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Sample records for first-line protease inhibitors

  1. HIV Protease Inhibitors Do Not Cause the Accumulation of Prelamin A in PBMCs from Patients Receiving First Line Therapy: The ANRS EP45 “Aging” Study

    PubMed Central

    Perrin, Sophie; Cremer, Jonathan; Faucher, Olivia; Reynes, Jacques; Dellamonica, Pierre; Micallef, Joëlle; Solas, Caroline; Lacarelle, Bruno; Stretti, Charlotte; Kaspi, Elise; Robaglia-Schlupp, Andrée; Tamalet, Corine Nicolino-Brunet Catherine; Lévy, Nicolas; Poizot-Martin, Isabelle; Cau, Pierre; Roll, Patrice

    2012-01-01

    Background The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes. Methods 35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years) containing boosted protease inhibitors (PI/r) (comprising lopinavir/ritonavir in 65% of patients) were recruited together with 49 seronegative age- and sex-matched control subjects (http://clinicaltrials.gov/, NCT01038999). In more than 88% of patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm3. Prelamin A processing in peripheral blood mononuclear cells (PBMCs) from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L). Results Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L) but not in plasma (60–80 g/L) or in medium supplemented with BSA (40 g/L), both of which contain a high protein concentration. Conclusions Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells. ClinicalTrials.gov NCT01038999 http://clinicaltrials.gov/ct2/show/NCT01038999. PMID

  2. Protease Inhibitor Resistance Analysis in the MONARK Trial Comparing First-Line Lopinavir-Ritonavir Monotherapy to Lopinavir-Ritonavir plus Zidovudine and Lamivudine Triple Therapy▿

    PubMed Central

    Delaugerre, Constance; Flandre, Philippe; Chaix, Marie Laure; Ghosn, Jade; Raffi, François; Dellamonica, Pierre; Jaeger, H.; Shürmann, D.; Cohen-Codar, Isabelle; Ngo Van, Philippe; Norton, Michael; Taburet, Anne-Marie; Delfraissy, Jean-François; Rouzioux, Christine

    2009-01-01

    The MONARK study was a pilot randomized trial comparing the safety and efficacy of lopinavir-ritonavir (LPV/r) monotherapy to those of LPV/r-zidovudine-lamivudine triple therapy for antiretroviral-naïve human immunodeficiency virus type 1 (HIV-1)-infected patients. Resistance testing was performed at the time of initial screening and at the time of virological failure (defined to include low-level viremia with >50 and <400 HIV-1 virus RNA copies/ml of plasma). Changes from the baseline sequences, including mutations noted on the 2008 International AIDS Society—USA list of resistance-associated protease mutations, were considered. Drug resistance testing was performed for 38 patients (5 of 53 on triple therapy and 33 of 83 on monotherapy). By week 96 (W96), virus samples from 18 of 33 patients in the monotherapy arm showed changes from baseline sequences, and 5 of these patients had viruses with major protease inhibitor (PI) resistance-associated mutations (M46I at W40, L76V at W48, M46I and L76V at W48, L10F and V82A at W72, and L76V at W84). Data on virus phenotypes detected at the time of initial screening and the time of virological failure were available for four patients in whom major PI resistance mutations developed, and these data revealed a mean increase of 2.2-fold (range, 0.75- to 4.6-fold) in the LPV 50% inhibitory concentration. All three patients in whom the L76V PI resistance mutation developed were infected with HIV-1 subtype CRF02_AG. In the triple-therapy group, no major PI resistance mutation was selected among the three patients with protease changes by W48. No association between the baseline CD4 cell count and the viral load, the W4 and final viral loads, or the final LPV trough concentration and the emergence of a major PI resistance mutation was found. Major PI resistance-associated mutations were detected in 5 (6%) of 83 patients treated with LPV/r monotherapy, suggesting that LPV/r monotherapy is an inappropriate first option. The

  3. Long-term effectiveness of initiating non-nucleoside reverse transcriptase inhibitor- versus ritonavir-boosted protease inhibitor-based antiretroviral therapy: implications for first-line therapy choice in resource-limited settings

    PubMed Central

    Lima, Viviane D; Hull, Mark; McVea, David; Chau, William; Harrigan, P Richard; Montaner, Julio SG

    2016-01-01

    Introduction In many resource-limited settings, combination antiretroviral therapy (cART) failure is diagnosed clinically or immunologically. As such, there is a high likelihood that patients may stay on a virologically failing regimen for a substantial period of time. Here, we compared the long-term impact of initiating non-nucleoside reverse transcriptase inhibitor (NNRTI)- versus boosted protease inhibitor (bPI)-based cART in British Columbia (BC), Canada. Methods We followed prospectively 3925 ART-naïve patients who started NNRTIs (N=1963, 50%) or bPIs (N=1962; 50%) from 1 January 2000 until 30 June 2013 in BC. At six months, we assessed whether patients virologically failed therapy (a plasma viral load (pVL) >50 copies/mL), and we stratified them based on the pVL at the time of failure ≤500 versus >500 copies/mL. We then followed these patients for another six months and calculated their probability of achieving subsequent viral suppression (pVL <50 copies/mL twice consecutively) and of developing drug resistance. These probabilities were adjusted for fixed and time-varying factors, including cART adherence. Results At six months, virologic failure rates were 9.5 and 14.3 cases per 100 person-months for NNRTI and bPI initiators, respectively. NNRTI initiators who failed with a pVL ≤500 copies/mL had a 16% higher probability of achieving subsequent suppression at 12 months than bPI initiators (0.81 (25th–75th percentile 0.75–0.83) vs. 0.72 (0.61–0.75)). However, if failing NNRTI initiators had a pVL >500 copies/mL, they had a 20% lower probability of suppressing at 12 months than pVL-matched bPI initiators (0.37 (0.29–0.45) vs. 0.46 (0.38–0.54)). In terms of evolving HIV drug resistance, those who failed on NNRTI performed worse than bPI in all scenarios, especially if they failed with a viral load >500 copies/mL. Conclusions Our results show that patients who virologically failed at six months on NNRTI and continued on the same regimen had a

  4. Peptidomimetic inhibitors of HIV protease.

    PubMed

    Randolph, John T; DeGoey, David A

    2004-01-01

    There are currently (July, 2002) six protease inhibitors approved for the treatment of HIV infection, each of which can be classified as peptidomimetic in structure. These agents, when used in combination with other antiretroviral agents, produce a sustained decrease in viral load, often to levels below the limits of quantifiable detection, and a significant reconstitution of the immune system. Therapeutic regimens containing one or more HIV protease inhibitors thus provide a highly effective method for disease management. The important role of protease inhibitors in HIV therapy, combined with numerous challenges remaining in HIV treatment, have resulted in a continued effort both to optimize regimens using the existing agents and to identify new protease inhibitors that may provide unique properties. This review will provide an overview of the discovery and clinical trials of the currently approved HIV protease inhibitors, followed by an examination of important aspects of therapy, such as pharmacokinetic enhancement, resistance and side effects. A description of new peptidomimetic compounds currently being investigated in the clinic and in preclinical discovery will follow. PMID:15193140

  5. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  6. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

  7. 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.

  8. HIV Protease Inhibitors and Obesity

    PubMed Central

    Anuurad, Erdembileg; Bremer, Andrew; Berglund, Lars

    2011-01-01

    Purpose of review To review the current scientific literature and recent clinical trials on HIV protease inhibitors (PIs) and their potential role in the pathogenesis of lipodystrophy and metabolic disorders. Recent findings HIV PI treatment may affect the normal stimulatory effect of insulin on glucose and fat storage. Further, chronic inflammation from HIV infection and PI treatment trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. This process leads to a pathologic cycle of lipodystrophy and lipotoxicity, a pro-atherogenic lipid profile, and a clinical phenotype of increased central body fat distribution similar to the metabolic syndrome. Summary PIs are a key component of antiretroviral therapy and have dramatically improved the life expectancy of HIV-infected individuals. However, they are also associated with abnormalities in glucose/lipid metabolism and body fat distribution. Further studies are needed to better define the pathogenesis of PI-associated metabolic and body fat changes and their potential treatment. PMID:20717021

  9. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

  10. 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

  11. 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

  12. Serine protease inhibitors of parasitic helminths.

    PubMed

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  13. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models. PMID:25493637

  14. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models.

  15. New directions for protease inhibitors directed drug discovery.

    PubMed

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  16. A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.

    PubMed

    Wan, Hu; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Lee, Kyung Yong; Jin, Byung Rae

    2014-01-01

    Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki=6.57nM) and proteinase K (Ki=7.11nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions.

  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. Risk Factors for Incident Diabetes in a Cohort Taking First-Line Nonnucleoside Reverse Transcriptase Inhibitor-Based Antiretroviral Therapy.

    PubMed

    Karamchand, Sumanth; Leisegang, Rory; Schomaker, Michael; Maartens, Gary; Walters, Lourens; Hislop, Michael; Dave, Joel A; Levitt, Naomi S; Cohen, Karen

    2016-03-01

    Efavirenz is the preferred nonnucleoside reverse transcriptase inhibitor (NNRTI) in first-line antiretroviral therapy (ART) regimens in low- and middle-income countries, where the prevalence of diabetes is increasing. Randomized control trials have shown mild increases in plasma glucose in participants in the efavirenz arms, but no association has been reported with overt diabetes. We explored the association between efavirenz exposure and incident diabetes in a large Southern African cohort commencing NNRTI-based first-line ART. Our cohort included HIV-infected adults starting NNRTI-based ART in a private sector HIV disease management program from January 2002 to December 2011. Incident diabetes was identified by the initiation of diabetes treatment. Patients with prevalent diabetes were excluded. We included 56,298 patients with 113,297 patient-years of follow-up (PYFU) on first-line ART. The crude incidence of diabetes was 13.24 per 1000 PYFU. Treatment with efavirenz rather than nevirapine was associated with increased risk of developing diabetes (hazard ratio 1.27 (95% confidence interval (CI): 1.10-1.46)) in a multivariate analysis adjusting for age, sex, body mass index, baseline CD4 count, viral load, NRTI backbone, and exposure to other diabetogenic medicines. Zidovudine and stavudine exposure were also associated with an increased risk of developing diabetes. We found that treatment with efavirenz, as well as stavudine and zidovudine, increased the risk of incident diabetes. Interventions to detect and prevent diabetes should be implemented in ART programs, and use of antiretrovirals with lower risk of metabolic complications should be encouraged. PMID:26945366

  19. Risk Factors for Incident Diabetes in a Cohort Taking First-Line Nonnucleoside Reverse Transcriptase Inhibitor-Based Antiretroviral Therapy

    PubMed Central

    Karamchand, Sumanth; Leisegang, Rory; Schomaker, Michael; Maartens, Gary; Walters, Lourens; Hislop, Michael; Dave, Joel A.; Levitt, Naomi S.; Cohen, Karen

    2016-01-01

    Abstract Efavirenz is the preferred nonnucleoside reverse transcriptase inhibitor (NNRTI) in first-line antiretroviral therapy (ART) regimens in low- and middle-income countries, where the prevalence of diabetes is increasing. Randomized control trials have shown mild increases in plasma glucose in participants in the efavirenz arms, but no association has been reported with overt diabetes. We explored the association between efavirenz exposure and incident diabetes in a large Southern African cohort commencing NNRTI-based first-line ART. Our cohort included HIV-infected adults starting NNRTI-based ART in a private sector HIV disease management program from January 2002 to December 2011. Incident diabetes was identified by the initiation of diabetes treatment. Patients with prevalent diabetes were excluded. We included 56,298 patients with 113,297 patient-years of follow-up (PYFU) on first-line ART. The crude incidence of diabetes was 13.24 per 1000 PYFU. Treatment with efavirenz rather than nevirapine was associated with increased risk of developing diabetes (hazard ratio 1.27 (95% confidence interval (CI): 1.10–1.46)) in a multivariate analysis adjusting for age, sex, body mass index, baseline CD4 count, viral load, NRTI backbone, and exposure to other diabetogenic medicines. Zidovudine and stavudine exposure were also associated with an increased risk of developing diabetes. We found that treatment with efavirenz, as well as stavudine and zidovudine, increased the risk of incident diabetes. Interventions to detect and prevent diabetes should be implemented in ART programs, and use of antiretrovirals with lower risk of metabolic complications should be encouraged. PMID:26945366

  20. Gag-Protease Sequence Evolution Following Protease Inhibitor Monotherapy Treatment Failure in HIV-1 Viruses Circulating in East Africa.

    PubMed

    Sutherland, Katherine A; Goodall, Ruth L; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Thiltgen, Geant; Gilks, Charles F; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Dunn, David; Gupta, Ravindra K

    2015-10-01

    Around 2.5 million HIV-infected individuals failing first-line therapy qualify for boosted protease inhibitor (bPI)-based second-line therapy globally. Major resistance mutations are rarely present at treatment failure in patients receiving bPI and the determinants of failure in these patients remain unknown. There is evidence that Gag can impact PI susceptibility. Here, we have sequenced Gag-Protease before and following failure in 23 patients in the SARA trial infected with subtypes A, C, and D viruses. Before bPI, significant variation in Protease and Gag was observed at positions previously associated with PI exposure and resistance including Gag mutations L449P, S451N, and L453P and Protease K20I and L63P. Following PI failure, previously described mutations in Protease and Gag were observed, including those at the cleavage sites such as R361K and P453L. However, the emergence of clear genetic determinants of therapy failure across patients was not observed. Larger Gag sequence datasets will be required to comprehensively identify mutational correlates of bPI failure across subtypes. PMID:26258548

  1. Positioning of HIV-protease inhibitors in clinical practice.

    PubMed

    Andreoni, M; Perno, C F

    2012-01-01

    The availability of more than 20 drugs for the treatment of HIV infection, and the success of the current antiretroviral regimens, should not overlook the difficulty of long-term maintaining the control of viral replication. The therapy needs to be continued for decades, if not for lifetime, and there are clear evidences that, even in patients fully suppressed for many years, HIV starts again its replication cycles in case antiviral pressure is removed. The development of resistance is a natural event at the time of virological failure, that needs to be taken into account in the global strategy against HIV in each particular patient. Taking all together, therapeutic regiments must be embedded, since the beginning, in a long-term strategy whose main task is the stable control of the replication of HIV. To do so, the choice of the first antiviral regimen has to be highly appropriate to keep the virus in check, and at the same time maintain future therapeutic options. Change of therapy at the time of failure has to be also appropriate, in term of timing, diagnostic strategy, and selection of drugs. Under these circumstances, the use of protease inhibitors in the first line acquires a strong rationale, that balances the greater pure potency of non-nucleoside reverse transcriptase inhibitors (NNRTI), and makes them a valuable options for many patients that need to start antiviral therapy.

  2. Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Sutherland, Katherine; Parry, Chris M.; Cane, Patricia A.; Coovadia, Ashraf; Kuhn, Louise; Hunt, Gillian

    2016-01-01

    Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of

  3. Cystatin protease inhibitors and immune functions.

    PubMed

    Zavasnik-Bergant, Tina

    2008-05-01

    Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. They are wide spread in all living organisms (mammals, nematodes, arthropods etc.) and are involved in various biological processes where they regulate normal proteolysis and also take part in disease pathology. Many cystatins show changes in expression and/or localization, as well as changes in secretion, following certain stimuli acting on immune cells. In immune cells, cystatins interfere with antigen processing and presentation, phagocytosis, expression of cytokines and nitric oxide and these ways modify the immune response. Further, it has been suggested that cystatin-type molecules secreted from parasites down-modulate the host immune response. Precise understanding of the regulatory roles on proteolytic enzymes of endogenous and exogenous cystatins, such as those from parasites, will provide us with valuable insight into how immune response could be modulated to treat a specific disease. This review covers some specific functions of individual cystatins, with a particular focus on the relevance of cystatins to the immune response.

  4. Design of translactam HCMV protease inhibitors as potent antivirals.

    PubMed

    Borthwick, Alan D

    2005-07-01

    Human cytomegalovirus (HCMV) is an important pathogen for which there is a significant unmet medical need. New HCMV antivirals, active against novel molecular targets, are undoubtedly needed as the currently available drugs ganciclovir, cidofovir, and foscarnet, which are all viral DNA inhibitors, suffer from limited effectiveness, mainly due to the development of drug resistance, poor bioavailability, and toxicity. One of the newer molecular targets that has been exploited in the search for better drug candidates is HCMV protease. Our deltaAla HCMV protease (wild type variant with the internal cleavage site deleted) was cloned and expressed in E. coli. This viral enzyme was used to develop HCMV protease assays to evaluate potential inhibitors. The chirally pure (SRS)-alpha-methyl pyrrolidine-5,5-trans-lactam template was synthesized, which together with the natural substrate requirements of HCMV protease and detailed SAR, was used to design potent and selective mechanism based inhibitors of HCMV protease. The mechanism of action of these inhibitors of HCMV protease was investigated by ESI/MS, and the X-ray crystal structure of the HCMV protease was used to refine our selective viral enzyme inhibitors to obtain plasma stable antivirals. A novel ELISA antiviral assay was developed which, together with a cytotoxicity assay, enabled us to discover anti-HCMV drug candidates equivalent in potency to ganciclovir that had good pharmacokinetics in the dog and good brain and ocular penetration in the guinea pig.

  5. Improving Viral Protease Inhibitors to Counter Drug Resistance.

    PubMed

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

    2016-07-01

    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

  6. 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

  7. 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.

  8. Hepatitis C virus NS3 protease is activated by low concentrations of protease inhibitors.

    PubMed

    Dahl, Göran; Arenas, Omar Gutiérrez; Danielson, U Helena

    2009-12-01

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

  9. Predictors of virologic response to ritonavir-boosted protease inhibitors.

    PubMed

    Marcelin, Anne-Genevieve; Flandre, Philippe; Peytavin, Gilles; Calvez, Vincent

    2005-01-01

    The primary mechanism of resistance to protease inhibitors involves the stepwise accumulation of mutations that alter and block the substrate binding site of HIV protease. The large degree of cross-resistance among the different protease inhibitors is a source of considerable concern for the management of patients after treatment failure. Although the output of HIV-resistance tests has been based on therapeutically arbitrary criteria, there is now an ongoing move towards correlating test interpretation with virologic outcomes on treatment. This approach is undeniably superior, in principle, for tests intended to guide drug choices. However, the predictive accuracy of a given stratagem that links genotype or phenotype to drug response is strongly influenced by the study design, data capture and the analytical methodology used to derive it. There is no definitively superior methodology for generating a genotype-response association for use in interpreting a resistance test, and the various approaches used to date all have their strengths and weaknesses. Combining the information of therapeutic drug monitoring and resistance tests is likely to be of greatest clinical utility in antiretroviral-experienced patients harboring HIV strains with reduced susceptibility. The combination of pharmacologic and virologic parameters as a predictor of the virologic response has been merged into the parameter known as "inhibitory quotient". This article discusses the potential interest of the use of inhibitory quotients as an approach for enhancing the potency and durability of boosted protease inhibitors against protease inhibitor-resistant viruses. PMID:16425962

  10. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative.

    PubMed

    Bijina, B; Chellappan, Sreeja; Krishna, Jissa G; Basheer, Soorej M; Elyas, K K; Bahkali, Ali H; Chandrasekaran, M

    2011-07-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  11. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

    PubMed Central

    Bijina, B.; Chellappan, Sreeja; Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Bahkali, Ali H.; Chandrasekaran, M.

    2011-01-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  12. 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…

  13. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    PubMed

    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

  14. Additivity in the Analysis and Design of HIV Protease Inhibitors

    PubMed Central

    Jorissen, Robert N.; Kiran Kumar Reddy, G. S.; Ali, Akbar; Altman, Michael D.; Chellappan, Sripriya; Anjum, Saima G.; Tidor, Bruce; Schiffer, Celia A.; Rana, Tariq M.; Gilson, Michael K.

