, this bacterial protease inhibitor in an oral vaccine formulation confers mucosal protection and reduces parasite loads after oral challenge with virulent Toxoplasma gondii.

  10. Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo

    PubMed Central

    Vermeire, Jon J.; Suzuki, Brian M.; Caffrey, Conor R.

    2016-01-01

    Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite’s resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck’s cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite’s CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN’s somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole. PMID:27384569

  11. Selective Dual Inhibitors of the Cancer-Related Deubiquitylating Proteases USP7 and USP47.

    PubMed

    Weinstock, Joseph; Wu, Jian; Cao, Ping; Kingsbury, William D; McDermott, Jeffrey L; Kodrasov, Matthew P; McKelvey, Devin M; Suresh Kumar, K G; Goldenberg, Seth J; Mattern, Michael R; Nicholson, Benjamin

    2012-10-11

    Inhibitors of the cancer-related cysteine isopeptidase human ubiquitin-specific proteases 7 (USP7) and 47 (USP47) are considered to have potential as cancer therapeutics, owing to their ability to stabilize the tumor suppressor p53 and to decrease DNA polymerase β (Polβ), both of which are potential anticancer effects. A new class of dual small molecule inhibitors of these enzymes has been discovered. Compound 1, a selective inhibitor of USP7 and USP47 with moderate potency, demonstrates inhibition of USP7 in cells and induces elevated p53 and apoptosis in cancer cell lines. Compound 1 has been shown to demonstrate modest activity in human xenograft multiple myeloma and B-cell leukemia in vivo models. This activity may be the result of dual inhibition of USP7 and USP47. To address issues regarding potency and developability, analogues of compound 1 have been synthesized and tested, leading to improvements in potency, solubility, and metabolic reactivity profile. Further optimization is expected to yield preclinical candidates and, ultimately, clinical candidates for the treatment of multiple myeloma, prostate cancer, and other cancers.

  12. Analysis of the structure of calpain-10 and its interaction with the protease inhibitor SNJ-1715.

    PubMed

    da Silva, Ronaldo Correia; de Alencar, Nelson Alberto N; Alves, Cláudio Nahum; Lameira, Jerônimo

    2013-10-01

    Calpain-10 (CAPN10) is a cysteine protease that is activated by intracellular calcium (Ca(2+)) and known to be involved in diseases such as cancer, heart attack, and stroke. A role for the CAPN10 gene in diabetes mellitus type II was recently identified. Hyper activation of the enzyme initiates a series of destructive cycles that can cause irreversible damage to cells. The development of inhibitors may be useful as therapeutic agents for a number of calpainopathies. In this paper, we have used the homology modelling technique to determine the 3D structure of calpain-10 from Homo sapiens. The model of calpain-10 obtained by homology modelling suggests that its active site is conserved among family members and the main interactions are similar to those observed for μ-calpain. Structural analysis revealed that there are small differences in the charge distribution and molecular surface of the enzyme. These differences are probably less dependent on calcium for calpain-10 than they are for μ-calpain. In addition, the ion pair Cys(-)/His(+) formation was observed using of Molecular Dynamics (MD) simulations that were based upon hybrid quantum mechanical/molecular mechanical (QM/MM) approaches. Finally, the binding of the SNJ-1715 inhibitor to calpain-10 was investigated in order to further understand the mechanism of inhibition of calpain-10 by this inhibitor at the molecular level.

  13. Development of Broad-Spectrum Halomethyl Ketone Inhibitors Against Coronavirus Main Protease 3CL(pro)

    SciTech Connect

    Bacha,U.; Barilla, J.; Gabelli, S.; Kiso, Y.; Amzel, L.; Freire, E.

    2008-01-01

    Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CLpro (Mpro), which is highly conserved among all coronaviruses. Here 3CLpro derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CLpro with Ki's as low as 300 nm. The crystal structure of the complex of one of the compounds with 3CLpro indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.

  14. [Presence of lectins, tannins and protease inhibitors in venezuelan marine algae].