    2009-01-01

    We explore the applicability of an additive treatment of substituent effects to the analysis and design of HIV protease inhibitors. Affinity data for a set of inhibitors with a common chemical framework were analyzed to provide estimates of the free energy contribution of each chemical substituent. These estimates were then used to design new inhibitors, whose high affinities were confirmed by synthesis and experimental testing. Derivations of additive models by least-squares and ridge-regression methods were found to yield statistically similar results. The additivity approach was also compared with standard molecular descriptor-based QSAR; the latter was not found to provide superior predictions. Crystallographic studies of HIV protease-inhibitor complexes help explain the perhaps surprisingly high degree of substituent additivity in this system, and allow some of the additivity coefficients to be rationalized on a structural basis. PMID:19193159

  15. A Critical Review of Trials of First-Line BCR-ABL Inhibitor Treatment in Patients With Newly Diagnosed Chronic Myeloid Leukemia in Chronic Phase

    PubMed Central

    Jabbour, Elias; Lipton, Jeffrey H.

    2015-01-01

    The characteristic expression of the constitutively active oncoprotein, BCR-ABL tyrosine kinase, in chronic myeloid leukemia (CML) was the basis for the development of BCR-ABL tyrosine kinase inhibitors for treatment. Three BCR-ABL inhibitors, imatinib, nilotinib, and dasatinib, have been approved by the US Food and Drug Administration for first-line treatment of patients with newly diagnosed CML in chronic phase (CML-CP). This article reviews the key phase III clinical trials supporting the use of first-line imatinib, nilotinib, and dasatinib in patients with CML-CP as well as findings of supportive phase II studies. At the time of its approval in 2001, imatinib induced unprecedented response rates in patients with CML-CP; however, resistance and intolerance to imatinib prevent 20% to 30% of patients from deriving full therapeutic benefit. Nilotinib and dasatinib, both approved in 2010 for first-line CML-CP treatment, are more potent than imatinib and less susceptible to imatinib resistance mechanisms. Comparative clinical trials of each agent with imatinib have shown that they are associated with significantly deeper and more rapid responses than standard-dose imatinib, without compromising safety. Given that evidence suggests achievement of an early response is predictive of improved long-term outcomes, earlier use of these compounds may lead to more rapid, deeper responses corresponding with improvements in patient outcome. Although future studies will benefit from more uniform definitions of endpoints and methods of analysis, data from published studies of first-line BCR-ABL inhibitor treatment for patients with newly diagnosed CML-CP support the use of either dasatinib or nilotinib in place of imatinib. PMID:24095296

  16. Protease Inhibitors in View of Peptide Substrate Databases

    PubMed Central

    2016-01-01

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  17. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles.

  18. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles. PMID:26955746

  19. Interaction of proteases with legume seed inhibitors. Molecular features.

    PubMed

    de Seidl, D S

    1996-12-01

    After having found that raw black beans (Phaseolus vulgaris) were toxic, while the cooked ones constitute the basic diet of the underdeveloped peoples of the world, in the sixties, our research directed by Dr. Jaffé, concentrated mainly around the detection and identification of the heat labile toxic factors in legume seeds. A micromethod for the detection of protease inhibitors (PI) in individual seeds was developed, for the purpose of establishing that the multiple trypsin inhibitors (TI) found in the Cubagua variety were expressions of single seeds and not a mixture of a non homogenous bean lot. Six isoinhibitors were isolated and purified, all of which were "double-headed" and interacted with trypsin (T) and chymotrypsin (CHT) independently and simultaneously, as shown by electrophoresis of their binary and ternary complexes with each and both enzymes. However, their affinity for the enzymes, including elastases, was rather variable, as well as their amino acid composition which consisted of 51 units for inhibitor V, the smallest, and 83 amino acids for inhibitor I, the largest. A low molecular weight protein fraction that inhibited subtilisin (S), but recognized neither T, CHT nor pancreatic elastase was detected in 63 varieties of Phaseolus vulgaris as well as in broad beans (Vicia faba), chick peas (Cicer arietinum), jack beans (Canavalia ensiformis), kidney beans (Vigna aureus), etc., It was absent though, in soybeans (Glycine max), lentils (Lens culinaris), green peas (Pisum sativum), cowpea (Vigna sinensis) and lupine seeds (Lupinus sp). Subtilisin inhibitors (SI) were isolated from black beans, broad beans, chick peas and jack beans. Their Mr is between 8-9KD and they show a rather high stability in the presence of denaturing agents. They are specific toward microbial proteases, in addition to subtilisins, Carlsberg and BPN', they inhibit the alkaline protease from Tritirachium album (Protease K), from Aspergillus oryzae and one isolated from

  20. Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens.

    PubMed

    Park, Hyun Bong; Crawford, Jason M

    2016-08-01

    Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey's analysis. Compounds 1-4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9 μm. PMID:27353165

  1. Systematic review of aromatase inhibitors in the first-line treatment for hormone sensitive advanced or metastatic breast cancer.

    PubMed

    Riemsma, Rob; Forbes, C A; Kessels, A; Lykopoulos, K; Amonkar, M M; Rea, D W; Kleijnen, J

    2010-08-01

    To undertake a systematic review of three first-line treatments (letrozole, anastrozole and exemestane) for hormone sensitive advanced or metastatic breast cancer (MBC) in post-menopausal women. We searched six databases from inception up to January 2009 for relevant trials regardless of language or publication status. Randomised controlled clinical trials assessing the safety and efficacy of first-line AIs for post-menopausal women with hormone receptor-positive (HR+, i.e. ER+ and/or PgR+) with or without ErbB2 (HER2)-positive MBC, who have not received prior therapy for advanced or metastatic disease were included. Where meta-analysis using direct or indirect comparisons was considered unsuitable for some or all of the data, we employed a narrative synthesis method. Four studies (25 papers) met the inclusion criteria. From the available evidence, it was possible to directly compare the three AIs with tamoxifen. In addition, by using a network meta-analysis it was possible to compare the three AIs with each other. Based on direct evidence, letrozole seemed to be significantly better than tamoxifen in terms of time-to-progression (TTP) (HR = 0.70 (95% CI: 0.60, 0.82)), objective response rate (RR = 0.65 (95% CI: 0.52, 0.82)) and quality-adjusted time without symptoms or toxicity (Q-Twist difference = 1.5; P < 0.001). Exemestane seemed significantly superior to tamoxifen in terms of objective response rate (RR = 0.68 (95% CI: 0.53, 0.89)). Anastrozole seemed significantly superior to tamoxifen in terms of TTP in one trial (HR = 1.42 (95% CI: 1.15, NR)), but not in the other (HR = 1.01 (95% CI: 0.87, NR)). In terms of adverse events, no significant differences were found between letrozole and tamoxifen. Tamoxifen was associated with significantly more serious adverse events in comparison with exemestane (OR = 0.61 (95% CI: 0.38, 0.97)); while exemestane was associated with significantly more arthralgia in comparison with tamoxifen (OR = 2.33 (95% CI: 1.07, 5

  2. Elimination of protease-inhibitor complexes from the arthritic joint.

    PubMed

    Ekerot, L; Ohlsson, K; Necking, L

    1985-01-01

    In the rheumatic joint, destructive proteolytic enzymes are released and counteracted by complexation to the predominant inhibitors alpha 2-macroglobulin and alpha 1-antitrypsin. The articular elimination of these complexes is thought to be of decisive importance in the protease-inhibitor interplay influencing the inhibitory capacity of the synovial fluid. Protease-alpha 2-macroglobulin complexes showed an intraarticular half-life shorter than 2 hours in arthritis and were eliminated by various routes including phagocytosis in cells of the synovial membrane and in regional lymph nodes, in addition to haematogenous resorption and uptake in the liver. The phagocytosed complexes were degraded to low-molecular-weight metabolites excretable in the urine. The articular elimination of elastase-alpha 1-antitrypsin complexes seemed to be an equivalent process, but an additional intrasynovial dissociation of the complexes was also indicated. Thus the intraarticularly released elastase seemed to be bound and eliminated in complex with alpha 2-macroglobulin. PMID:2414244

  3. Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

    PubMed Central

    Smith, Robert A.; Gottlieb, Geoffrey S.

    2015-01-01

    ABSTRACT Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCE Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine

  4. Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES.

    PubMed

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

    2016-09-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu(15) within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu(15)/Arg(15)) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu(15) in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

  5. Primary structural analysis of sulfhydryl protease inhibitors from pineapple stem.

    PubMed

    Reddy, M N; Keim, P S; Heinrikson, R L; Kezdy, F J

    1975-03-10

    Pineapple stem acetone powder provides a rich source of the sulfhydryl protease bromelain and of a family of compositionally similar but chromatographically distinct polypeptide inihibtors of this enzyme. The isoinhibitors have molecular weights of 5600, and they contain five disulfide bonds and about 50 amino acids each (Perlstein, S. H., AND Kezdy, F.J. (1973) J. Supramol. Struct. 1, 249-254). Primary structural analysis of one of the seven inhibitor fractions (VII) revealed extensive microheterogeneity. Each of the inhibitor molecules in Fraction VII was shown to be composed of two peptide chains joined by disulfide bonds. These chains, designated A and B on the basis of size, comprise 41 and 10-11 residues, respectively, and the amino acid sequence of one of each are given below: (see article for formular). On the basis of ionization properties and yields of the A and B chains, it would appear that one of the major inhibitor species in Fraction VII is the covalently linked complex of the two chains shown, namely [A-1, B-2]. The second major inhibitor component of Fraction VII is identical in structure with [A-1, B-2i1 except that residues 1 and 8 in the A chain are pyroglutamate and threonine, respectively, and in the B chain glutamine 11 is replaced by arginine. The third inhibitor in Fraction VII is a minor constituent identical with the second, except that residue 1 in the A chain is glutamate rather than pyroglutamate. This microheterogeneity in the inhibitors of Fraction VII is further increased by the fact that B chains may lack threonine 1, in which case they are decapeptides beginning with alanine. On the basis of the striking homology of the cysteine residues with those of other protease inhibitors, it is proposed that the bromelain inhibitors are generated enzymatically from single chain precursors by excision of a "bridge" paptide which links the NH-2 termal A chain to the COOH-terminal B chain.

  6. Comparative safety of BRAF and MEK inhibitors (vemurafenib, dabrafenib and trametinib) in first-line therapy for BRAF-mutated metastatic melanoma

    PubMed Central

    Cebollero, Ana; Puértolas, Teresa; Pajares, Isabel; Calera, Lourdes; Antón, Antonio

    2016-01-01

    A retrospective observational study was conducted on patients diagnosed with serine/threonine-protein kinase B-Raf (BRAF)-mutated metastatic melanoma, who underwent first-line therapy with BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors (vemurafenib, dabrafenib or a combination of dabrafenib and trametinib) at the Miguel Servet University Hospital (Zaragoza, Spain) between November, 2011 and August, 2015. The aim of this study was to analyse the toxicity produced by BRAF and MEK inhibitors. The most common toxicities were similar to those published in clinical trials, particularly arthralgia, alopecia and photosensitivity in the vemurafenib group; asthenia, hyperkeratosis and dry skin in the dabrafenib group; and diarrhoea and dry skin in the dabrafenib plus trametinib group. Toxicities that had not been described in clinical trials were also identified. Thus, the present study confirmed that the results obtained in clinical trials are similar to those obtained in clinical practice. PMID:27699043

  7. Comparative safety of BRAF and MEK inhibitors (vemurafenib, dabrafenib and trametinib) in first-line therapy for BRAF-mutated metastatic melanoma

    PubMed Central

    Cebollero, Ana; Puértolas, Teresa; Pajares, Isabel; Calera, Lourdes; Antón, Antonio

    2016-01-01

    A retrospective observational study was conducted on patients diagnosed with serine/threonine-protein kinase B-Raf (BRAF)-mutated metastatic melanoma, who underwent first-line therapy with BRAF and mitogen-activated protein kinase kinase (MEK) inhibitors (vemurafenib, dabrafenib or a combination of dabrafenib and trametinib) at the Miguel Servet University Hospital (Zaragoza, Spain) between November, 2011 and August, 2015. The aim of this study was to analyse the toxicity produced by BRAF and MEK inhibitors. The most common toxicities were similar to those published in clinical trials, particularly arthralgia, alopecia and photosensitivity in the vemurafenib group; asthenia, hyperkeratosis and dry skin in the dabrafenib group; and diarrhoea and dry skin in the dabrafenib plus trametinib group. Toxicities that had not been described in clinical trials were also identified. Thus, the present study confirmed that the results obtained in clinical trials are similar to those obtained in clinical practice.

  8. 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. PMID:25462990

  9. Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

    PubMed

    Hope-Onyekwere, Nnadozie Stanley; Ogueli, Godwin Ifeanyi; Cortelazzo, Alessio; Cerutti, Helena; Cito, Annarita; Aguiyi, John C; Guerranti, Roberto

    2012-12-01

    The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay. PMID:22447581

  10. Elastase and alpha 1-protease inhibitor in burn wound exudates.

    PubMed

    Prager, M D; Herring, M; Germany, B; Baxter, C R

    1991-01-01

    By degrading antithrombin III, polymorphonuclear neutrophil (PMN) elastase can become a procoagulant. Because intravascular coagulation may accompany severe burn injury, this study examined burn wound exudates for PMN elastase and its physiologic inhibitor, plasma alpha 1-protease inhibitor (alpha 1-PI), as a step in evaluating their contributions to coagulopathy in patients with burns. Each of the nine exudates examined were inhibitory for PMN elastase. Chromatographic characterization of the inhibitor indicated that it was alpha 1-PI; its elution volume for four exudates was identical to that of pure alpha 1-PI. Confirmation of the inhibitor's identity was achieved by reaction of anti-alpha 1-PI antibody with each exudate and with inhibitory chromatographic fractions of exudates with the most inhibitory activity. Inhibitor potency, determined from dose-response curves against a standard PMN elastase activity, varied twentyfold among exudates. Only one exudate had catalytic activity with the PMN elastase substrate. Although this enzyme had elastase-like properties, it appeared to differ from PMN elastase. The presence of alpha 1-PI in the wound exudate suggests that this inhibitor may act to diminish fibrin formation from the level that might otherwise have been seen if excess elastase were free to degrade antithrombin III.

  11. 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.

  12. Peptidyl cyclopropenones: Reversible inhibitors, irreversible inhibitors, or substrates of cysteine proteases?

    PubMed Central

    Cohen, Meital; Bretler, Uriel; Albeck, Amnon

    2013-01-01

    Peptidyl cyclopropenones were previously introduced as selective cysteine protease reversible inhibitors. In the present study we synthesized one such peptidyl cyclopropenone and investigated its interaction with papain, a prototype cysteine protease. A set of kinetics, biochemical, HPLC, MS, and 13C-NMR experiments revealed that the peptidyl cyclopropenone was an irreversible inhibitor of the enzyme, alkylating the catalytic cysteine. In parallel, this cyclopropenone also behaved as an alternative substrate of the enzyme, providing a product that was tentatively suggested to be either a spiroepoxy cyclopropanone or a gamma-lactone. Thus, a single family of compounds exhibits an unusual variety of activities, being reversible inhibitors, irreversible inhibitors and alternative substrates towards enzymes of the same family. PMID:23553793

  13. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

    PubMed

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease. PMID

  14. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.

    PubMed Central

    Kaplan, A H; Michael, S F; Wehbie, R S; Knigge, M F; Paul, D A; Everitt, L; Kempf, D J; Norbeck, D W; Erickson, J W; Swanstrom, R

    1994-01-01

    Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities. Images PMID:8202533

  15. [Prospects for the design of new therapeutically significant protease inhibitors based on knottins and sunflower seed trypsin inhibitor (SFTI 1)].

    PubMed

    Kuznetsova, S S; Kolesanova, E F; Talanova, A V; Veselovsky, A V

    2016-05-01

    Plant seed knottins, mainly from the Cucurbitacea family, and sunflower seed trypsin inhibitor (SFTI 1) are the most low-molecular canonical peptide inhibitors of serine proteases. High efficiency of inhibition of various serine proteases, structure rigidity together with the possibility of limited variations of amino acid sequences, high chemical stability, lack of toxic properties, opportunity of production by either chemical synthesis or use of heterologous expression systems make these inhibitors attractive templates for design of new compounds for regulation of therapeutically significant serine protease activities. Hence the design of such compounds represents a prospective research field. The review considers structural characteristics of these inhibitors, their properties, methods of preparation and design of new analogs. Examples of successful employment of natural serine protease inhibitors belonging to knottin family and SFTI 1 as templates for the design of highly specific inhibitors of certain proteases are given. PMID:27562989

  16. The triple threat of HIV-1 protease inhibitors.

    PubMed

    Potempa, Marc; Lee, Sook-Kyung; Wolfenden, Richard; Swanstrom, Ronald

    2015-01-01

    Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy. PMID:25778681

  17. Structural and thermodynamic basis of resistance to HIV-1 protease inhibition: implications for inhibitor design.

    PubMed

    Velazquez-Campoy, Adrian; Muzammil, Salman; Ohtaka, Hiroyasu; Schön, Arne; Vega, Sonia; Freire, Ernesto

    2003-12-01

    One of the most serious side effects associated with the therapy of HIV-1 infection is the appearance of viral strains that exhibit resistance to protease inhibitors. At the molecular level, resistance to protease inhibition predominantly takes the form of mutations within the protease molecule that preferentially lower the affinity of protease inhibitors with respect to protease substrates, while still maintaining a viable catalytic activity. Mutations associated with drug resistance occur within the active site cavity as well as distal sites. Active site mutations affect directly inhibitor/protease interactions while non-active site mutations affect inhibitor binding through long range cooperative perturbations. The effects of mutations associated with drug resistance are compounded by the presence of naturally occurring polymorphisms, especially those observed in non-B subtypes of HIV-1. The binding thermodynamics of all clinical inhibitors against the wild type protease, drug resistant mutations and non-B subtype HIV-1 proteases has been determined by high sensitivity isothermal titration calorimetry. In conjunction with structural information, these data have provided a precise characterization of the binding mechanism of different inhibitors and their response to mutations. Inhibitors that exhibit extremely high affinity and low susceptibility to the effects of mutations share common features and binding determinants even if they belong to different chemical scaffolds. These binding determinants define a set of rules and constraints for the design of better HIV-1 protease inhibitors.

  18. Fullerene-based inhibitors of HIV-1 protease.

    PubMed

    Strom, T Amanda; Durdagi, Serdar; Ersoz, Suha Salih; Salmas, Ramin Ekhteiari; Supuran, Claudiu T; Barron, Andrew R

    2015-12-01

    A series of Fmoc-Phe(4-aza-C60)-OH of fullerene amino acid derived peptides have been prepared by solid phase peptide synthesis, in which the terminal amino acid, Phe(4-aza-C60)-OH, is derived from the dipolar addition to C60 of the Fmoc-Nα-protected azido amino acids derived from phenylalanine: Fmoc-Phe(4-aza-C60)-Lys3-OH (1), Fmoc-Phe(4-aza-C60)-Pro-Hyp-Lys-OH (2), and Fmoc-Phe(4-aza-C60)-Hyp-Hyp-Lys-OH (3). The inhibition constant of our fullerene aspartic protease PRIs utilized FRET-based assay to evaluate the enzyme kinetics of HIV-1 PR at various concentrations of inhibitors. Simulation of the docking of the peptide Fmoc-Phe-Pro-Hyp-Lys-OH overestimated the inhibition, while the amino acid PRIs were well estimated. The experimental results show that C60-based amino acids are a good base structure in the design of protease inhibitors and that their inhibition can be improved upon by the addition of designer peptide sequences.

  19. 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

  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. Advances in the development of SUMO specific protease (SENP) inhibitors

    PubMed Central

    Kumar, Ashutosh; Zhang, Kam Y.J.

    2015-01-01

    Sumoylation is a reversible post-translational modification that involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to their substrate proteins. Prior to their conjugation, SUMO proteins need to be proteolytically processed from its precursor form to mature or active form. SUMO specific proteases (SENPs) are cysteine proteases that cleave the pro or inactive form of SUMO at C-terminus using its hydrolase activity to expose two glycine residues. SENPs also catalyze the de-conjugation of SUMO proteins using their isopeptidase activity, which is crucial for recycling of SUMO from substrate proteins. SENPs are important for maintaining the balance between sumoylated and unsumoylated proteins required for normal cellular physiology. Several studies reported the overexpression of SENPs in disease conditions and highlighted their role in the development of various diseases, especially cancer. In this review, we will address the current biological understanding of various SENP isoforms and their role in the pathogenesis of different cancers and other diseases. We will then discuss the advances in the development of protein-based, peptidyl and small molecule inhibitors of various SENP isoforms. Finally, we will summarize successful examples of computational screening that allowed the identification of SENP inhibitors with therapeutic potential. PMID:25893082

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

    PubMed Central

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

    2013-01-01

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

  3. 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. PMID:27437081

  4. 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.

  5. 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. PMID:27608950

  6. 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.

  7. Oral candidiasis in HIV+ patients under treatment with protease inhibitors.

    PubMed

    Witzel, Andréa Lusvarghi; Silveira, Fernando Ricardo Xavier da; Pires, Maria de Fátima Costa; Lotufo, Mônica Andrade

    2008-01-01

    The purpose of this work was to evaluate the influence of Protease Inhibitors (PI) on the occurrence of oral candidiasis in 111 HIV+ patients under PI therapy (Group A). The controls consisted of 56 patients that were not using PI drugs (Group B) and 26 patients that were not using any drugs for HIV therapy (Group C). The patient's cd4 cell counts were taken in account for the correlations. One hundred and ninety three patients were evaluated. The PI did not affect the prevalence of oral candidiasis (p = 0.158) or the frequency of C. albicans isolates (p = 0.133). Patients with lower cd4 cell counts showed a higher frequency of C. albicans isolates (p = 0.046) and a greater occurrence of oral candidiasis (p = 0.036).

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

    PubMed

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

    2015-11-01

    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.

  9. 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

  10. Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

    PubMed Central

    Bagdasarian, A; Wheeler, J; Stewart, G J; Ahmed, S S; Colman, R W

    1981-01-01

    Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin. Images PMID:6161137

  11. 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. PMID:26938078

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

    PubMed Central

    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. Trial Registration ClinicalTrials.gov NCT02545400 ClinicalTrials.gov NCT02545335 PMID:26938078

  13. SjAPI, the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

    PubMed Central

    Yang, Weishan; Cao, Zhijian; Zhuo, Renxi; Li, Wenxin; Wu, Yingliang

    2013-01-01

    Background 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. Principal Findings 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. Conclusions/Significance 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

  14. Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors.

    PubMed

    Liao, Jiahn-Haur; Ihara, Kentaro; Kuo, Chiao-I; Huang, Kai-Fa; Wakatsuki, Soichi; Wu, Shih-Hsiung; Chang, Chung-I

    2013-08-01

    The Lon proteases are a unique family of chambered proteases with a built-in AAA+ (ATPases associated with diverse cellular activities) module. Here, crystal structures of a unique member of the Lon family with no intrinsic ATPase activity in the proteolytically active form are reported both alone and in complexes with three covalent inhibitors: two peptidomimetics and one derived from a natural product. This work reveals the unique architectural features of an ATP-independent Lon that selectively degrades unfolded protein substrates. Importantly, these results provide mechanistic insights into the recognition of inhibitors and polypeptide substrates within the conserved proteolytic chamber, which may aid the development of specific Lon-protease inhibitors.

  15. 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

  16. Protease inhibitors from marine venomous animals and their counterparts in terrestrial venomous animals.

    PubMed

    Mourão, Caroline B F; Schwartz, Elisabeth F

    2013-06-14

    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.

  17. A family of serine protease inhibitors (serpins) in the cattle tick Rhipicephalus (Boophilus) microplus.

    PubMed

    Tirloni, Lucas; Seixas, Adriana; Mulenga, Albert; Vaz, Itabajara da Silva; Termignoni, Carlos

    2014-02-01

    Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential roles in many organisms. In arthropods these proteins are involved in innate immune system, morphogenesis and development. In mammals serpins regulate pathways that are essential to life such as blood coagulation, fibrinolysis, inflammation and complement activation, some of which are considered the host's first line of defense to hematophagous and/or blood dueling parasites. Thus, it is hypothesized that ticks use serpins to evade host defense, facilitating parasitism. This study describes eighteen full-length cDNA sequences encoding serpins identified in Rhipicephalus (Boophilus) microplus, here named RmS 1-18 (R. microplus serpin). Spatial and temporal transcriptional profiling demonstrated that R. microplus serpins are transcribed during feeding, suggesting their participation in tick physiology regulation. We speculate that the majority of R. microplus serpins are conserved in other ticks, as indicated by phylogeny analysis. Over half of the 18 RmSs are putatively functional in the extracellular environment, as indicated by putative signal peptides on 11 of 18 serpins. Comparative modeling and structural-based alignment revealed that R. microplus serpins in this study retain the consensus secondary of typical serpins. This descriptive study enlarges the knowledge on the molecular biology of R. microplus, an important tick species.

  18. New, potent P1/P2-morpholinone-based HIV-protease inhibitors.

    PubMed

    Kazmierski, Wieslaw M; Furfine, Eric; Spaltenstein, Andrew; Wright, Lois L

    2006-10-01

    We have developed efficient synthesis of morpholinone-based cyclic mimetics of the P1/P2 portion of the HIV-1 protease inhibitor Amprenavir. This effort led to discovery of allyl- and spiro-cyclopropyl-P2-substituted inhibitors 17 and 31, both 500 times more potent than the parent inhibitor 1. These results support morpholinones as novel mimetics of the P1/P2 portion of Amprenavir and potentially of other HIV-protease inhibitors, and thus provide a novel medicinal chemistry template for optimization toward more potent and drug-like inhibitors. PMID:16904316

  19. Effects of cysteine protease inhibitors on oviposition rate of the western flower thrips, Frankliniella occidentalis.

    PubMed

    Annadana, S; Peters, J; Gruden, K; Schipper, A; Outchkourov, N S; Beekwilder, M J.; Udayakumar, M; Jongsma, M A.

    2002-07-01

    Proteolytic activity in whole insect extracts of the western flower thrips, Frankliniella occidentalis, was found to belong predominantly to the class of cysteine proteases. The pH optimum of the general proteolytic activity was determined to be 3.5, which is low when compared to other insects using cysteine proteases for protein digestion. The proteinaceous cysteine protease inhibitors chicken cystatin, potato cystatin and sea anemone equistatin inhibited in vitro more than 90% of the protease activity. To test in vivo the biological effect of such inhibitors on the oviposition rate of western flower thrips, recombinant potato cystatin and equistatin were fed to adult females. A gradual reduction in oviposition rate to about 45% of control was observed when reared on these PIs for a period of 5 days, with no increase in mortality. These results are discussed in the light of the application of protease inhibitors in transgenic plants to control this insect pest.

  20. Phenylalanine and Phenylglycine Analogues as Arginine Mimetics in Dengue Protease Inhibitors.

    PubMed

    Weigel, Lena F; Nitsche, Christoph; Graf, Dominik; Bartenschlager, Ralf; Klein, Christian D

    2015-10-01

    Dengue virus is an increasingly global pathogen. One of the promising targets for antiviral drug discovery against dengue and related flaviviruses such as West Nile virus is the viral serine protease NS2B-NS3. We here report the synthesis and in vitro characterization of potent peptidic inhibitors of dengue virus protease that incorporate phenylalanine and phenylglycine derivatives as arginine-mimicking groups with modulated basicity. The most promising compounds were (4-amidino)-L-phenylalanine-containing inhibitors, which reached nanomolar affinities against dengue virus protease. The type and position of the substituents on the phenylglycine and phenylalanine side chains has a significant effect on the inhibitory activity against dengue virus protease and selectivity against other proteases. In addition, the non-natural, basic amino acids described here may have relevance for the development of other peptidic and peptidomimetic drugs such as inhibitors of the blood clotting cascade.

  1. 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

  2. Cyanobacterial Protease Inhibitor Microviridin J Causes a Lethal Molting Disruption in Daphnia pulicaria

    PubMed Central

    Rohrlack, Thomas; Christoffersen, Kirsten; Kaebernick, Melanie; Neilan, Brett A.

    2004-01-01

    Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton. PMID:15294849

  3. Cyanobacterial protease inhibitor microviridin J causes a lethal molting disruption in Daphnia pulicaria.

    PubMed

    Rohrlack, Thomas; Christoffersen, Kirsten; Kaebernick, Melanie; Neilan, Brett A

    2004-08-01

    Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.

  4. Nucleoside reverse transcriptase inhibitors prevent HIV protease inhibitor-induced atherosclerosis by ubiquitination and degradation of protein kinase C.

    PubMed

    Bradshaw, Emily L; Li, Xiang-An; Guerin, Theresa; Everson, William V; Wilson, Melinda E; Bruce-Keller, Annadora J; Greenberg, Richard N; Guo, Ling; Ross, Stuart A; Smart, Eric J

    2006-12-01

    HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Calpha (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.

  5. 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

  6. 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)

  7. Genetic Evidence Supporting the Association of Protease and Protease Inhibitor Genes with Inflammatory Bowel Disease: A Systematic Review

    PubMed Central

    Bekkering, Geertruida E.; Nüesch, Eveline; Mendes, Camila T.; Schmied, Stefanie; Wyder, Stefan; Kellen, Eliane; Villiger, Peter M.; Rutgeerts, Paul; Vermeire, Séverine; Lottaz, Daniel

    2011-01-01

    As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family. PMID:21931648

  8. 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. PMID:26800491

  9. Redistribution of human immunodeficiency virus type 1 variants resistant to protease inhibitors after a protease inhibitor-sparing regimen.

    PubMed

    Gianotti, Nicola; Seminari, Elena; Lazzarin, Adriano; Boeri, Enzo; Clementi, Massimo; Danise, Anna; Salpietro, Stefania; Fusetti, Giuliana; Castagna, Antonella

    2005-06-01

    The redistribution of mutations related to protease inhibitor (PI) resistance after a PI-sparing regimen in human immunodeficiency virus (HIV)-infected, highly PI-experienced patients was prospectively assessed. Twenty-five patients failing a PI-including regimen were given PI-sparing antiretroviral therapy, and then followed for 24 weeks after PI resumption. Genotyping was performed by direct sequencing before and during the PI-sparing regimen. The median (interquartile range, IQR) baseline CD4+ T-lymphocyte count was 198 (120-255) cells/microl, and the median HIV-RNA level was 82,000 (41,000-300,000) copies/ml. Patients had experienced a median of 4.5 (4-5.25) PIs. The median number of PI mutations was eight (6-9). The PI-sparing regimen consisted of a median of three (3-4) drugs and lasted for a median of 53 (24-67) weeks. At the end of the study, the median number of PI mutations was 6.5 (6-9). The median change in the number of PI mutations was -1 (IQR from -1 to 0). A reduction from baseline was observed in 13 cases (52%); nine (36%) showed no change and three (12%) showed an increased number of PI substitutions. In highly PI-experienced patients, a PI-sparing regimen may lead to a reduction, no change, or increase in the number of PI mutations. The reduction is negligible in most cases.

  10. Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Coovadia, Ashraf; Kuhn, Louise; Abrams, Elaine J.; Strehlau, Renate; Morris, Lynn

    2015-01-01

    Abstract Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection. PMID:25919760

  11. Fluorometric CCHFV OTU protease assay with potent inhibitors.

    PubMed

    Kocabas, Fatih; Aslan, Galip S

    2015-10-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay.

  12. Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

    PubMed

    Edwards, J V; Bopp, A F; Batiste, S; Ullah, A J; Cohen, I K; Diegelmann, R F; Montante, S J

    1999-01-01

    A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on

  13. 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.

  14. 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.

  15. Impact of protease inhibitors on intracellular concentration of tenofovir-diphosphate among HIV-1 infected patients

    PubMed Central

    Lahiri, Cecile D.; Tao, Sijia; Jiang, Yong; Sheth, Anandi N.; Acosta, Edward P.; Marconi, Vincent C.; Armstrong, Wendy S.; Schinazi, Raymond F.; Vunnava, Aswani; Sanford, Sara; Ofotokun, Ighovwerha

    2015-01-01

    Intracellular nucleoside reverse transcriptase inhibitor (NRTI) concentrations are associated with plasma HIV-1 response. Coadministration of protease inhibitors with NRTIs can affect intra-cellular concentrations due to protease inhibitor inhibition of efflux transporters. Tenofovir-diphosphate (TFV-DP) concentrations within peripheral blood mononuclear cells were compared among individuals receiving either atazanavir or darunavir-based regimens. There was a trend towards higher TFV-DP concentrations among women and among participants receiving atazanavir. TFV-DP intracellular concentrations were positively associated with undetectable plasma HIV-1 RNA. PMID:25870991

  16. 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

  17. 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.

  18. 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

  19. 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.

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

    PubMed

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

    2015-12-01

    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

  1. 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

  2. Cure of Hookworm Infection with a Cysteine Protease Inhibitor

    PubMed Central

    Vermeire, Jon J.; Lantz, Lorine D.; Caffrey, Conor R.

    2012-01-01

    Background Hookworm disease is a major global health problem and principal among a number of soil-transmitted helminthiases (STHs) for the chronic disability inflicted that impacts both personal and societal productivity. Mass drug administration most often employs single-dose therapy with just two drugs of the same chemical class to which resistance is a growing concern. New chemical entities with the appropriate single-dose efficacy are needed. Methods and Findings Using various life-cycle stages of the hookworm Ancylostoma ceylanicum in vitro and a hamster model of infection, we report the potent, dose-dependent cidal activities of the peptidyl cysteine protease inhibitors (CPIs) K11002 (4-mopholino-carbonyl-phenylalanyl-homophenylalanyl- vinyl sulfone phenyl) and K11777 (N-methylpiperazine-phenylalanyl-homophenylalanyl-vinylsulfone phenyl). The latter is in late pre-clinical testing for submission as an Investigational New Drug (IND) with the US Federal Drug Administration as an anti-chagasic. In vitro, K11002 killed hookworm eggs but was without activity against first-stage larvae. The reverse was true for K11777 with a larvicidal potency equal to that of the current anti-hookworm drug, albendazole (ABZ). Both CPIs produced morbidity in ex vivo adult hookworms with the activity of K11777 again being at least the equivalent of ABZ. Combinations of either CPI with ABZ enhanced morbidity compared to single compounds. Strikingly, oral treatment of infected hamsters with 100 mg/kg K11777 b.i.d. (i.e., a total daily dose of 200 mg/kg) for one day cured infection: a single 100 mg/kg treatment removed >90% of worms. Treatment also reversed the otherwise fatal decrease in blood hemoglobin levels and body weights of hosts. Consistent with its mechanism of action, K11777 decreased by >95% the resident CP activity in parasites harvested from hamsters 8 h post-treatment with a single 100 mg/kg oral dose. Conclusion A new, oral single-dose anthelmintic that is active in an

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

    SciTech Connect

    Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

    2006-01-01

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

  4. 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.

  5. Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18

    PubMed Central

    Guo, Peng-Chao; Dong, Zhaoming; Zhao, Ping; Zhang, Yan; He, Huawei; Tan, Xiang; Zhang, Weiwei; Xia, Qingyou

    2015-01-01

    Serpins generally serve as inhibitors that utilize a mobile reactive center loop (RCL) as bait to trap protease targets. Here, we present the crystal structure of serpin18 from Bombyx mori at 1.65 Å resolution, which has a very short and stable RCL. Activity analysis showed that the inhibitory target of serpin18 is a cysteine protease rather than a serine protease. Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments. This activity differs from previously reported modes of inhibition for serpins. Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18. Furthermore, one physiological target of serpin18, fibroinase, was identified, which enables us to better define the potential role for serpin18 in regulating fibroinase activity during B. mori development. PMID:26148664

  6. 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.

  7. Model system for high-throughput screening of novel human immunodeficiency virus protease inhibitors in Escherichia coli.

    PubMed

    Cheng, Ting-Jen; Brik, Ashraf; Wong, Chi-Huey; Kan, Chen-Chen

    2004-07-01

    Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS. To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system. This E. coli-based system involves coexpression of an engineered beta-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain. Autoprocessing of the HIV protease precursor releases the mature HIV protease. Subsequently, the HIV protease cleaves beta-galactosidase, resulting in a loss of the beta-galactosidase activity, which can be detected in high-throughput screens. Using Food and Drug Administration-approved HIV protease inhibitors, this E. coli-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity. The usefulness of the E. coli-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores. A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo E. coli-based system. This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains. This simple yet efficient E. coli system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases.

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

    NASA Astrophysics Data System (ADS)

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

    2000-10-01

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

  9. Study of protein complexes via homology modeling, applied to cysteine proteases and their protein inhibitors.

    PubMed

    Tastan Bishop, Ozlem; Kroon, Matthys

    2011-12-01

    This paper develops and evaluates large-scale calculation of 3D structures of protein complexes by homology modeling as a promising new approach for protein docking. The complexes investigated were papain-like cysteine proteases and their protein inhibitors, which play numerous roles in human and parasitic metabolisms. The structural modeling was performed in two parts. For the first part (evaluation set), nine crystal structure complexes were selected, 1325 homology models of known complexes were rebuilt by various templates including hybrids, allowing an analysis of the factors influencing the accuracy of the models. The important considerations for modeling the interface were protease coverage and inhibitor sequence identity. In the second part (study set), the findings of the evaluation set were used to select appropriate templates to model novel cysteine protease-inhibitor complexes from human and malaria parasites Plasmodium falciparum and Plasmodium vivax. The energy scores, considering the evaluation set, indicate that the models are of high accuracy. PMID:21365221

  10. Synergistic binding of inhibitors to the protease from HIV type 1.

    PubMed Central

    Asante-Appiah, E; Chan, W W

    1996-01-01

    Inhibition of the protease in HIV is a potentially useful approach for the treatment of AIDS. In the course of evaluating inhibitors of the HIV-1 protease, we observed a strong synergism between certain inhibitors that might be expected to bind to different sites in this enzyme. The binding affinity of carbobenzyloxyisoleucinylphenylalaninol, for example, is increased 125-fold in the presence of carbobenzyloxyglutaminylisoamylamide. These synergistic effects between inhibitors have specific structural requirements that correlate well with the known substrate preference of the enzyme. The modular basis for this phenomenon remains to be elucidated but it could involve substrate-induced conformational change as part of the reaction mechanism. Similar effects have been reported previously for several zinc proteases. Thus this work extends the observation to a different class of enzymes and suggests that the phenomenon might be widespread. PMID:8670094

  11. Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

    PubMed

    Papayannopoulos, I A; Biemann, K

    1992-02-01

    The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

  12. Effect of protease inhibitors on pulmonary bioavailability of therapeutic proteins and peptides in the rat.

    PubMed

    Amancha, Kiran Prakash; Hussain, Alamdar

    2015-02-20

    The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

  13. NS3 protease inhibitors for treatment of chronic hepatitis C: Efficacy and safety

    PubMed Central

    Bakulin, Igor; Pasechnikov, Victor; Varlamicheva, Anna; Sannikova, Irina

    2014-01-01

    A new treatment paradigm for hepatitis C is that the treatment must include an existing direct-acting antiviral agent, namely, a protease inhibitor (PI) combined with PEGylated interferon-α and ribavirin. The currently marketed PIs and PIs in clinical trials have different mechanisms of action. The development of new PIs aims for an improved safety profile and higher effectiveness. This article reviews NS3/4A protease inhibitors, focusing on major criteria such as their effectiveness and safety. Specific attention is paid to dosing regimens and adverse event profiles of PIs administered in clinical settings. PMID:24868326

  14. 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. PMID:18952157

  15. Probing Multidrug-Resistance and Protein-Ligand Interactions with Oxatricyclic Designed Ligands in HIV-1 Protease Inhibitors

    SciTech Connect

    Ghosh, Arun K.; Xu, Chun-Xiao; Rao, Kalapala V.; Baldridge, Abigail; Agniswamy, Johnson; Wang, Yuan-Fang; Weber, Irene T.; Aoki, Manabu; Miguel, Salcedo Pedro; Amano, Masayuki; Mitsuya, Hiroaki

    2010-10-29

    We report the design, synthesis, biological evaluation, and X-ray crystallographic analysis of a new class of HIV-1 protease inhibitors. Compound 4 proved to be an extremely potent inhibitor toward various multidrug-resistant HIV-1 variants, representing a near 10-fold improvement over darunavir (DRV). Compound 4 also blocked protease dimerization with at least 10-fold greater potency than DRV.