    PubMed

    Perez-Lorenzo, S; Levy-Benshimol, A; Gomez-Acevedo, S

    1998-01-01

    The presence of lectins, tannins and protease inhibitors was studied in 27 algae species collected at four Venezuelan coral rift sites. Among the species studied, only six had hemagglutinating activity, apparently due to their lectin content. Higher hemagglutinating titers were obtained when the extracts were tested on pronase-treated erythrocytes. Hemagglutination was inhibited by simple sugars and by bovine submaxillary gland mucine. GaINAc was the only inhibitor of the hemagglutination caused by Grateulopia filicina extracts. None of the compounds tested inhibited the hemagglutination caused by Halimeda opuntia. The polyvinylpolypirrolidone treatment abolished the hemagglutinating activity of both brown and red algae. However, in Grateulopia filicina and Hypnea cervicornis (Rhodophyta) hemagglutinating activity persisted after the polyvinylpolypirrolidone treatment, presumably due to the presence of true lectins in those algae. Tannin content (presumably phlorotannins) was higher in the Phaeophyta as compared to the Rhodophyta. The brown alga Padina gymnospora had the higher content of these polyphenols. Trypsin inhibitors were detected, in minute ammounts, only in Padina gymnospora (Phaeophyta) and Acantophora spicifera (Rhodophyta). No subtilisin inhibition was observed whatsoever.

  15. Effects of the aspartic protease inhibitor from Lupinus bogotensis seeds on the growth and development of Hypothenemus hampei: an inhibitor showing high homology with storage proteins.

    PubMed

    Molina, Diana; Patiño, Luisa; Quintero, Mónica; Cortes, José; Bastos, Sara

    2014-02-01

    The coffee berry borer Hypothenemus hampei is a pest that causes great economic damage to coffee grains worldwide. Because the proteins consumed are digested by aspartic proteases in the insect's midgut, the inhibition of these proteases by transferring a gene encoding an aspartic protease inhibitor from Lupinus bogotensis Benth. to coffee plants could provide a promising strategy to control this pest. Five aspartic protease inhibitors from L. bogotensis (LbAPI) were accordingly purified and characterized. The gene encoding the L. bogotensis aspartic protease inhibitor (LbAPI), with the highest inhibitory activity against H. hampei, was expressed in Escherichia coli and the purified recombinant protein (rLbAPI), with a molecular mass of 15 kDa, was subsequently assessed for its ability to inhibit the aspartic protease activity present in the H. hampei midgut in vitro, as well as its effects on the growth and development of H. hampei in vivo. The in vitro experiments showed that rLbAPI was highly effective against aspartic proteases from H. hampei guts, with a half maximal inhibitory concentration (IC50) of 2.9 μg. The in vivo experiments showed that the concentration of rLbAPI (w/w) in the artificial diet necessary to cause 50% mortality (LD50) of the larvae was 0.91%. The amino acid sequence of LbAPI had high homology (52-80%) to the seed storage proteins, vicilin and β-conglutin, suggesting that this protein was generated by evolutionary events from a β-conglutin precursor. Based on these results, LbAPI may have a dual function as storage protein, and as defense protein against H. hampei. These results provide a promising alternative to obtain a coffee plant resistant to H. hampei.

  16. Binding of phosphinate and phosphonate inhibitors to aspartic proteases: a first-principles study.

    PubMed

    Vidossich, Pietro; Carloni, Paolo

    2006-01-26

    Phosphinate and phosphonate derivatives are potent inhibitors of aspartic proteases (APs). The affinity for the enzyme might be caused by the presence of low barrier hydrogen bonds between the ligand and the catalytic Asp dyad in the cleavage site. We have used density functional theory calculations along with hybrid quantum mechanics/molecular mechanics Car-Parrinello molecular dynamics simulations to investigate the hydrogen-bonding pattern at the binding site of the complexes of human immunodeficiency virus type-1 AP and the eukaryotic endothiapepsin and penicillopepsin. Our calculations are in fair agreement with the NMR data available for endothiapepsin (Coates et al. J. Mol. Biol. 2002, 318, 1405-1415) and show that the most stable active site configuration is the diprotonated, negatively charged form. In the viral complex both protons are located at the catalytic Asp dyad, while in the eukaryotic complexes the proton shared by the closest oxygen atoms is located at the phosphinic/phosphonic group.

  17. Complexity in modeling and understanding protonation states: computational titration of HIV-1-protease-inhibitor complexes.

    PubMed

    Tripathi, Ashutosh; Fornabaio, Micaela; Spyrakis, Francesca; Mozzarelli, Andrea; Cozzini, Pietro; Kellogg, Glen E

    2007-11-01

    The computational-titration (CT) algorithm based on the 'natural' Hydropathic INTeractions (HINT) force field is described. The HINT software model is an empirical, non-Newtonian force field derived from experimentally measured partition coefficients for solvent transfer between octanol and H(2)O (log P(o/w)). The CT algorithm allows the identification, modeling, and optimization of multiple protonation states of residues and ligand functional groups at the protein-ligand active site. The importance of taking into account pH and ionization states of residues, which strongly affect the process of ligand binding, for correctly predicting binding free energies is discussed. The application of the CT protocol to a set of six cyclic inhibitors in their complexes with HIV-1 protease is presented, and the advance of HINT as a virtual-screening tool is outlined.