  16. 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.

  17. 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

  18. 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. PMID:20805393

  19. Salmon blood plasma: effective inhibitor of protease-laden Pacific whiting surimi and salmon mince.

    PubMed

    Fowler, Matthew R; Park, Jae W

    2015-06-01

    The effect of salmon plasma (SP) from Chinook salmon on proteolytic inhibition was investigated. SP was found to inhibit both cysteine and serine proteases as well as protease extracted from Pacific whiting muscle. SP was found to contain a 55kDa cysteine protease inhibitor through SDS-PAGE inhibitor staining. Freeze dried salmon plasma (FSP) and salmon plasma concentrated by ultrafiltration (CSP) were tested for their ability to inhibit autolysis in Pacific whiting surimi and salmon mince at concentrations of 0.25%, 0.5%, 1%, and 2%. Pacific whiting surimi autolysis was inhibited by an average of 89% regardless of concentration while inhibition of salmon mince autolysis increased with concentration (p<0.05). CSP performed slightly better than FSP at inhibiting salmon mince autolysis (p<0.05). Serine protease inhibition decreased when SP heated above 40°C but was stable across a broad NaCl and pH range. Cysteine protease inhibitors exhibited good temperature, NaCl, and pH stability. PMID:25624255

  20. 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.

  1. 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.

  2. Inhibition of Norovirus 3CL Protease by Bisulfite Adducts of Transition State Inhibitors

    PubMed Central

    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 U.S. 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 ED50 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. PMID:23218713

  3. 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.

  4. 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. PMID:18001685

  5. 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.

  6. Docking analysis of gallic acid derivatives as HIV-1 protease inhibitors.

    PubMed

    Singh, Anjali; Pal, Tapan Kumar

    2015-01-01

    HIV-1 Protease (HIV-1 PR) enzymes are essential for accurate assembly and maturation of infectious HIV retroviruses. The significant role of HIV-1 protease in viral replication has made it a potential drug target. In the recent past, phytochemical Gallic Acid (GA) derivatives have been screened for protease inhibitor activity. The present work aims to design and evaluate potential GA-based HIV-1 PR phytoinhibitors by docking approach. The ligands were prepared by ChemDraw and docking was performed in HEX software. In this present study, one of the GA analogues (GA4) emerged as a potent drug candidate for HIV-1 PR inhibition, and docking results showed it to be comparable with anti-HIV drugs, darunavir and amprenavir. The GA4 derivative provided a lead for designing more effective HIV-1 PR inhibitors.

  7. 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

  8. 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…

  9. 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. PMID:26071808

  10. 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.

  11. 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.

  12. 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. PMID:27329566

  13. 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. PMID:25524889

  14. 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.

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

    PubMed

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

    2015-12-01

    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.

  16. 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

  17. 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. PMID:26645142

  18. 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.

  19. 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.

  20. Peripheral administration of a serine protease inhibitor blocks kindling.

    PubMed

    Hoffman, K B; Hwee, V; Larson, J; Lynch, G

    2000-04-01

    An inhibitor of tissue plasminogen activator (tPA) was tested for its effects on the rapid kindling induced by a series of afterdischarges (ADs) triggered in hippocampus over a 3-h period. Rats injected with vehicle prior to the session had prolonged ADs in tests carried out 10 days later. This was not the case for animals treated with the inhibitor. These findings support the hypothesis that activity-driven proteolysis contributes importantly to the production of long-lasting physiological changes.

  1. Synergistic Inhibitor Binding to the Papain-Like Protease of Human SARS Coronavirus – Mechanistic and Inhibitor Design Implications

    PubMed Central

    Lee, Hyun; Cao, Shuyi; Hevener, Kirk E.; Truong, Lena; Gatuz, Joseph L.; Patel, Kavankumar; Ghosh, Arun K.; Johnson, Michael E.

    2014-01-01

    We have previously developed two potent chemical classes that inhibit the essential papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, we applied a novel approach to identify small fragments that act synergistically with these inhibitors. A fragment library was screened in combination with four previously developed lead inhibitors by fluorescence-based enzymatic assays. Several fragment compounds synergistically enhanced the inhibitory activity of the lead inhibitors by approximately an order of magnitude. Surface plasmon resonance (SPR) measurements showed that three fragments bind specifically to the PLpro enzyme. Mode of inhibition, computational solvent mapping, and molecular docking studies suggest that these fragments bind adjacent to the binding site of the lead inhibitors and further stabilize the inhibitor-bound state. We propose potential next generation compounds based upon a computational, fragment-merging approach. This approach provides an alternative strategy for lead optimization in cases where direct co-crystallization is difficult. PMID:23788528

  2. Blockage of skin invasion by schistosome cercariae by serine protease inhibitors.

    PubMed

    Lim, K C; Sun, E; Bahgat, M; Bucks, D; Guy, R; Hinz, R S; Cullander, C; McKerrow, J H

    1999-03-01

    Invasion of skin by schistosome cercariae is facilitated by a serine protease secreted from the acetabular cells of cercariae in response to skin lipid. Specific inhibitors of the protease, when applied to human skin in formulations designed to retain the inhibitor on and in the upper stratum corneum layers, block cercarial invasion of human skin. Both peptide-based, irreversible inhibitors and non-peptide, reversible inhibitors block cercarial invasion when applied in a propylene glycol:isopropyl alcohol (3:1) formulation in vitro. Arrest of cercarial invasion could be achieved even after immersion of treated skin in water for 2 hr. Peptide-based irreversible inhibitors in the presence of three different Topicare Delivery Compounds optimized arrest of cercarial invasion. The three Topicare Delivery Compounds applied alone prevented 80-100% of cercarial invasion. With inclusion of the inhibitor, there was 97-100% inhibition in vitro. The optimal formulation with inhibitor was then applied to the tails of BALB/c mice, and the mice were exposed to 120 cercariae by tail immersion. With the carrier lotion alone, there was a 50% reduction in worm burden and a 70% reduction in egg burden. When inhibitor was included, an 80% reduction in worm burden and a 92% reduction in egg burden was observed.

  3. Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease.

    PubMed

    Cai, Yufeng; Myint, Wazo; Paulsen, Janet L; Schiffer, Celia A; Ishima, Rieko; Kurt Yilmaz, Nese

    2014-08-12

    Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput. 2012, 8, 3452-3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms. PMID:25136270

  4. Bis-Tetrahydrofuran: a Privileged Ligand for Darunavir and a New Generation of HIV Protease Inhibitors That Combat Drug Resistance

    SciTech Connect

    Ghosh, Arun K.; Sridhar, Perali Ramu; Kumaragurubaran, Nagaswamy; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki

    2008-06-06

    Two inhibitors that incorporate bis-THF as an effective high-affinity P{sub 2} ligand for the HIV-1 protease substrate binding site maintain impressive potency against mutant strains resistant to currently approved protease inhibitors. Crystallographic structures of protein-ligand complexes help to explain the superior antiviral property of these inhibitors and their potency against a wide spectrum of HIV-1 strains.

  5. Purification and properties of two protease inhibitors from rat skin inhibiting papain and other SH-proteases.

    PubMed

    Järvinen, M

    1976-01-01

    Two papain inhibitors, I1 and I2, from rat skin extract were purified by affinity chromatography on KSCN-modified papain-agarose gel and by gel filtration on Sephadex G-100. I1 had a molecular weight of 74 000, a pI of 4.6, and it contained 4% of carbohydrates. I1 inhibited papain, ficin, bromelain, rat skin benzoylarginine-2-naphthylamide hydrolase, and to a minor extent, rat skin cathepsin C and bovine trypsin. Bovine chymotrypsin or rat skin cathepsin D were not inhibited and benzoylarginine-2-naphthylamide hydrolase was inhibited only at alkaline pH. An inhibitor corresponding to I1 was present in various rat tissues and also in serum. A similar inhibitor was present in the skin of cat, rabbit, guinea pig, and man. I2 had a molecular weight of 13 400, a pI of 4.9 and it contained no carbohydrates. I2 inhibited all thiol proteases tested, but not trypsin, chymotrypsin, or rat skin cathepsin D. I2 formed an equimolar complex with papain and benzoylarginine-2-naphthylamide hydrolase. I2 was present in rat skin, muscle, lung, and small intestine, but not in kidney, liver, or serum. A similar inhibitor was found in skin extracts of cat, rabbit, guinea pig, and man.

  6. A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization

    PubMed Central

    Wolf, Eliane V.; Zeißler, Annett; Vosyka, Oliver; Zeiler, Evelyn; Sieber, Stephan; Verhelst, Steven H. L.

    2013-01-01

    Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases. PMID:23991088

  7. 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.

  8. 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.

  9. Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors

    SciTech Connect

    Wong, R.L.; Gutowski, J.K.; Katz, M.; Goldfarb, R.H.; Cohen, S.

    1987-01-01

    Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-..cap alpha..-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These finding are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor the authors have called ADR may be a protease itself.

  10. Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

    PubMed

    Prasad, J V; Boyer, F E; Domagala, J M; Ellsworth, E L; Gajda, C; Hamilton, H W; Hagen, S E; Markoski, L J; Steinbaugh, B A; Tait, B D; Humblet, C; Lunney, E A; Pavlovsky, A; Rubin, J R; Ferguson, D; Graham, N; Holler, T; Hupe, D; Nouhan, C; Tummino, P J; Urumov, A; Zeikus, E; Zeikus, G; Gracheck, S J; Erickson, J W

    1999-12-01

    With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

  11. 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

  12. 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

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

    PubMed

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

    2015-01-01

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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  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. 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

  3. Protease inhibitors and indoleamines selectively inhibit cholinesterases in the histopathologic structures of Alzheimer disease.

    PubMed Central

    Wright, C I; Guela, C; Mesulam, M M

    1993-01-01

    Neurofibrillary tangles and amyloid plaques express acetylcholinesterase and butyrylcholinesterase activity in Alzheimer disease. We previously reported that traditional acetylcholinesterase inhibitors such as BW284C51, tacrine, and physostigmine were more potent inhibitors of the acetylcholinesterase in normal axons and cell bodies than of the acetylcholinesterase in plaques and tangles. We now report that the reverse pattern is seen with indoleamines (such as serotonin and its precursor 5-hydroxytryptophan), carboxypeptidase inhibitor, and the nonspecific protease inhibitor bacitracin. These substances are more potent inhibitors of the cholinesterases in plaques and tangles than of those in normal axons and cell bodies. These results show that the enzymatic properties of plaque and tangle-associated cholinesterases diverge from those of normal axons and cell bodies. The selective susceptibility to bacitracin and carboxypeptidase inhibitor indicates that the catalytic sites of plaque and tangle-bound cholinesterases are more closely associated with peptidase or protease-like properties than the catalytic sites of cholinesterases in normal axons and cell bodies. This shift in enzymatic affinity may lead to the abnormal protein processing that is thought to play a major role in the pathogenesis of Alzheimer disease. The availability of pharmacological and dietary means for altering brain indoleamines raises therapeutic possibilities for inhibiting the abnormal cholinesterase activity associated with Alzheimer disease. Images PMID:8421706

  4. Anthracene-based inhibitors of dengue virus NS2B-NS3 protease.

    PubMed

    Tomlinson, Suzanne M; Watowich, Stanley J

    2011-02-01

    Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA (CDC, 2010), Australia (Hanna et al., 2009), Taiwan (Kuan et al., 2010) and France (La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B-NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture (Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four "second series" analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ∼2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors.

  5. 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.

  6. Potential protease inhibitors based on a functionalized cyclic sulfamide scaffold.

    PubMed

    Zhong, Jiaying; Gan, Xiangdong; Alliston, Kevin R; Lai, Zhong; Yu, Hongyi; Groutas, Christopher S; Wong, Tzutshin; Groutas, William C

    2004-01-01

    Exploratory studies related to the design and synthesis of functionalized cyclic sulfamides (I) as potential inhibitors of proteolytic enzymes were carried out. The structural motif and three diversity sites embodied in the scaffold render it amenable to combinatorial parallel synthesis and the facile generation of lead discovery prospecting libraries. The scaffold was readily assembled starting with (DL) serine methyl ester, and a series of compounds was generated and screened against human leukocyte elastase. Modification of the P(1) recognition element, believed to be accommodated at the primary specificity site (S(1) subsite) of the enzyme, yielded compounds that inhibited the enzyme by an apparent hyperbolic partial mixed-type inhibition. PMID:15244417

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

    PubMed

    Vlasova, T I; Vanyushin, B F

    1998-06-01

    Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage. PMID:9635138

  12. 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.

  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. Synthesis and Biological Evaluation of Botulinum Neurotoxin A Protease Inhibitors

    PubMed Central

    Li, Bing; Pai, Ramdas; Cardinale, Steven C.; Butler, Michelle M.; Peet, Norton P.; Moir, Donald T.; Bavari, Sina; Bowlin, Terry L.

    2010-01-01

    NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogs have been synthesized and shown to inhibit BoNT/A LC in a FRET-based enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in these two series is compound 12 (IC50 = 2.5 µM, FRET assay), which is 4.4-fold more potent than the lead structure, and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase. Structure-activity relationship studies have revealed structural features important to potency and enzyme specificity. PMID:20155918

  15. 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

  16. Structural and Inhibitor Studies of Norovirus 3C-like Proteases

    PubMed Central

    Takahashi, Daisuke; Kim, Yunjeong; Lovell, Scott; Prakash, Om; Groutas, William C; Chang, Kyeong-Ok

    2013-01-01

    Noroviruses have a single-stranded, positive sense 7–8 kb RNA genome, which encodes a polyprotein precursor processed by a virus-encoded 3C-like cysteine protease (3CLpro) to generate mature non-structural proteins. Because processing of the polyprotein is essential for virus replication, norovirus 3CLpro has been targeted for the discovery of anti-norovirus small molecule therapeutics. Thus, we performed functional, structural and inhibition studies of norovirus 3CLpro with fluorescence resonance energy transfer (FRET) assay, X-ray crystallography, and NMR spectroscopy with a synthetic protease inhibitor. Three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murine norovirus-1 (MNV-1, genogroup V) were optimized for a FRET assay, and compared for the inhibitory activities of a synthetic protease inhibitor (GC376). The apo 3D structures of NV 3CLpro determined with X-ray crystallography and NMR spectroscopy were further analyzed. In addition, the binding mode of NV 3CLpro-GC376 was compared with X-ray crystallography and NMR spectroscopy. The results of this report provide insight into the interaction of NV 3CLpro with substrate/inhibitor for better understanding of the enzyme and antiviral drug development. PMID:24055466

  17. Distribution of the human intracellular serpin protease inhibitor 8 in human tissues.

    PubMed

    Strik, Merel C; Bladergroen, Bellinda A; Wouters, Dorine; Kisiel, Walter; Hooijberg, Jan Hendrik; Verlaan, Angelique R; Hordijk, Peter L; Schneider, Pascal; Hack, C Erik; Kummer, J Alain

    2002-11-01

    Ovalbumin-like serine protease inhibitors are mainly localized intracellularly and their in vivo functions are largely unknown. To elucidate their physiological role(s), we studied the expression of one of these inhibitors, protease inhibitor 8 (PI-8), in normal human tissues by immunohistochemistry using a PI-8-specific monoclonal antibody. PI-8 was strongly expressed in the nuclei of squamous epithelium of mouth, pharynx, esophagus, and epidermis, and by the epithelial layer of skin appendages, particularly by more differentiated epithelial cells. PI-8 was also expressed by monocytes and by neuroendocrine cells in the pituitary gland, pancreas, and digestive tract. Monocytes showed nuclear and cytoplasmic localization of PI-8, whereas neuroendocrine cells showed only cytoplasmic staining. In vitro nuclear localization of PI-8 was confirmed by confocal analysis using serpin-transfected HeLa cells. Furthermore, mutation of the P(1) residue did not affect the subcellular distribution pattern of PI-8, indicating that its nuclear localization is independent of the interaction with its target protease. We conclude that PI-8 has a unique distribution pattern in human tissues compared to the distribution patterns of other intracellular serpins. Additional studies must be performed to elucidate its physiological role.

  18. Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

    PubMed

    Kummer, J A; Micheau, O; Schneider, P; Bovenschen, N; Broekhuizen, R; Quadir, R; Strik, M C M; Hack, C E; Tschopp, J

    2007-08-01

    Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

  19. High-level expression and characterization of two serine protease inhibitors from Trichinella spiralis.

    PubMed

    Zhang, Zhaoxia; Mao, Yixian; Li, Da; Zhang, Yvhan; Li, Wei; Jia, Honglin; Zheng, Jun; Li, Li; Lu, Yixin

    2016-03-30

    Serine protease inhibitors (SPIs) play important roles in tissue homeostasis, cell survival, development, and host defense. So far, SPIs have been identified from various organisms, such as animals, plants, bacteria, poxviruses, and parasites. In this study, two SPIs (Tsp03044 and TspAd5) were identified from the genome of Trichinella spiralis and expressed in Escherichia coli. Sequence analysis revealed that these two SPIs contained essential structural motifs, which were well conserved within the tumor-infiltrating lymphocytes (TIL) and serpin superfamily. Based on protease inhibition assays, the recombinant Tsp03044 showed inhibitory effects on trypsin, α-chymotrypsin, and pepsin, while the recombinant TspAd5 could effectively inhibit the activities of α-chymotrypsin and pepsin. Both these inhibitors showed activity between 28 and 48 °C. The expression levels of the two SPIs were also determined at different developmental stages of the parasite with real-time PCR. Our results indicate that Tsp03044 and TspAd5 are functional serine protease inhibitors. PMID:26921036

  20. Protease inhibitor from insect silk-activities of derivatives expressed in vitro and in transgenic potato.

    PubMed

    Kodrík, Dalibor; Kludkiewicz, Barbara; Navrátil, Oldřich; Skoková Habuštová, Oxana; Horáčková, Vendulka; Svobodová, Zdeňka; Vinokurov, Konstantin S; Sehnal, František

    2013-09-01

    Several recombinant derivatives of serine protease inhibitor called silk protease inhibitor 2 (SPI2), which is a silk component in Galleria mellonella (Lepidoptera, Insecta), were prepared in the expression vector Pichia pastoris. Both the native and the recombinant protease inhibitors were highly active against subtilisin and proteinase K. The synthetic SPI2 gene with Ala codon in the P1 position was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was inserted into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens that was then used for genetic transformation of the potato variety Velox. The transgene expression was monitored with the aid of ELISA employing polyclonal antibody against natural SPI2. In vitro tests showed increased resistance to the late blight Phytophthora infestans in several transformed lines. No effect was seen on the growth, mortality, life span or reproduction of Spodoptera littoralis (Lepidoptera, Insecta) caterpillars, while feeding on transformed potato plants expressing the fusion protein, indicating that the transformed potatoes may be harmless to non-target organisms.

  1. 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

  2. Synthesis, antiviral activity, and bioavailability studies of gamma-lactam derived HIV protease inhibitors.

    PubMed

    Hungate, R W; Chen, J L; Starbuck, K E; Vacca, J P; McDaniel, S L; Levin, R B; Dorsey, B D; Guare, J P; Holloway, M K; Whitter, W

    1994-09-01

    Incorporation of a gamma-lactam in hydroxyethylene isosteres results in modest inhibitors of HIV-1 protease. Additional structural activity studies have produced significantly more potent inhibitors with the introduction of the trisubstituted cyclopentane (see compound 20) as the optimum substituent for the C-terminus. This new amino acid amide surrogate can be readily prepared in large scale from (R)-pulegone. Optimized compounds (36) and (60) are potent antiviral agents and are well absorbed (15-20%) in a dog model after oral administration. PMID:7712123

  3. Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

    PubMed

    Ferry, N; Jouanin, L; Ceci, L R; Mulligan, E A; Emami, K; Gatehouse, J A; Gatehouse, A M R

    2005-01-01

    Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.

  4. 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.

  5. Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver.

    PubMed

    Shah, Aaliya; Priyadarshini, Medha; Khan, Mohd Shahnawaz; Aatif, Mohammad; Amin, Fakhra; Bano, Bilqees

    2013-10-01

    Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.

  6. Triggering HIV polyprotein processing by light using rapid photodegradation of a tight-binding protease inhibitor.

    PubMed

    Schimer, Jiří; Pávová, Marcela; Anders, Maria; Pachl, Petr; Šácha, Pavel; Cígler, Petr; Weber, Jan; Majer, Pavel; Řezáčová, Pavlína; Kräusslich, Hans-Georg; Müller, Barbara; Konvalinka, Jan

    2015-01-01

    HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro. Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time. PMID:25751579

  7. De novo design of caseinolytic protein proteases inhibitors based on pharmacophore and 2D molecular fingerprints.

    PubMed

    Wu, Guanzhong; Zhang, Zhen; Chen, Hong; Lin, Kejiang

    2015-06-01

    Caseinolytic protein proteases (ClpP) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Inhibitors of ClpP can significantly attenuate the capability to produce virulence factors of the bacteria. In this work, we developed a workflow to expand the chemical space of potential ClpP inhibitors based on a set of β-lactones. In our workflow, an artificial pharmacophore model was generated based on HipHop and HYPOGEN method. A de novo compound library based on molecular fingerprints was constructed and virtually screened by the pharmacophore model. The results were further investigated by molecular docking study. The workflow successfully achieved potential ClpP inhibitors. It could be applied to design more novel potential ClpP inhibitors and provide theoretical basis for the further optimization of the hit compounds. PMID:25937012

  8. De novo design of caseinolytic protein proteases inhibitors based on pharmacophore and 2D molecular fingerprints.

    PubMed

    Wu, Guanzhong; Zhang, Zhen; Chen, Hong; Lin, Kejiang

    2015-06-01

    Caseinolytic protein proteases (ClpP) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Inhibitors of ClpP can significantly attenuate the capability to produce virulence factors of the bacteria. In this work, we developed a workflow to expand the chemical space of potential ClpP inhibitors based on a set of β-lactones. In our workflow, an artificial pharmacophore model was generated based on HipHop and HYPOGEN method. A de novo compound library based on molecular fingerprints was constructed and virtually screened by the pharmacophore model. The results were further investigated by molecular docking study. The workflow successfully achieved potential ClpP inhibitors. It could be applied to design more novel potential ClpP inhibitors and provide theoretical basis for the further optimization of the hit compounds.

  9. 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

  10. Discovery of novel P3-oxo inhibitor of hepatitis C virus NS3/4A serine protease.

    PubMed

    Duan, Maosheng; Kazmierski, Wieslaw; Crosby, Renae; Gartland, Margaret; Ji, Jinjing; Tallant, Matt; Wang, Amy; Hamatake, Robert; Wright, Lois; Wu, Min; Zhang, Yong-Kang; Ding, Charles Z; Li, Xianfeng; Liu, Yang; Zhang, Suoming; Zhou, Yasheen; Plattner, Jacob J; Baker, Stephen J

    2012-04-15

    A novel series of P3 oxo-modified macrocyclic hepatitis C virus NS3/4A serine protease inhibitor was designed, synthesized and biologically evaluated. The hydroxy-substituted inhibitor 10 demonstrated high potency in genotype 1a and 1b replicon and in the panel of HCV protease mutants. Interestingly, the t-butyl carbonate analog 9c, while not the most potent one in this series, exhibited a virtually flat potency profile in the panel of HCV protease mutants, thus providing opportunity for further optimization. PMID:22425454

  11. 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.

  12. 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. PMID:20513059

  13. 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.

  14. Inhibitors of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura.

    PubMed

    Tsai, H M; Li, A; Rock, G

    2001-01-01

    Thrombotic thrombocytopenic purpura (TTP), characterized by platelet thrombi in the arterioles and capillaries, is associated with antibodies that inhibit the activity of von Willebrand factor (vWF)-cleaving protease. Using a modified Bethesda method, we studied the inhibitor titers in patients who participated in the trial conducted by the Canadian Apheresis Group. Among the 41 patients investigated, the inhibitor titers at presentation were 1.4 +/- 1.7 U/mL (range -0.2-6.2 U/mL). Thirty-one patients (76%) had a titer > or = 0.2 U/mL, 8 patients (20%) had a titer > or = 2.0 U/mL but none had a titer > or = 10 U/mL. Among the 33 patients with an inhibitor titer < 2.0 U/mL (low titer group) and the 8 patients with an inhibitor titer > or = 2 U/mL (high titer group), 20 (61%) and 8 (100%) respectively had a platelet count < 25x10(9)/L (P = 0.04). Neurological abnormalities were among the presenting problems in 19 (58%) of the low titer and 6 (75%) of the high titer groups. Among the 23 patients who were randomized to plasma exchange, 5 patients had an inhibitor titer > or = 2 U/mL and none responded at the end of the first treatment cycle, while 8 of the 18 patients (44%) with a titer < 2 U/mL responded. This study shows that inhibitors of vWF-cleaving protease are of low titers in most cases of acquired TTP. A higher inhibitor titer is associated with a more advanced stage of the disease and may delay the response to plasma exchange.

  15. 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

  16. 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. PMID:27575432

  17. Pulsed EPR Characterization of HIV-1 Protease Conformational Sampling and Inhibitor-Induced Population Shifts

    PubMed Central

    Liu, Zhanglong; Casey, Thomas M.; Blackburn, Mandy E.; Huang, Xi; Pham, Linh; de Vera, Ian Mitchelle S.; Carter, Jeffrey D.; Kear-Scott, Jamie L.; Veloro, Angelo M.; Galiano, Luis; Fanucci, Gail E.

    2015-01-01

    The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed “curled/tucked”, “closed”, “semi-open” and “wide-open” conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function. PMID:26489725

  18. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    PubMed

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing. PMID:27386922

  19. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    PubMed

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing.

  20. Inactivation of C-1 inhibitor by proteases: demonstration by a monoclonal antibody of a neodeterminant on inactivated, non-complexed C-1 inhibitor.

    PubMed

    Nuijens, J H; Huijbregts, C C; van Mierlo, G M; Hack, C E

    1987-07-01

    Monoclonal antibodies were raised against kallikrein-C-1 inhibitor and factor XIIa-C-1 inhibitor complexes. One of the monoclonal antibodies (KII) appeared to react predominantly with C-1 inhibitor complexes in an ELISA. However, the apparent binding of KII to C-1 inhibitor complexes was probably due to the presence of proteolytically inactivated C-1 inhibitor in the complex mixture used for the coating:KII did not bind either kallikrein-C-1 inhibitor or factor XIIa-C-1 inhibitor complexes generated in plasma by dextran sulphate. SDS-PAGE analysis of C-1 inhibitor incubated with proteases revealed that KII-reactive C-1 inhibitor has a lower molecular weight than native C-1 inhibitor. We propose that the determinant that reacts with KII is exposed after cleavage of C-1 inhibitor in its reactive site. The monoclonal antibody KII will enable us to study the inactivation of C-1 inhibitor in human inflammatory disease.

  1. 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.

  2. The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin.

    PubMed

    Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin; Vogel, Lotte K; Kataoka, Hiroaki; Bugge, Thomas H

    2014-08-01

    The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.

  3. 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. PMID:21615062

  4. 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.

  5. 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

  6. Structure-based design of inhibitors of the aspartic protease endothiapepsin by exploiting dynamic combinatorial chemistry.

    PubMed

    Mondal, Milon; Radeva, Nedyalka; Köster, Helene; Park, Ahyoung; Potamitis, Constantinos; Zervou, Maria; Klebe, Gerhard; Hirsch, Anna K H

    2014-03-17

    Structure-based design (SBD) can be used for the design and/or optimization of new inhibitors for a biological target. Whereas de novo SBD is rarely used, most reports on SBD are dealing with the optimization of an initial hit. Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify bioactive ligands given that it enables the target to direct the synthesis of its strongest binder. We have designed a library of potential inhibitors (acylhydrazones) generated from five aldehydes and five hydrazides and used DCC to identify the best binder(s). After addition of the aspartic protease endothiapepsin, we characterized the protein-bound library member(s) by saturation-transfer difference NMR spectroscopy. Cocrystallization experiments validated the predicted binding mode of the two most potent inhibitors, thus demonstrating that the combination of de novo SBD and DCC constitutes an efficient starting point for hit identification and optimization.

  7. Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor.

    PubMed

    Yanuar, Arry; Suhartanto, Heru; Munim, 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.

  8. Baseline, Trend, and Normalization of Carcinoembryonic Antigen as Prognostic Factors in Epidermal Growth Factor Receptor-Mutant Nonsmall Cell Lung Cancer Patients Treated With First-Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

    PubMed Central

    Chen, Yu-Mu; Lai, Chien-Hao; Chang, Huang-Chih; Chao, Tung-Ying; Tseng, Chia-Cheng; Fang, Wen-Feng; Wang, Chin-Chou; Chung, Yu-Hsiu; Huang, Kuo-Tung; Chen, Hung-Cheng; Chang, Ya-Chun; Lin, Meng-Chih

    2015-01-01

    Abstract Among epidermal growth factor receptor (EGFR) mutation status unknown nonsmall cell lung cancer (NSCLC) patients, those with higher carcinoembryonic antigen (CEA) level are more likely to response to EGFR-tyrosine kinase inhibitors (TKIs) because they tend to have mutant epidermal growth factor receptor (EGFR). However, patients with higher CEA also have more tumor burden. With the above paradoxical evidence, it is prudent to understand the prognostic significance of baseline CEA in patients with EGFR-mutant NSCLC treated with first-line EGFR-TKIs. The clinical significance of the trend in CEA after treatment and the impact of CEA normalization during EGFR-TKI therapy are also unknown and potentially important. A total of 241 patients who received first-line EGFR-TKIs were included. As to baseline CEA, patients were divided into normal, low, and high baseline CEA by cut point determined by receiver operating characteristic curves. As to CEA responses, patients were divided into 3 groups accordingly to their amount of CEA change after taking TKIs. In group A, 1-month follow-up CEA level decreased more than 35% with nadir CEA normalization; in group B, 1-month follow-up CEA level decreased more than 35% without nadir CEA normalization; and in group C, 1-month follow-up CEA level decreased less than 35% or increased. Patients with higher baseline CEA levels had shorter progression-free survival (PFS) and overall survival (OS) (CEA > 32 vs 5–32 vs <5 ng/mL, PFS = 8.8 vs 11.3 vs 14.4 months, respectively, P < 0.001; OS = 17.8 vs 22.0 vs 27.9 months, respectively, P = 0.01). For trend and CEA normalization in groups A, B, and C, PFS was 14.3, 10.6, and 7.1 months, respectively (P < 0.001); OS was 29.7, 20.0, and 16.2 months, respectively (P < 0.001). Baseline, trend, and normalization of CEA levels are potential prognostic markers for patients with EGFR-mutant advanced NSCLC treated with first line EGFR-TKIs. PMID:26683939

  9. 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.

  10. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro.

    PubMed

    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.

  11. 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

  12. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro.

    PubMed

    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

  13. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Thiol containing compounds and amino acid hydroxamates as reversible synthetic inhibitors of Astacus protease.

    PubMed

    Wolz, R L; Zeggaf, C; Stöcker, W; Zwilling, R

    1990-09-01

    Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish. Effective inhibition was demonstrated for several simple thiol containing compounds and a series of amino acid hydroxamates. Both classes of inhibitors had ID50 values ranging from 10(-2) to 10(-4) M for inhibition of hydrolysis of succinyl-Ala-Ala-Ala-p-nitroanilide. Tyrosine hydroxamate was found to be the most effective inhibitor with an ID50 of 175 microM and the mode of inhibition by this compound was determined to be of the simple noncompetitive type. In contrast to the other inhibitors tested, cysteine was seen to partially inactivate the enzyme in a time-dependent manner. The kinetics of this process was studied in detail using progress curve analysis. It was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme. In the presence of the strong zinc scavenger EDTA, cysteine can, in effect, function as a catalyst in transferring the metal from the protein to the secondary chelator at a rate 10,000 times faster than the rate of unassisted zinc dissociation. The series of amino acid hydroxamates served as probes into the microenvironment of the active site. Possible binding modes of the inhibitors are discussed on the basis of the relationship between the chemical nature of the inhibitor side chains and the strength of inhibition.

  16. 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.

  17. Rezolsta® (Darunavir/Cobicistat): First Boosted Protease Inhibitor Co-formulated with Cobicistat.

    PubMed

    Curran, Adrián; Pérez-Valero, Ignacio; Moltó, José

    2015-01-01

    Rezolsta® (darunavir/cobicistat) is the first boosted protease inhibitor in a fixed-dose combination to be approved for the treatment of HIV infection. It contains darunavir, a protease inhibitor with a well-known safety and efficacy profile, and the new pharmacokinetic enhancer cobicistat. The convenience of this combination makes boosted darunavir easier to take, thus improving adherence. Exposure to darunavir is equivalent when it is administered with cobicistat or with ritonavir. Darunavir/cobicistat-based antiretroviral therapy has shown considerable efficacy and good tolerability in several clinical trials. Data from the single-arm, open-label, phase III GS-US-216-130 trial showed virological efficacy rates comparable to those from ARTEMIS and ODIN. Darunavir/cobicistat was well tolerated; most adverse events were mild and consisted of gastrointestinal disturbances. Cobicistat inhibits transporters of creatinine in kidney tubules, thus causing a minimal and reversible reduction in estimated glomerular filtration rate. Like ritonavir, cobicistat is a strong CYP3A4 inhibitor and, as such, shares most of its drug interactions. However, inhibition by cobicistat seems to be more specific than with ritonavir, and cobicistat has no inducer effect; therefore, differences in its drug interaction profile may be observed. PMID:26035169

  18. Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

    PubMed

    Kuhar, Kalika; Mittal, Anuradha; Kansal, Rekha; Gupta, Vijay Kumar

    2014-02-01

    Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to -14-fold with -84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100 degrees C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50 degrees C and 37 degrees C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

  19. Initial therapy with protease inhibitor-sparing regimens: evaluation of nevirapine and delavirdine.

    PubMed

    Conway, B

    2000-06-01

    We have compared the results (on-treatment analyses) of 2 randomized clinical trials of protease inhibitor-sparing regimens in drug-naive patients. In the INCAS (Italy, Netherlands, Canada, Australia) study, the mean decrease in plasma viral load over 52 weeks was 2.2 log(10) copies/mL in 40 patients who were receiving zidovudine/didanosine/nevirapine (18 [45%] had maximal suppression), with a mean increase in CD4 T cell counts of 139 cells/microL. In protocol 0021 Part II, the mean decrease in plasma viral load over 52 weeks was 2.1 log(10) copies/mL in 34 patients who were receiving zidovudine/lamivudine/delavirdine (20 [59%] had maximal suppression), with a mean increase in CD4 T cell counts of 88 cells/microL. The virologic and immunologic efficacy of the 2 triple-drug regimens are similar. Until results of long-term studies are available to establish whether a preferred approach to initial therapy exists, nonnucleoside reverse transcriptase inhibitors may be a valuable alternative to protease inhibitors in the initial therapy of antiretroviral-naive, moderately immunosuppressed patients.

  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. 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

  2. HIV protease inhibitors disrupt astrocytic glutamate transporter function and neurobehavioral performance

    PubMed Central

    Vivithanaporn, Pornpun; Asahchop, Eugene L.; Acharjee, Shaona; Baker, Glen B.; Power, Christopher

    2016-01-01

    Objective: The neurotoxic actions of the HIV protease inhibitors, amprenavir (APV) and lopinavir (LPV) were investigated. Design: 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. Methods: 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. Results: 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). Conclusion: 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. PMID:26558720

  3. Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.

    PubMed

    Birkus, Gabriel; Bam, Rujuta A; Willkom, Madeleine; Frey, Christian R; Tsai, Luong; Stray, Kirsten M; Yant, Stephen R; Cihlar, Tomas

    2016-01-01

    Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF. PMID:26503655

  4. 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.

  5. A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

    PubMed

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

    2014-01-01

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

  6. A Synthesis of a Spirocyclic Macrocyclic Protease Inhibitor for the Treatment of Hepatitis C.

    PubMed

    Chung, Cheol K; Cleator, Ed; Dumas, Aaron M; Hicks, Jacqueline D; Humphrey, Guy R; Maligres, Peter E; Nolting, Andrew F; Rivera, Nelo; Ruck, Rebecca T; Shevlin, Michael

    2016-03-18

    The development of a convergent and highly stereoselective synthesis of an HCV NS3/4a protease inhibitor possessing a unique spirocyclic and macrocyclic architecture is described. A late-stage spirocyclization strategy both enabled rapid structure-activity relationship studies in the drug discovery phase and simultaneously served as the basis for the large scale drug candidate preparation for clinical use. Also reported is the discovery of a novel InCl3-catalyzed carbonyl reduction with household aluminum foil or zinc powder as the terminal reductant. PMID:26950496

  7. 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.

  8. 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

  9. Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes.

    PubMed

    Williams, Andrew R; Zakutansky, Sara E; Miura, Kazutoyo; Dicks, Matthew D J; Churcher, Thomas S; Jewell, Kerry E; Vaughan, Aisling M; Turner, Alison V; Kapulu, Melissa C; Michel, Kristin; Long, Carole A; Sinden, Robert E; Hill, Adrian V S; Draper, Simon J; Biswas, Sumi

    2013-10-01

    The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2. Antibodies against Anopheles gambiae serpin-2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.

  10. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor

    PubMed Central

    2012-01-01

    A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1–3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species. PMID:24900473

  11. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor.