  18. Antibacterial Activity of Fistulin: A Protease Inhibitor Purified from the Leaves of Cassia fistula.

    PubMed

    Arulpandi, I; Sangeetha, R

    2012-01-01

    Plant protease inhibitors (PPIs) are one of the important components of a plant's defense machinery. PPIs are active against the insects and microbes which invade the plant. Cassia species possess anti-insecticidal and antimicrobial properties and this study was aimed at investigating the antibacterial efficacy of a PPI present in the leaves of Cassia fistula. A PPI, fistulin, was isolated from the leaves of C. fistula and purified by gel filtration chromatography. The antibacterial activity of the purified fistulin was studied against five bacterial strains, namely, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli. The PPI was found to be very active against S. aureus, E. coli, B. subtilis, and K. pneumonia, and its efficacy was comparable to the standard drug, streptomycin sulphate.

  19. Development and evaluation of a highly reliable assay for SUMO-specific protease inhibitors.

    PubMed

    Xie, Wenjuan; Wang, Zhongli; Zhang, Jianchen; Wang, Lie; Zhao, Yaxue; Zhou, Huchen

    2016-05-01

    SUMOylation, as a post-translational modification of proteins, plays essential regulatory roles in a variety of pathological conditions. In the dynamic process of SUMOylation and deSUMOylation, SENPs (SUMO-specific proteases), in charge of deconjugation of SUMO (small ubiquitin-related modifier) from substrate proteins, have recently been found to be potential therapeutic targets for cancer treatment. A reliable and practical assay is much needed to accelerate the discovery of SENPs inhibitors. We established a quantitative assay based on readily available SDS-PAGE-Coomassie system using RanGAP-SUMO as the substrate, thus avoiding the use of expensive fluorescent dyes or the error-prone fluorescent reporter. Its reproducibility and reliability were also evaluated in this report. Copyright © 2016. Published by Elsevier Ltd.

  20. Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash

    PubMed Central

    Pierce Campbell, Christine M.; Guan, Wei; Sprung, Robert; Koomen, John M.; O’Keefe, Michael T.; Ingles, Donna J.; Abrahamsen, Martha; Giuliano, Anna R.

    2014-01-01

    Background Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake. Methods Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit. Sample suitability was assessed and quality control analyses were conducted. Results Salivary SLPI was successfully recovered from oral gargles with low intra-assay and high inter-individual variability. Initial measurements showed that salivary SLPI varied considerably across individuals, and that SLPI was inversely associated with age. Conclusions This optimized assay can be used to examine the role of SLPI in the acquisition of oral HPV and other infections. PMID:24140751

  1. 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

  2. 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

  3. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    USDA-ARS?s Scientific Manuscript database

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  4. Inhibitors of hepatitis C virus NS3.4A protease 1. Non-charged tetrapeptide variants.

    PubMed

    Perni, Robert B; Britt, Shawn D; Court, John C; Courtney, Lawrence F; Deininger, David D; Farmer, Luc J; Gates, Cynthia A; Harbeson, Scott L; Kim, Joseph L; Landro, James A; Levin, Rhonda B; Luong, Yu-Ping; O'Malley, Ethan T; Pitlik, Janos; Rao, B Govinda; Schairer, Wayne C; Thomson, John A; Tung, Roger D; Van Drie, John H; Wei, Yunyi

    2003-11-17

    Tetrapeptide-based peptidomimetic compounds have been shown to effectively inhibit the hepatitis C virus NS3.4A protease without the need of a charged functionality. An aldehyde is used as a prototype reversible electrophilic warhead. The SAR of the P1 and P2 inhibitor positions is discussed.