    PubMed

    Harper, Steven; McCauley, John A; Rudd, Michael T; Ferrara, Marco; DiFilippo, Marcello; Crescenzi, Benedetta; Koch, Uwe; Petrocchi, Alessia; Holloway, M Katharine; Butcher, John W; Romano, Joseph J; Bush, Kimberly J; Gilbert, Kevin F; McIntyre, Charles J; Nguyen, Kevin T; Nizi, Emanuela; Carroll, Steven S; Ludmerer, Steven W; Burlein, Christine; DiMuzio, Jillian M; Graham, Donald J; McHale, Carolyn M; Stahlhut, Mark W; Olsen, David B; Monteagudo, Edith; Cianetti, Simona; Giuliano, Claudio; Pucci, Vincenzo; Trainor, Nicole; Fandozzi, Christine M; Rowley, Michael; Coleman, Paul J; Vacca, Joseph P; Summa, Vincenzo; Liverton, Nigel J

    2012-04-12

    A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

  12. 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

  13. Molecular basis for increased susceptibility of isolates with atazanavir resistance-conferring substitution I50L to other protease inhibitors.

    PubMed

    Yanchunas, Joseph; Langley, David R; Tao, Li; Rose, Ronald E; Friborg, Jacques; Colonno, Richard J; Doyle, Michael L

    2005-09-01

    Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2' (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.

  14. 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

  15. Serine protease inhibitors block priming of monocytes for enhanced release of superoxide.

    PubMed Central

    Megyeri, P; Pabst, K M; Pabst, M J

    1995-01-01

    Monocytes freshly isolated from human blood produced large amounts of superoxide when triggered by phorbol ester. After monocytes were cultured for 18-24 hr in endotoxin-free, non-adherent conditions, they produced low amounts of superoxide. Addition of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), or platelet-activating factor (PAF) at the beginning of culture 'primed' the monocytes, causing them to maintain a high superoxide response for at least 96 hr. Also, in response to LPS, monocytes secreted TNF-alpha. The ability of LPS, IFN-gamma, TNF-alpha or PAF to maintain the high superoxide response was blocked by addition of inhibitors of serine proteases, either 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) or 3,4-dichloroisocoumarin. AEBSF was most effective at 200 microns, and required 6 hr for maximum effect. AEBSF did not affect phorbol-triggered superoxide release by unprimed monocytes. AEBSF did not affect cell viability, nor did it interfere with the TNF-alpha secretion in response to LPS. An analogue of AEBSF that lacked ability to inhibit proteases did not affect monocyte responses. 3,4-Dichloroisocoumarin blocked priming at a low concentration, 1 microM. We conclude that activity of a monocyte serine protease is required to maintain the high superoxide response in monocytes primed with LPS, IFN-gamma, TNF-alpha, or PAF. PMID:8567031

  16. Enhanced permeation, leaf retention, and plant protease inhibitor activity with bicontinuous microemulsions.

    PubMed

    Tamhane, Vaijayanti A; Dhaware, Deepika G; Khandelwal, Neha; Giri, Ashok P; Panchagnula, Venkateswarlu

    2012-10-01

    Bicontinuous microemulsions (BCMEs) have excellent solubulizing properties along with low interfacial tension and aqueous content that can be controlled. In this work, water soluble plant protease inhibitor (PI), well characterized for its activity against insect pests, was incorporated into a BCME system and explored for permeation on hydrophobic leaf surfaces and protease inhibition activity. The bicontinuous nature of the microemulsion containing water:2-propanol:1-butanol (55:35:10 w/w) was characterized using conductivity and self-diffusion coefficient measurements. The PI was soluble in the water-rich bicontinuous domains, stable in the microemulsions, and protease inhibition activity was retained for a prolonged duration. The microemulsions ensured greater wettability and a wider spread of the PI on hydrophobic leaf surfaces as revealed by contact angle measurements. Significantly, trypsin inhibition activity assays of the PI recovered from the leaves after delivery from the microemulsion indicated a significant increase in the PI retention on the leaf. This BCME enabled greater leaf permeation and retention of the PI can be attributed to a temporary disruption of the waxy leaf surface followed by self-repair without causing any long term damage to the plant.

  17. Expression and Characterization of Recombinant Human Secretory Leukocyte Protease Inhibitor (SLPI) Protein from Pichia pastoris

    PubMed Central

    Li, Zhiguo; Moy, Allison; Sohal, Kirti; Dam, Carolyn; Kuo, Peter; Ulrich, Beau; Whittaker, James; Whittaker, Mei; Düzgünes, Nejat; Konopka, Kryatyna; Franz, Andreas H.; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P.

    2009-01-01

    The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7 kD peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The posttransformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications. PMID:19505578

  18. 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

  19. Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease.

    PubMed

    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

  20. Modulation of tissue factor and tissue factor pathway inhibitor-1 by neutrophil proteases.

    PubMed

    Steppich, Birgit A; Seitz, Isabell; Busch, Gabi; Stein, Andreas; Ott, Ilka

    2008-12-01

    During systemic inflammation, neutrophil activation is accompanied by endothelial cell damage and hypercoagulability. Activated neutrophils release serine proteases that participate in tissue injury. We sought to investigate the effects of neutrophil proteases on proinflammatory and procoagulant changes in endothelial cells. The effects of elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3) on expression of tissue factor (TF) and tissue factor pathway inhibitor-1 (TFPI) were examined in human umbilical vein endothelial cells. Flow cytometry demonstrated that these proteases proteolytically degraded endothelial cell-bound TFPI. TFPI mRNA expression was reduced by HNE and CG. PR3, but not HNE or CG, increased surface expression of TF and TF mRNA. Yet, increased TF expression did not enhance TF activity suggesting induction of encrypted TF. Using antibodies and siRNA to inhibit and silence PAR-1 and PAR-2, we observed that PR3 upregulation of TF is at least in part mediated by PAR-1. Although CG and HNE cleaved PAR-1, antibody reactivity to the PAR-1 hirudin-like sequence demonstrated inactivating cleavage, accounting for the selective ability of PR3 to induce PAR-1-mediated procoagulant effects. This was supported by induction of p42/44 MAPK by PR3. In conclusion, PR3 degradation of TFPI increases the procoagulant activity of endothelial cells. Release of PR3 after neutrophil activation may represent an important step in neutrophil-mediated vascular injury. PMID:19132232

  1. Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms.

    PubMed

    Carrillo, Laura; Herrero, Ignacio; Cambra, Inés; Sánchez-Monge, Rosa; Diaz, Isabel; Martinez, Manuel

    2011-10-01

    Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed. PMID:21482127

  2. Inhibitors of HIV-1 protease: a major success of structure-assisted drug design.

    PubMed

    Wlodawer, A; Vondrasek, J

    1998-01-01

    Retroviral protease (PR) from the human immunodeficiency virus type 1 (HIV-1) was identified over a decade ago as a potential target for structure-based drug design. This effort was very successful. Four drugs are already approved, and others are undergoing clinical trials. The techniques utilized in this remarkable example of structure-assisted drug design included crystallography, NMR, computational studies, and advanced chemical synthesis. The development of these drugs is discussed in detail. Other approaches to designing HIV-1 PR inhibitors, based on the concepts of symmetry and on the replacement of a water molecule that had been found tetrahedrally coordinated between the enzyme and the inhibitors, are also discussed. The emergence of drug-induced mutations of HIV-1 PR leads to rapid loss of potency of the existing drugs and to the need to continue the development process. The structural basis of drug resistance and the ways of overcoming this phenomenon are mentioned.

  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. Heterodera glycines cysts contain an extensive array of endoproteases as well as inhibitors of proteases in H. glycines and Meloidogyne incognita infective juvenile stages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  5. Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

    PubMed Central

    Pflughoeft, Kathryn J.; Swick, Michelle C.; Engler, David A.; Yeo, Hye-Jeong

    2014-01-01

    The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself. PMID:24214942

  6. Crystal structure of a novel synthetic inhibitor of HIV-1 protease

    NASA Astrophysics Data System (ADS)

    Hilgeroth, Andreas; Tykarska, Ewa; Jaskolski, Mariusz

    2002-02-01

    The crystal structure of a novel non-peptidic HIV-1 protease inhibitor derived by simple solid-state dimerization of 4-aryl-1,4-dihydropyridines, reveals a strained central cage and the conformation of its phenyl, benzyl, and hydroxymethylene substituents. The polycyclic cage includes two nearly flat cyclobutane rings and four fused piperidine rings in boat conformations. The cage geometry reveals two unexpected features, namely marked distortions of the valence angles in every second piperidine and a shortening of one of the cyclobutane bonds. The molecule displays exact centrosymmetry, but the central cage and the hydroxymethylene substituents also approximate the C2-symmetry of the target enzyme. The two independent hydroxyl groups are involved in intermolecular hydrogen bonding, one as a donor, the other as an acceptor. The disposition of the hydroxyl groups in the molecular framework is compatible with the dual role of the inhibitor in the active-site cavity of HIV-1 protease, whereby one OH group is hydrogen-bonded to the catalytic aspartates, whereas another one provides an interface to the locked flaps of the enzyme.

  7. Benzophenone derivatives as cysteine protease inhibitors and biological activity against Leishmania(L.) amazonensis amastigotes.

    PubMed

    de Almeida, Letícia; Alves, Karina Ferreira; Maciel-Rezende, Claudia Mara; Jesus, Larissa de Oliveira Passos; Pires, Francieli Ribeiro; Junior, Claudio Viegas; Izidoro, Mario Augusto; Júdice, Wagner Alves de Souza; dos Santos, Marcelo Henrique; Marques, Marcos José

    2015-10-01

    The leishmanicidal potential of benzophenones has been described, some of them highlighting their potential as cysteine protease inhibitors. Therefore, this work described leishmanicidal activity of nine benzophenone derivatives (1a-c;2a-c;3a-c) against intramacrophage amastigote forms of Leishmania(L.)amazonensis (IC50) and the cytotoxic effect on murine peritoneal macrophages (CC50). The derivative 1c exhibited a selectivity index SI (CC50/IC50) of 6.7, besides cytotoxicity lower than Amphotericin B (p< 0.05). Moreover it showed inhibitory activity against papain (42.8±0.3, p<0.05), and when tested on trypanosomatids cysteine proteases 1c also proved to be a potent inhibitor of rCPB2.8, rCPB3.0 and cruzain, showing non-competitive inhibition mechanism by enzymatic assays in vitro.So, benzophenone 1c is interesting drug candidate prototype, with a multi-target directed mode of action, inhibiting rCPB2.8, rCPB3.0 and cruzain.

  8. Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease.

    PubMed

    Meyer, Michael; Leptihn, Sebastian; Welz, Max; Schaller, Andreas

    2016-09-01

    Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans SBT3 was inhibited by SBT3PP with a Kd of 74 nm for the enzyme-inhibitor complex. With a melting point of 87 °C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall. PMID:27451395

  9. 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.

  10. 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

  11. Conformation of inhibitor-free HIV-1 protease derived from NMR spectroscopy in a weakly oriented solution

    PubMed Central

    Roche, Julien; Louis, John M.; Bax, Ad

    2014-01-01

    Flexibility of the glycine-rich flaps is known to be essential for catalytic activity of the HIV-1 protease, but their exact conformations at the different stages of the enzymatic pathway remain subject to much debate. While hundreds of crystal structures of protease-inhibitor complexes have been solved, only about a dozen inhibitor-free protease structures have been reported. These apo-structures reveal a large diversity of flap conformations, ranging from closed, to semi-open and wide-open. To evaluate the average structure in solution, we measured residual dipolar couplings (RDCs) and compared these to values calculated for crystal structures representative of the closed, semi-open and wide-open states. The RDC data clearly indicate that the inhibitor-free protease, on average, adopts a closed conformation in solution that is very similar to the inhibitor-bound state. By contrast, a highly drug-resistant protease mutant, PR20, adopts the wide-open flap conformation. PMID:25470009

  12. In vitro Isolation and Identification of Human Immunodeficiency Virus (HIV) Variants with Reduced Sensitivity to C-2 Symmetrical Inhibitors of HIV Type 1 Protease

    NASA Astrophysics Data System (ADS)

    Otto, M. J.; Garber, S.; Winslow, D. L.; Reid, C. D.; Aldrich, P.; Jadhav, P. K.; Patterson, C. E.; Hodge, C. N.; Cheng, Y.-S. E.

    1993-08-01

    Protease inhibitors are another class of compounds for treatment of human immunodeficiency virus (HIV)-caused disease. The emergence of resistance to the current anti-HIV drugs makes the determination of potential resistance to protease inhibitors imperative. Here we describe the isolation of an HIV type 1 (HIV-1) resistant to an HIV-protease inhibitor. Serial passage of HIV-1 (strain RF) in the presence of the inhibitor, [2-pyridylacetylisoleucylphenylalanyl-psi(CHOH)]_2 (P9941), failed to yield a stock of virus with a resistance phenotype. However, variants of the virus with 6- to 8-fold reduced sensitivity to P9941 were selected by using a combination of plaque assay and endpoint titration. Genetic analysis and computer modeling of the variant proteases revealed a single change in the codon for amino acid 82 (Val -> Ala), which resulted in a protease with lower affinity and reduced sensitivity to this inhibitor and certain, but not all, related inhibitors.

  13. In vitro isolation and identification of human immunodeficiency virus (HIV) variants with reduced sensitivity to C-2 symmetrical inhibitors of HIV type 1 protease.

    PubMed

    Otto, M J; Garber, S; Winslow, D L; Reid, C D; Aldrich, P; Jadhav, P K; Patterson, C E; Hodge, C N; Cheng, Y S

    1993-08-15

    Protease inhibitors are another class of compounds for treatment of human immunodeficiency virus (HIV)-caused disease. The emergence of resistance to the current anti-HIV drugs makes the determination of potential resistance to protease inhibitors imperative. Here we describe the isolation of an HIV type 1 (HIV-1) resistant to an HIV-protease inhibitor. Serial passage of HIV-1 (strain RF) in the presence of the inhibitor, [2-pyridylacetylisoleucylphenylalanyl-psi (CHOH)]2 (P9941), failed to yield a stock of virus with a resistance phenotype. However, variants of the virus with 6- to 8-fold reduced sensitivity to P9941 were selected by using a combination of plaque assay and endpoint titration. Genetic analysis and computer modeling of the variant proteases revealed a single change in the codon for amino acid 82 (Val-->Ala), which resulted in a protease with lower affinity and reduced sensitivity to this inhibitor and certain, but not all, related inhibitors.

  14. 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.

  15. 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.

  16. Early antiretroviral therapy: rationale, protease inhibitor-sparing regimens and once daily dosing.

    PubMed

    Gatell, J M

    1998-01-01

    In 1998 it seems reasonable and widely accepted that all human immunodeficiency virus type 1 (HIV-1)-infected patients willing to be treated may benefit from receiving antiretroviral therapy. Only those with undetectable plasma HIV-1 RNA, normal CD4 lymphocyte counts and lack of markers of immunological system activation may be possible exceptions. The rationale supporting the early initiation of antiretroviral therapy are (i) data on viral dynamics; (ii) preliminary data pointing toward a better and a quicker restoration of immune function when treatment is initiated in very early stages (during or within a few weeks or months of acute symptomatic or asymptomatic HIV-1 infection); (iii) the lack of a stable viral load set-point even in patients in the early stages (CD4 > 500 cells/mm3) who have a very low viral load (< 5000 copies/ml); (iv) the relatively high likelihood of clinical progression at mid-term of the approximately 50-75% of patients in very early disease stages (CD4 > 500 cells/mm3) who have a plasma viral load above 5000 to 10,000 HIV-1 RNA copies/ml; (v) data from the Spanish Earth-1 study, which used a composite endpoint (virological, immunological or clinical progression), demonstrating that even in these very early stages of HIV-1 disease any antiretroviral therapy (double or triple combination) was better than no treatment. Even in early disease stages, a triple combination is needed to achieve a durable and profound virological and immunological response. In addition, the combination of stavudine plus didanosine has several advantages and can be considered one of the best double nucleoside combinations to combine with a protease inhibitor or with a non-nucleoside reverse transcriptase inhibitor. The INCAS study and the preliminary results of the ongoing Spanish SCAN study have demonstrated the possibility of protease inhibitor-sparing combinations for initial antiretroviral treatment, at least in selected patient subsets, such as those with a

  17. Relationships between structure and interaction kinetics for HIV-1 protease inhibitors.

    PubMed

    Markgren, Per-Olof; Schaal, Wesley; Hämäläinen, Markku; Karlén, Anders; Hallberg, Anders; Samuelsson, Bertil; Danielson, U Helena

    2002-12-01

    The interaction between HIV-1 protease and 58 structurally diverse transition-state analogue inhibitors has been analyzed by a surface plasmon resonance based biosensor. Association and dissociation rate constants and affinities were determined and displayed as k(on)-k(off)-K(D) maps. It was shown that different classes of inhibitors fall into distinct clusters in these maps. Significant changes in association and dissociation rates were found as a result of modifying the P1/P1' or P2/P2' side chains of a linear lead compound. Similarly, cyclic urea and cyclic sulfamide inhibitors displayed different kinetic features and the affinities of both classes of cyclic compounds were limited by fast dissociation rates. These results confirm that association and dissociation rates are important features of drug-target interactions and indicate that optimization of inhibitor efficacy may be guided by aiming for high association and low dissociation rates rather than high affinity alone. The present approach thus provides a new tool for structure-interaction kinetic analysis and drug discovery. PMID:12459011

  18. 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.

  19. 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.

  20. Coarse-grained molecular dynamics of ligands binding into protein: The case of HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Li, Dechang; Liu, Ming S.; Ji, Baohua; Hwang, Kehchih; Huang, Yonggang

    2009-06-01

    Binding dynamics and pathways of ligands or inhibitors to target proteins are challenging both experimental and theoretical biologists. A dynamics understanding of inhibitors interacting with protein is essential for the design of novel potent drugs. In this work we applied a coarse-grained molecular dynamics method for simulating inhibitors entering the binding cavity of human immunodeficiency virus type 1 protease (PR). It shows that the coarse-grained dynamics, consistent with the experimental results, can capture the essential molecular dynamics of various inhibitors binding into PR. The primary driving force for the binding processes is the nonbond interaction between inhibitors and PR. The size and topology of inhibitors and the interacting strength between inhibitors and PR have great influence on the binding mode and processes. The interaction strength between the PR and various inhibitors is also analyzed by atomistic molecular mechanics and Poisson-Boltzmann solvation area method.

  1. Protease inhibitors effectively block cell-to-cell spread of HIV-1 between T cells

    PubMed Central

    2013-01-01

    Background The Human Immunodeficiency Virus type-1 (HIV-1) spreads by cell-free diffusion and by direct cell-to-cell transfer, the latter being a significantly more efficient mode of transmission. Recently it has been suggested that cell-to-cell spread may permit ongoing virus replication in the presence of antiretroviral therapy (ART) based on studies performed using Reverse Transcriptase Inhibitors (RTIs). Protease Inhibitors (PIs) constitute an important component of ART; however whether this class of inhibitors can suppress cell-to-cell transfer of HIV-1 is unexplored. Here we have evaluated the inhibitory effect of PIs during cell-to-cell spread of HIV-1 between T lymphocytes. Results Using quantitative assays in cell line and primary cell systems that directly measure the early steps of HIV-1 infection we find that the PIs Lopinavir and Darunavir are equally potent against both cell-free and cell-to-cell spread of HIV-1. We further show that a protease resistant mutant maintains its resistant phenotype during cell-to-cell spread and is transmitted more efficiently than wild-type virus in the presence of drug. By contrast we find that T cell-T cell spread of HIV-1 is 4–20 fold more resistant to inhibition by the RTIs Nevirapine, Zidovudine and Tenofovir. Notably, varying the ratio of infected and uninfected cells in co-culture impacted on the degree of inhibition, indicating that the relative efficacy of ART is dependent on the multiplicity of infection. Conclusions We conclude that if the variable effects of antiviral drugs on cell-to-cell virus dissemination of HIV-1 do indeed impact on viral replication and maintenance of viral reservoirs this is likely to be influenced by the antiviral drug class, since PIs appear particularly effective against both modes of HIV-1 spread. PMID:24364896

  2. 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

    2015-07-01

    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.

  3. Role of the backbone conformation at position 7 in the structure and activity of marinostatin, an ester-linked serine protease inhibitor.

    PubMed

    Taichi, Misako; Yamazaki, Toshimasa; Nishiuchi, Yuji

    2012-09-01

    Rational design of inhibitors: The cis-amide backbone at position 7 in the serine protease inhibitor marinostatin was replaced with an E or Z olefin. The E olefin analogue was not active, but the Z analogue was. The cis conformation might play a critical role in organizing a canonical structure for binding to proteases.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Insights From Atomic-Resolution X-Ray Structures Of Chemically-Synthesized Hiv-1 Protease In Complex With Inhibitors

    PubMed Central

    Johnson, Erik C.B.; Malito, Enrico; Shen, Yuequan; Pentelute, Brad; Rich, Dan; Florián, Jan; Tang, Wei-Jen; Kent, Stephen B.H.

    2007-01-01

    Summary The HIV-1 protease is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 protease has one catalytic site formed by the homodimeric enzyme. We have chemically synthesized fully active HIV-1 protease using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 protease formed crystals that diffracted to 1.04 and 1.2Å resolution, respectively. These atomic resolution structures revealed additional structural details of the HIV-1 protease interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process. PMID:17869270

  6. The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

    PubMed

    Rocha-Pereira, J; Nascimento, M S J; Ma, Q; Hilgenfeld, R; Neyts, J; Jochmans, D

    2014-08-01

    Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

  7. Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

    PubMed

    de Sousa, Lorena Ramos Freitas; Wu, Hongmei; Nebo, Liliane; Fernandes, João Batista; da Silva, Maria Fátima das Graças Fernandes; Kiefer, Werner; Kanitz, Manuel; Bodem, Jochen; Diederich, Wibke E; Schirmeister, Tanja; Vieira, Paulo Cezar

    2015-02-01

    NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors.

  8. In vivo and in vitro inhibition of Spodoptera littoralis gut-serine protease by protease inhibitors isolated from maize and sorghum seeds.

    PubMed

    El-latif, Ashraf Oukasha Abd

    2014-11-01

    Seeds of cereals (Gramineae) are a rich source of serine proteinase inhibitors of most of the several inhibitor families. In the present study, trypsin and chymotrypsin inhibitory activities was detected in the seed flour extracts of three varieties of maize (Zea maize) and six varieties of sorghum (Sorghum bicolor). The maize variety, Hi Teck 2031 and the sorghum variety, Giza 10 were found to have higher trypsin and chymotrypsin inhibitory potentials compared to other tested varieties for which they have been selected for further purification studies using ammonium sulfate fractionation and DEAE-Sephadex A-25 column. Maize and sorghum purified proteins showed a single band on SDS-PAGE corresponding to molecular mass of 20.0 and 15.2 kDa for maize and sorghum PIs respectively. The purified inhibitors were stable at temperature below 60 °C and were active at wide range of pH from 2 to 12 pH. The kinetic analysis revealed non-competitive 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 S.littoralis where maize PI was more effective than sorghum PI. It may be concluded that maize and sorghum protease inhibitor gene(s) could be potential targets for future studies in developing insect resistant transgenic plants.

  9. 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.

  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. Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo.

    PubMed

    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

  12. 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.

  13. Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

    PubMed

    Smid, Ida; Gruden, Kristina; Buh Gašparič, Meti; Koruza, Katarina; Petek, Marko; Pohleven, Jure; Brzin, Jože; Kos, Janko; Zel, Jana; Sabotič, Jerica

    2013-12-26

    Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests. PMID:24295324

  14. Inhibition of the growth of colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom.

    PubMed

    Smid, Ida; Gruden, Kristina; Buh Gašparič, Meti; Koruza, Katarina; Petek, Marko; Pohleven, Jure; Brzin, Jože; Kos, Janko; Zel, Jana; Sabotič, Jerica

    2013-12-26

    Proteins from higher fungi have attracted interest because of their exceptional characteristics. Macrocypins, cysteine protease inhibitors from the parasol mushroom Macrolepiota procera , were evaluated for their adverse effects and their mode of action on the major potato pest Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). They were shown to reduce larval growth when expressed in potato or when their recombinant analogues were added to the diet. Macrocypins target a specific set of digestive cysteine proteases, intestains. Additionally, protein-protein interaction analysis revealed potential targets among other digestive enzymes and proteins related to development and primary metabolism. No effect of dietary macrocypins on gene expression of known adaptation-related digestive enzymes was observed in CPB guts. Macrocypins are the first fungal protease inhibitors to be reported as having a negative effect on growth and development of CPB larvae and could also be evaluated as control agents for other pests.

  15. Selective Dual Inhibitors of the Cancer-Related Deubiquitylating Proteases USP7 and USP47

    PubMed Central

    2012-01-01

    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. PMID:24900381

  16. Computational Design of Protein-Based Inhibitors of Plasmodium vivax Subtilisin-Like 1 Protease

    PubMed Central

    Bastianelli, Giacomo; Bouillon, Anthony; Nguyen, Christophe; Le-Nguyen, Dung

    2014-01-01

    Background Malaria remains a major global health concern. The development of novel therapeutic strategies is critical to overcome the selection of multiresistant parasites. The subtilisin-like protease (SUB1) involved in the egress of daughter Plasmodium parasites from infected erythrocytes and in their subsequent invasion into fresh erythrocytes has emerged as an interesting new drug target. Findings Using a computational approach based on homology modeling, protein–protein docking and mutation scoring, we designed protein–based inhibitors of Plasmodium vivax SUB1 (PvSUB1) and experimentally evaluated their inhibitory activity. The small peptidic trypsin inhibitor EETI-II was used as scaffold. We mutated residues at specific positions (P4 and P1) and calculated the change in free-energy of binding with PvSUB1. In agreement with our predictions, we identified a mutant of EETI-II (EETI-II-P4LP1W) with a Ki in the medium micromolar range. Conclusions Despite the challenges related to the lack of an experimental structure of PvSUB1, the computational protocol we developed in this study led to the design of protein-based inhibitors of PvSUB1. The approach we describe in this paper, together with other examples, demonstrates the capabilities of computational procedures to accelerate and guide the design of novel proteins with interesting therapeutic applications. PMID:25343504

  17. 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.

  18. Algorithm to design inhibitors using stereochemically mixed l,d polypeptides: Validation against HIV protease.

    PubMed

    Gupta, Pooja; Durani, Susheel

    2015-11-01

    Polypeptides have potential to be designed as drugs or inhibitors against the desired targets. In polypeptides, every chiral α-amino acid has enantiomeric structural possibility to become l or d amino acids and can be used as design monomer. Among the various possibilities, use of stereochemistry as a design tool has potential to determine both functional specificity and metabolic stability of the designed polypeptides. The polypeptides with mixed l,d amino acids are a class of peptidomimitics, an attractive drug like molecules and also less susceptible to proteolytic activities. Therefore in this study, a three step algorithm is proposed to design the polypeptides against desired drug targets. For this, all possible configurational isomers of mixed l,d polyleucine (Ac-Leu8-NHMe) structure were randomly modeled with simulated annealing molecular dynamics and the resultant library of discrete folds were scored against HIV protease as a model target. The best scored folds of mixed l,d structures were inverse optimized for sequences in situ and the resultant sequences as inhibitors were validated for conformational integrity using molecular dynamics. This study presents and validates an algorithm to design polypeptides of mixed l,d structures as drugs/inhibitors by inverse fitting them as molecular ligands against desired target.

  19. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection.

    PubMed

    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

  20. 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

  1. Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection.

    PubMed

    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.

  2. 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. PMID:25054490

  3. 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. PMID:27032332

  4. SARS-CoV protease inhibitors design using virtual screening method from natural products libraries.

    PubMed

    Liu, Bing; Zhou, Jiaju

    2005-04-15

    Two natural products databases, the marine natural products database (MNPD) and the traditional Chinese medicines database (TCMD), were used to find novel structures of potent SARS-CoV protease inhibitors through virtual screening. Before the procedure, the databases were filtered by Lipinski's ROF and Xu's extension rules. The results were analyzed by statistic methods to eliminate the bias in target-based database screening toward higher molecular weight compounds for enhancing the hit rate. Eighteen lead compounds were recommended by the screening procedure. They were useful for experimental scientists in prioritizing drug candidates and studying the interaction mechanism. The binding mechanism was also analyzed between the best screening compound and the SARS protein. PMID:15693056

  5. Effect of corticosteroids on sputum sol-phase protease inhibitors in chronic obstructive pulmonary disease.

    PubMed Central

    Wiggins, J; Elliott, J A; Stevenson, R D; Stockley, R A

    1982-01-01

    Corticosteroids caused a reduction in the ratio of sol-phase sputum concentration to serum concentration of albumin in 12 patients with chronic obstructive bronchitis, suggesting a reduction in protein transudation. Alpha-1-antitrypsin values followed the same pattern as those of albumin in both the control and treatment periods, confirming the similar behaviour of the two proteins. The alpha 1-antichymotrypsin ratios were on average three times higher than those of albumin in the control period, confirming the presence of local mechanisms in the lung for preferentially concentrating this protein. The sputum-to-serum ratio of alpha 1-antichymotrypsin, however, rose during steroid treatment with the result that there was a selective increase in this protease inhibitor, which may be of potential benefit to such patients. PMID:6984237

  6. Isoforms of agrin are widely expressed in the developing rat and may function as protease inhibitors.

    PubMed

    Biroc, S L; Payan, D G; Fisher, J M

    1993-09-17

    The agrin family of extracellular matrix proteins may be important in the formation of the neuromuscular junction. Using in situ hybridization with a probe recognizing all agrin isoforms, we demonstrate that it is widely expressed during mammalian embryogenesis. In the developing rat, particularly high levels of expression are found in the dorsal root and cranial ganglia, gut, whisker rudiments, penis, snout, teeth, retina, hippocampus, cerebral cortex and the lining of brain ventricles. Functional analysis of the recombinant rat protein shows that it is a potent inhibitor of the proteases trypsin, chymotrypsin and plasmin but not thrombin or the plasminogen activators. We conclude that agrin and its isoforms may play multiple roles in mammalian development including the regulation of proteolysis in the extracellular matrix.

  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. 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.

  9. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    PubMed

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone. PMID:25463299

  10. Structural characterization and expression analysis of a novel cysteine protease inhibitor from Haliotis discus hannai Ino.

    PubMed

    Ding, Jianfeng; Li, Li; Zhang, Guofan

    2015-02-01

    The sequence of the cysteine protease inhibitor gene of Haliotis discus hannai (designated HdCpi) was determined using the RACE method. The full-length HdCpi cDNA is 1049 bp long, and contains an open reading frame of 813 bp, encoding a 271-amino-acid protein with a calculated molecular mass of 29.83 kDa and an isoelectric point of 8.57. The deduced amino acid sequence of HdCpi contains two cystatin-like domains, and each has the structural features of the cystatin family, including three evolutionarily conserved motifs known to interact with the active sites of cysteine peptidases: the Gly residue at the N-terminus (Gly(65) and Gly(160)), the Gln-X-Val-X-Gly motif (Q(106)IVSG(110) and Q(202)VVAG(206)), and the less conserved motif at the C-terminus (S(136)W(137) and A(254)W(255)). Many putative transcription-factor-binding sites involved in the immune system and cancer occur in the promoter region of HdCpi. Quantitative real-time RT-PCR detected HdCpi expression in all the tissues examined and in the gills of abalone challenged with the bacterium Vibrio anguillarum. HdCpi transcripts were expressed in the mantle, gill, digestive tract, hemocytes, and muscle, and increased HdCpi expression was observed after bacterial stimulation. These results suggest that HdCpi is a biologically active protease inhibitor that is likely to be involved in the antibacterial response of the abalone.

  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. Human immunodeficiency virus type 1 protease inhibitors irreversibly block infectivity of purified virions from chronically infected cells.

    PubMed

    Lambert, D M; Petteway, S R; McDanal, C E; Hart, T K; Leary, J J; Dreyer, G B; Meek, T D; Bugelski, P J; Bolognesi, D P; Metcalf, B W

    1992-05-01

    Synthetic peptide analog inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were used to study the effects of inhibition of polyprotein processing on the assembly, structure, and infectivity of virions released from a T-cell line chronically infected with HIV-1. Inhibition of proteolytic processing of both Pr55gag and Pr160gag-pol was observed in purified virions from infected T cells after treatment. Protease inhibition was evident by the accumulation of precursors and processing intermediates of Pr55gag and by corresponding decreases in mature protein products. Electron microscopy revealed that the majority of the virion particles released from inhibitor-treated cells after a 24-h treatment had an immature or aberrant capsid morphology. This morphological change correlated with the inhibition of polyprotein processing and a loss of infectivity. The infectivity of virion particles purified from these chronically infected cell cultures was assessed following treatment with the inhibitor for 1 to 3 days. Virions purified from cultures treated with inhibitor for 1 or 2 days demonstrated a 95- to 100-fold reduction in virus titers, and treatment for 3 days resulted in complete loss of detectable infectivity. The fact that virions from treated cultures were unable to establish infection over the 7- to 10-day incubation period in the titration experiments strongly suggests that particles produced by inhibitor-treated cells were unable to reactivate to an infectious form when they were purified away from exogenous protease inhibitor. Thus, a block of HIV-1 protease processing of viral polyproteins by specific inhibitors results in a potent antiviral effect characterized by the production of noninfectious virions with altered protein structures and immature morphologies.

  13. Development of Trypsin-Like Serine Protease Inhibitors as Therapeutic Agents: Opportunities, Challenges, and their Unique Structure-Based Rationales.

    PubMed

    Liang, Guyan; Bowen, J Phillip

    2016-01-01

    There has been a revolution in the development of effective, small-molecule anticoagulants and antiplatelet agents. Numerous trypsin-like serine proteases have been under active pursuit as therapeutic targets. Important examples include thrombin, factor VIIa, factor Xa, and β-tryptase with indications ranging from thrombosis and inflammation to asthma and chronic obstructive pulmonary disease (COPD). Trypsin-like serine proteases exhibit a highly similar tertiary folding pattern, especially for the region near the substrate binding pocket that includes the conserved catalytic triad consisting of histidine 57, aspartic acid 102, and serine 195. A rich collection of X-ray structures for many trypsin-like serine proteases is available, which greatly facilitated the optimization of small organic inhibitors as therapeutic agents. The present review surveyed those inhibitors disclosed in peer-reviewed scientific journals and patent publications with a special focus on structural features and protein-inhibitor interactions that implicated the inhibitor optimization process. The role played by the residue 190 of trypsin-like serine proteases is critical. While many inhibitors without a basic group have progressed into the clinic for ones with alanine 190, the task for those with serine 190 remains extremely challenging, if not impossible. In addition to warfarin, heparin, and low molecular weight heparins (LMWHs), treatment options have expanded with the development and approval of the new oral anticoagulants (NOACs). The NOACs are superior to vitamin K antagonists in terms of rapid onset, pharmacokinetics, drug/food interactions, and regular coagulation monitoring; but one serious drawback is the lack of an effective antidote at this time. Apixaban (Eliquis), rivaroxaban (Xarelto), and edoxaban (Savaysa) are the new Xa inhibitors that have been recently approved by the U.S. FDA and are in current clinical practice. These drugs bind to the active site of factor Xa (f

  14. 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

  15. Inferring selection in the Anopheles gambiae species complex: an example from immune-related serine protease inhibitors

    PubMed Central

    Obbard, Darren J; Welch, John J; Little, Tom J

    2009-01-01

    Background Mosquitoes of the Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution. Methods Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the An. gambiae species complex in both East and West Africa. Results Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes. Conclusion It is well known that phylogenetic and population history in the An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed. PMID:19497100

  16. Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings

    PubMed Central

    Wallis, Carole L.; Aga, Evgenia; Ribaudo, Heather; Saravanan, Shanmugam; Norton, Michael; Stevens, Wendy; Kumarasamy, Nagalingeswaran; Bartlett, John; Katzenstein, David

    2014-01-01

    Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania. Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests. Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site. Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance. PMID:24795328

  17. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  18. Extreme multidrug resistant HIV-1 protease with 20 mutations is resistant to novel protease inhibitors with P1'-pyrrolidinone or P2-tris-tetrahydrofuran.

    PubMed

    Agniswamy, Johnson; Shen, Chen-Hsiang; Wang, Yuan-Fang; Ghosh, Arun K; Rao, Kalapala Venkateswara; Xu, Chun-Xiao; Sayer, Jane M; Louis, John M; Weber, Irene T

    2013-05-23

    Extreme drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with the clinical inhibitor amprenavir (1) and two potent antiviral investigational inhibitors GRL-02031 (2) and GRL-0519 (3). Clinical inhibitors are >1000-fold less active on PR20 than on wild-type enzyme, which is consistent with dissociation constants (KL) from isothermal titration calorimetry of 40 nM for 3, 178 nM for amprenavir, and 960 nM for 2. High resolution crystal structures of PR20-inhibitor complexes revealed altered interactions compared with the corresponding wild-type PR complexes in agreement with relative inhibition. Amprenavir lacks interactions due to PR20 mutations in the S2/S2' subsites relative to PR. Inhibitors 2 and 3 lose interactions with Arg8' in PR20 relative to the wild-type enzyme because Arg8' shifts to interact with mutated L10F side chain. Overall, inhibitor 3 compares favorably with darunavir in affinity for PR20 and shows promise for further development.

  19. Receptor-independent 4D-QSAR analysis of a set of norstatine derived inhibitors of HIV-1 protease.

    PubMed

    Senese, Craig L; Hopfinger, A J

    2003-01-01

    A training set of 27 norstatine derived inhibitors of HIV-1 protease, based on the 3(S)-amino-2(S)-hydroxyl-4-phenylbutanoic acid core (AHPBA), for which the -log IC(50) values were measured, was used to construct 4D-QSAR models. Five unique RI-4D-QSAR models, from two different alignments, were identified (q(2) = 0.86-0.95). These five models were used to map the atom type morphology of the lining of the inhibitor binding site at the HIV protease receptor site as well as predict the inhibition potencies of seven test set compounds for model validation. The five models, overall, predict the -log IC(50) activity values for the test set compounds in a manner consistent with their q(2) values. The models also correctly identify the hydrophobic nature of the HIV protease receptor site, and inferences are made as to further structural modifications to improve the potency of the AHPBA inhibitors of HIV protease. The finding of five unique, and nearly statistically equivalent, RI-4D-QSAR models for the training set demonstrates that there can be more than one way to fit structure-activity data even within a given QSAR methodology. This set of unique, equally good individual models is referred to as the manifold model.

  20. Serine protease inhibitor antithrombin III and its messenger RNA in the pathogenesis of Alzheimer's disease.

    PubMed Central

    Kalaria, R. N.; Golde, T.; Kroon, S. N.; Perry, G.