  5. Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death

    PubMed Central

    de Koning, Pieter J. A.; Kummer, J. Alain; de Poot, Stefanie A. H.; Quadir, Razi; Broekhuizen, Roel; McGettrick, Anne F.; Higgins, Wayne J.; Devreese, Bart; Worrall, D. Margaret; Bovenschen, Niels

    2011-01-01

    Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e. GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death. Expression of intracellular serine protease inhibitors (serpins) is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by tumor cells renders them resistant to GrB-induced apoptosis. In contrast to GrB, however, no physiological intracellular inhibitors are known for the other four human granzymes. In the present study, we show that SERPINB4 formed a typical serpin-protease SDS-stable complex with both recombinant and native human GrM. Mutation of the P2-P1-P1′ triplet in the SERPINB4 reactive center loop completely abolished complex formation with GrM and N-terminal sequencing revealed that GrM cleaves SERPINB4 after P1-Leu. SERPINB4 inhibited GrM activity with a stoichiometry of inhibition of 1.6 and an apparent second order rate constant of 1.3×104 M−1s−1. SERPINB4 abolished cleavage of the macromolecular GrM substrates α-tubulin and nucleophosmin. Overexpression of SERPINB4 in tumor cells inhibited recombinant GrM-induced as well as NK cell-mediated cell death and this inhibition depended on the reactive center loop of the serpin. As SERPINB4 is highly expressed by squamous cell carcinomas, our results may represent a novel mechanism by which these tumor cells evade cytotoxic lymphocyte-induced GrM-mediated cell death. PMID:21857942

  6. Shear stress-induced Ets-1 modulates protease inhibitor expression in microvascular endothelial cells.

    PubMed

    Milkiewicz, Malgorzata; Uchida, Cassandra; Gee, Eric; Fudalewski, Tomasz; Haas, Tara L

    2008-11-01

    Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4, and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm(2)) for 0.5, 2, and 24 h. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2 h shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1, and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.

  7. Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

    SciTech Connect

    Tie, Yunfeng; Wang, Yuan-Fang; Boross, Peter I.; Chiu, Ting-Yi; Ghosh, Arun K.; Tozser, Jozsef; Louis, John M.; Harrison, Robert W.; Weber, Irene T.

    2012-03-15

    Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR{sub 2}) than on HIV-1 protease (PR{sub 1}). We solved the crystal structure of PR{sub 2} with APV at 1.5 {angstrom} resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR{sub 1} mutant (PR{sub 1M}) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR{sub 2}. PR{sub 1M} more closely resembled PR{sub 2} than PR{sub 1} in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR{sub 1M} with APV, DRV, and SQV were compared with available PR{sub 1} and PR{sub 2} complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR{sub 1M} and PR{sub 1}, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR{sub 1M}. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR{sub 1M} and PR{sub 2} relative to the strong hydrogen bonds observed in PR{sub 1}, consistent with 15- and 19-fold weaker inhibition, respectively. Overall, PR{sub 1M} partially replicates the specificity of PR{sub 2} and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2.

  8. 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

  9. Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors.

    PubMed

    Tie, Yunfeng; Wang, Yuan-Fang; Boross, Peter I; Chiu, Ting-Yi; Ghosh, Arun K; Tozser, Jozsef; Louis, John M; Harrison, Robert W; Weber, Irene T

    2012-03-01

    Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR₂) than on HIV-1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR(1M) ) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR(1M) more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR(1M) with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR(1M) and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR(1M). Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR₁ (M) and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15- and 19-fold weaker inhibition, respectively. Overall, PR(1M) partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2. Copyright © 2012 The Protein Society.

  10. Identification of the protease inhibitor miraziridine A in the Red sea sponge Theonella swinhoei

    PubMed Central

    Tabares, Paula; Degel, Björn; Schaschke, Norbert; Hentschel, Ute; Schirmeister, Tanja

    2012-01-01

    Background: Miraziridine A, a natural peptide isolated from a marine sponge, is a potent cathepsin B inhibitor with a second-order rate constant of 1.5 × 104 M-1 s-1. In the present study, miraziridine A was isolated from the Red Sea sponge Theonella swinhoei on the basis of chromatographic and spectrometric techniques. We conclude that T. swinhoei from the Red Sea represents an alternative source of the aziridinylpeptide miraziridine A to the previously identified Theonella mirabilis from Japan. We confirmed that the metabolite is produced by marine sponges from different geographical locations. Context: Marine sponges have been proven to be a rich source of secondary metabolites exhibiting a huge diversity of biological activities, including antimicrobial, antitumor and immunomodulatory activities. Theonella species (order Lithistida, Demospongiae) have been shown to be a source of anti-protease and anti-HIV secondary metabolites. Aims: To identify the protease inhibitor mirazirine A in the marine sponge Theonella swinhoei. Material and Methods: The marine sponge Theonella swinhoei was collected by SCUBA diving in the Red Sea in Eilat (Israel). Sponge material was lyophilized and further extracted successively with cyclohexane, dichloromethane and methanol to obtain three crude extracts. LC-MS analysis was performed to confirm the presence of Miraziridine A in the dichloromethane fraction. Results: In the present study, miraziridine A was isolated from the Red Sea sponge T. swinhoei on the basis of chromatographic and spectrophotometric techniques. Conclusions: We conclude that T. swinhoei from the Red Sea represents an alternative source of the aziridinylpeptide miraziridine A to the previously identified Theonella mirabilis from Japan. PMID:22224064

  11. The HIV-protease inhibitor saquinavir reduces proliferation, invasion and clonogenicity in cervical cancer cell lines

    PubMed Central

    Bandiera, Elisabetta; Todeschini, Paola; Romani, Chiara; Zanotti, Laura; Erba, Eugenio; Colmegna, Benedetta; Bignotti, Eliana; Santin, Alessandro Davide; Sartori, Enrico; Odicino, Franco Edoardo; Pecorelli, Sergio; Tassi, Renata Alessandra; Ravaggi, Antonella

    2016-01-01

    Innovative therapies in cervical cancer (CC) remain a priority. Recent data indicate that human immunodeficiency virus (HIV)-protease inhibitors used in highly active antiretroviral therapy can exert direct antitumor activities also in HIV-free preclinical and clinical models. The aim of the present study was to evaluate the antineoplastic effects of various HIV-protease inhibitors (indinavir, ritonavir and saquinavir) on primary and established CC cell lines. Two CC cell lines established in our laboratory and four commercially available CC cell lines were treated with indinavir, ritonavir and saquinavir at different concentrations and for different times. Proliferation, clonogenicity and radiosensitivity were evaluated by crystal violet staining. Proteasomal activities were assessed using a cell-based assay and immunoblotting. Cell cycle was analyzed by propidium iodide staining and flow cytometric analysis. Invasion was tested with Matrigel chambers. A t-test for paired samples was used for statistical analysis. In all cell lines, saquinavir was more effective than ritonavir in reducing cell proliferation and inhibiting proteasomal activities (P≤0.05). Conversely, indinavir exerted a negligible effect. The saquinavir concentrations required to modulate the proteasome activities were higher than those observed to be effective in inhibiting cell proliferation. In HeLa cells, saquinavir was strongly effective in inhibiting cell invasion and clonogenicity (P≤0.05) at concentrations much lower than those required to perturb proteasomal activities. Saquinavir did not contribute to increase the sensitivity of HeLa cells to X-rays. In conclusion, the present results demonstrate that saquinavir is able to significantly reduce cell proliferation, cell invasion and clonogenicity in a proteasome-independent manner in in vitro models of CC, and suggest that saquinavir could be a promising CC therapeutic agent. PMID:27698818

  12. Purification and characterization of alpha 1-antichymotrypsin-like protease inhibitor that regulates prohormone thiol protease involved in enkephalin precursor processing.

    PubMed

    Hook, V Y; Purviance, R T; Azaryan, A V; Hubbard, G; Krieger, T J

    1993-09-25

    Evidence is presented showing that alpha 1-antichymotrypsin (ACT) inhibits a novel prohormone thiol protease (PTP) involved in processing the enkephalin precursor. Colocalization of ACT immunoreactivity with PTP within isolated secretory vesicles of bovine adrenal medulla and pituitary indicated that endogenous ACT could regulate PTP in vivo. The endogenous 60 kDa bovine ACT (bACT)-like protein was purified from pituitary by chromatography on DEAE-Sepharose, chromatofocusing, butyl-Sepharose, and Sephacryl S-200. Characterization showed that the bACT-like protein was a potent inhibitor of PTP (Ki,app value of 2.2 nM) as well as an effective inhibitor of chymotrypsin (Ki,app value of 2.3 nM). Furthermore, the bACT-like protein formed sodium dodecyl sulfate-stable complexes with chymotrypsin, which is typical of serpin protease inhibitors. Importantly, PTP formed sodium dodecyl sulfate-stable complexes with human ACT, suggesting that PTP's cleavage specificity may resemble the reactive center of ACT. PTP cleavage of enkephalin-containing peptides at the NH2-terminal side of paired basic residues (Lys-Arg, Arg-Arg, Lys-Lys), flanking the COOH terminus of (Met)enkephalin (Tyr-Gly-GLy-Phe-Met), indicates methionine at the P1 position. PTP cleavage of peptide-methylcoumarin amide and peptide-p-nitroanilide substrates demonstrated specificity for paired basic and monobasic residues, as well as a role for methionine in PTP's cleavage site. These results showing PTP's ability for processing at a methionine residue which resembles the P1 specificity of ACT are compatible with inhibition of PTP by ACT. These findings are the first demonstration of the involvement of a protease inhibitor in neuropeptide precursor processing. The known developmental regulation of ACT in brain and significant amounts of ACT in amyloid plaques of Alzheimer's disease suggest a possible role for PTP in the maturation of peptidergic neurons.