    1993-01-01

    The classical plasma protein antithrombin III (ATIII), an inhibitor of the blood coagulation cascade, is a member of the serpins that are gaining import in the nervous system. In this study, we examined the presence of ATIII in the pathological lesions of Alzheimer's disease (AD). Antibodies to ATIII consistently detected approximately 58-kd protein(s) on immunoblots of cerebral cortex and brain microvessels. Immunocytochemical studies showed ATIII reactivity within amyloid deposits, neurites associated with plaques, and neurofibrillary tangles in neocortex and hippocampus of virtually all the AD cases examined. In some cases, astrocytes were also stained, suggesting ATIII in these cells. ATIII immunoreactivity in neurofibrillary tangles was further defined by electron microscopy, which showed it to be associated with paired helical filaments. Using the polymerase chain reaction technique to amplify ATIII complementary DNA, we found low levels of messenger RNA expression, relative to liver, in control human brain samples, and these were increased in AD samples, particularly in the white matter. Our results suggest the increased presence of ATIII commensurate with astrogliosis and association with the neurofibrillary pathology of AD. We conclude that in concert with other amyloid-associated serine protease inhibitors, ATIII may play a role in the pathogenesis of cerebral amyloidosis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8362984

  1. Inhibitors of Serine Proteases in Regulating the Production and Function of Neutrophil Extracellular Traps

    PubMed Central

    Majewski, Pawel; Majchrzak-Gorecka, Monika; Grygier, Beata; Skrzeczynska-Moncznik, Joanna; Osiecka, Oktawia; Cichy, Joanna

    2016-01-01

    Neutrophil extracellular traps (NETs), DNA webs released into the extracellular environment by activated neutrophils, are thought to play a key role in the entrapment and eradication of microbes. However, NETs are highly cytotoxic and a likely source of autoantigens, suggesting that NET release is tightly regulated. NET formation involves the activity of neutrophil elastase (NE), which cleaves histones, leading to chromatin decondensation. We and others have recently demonstrated that inhibitors of NE, such as secretory leukocyte protease inhibitor (SLPI) and SerpinB1, restrict NET production in vitro and in vivo. SLPI was also identified as a NET component in the lesional skin of patients suffering from the autoinflammatory skin disease psoriasis. SLPI-competent NET-like structures (a mixture of SLPI with neutrophil DNA and NE) stimulated the synthesis of interferon type I (IFNI) in plasmacytoid dendritic cells (pDCs) in vitro. pDCs uniquely respond to viral or microbial DNA/RNA but also to nucleic acids of “self” origin with the production of IFNI. Although IFNIs are critical in activating the antiviral/antimicrobial functions of many cells, IFNIs also play a role in inducing autoimmunity. Thus, NETs decorated by SLPI may regulate skin immunity through enhancing IFNI production in pDCs. Here, we review key aspects of how SLPI and SerpinB1 can control NET production and immunogenic function. PMID:27446090

  2. Variation of proteins and proteinases in Entamoeba histolytica lysates containing a protease inhibitor.

    PubMed

    López-Revilla, R; Jiménez-Delgadillo, B; Canto-Ortiz, L; Chávez-Dueñas, L

    1992-01-01

    Sodium dodecyl sulfate (SDS)-lysates of E. histolytica trophozoites were analyzed by electrophoresis in simple and gelatin-containing ("substrate") SDS-polyacrylamide gels. In simple gels, boiled lysates with para hydroxymercuribenzoate (pHMB) had a complex pattern of apparently undegraded proteins; boiled lysates without pHMB showed a major 30 kDa and four minor (43, 46, 63 and 117 kDa) proteins, whereas unheated lysates displayed only the 117 kDa protein. Using substrate gels no gelatinases were detected in heated lysates; unheated lysates without pHMB showed a major 30 kDa and three minor (33, 46 and 68 kDa) gelatinases, whereas those with pHMB presented a major 56 kDa and two minor (70 and 105 kDa) gelatinases. Three caseinase peaks were separated by Sephadex G-75 chromatography from unheated lysates: peak I contained 46, 56 and 117 kDa pHMB-sensitive gelatinases and peaks II and III contained smaller pHMB-resistant caseinases. We conclude that proteins remaining in lysates after SDS-induced proteolysis appear to be mainly proteases relatively resistant to self-digestion whose type and amount changes with the conditions of lysis and the presence of inhibitors; this is exemplified by the finding of the major gelatinase of lysates with pHMB being larger (56 kDa) than in lysates lacking the inhibitor (30 kDa). PMID:1340329

  3. 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

  4. Free energy component analysis for drug design: a case study of HIV-1 protease-inhibitor binding.

    PubMed

    Kalra, P; Reddy, T V; Jayaram, B

    2001-12-01

    A theoretically rigorous and computationally tractable methodology for the prediction of the free energies of binding of protein-ligand complexes is presented. The method formulated involves developing molecular dynamics trajectories of the enzyme, the inhibitor, and the complex, followed by a free energy component analysis that conveys information on the physicochemical forces driving the protein-ligand complex formation and enables an elucidation of drug design principles for a given receptor from a thermodynamic perspective. The complexes of HIV-1 protease with two peptidomimetic inhibitors were taken as illustrative cases. Four-nanosecond-level all-atom molecular dynamics simulations using explicit solvent without any restraints were carried out on the protease-inhibitor complexes and the free proteases, and the trajectories were analyzed via a thermodynamic cycle to calculate the binding free energies. The computed free energies were seen to be in good accord with the reported data. It was noted that the net van der Waals and hydrophobic contributions were favorable to binding while the net electrostatics, entropies, and adaptation expense were unfavorable in these protease-inhibitor complexes. The hydrogen bond between the CH2OH group of the inhibitor at the scissile position and the catalytic aspartate was found to be favorable to binding. Various implicit solvent models were also considered and their shortcomings discussed. In addition, some plausible modifications to the inhibitor residues were attempted, which led to better binding affinities. The generality of the method and the transferability of the protocol with essentially no changes to any other protein-ligand system are emphasized.

  5. Structure-based Design of Potent HIV-1 Protease Inhibitors with Modified P1 - Biphenyl Ligands: Synthesis, Biological Evaluation, and Enzyme-inhibitor X-ray Structural studies

    PubMed Central

    Ghosh, Arun K.; Yu, Xufen; Osswald, Heather L.; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2016-01-01

    We report the design, synthesis, X-ray structural studies, and biological evaluation of a novel series of HIV-1 protease inhibitors. We designed a variety of functionalized biphenyl derivatives to make enhanced van der Waals interactions in the S1 subsite of HIV-1 protease. These biphenyl derivatives were conveniently synthesized using a Suzuki-Miyaura cross-coupling reaction as the key step. We examined the potential of these functionalized biphenyl-derived P1 ligands in combination with 3-(S)-tetrahydrofuranyl urethane and bis-tetrahydrofuranyl urethane as the P2 ligands. Inhibitor 21e, with a 2-methoxy-1, 1’-biphenyl derivative as P1 ligand and bis-THF as the P2 ligand, displayed the most potent enzyme inhibitory and antiviral activity. This inhibitor also exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray crystal structure of related Boc-derivative 17a-bound HIV-1 protease provided important molecular insight into the ligand-binding site interactions of the biphenyl core in the S1 subsite of HIV-1 protease. PMID:26107245

  6. Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

    PubMed Central

    Qu, Lin; Vongpunsawad, Sompong; Atmar, Robert L.; Prasad, B. V. Venkataram

    2014-01-01

    ABSTRACT Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and the leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here, we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At noncytotoxic concentrations, protease inhibitors ZnCl2 and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents. IMPORTANCE Human noroviruses are the leading cause of epidemic gastroenteritis worldwide. Currently, there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently noncultivatable in cell culture. Here, we report

  7. Crystal structure of the cysteine protease inhibitor 2 from Entamoeba histolytica: Functional convergence of a common protein fold

    SciTech Connect

    Casados-Vázquez, Luz E.; Lara-González, Samuel; Brieb, Luis G.

    2012-04-18

    Cysteine proteases (CP) are key pathogenesis and virulence determinants of protozoan parasites. Entamoeba histolytica contains at least 50 cysteine proteases; however, only three (EhCP1, EhCP2 and EhCP5) are responsible for approximately 90% of the cysteine protease activity in this parasite. CPs are expressed as inactive zymogens. Because the processed proteases are potentially cytotoxic, protozoan parasites have developed mechanisms to regulate their activity. Inhibitors of cysteine proteases (ICP) of the chagasin-like inhibitor family (MEROPS family I42) were recently identified in bacteria and protozoan parasites. E. histolytica contains two ICP-encoding genes of the chagasin-like inhibitor family. EhICP1 localizes to the cytosol, whereas EhICP2 is targeted to phagosomes. Herein, we report two crystal structures of EhICP2. The overall structure of EhICP2 consists of eight {beta}-strands and closely resembles the immunoglobulin fold. A comparison between the two crystal forms of EhICP2 indicates that the conserved BC, DE and FG loops form a flexible wedge that may block the active site of CPs. The positively charged surface of the wedge-forming loops in EhICP2 contrasts with the neutral surface of the wedge-forming loops in chagasin. We postulate that the flexibility and positive charge observed in the DE and FG loops of EhICP2 may be important to facilitate the initial binding of this inhibitor to the battery of CPs present in E. histolytica.

  8. The role of factor XIa (FXIa) catalytic domain exosite residues in substrate catalysis and inhibition by the Kunitz protease inhibitor domain of protease nexin 2.

    PubMed

    Su, Ya-Chi; Miller, Tara N; Navaneetham, Duraiswamy; Schoonmaker, Robert T; Sinha, Dipali; Walsh, Peter N

    2011-09-01

    To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions. PMID:21778227

  9. Atomic resolution crystal structure of HV-BBI protease inhibitor from amphibian skin in complex with bovine trypsin.

    PubMed

    Grudnik, Przemyslaw; Debowski, Dawid; Legowska, Anna; Malicki, Stanislaw; Golik, Przemyslaw; Karna, Natalia; Rolka, Krzysztof; Dubin, Grzegorz

    2015-03-01

    Protease inhibitors of the Bowman-Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV-BBI) in complex with trypsin. HV-BBI shares significant similarities in sequence with a previously described inhibitor from a diskless-fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher Ki against trypsin than HV-BBI. Comparative analysis of trypsin cocrystal structures of HV-BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI-1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease-binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin-like specificity. PMID:25546528

  10. Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity

    PubMed Central

    Koltai, Tomas

    2015-01-01

    Objective: To review the mechanisms of anti-cancer activity of nelfinavir and other protease inhibitors (PIs) based on evidences reported in the published literature. Methods: We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed. Conclusions: The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death. PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors. Beyond the phase I trials that have been recently completed, adequately powered and well-designed clinical trials are needed in the various cancer type settings, and specific trials where NFV is tested in association with other known anti-cancer pharmaceuticals should be sought, in order to find an appropriate place for NFV in cancer treatment. The analysis of controversies on the molecular mechanisms of NFV hints to the possibility that NFV works in a different way in tumor cells and in hepatocytes and adipocytes. PMID:26097685

  11. Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

    PubMed

    Reynolds, Simone L; Pike, Robert N; Mika, Angela; Blom, Anna M; Hofmann, Andreas; Wijeyewickrema, Lakshmi C; Kemp, Dave; Fischer, Katja

    2014-05-01

    Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics. PMID:24854034

  12. 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

  13. Chickpea (Cicer arietinum) and other plant-derived protease inhibitor concentrates inhibit breast and prostate cancer cell proliferation in vitro.

    PubMed

    Magee, Pamela J; Owusu-Apenten, Richard; McCann, Mark J; Gill, Chris I; Rowland, Ian R

    2012-01-01

    The soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), is currently showing great promise as a novel cancer chemopreventive agent. In contrast to the wealth of research conducted on this compound, the anticancer effects of protease inhibitors isolated from other leguminous sources have received limited attention. In the current study, 7 protease inhibitor concentrates (PICs) were isolated from various leguminous sources (including soybean) and characterized. The effects of PICs on the proliferation of breast and prostate cancer cells were investigated in vitro. Chickpea PIC significantly inhibited the viability of MDA-MB-231 breast cancer and PC-3 and LNCaP prostate cancer cells at all concentrations tested (25-400 μg/ml). In addition, kidney bean (200, 400 μg/ml), soybean (50, 100 μg/ml), and mungbean (100, 200 μg/ml) PICs inhibited LNCaP cell viability. These findings suggest that leguminous PICs may possess similar anticancer properties to that of soybean BBI and deserve further study as possible chemopreventive agents.

  14. 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.

  15. 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

    2016-01-01

    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

  16. Modulation of the Inhibitor Properties of Dipeptidyl (Acyloxy)methyl Ketones Toward the CaaX Proteases

    PubMed Central

    Dechert, Anne-Marie R.; MacNamara, James P.; Breevoort, Sarah R.; Hildebrandt, Emily R.; Hembree, Ned W.; Rea, Adam C.; McLain, Duncan E.; Porter, Stephen B.; Schmidt, Walter K.; Dore, Timothy M.

    2010-01-01

    Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A “warhead” free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease. PMID:20696584

  17. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

    SciTech Connect

    Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander

    2012-10-23

    Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.

  18. Antiparasitic effect of a fraction enriched in tight-binding protease inhibitors isolated from the Caribbean coral Plexaura homomalla.

    PubMed

    Salas-Sarduy, Emir; Cabrera-Muñoz, Aymara; Cauerhff, Ana; González-González, Yamile; Trejo, Sebastián A; Chidichimo, Agustina; Chávez-Planes, Maria de Los Angeles; Cazzulo, Juan José

    2013-11-01

    Malaria and American Trypanosomiasis constitute major global health problems. The continued emergence and spreading of resistant strains and the limited efficacy and/or safety of currently available therapeutic agents require a constant search for new sources of antiparasitic compounds. In the present study, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral Plexaura homomalla (Esper, 1792), functionally characterized and tested for their antiparasitic activity against Trypanosoma cruzi and Plasmodium falciparum. The resultant fraction was chromatographically enriched in tight-binding inhibitors active against Papain-like cysteine peptidases (92%) and Pepsin-like aspartyl peptidases (8%). Globally, the inhibitors present in the enriched fraction showed no competition with substrates and apparent Ki values of 1.99 and 4.81nM for Falcipain 2 and Cruzipain, the major cysteine peptidases from P. falciparum and T. cruzi, respectively. The inhibitor-enriched fraction showed promising antiparasitic activity in cultures. It reduced the growth of the chloroquine-resistant P. falciparum strain Dd2 (IC50=0.46μM) and promoted the apparent accumulation of trophozoites, both consistent with a blockade in the hemoglobin degradation pathway. At sub-micromolar concentrations, the inhibitor-enriched fraction reduced the infection of VERO cells by T. cruzi (CL Brener clone) trypomastigotes and interfered with intracellular differentiation and/or replication of the parasites. This study provides new scientific evidence that confirms P. homomalla as an excellent source of tight-biding protease inhibitors for different proteases with biomedical relevance, and suggests that either the individual inhibitors or the enriched fraction itself could be valuable as antiparasitic compounds. PMID:24090569

  19. Antiparasitic effect of a fraction enriched in tight-binding protease inhibitors isolated from the Caribbean coral Plexaura homomalla.

    PubMed

    Salas-Sarduy, Emir; Cabrera-Muñoz, Aymara; Cauerhff, Ana; González-González, Yamile; Trejo, Sebastián A; Chidichimo, Agustina; Chávez-Planes, Maria de Los Angeles; Cazzulo, Juan José

    2013-11-01

    Malaria and American Trypanosomiasis constitute major global health problems. The continued emergence and spreading of resistant strains and the limited efficacy and/or safety of currently available therapeutic agents require a constant search for new sources of antiparasitic compounds. In the present study, a fraction enriched in tight-binding protease inhibitors was isolated from the Caribbean coral Plexaura homomalla (Esper, 1792), functionally characterized and tested for their antiparasitic activity against Trypanosoma cruzi and Plasmodium falciparum. The resultant fraction was chromatographically enriched in tight-binding inhibitors active against Papain-like cysteine peptidases (92%) and Pepsin-like aspartyl peptidases (8%). Globally, the inhibitors present in the enriched fraction showed no competition with substrates and apparent Ki values of 1.99 and 4.81nM for Falcipain 2 and Cruzipain, the major cysteine peptidases from P. falciparum and T. cruzi, respectively. The inhibitor-enriched fraction showed promising antiparasitic activity in cultures. It reduced the growth of the chloroquine-resistant P. falciparum strain Dd2 (IC50=0.46μM) and promoted the apparent accumulation of trophozoites, both consistent with a blockade in the hemoglobin degradation pathway. At sub-micromolar concentrations, the inhibitor-enriched fraction reduced the infection of VERO cells by T. cruzi (CL Brener clone) trypomastigotes and interfered with intracellular differentiation and/or replication of the parasites. This study provides new scientific evidence that confirms P. homomalla as an excellent source of tight-biding protease inhibitors for different proteases with biomedical relevance, and suggests that either the individual inhibitors or the enriched fraction itself could be valuable as antiparasitic compounds.

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

    PubMed Central

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

    2012-01-01

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

  1. Pyrrolidine-5,5-trans-lactams as novel mechanism-based inhibitors of human cytomegalovirus protease. Part 3: potency and plasma stability.

    PubMed

    Borthwick, Alan D; Exall, Anne M; Haley, Terry M; Jackson, Deborah L; Mason, Andrew M; Weingarten, Gordon G

    2002-07-01

    Mechanism-based inhibitors of HCMV protease, which are stable to human plasma (> or = 20 h) and have single-figure potency in the microM range against HCMV protease, have been developed based on the dansylproline alpha-methyl pyrrolidine-5,5-trans-lactam nucleus.

  2. A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki

    PubMed Central

    Negulescu, Horia; Guo, Youzhong; Garner, Thomas P.; Goodwin, Octavia Y.; Henderson, Gregg; Laine, Roger A.; Macnaughtan, Megan A.

    2015-01-01

    Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys8 to Cys16) was identified. PMID:25978745

  3. [Dysmetabolic syndrome related to HIV-1 protease inhibitors. Review of the literature and personal data].

    PubMed

    Urso, R; Croce, G F; Tubili, C; De Marco, M; La Scala, P; Luglio, D; Narciso, P

    2000-02-01

    HIV-positive patients receiving antiretroviral therapy with HIV-1 protease-inhibitors (PI) frequently show insulin-resistance, impaired glucose tolerance, hypertriglyceridaemia and lipodystrophy (LD). LD has often been reported only after the beginning of PI therapy. Some authors link LD to HIV chronic infection, some others suggest that PIs increase pre-existent disturb. Preliminary data of an observational study drawn in IV day-hospital of Spallanzani Institute in Rome showed hypertriglyceridaemia in 36.4% and hyperglycaemia in 11.2% of patients treated with PI. Carr suggests that such drugs should have this lipid-increasing effect because of their inhibition of low density lipoprotein-receptor-related protein, cytoplasmic retinoic-acid binding protein type 1 and P450 3A cytochrome. This theory doesn't explain why both untreated patients and treated with only reverse transcriptase inhibitors show sometimes the same disorders. According to another hypothesis Tumor necrosis factor-alpha, through inhibition of lipoprotein-lipase, would determine high fat-storage in the adipose tissue. Cardiovascular risk factors have always to be assessed before starting a therapy with PI. Glycaemia, triglyceridaemia, cholesterolaemia have to be performed every three months during the treatment and, if necessary, C-Peptide and insulinaemia too. A treatment with lipid-lowering drugs is always recommended in patients with hypertriglyceridaemia > 500 mg/dl and/or hypercholesterolaemia LDL > 190 mg/dl in two following checks. Fibrates have proven to be effective in reducing hypertriglyceridaemia, but there is no certainty that such therapies could have good effects on the LD itself too.

  4. Bowman-Birk protease inhibitor from soybeans enhances cisplatin-induced cytotoxicity in human mesothelioma cells.

    PubMed

    Kashiwagi, Korehito; Virgona, Nantiga; Yamada, Jin; Sato, Ayami; Ota, Masako; Yazawa, Takuya; Yano, Tomohiro

    2011-07-01

    Malignant mesothelioma (MM) is an aggressive cancer with no effective treatment options. Enforced expression of the gap junction (GJ) component connexin 43 (Cx43) increases the sensitivity of MM cells to cisplatin. Bowman-Birk protease inhibitor (BBI) induces the restoration of Cx43 in several