  13. C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay.

    PubMed

    Ghannam, A; Sellier, P; Defendi, F; Favier, B; Charignon, D; López-Lera, A; López-Trascasa, M; Ponard, D; Drouet, C

    2015-09-01

    Controlling prekallikrein activation by C1 inhibitor (C1Inh) represents the most essential mechanism for angioedema patient protection. C1Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1Inh function based on contact-phase activation and correlation with angioedema diagnostic requirements. The contact phase was reconstituted using the purified components, with C1Inh standard or plasma sample. The kinetics of the amidase activity were monitored using Pro-Phe-Arg-pNA, independently of alpha2-macroglobulin. We prevented any interference from a possible high plasma kininogenase activity by preincubating the samples with protease inhibitor. Receiver operating characteristics (ROC) were used to calculate the assay's diagnostic performance. The calibration curve was built using C1Inh standard (threshold limit 0.10 × 10(-3) U, i.e., 0.2 pmol), and C1Inh function was quantified in the sample, with a reference interval established based on healthy individuals (n = 281; men: 0.61-1.10 U/ml, median: 0.85 U/ml; women: 0.42-1.08 U/ml, median: 0.74 U/ml). The median values of female donors were lower than those of the others due to estrogen, yet C1Inh function remained within the reference interval. The ROC curve calculation provided the following optimum diagnostic cutoff values: women 0.36 U/ml (area under curve [AUC]: 0.99; sensitivity: 93.48%; specificity: 99.37%); and men 0.61 U/ml (AUC: 1; sensitivity: 100.0%; specificity: 100.0%). The performance outcome provided features suitable for angioedema diagnostic or follow-up. Established by means of the kinin formation process, this assay should be preferred over the method based on a C1s protease target. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Minor mutations in HIV protease at baseline and appearance of primary mutation 90M in patients for whom their first protease-inhibitor antiretroviral regimens failed.

    PubMed

    Perno, Carlo Federico; Cozzi-Lepri, Alessandro; Forbici, Federica; Bertoli, Ada; Violin, Michela; Stella Mura, Maria; Cadeo, Giampiero; Orani, Anna; Chirianni, Antonio; De Stefano, Carlo; Balotta, Claudia; d'Arminio Monforte, Antonella

    2004-06-01

    The association between minor mutations in human immunodeficiency virus (HIV) protease at baseline and development of common primary mutation 90M at virological failure (conferring some resistance to all protease inhibitors [PIs]) was evaluated in 93 previously drug-naive patients experiencing failure of their first PI-based antiretroviral regimens. In logistic regression analysis, the probability of accumulating a new 90M mutation at virological failure was associated with the presence at baseline of minor mutation 36I (naturally occurring in approximately 25% of HIV clade B and in >80% of HIV non-clade-B viruses) (adjusted odds ratio, 13.5 [95% confidence interval, 1.89-95.6]; P=.009) and, possibly, of 10I/V. This suggests a potential role for the presence of 36I at baseline in predicting the appearance of 90M at virological failure.

  15. Broad-Spectrum Inhibitors against 3C-Like Proteases of Feline Coronaviruses and Feline Caliciviruses

    PubMed Central

    Shivanna, Vinay; Narayanan, Sanjeev; Prior, Allan M.; Weerasekara, Sahani; Hua, Duy H.; Kankanamalage, Anushka C. Galasiti; Groutas, William C.; Chang, Kyeong-Ok

    2015-01-01

    ABSTRACT Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats

  16. Broad-spectrum inhibitors against 3C-like proteases of feline coronaviruses and feline caliciviruses.

    PubMed

    Kim, Yunjeong; Shivanna, Vinay; Narayanan, Sanjeev; Prior, Allan M; Weerasekara, Sahani; Hua, Duy H; Kankanamalage, Anushka C Galasiti; Groutas, William C; Chang, Kyeong-Ok

    2015-05-01

    Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally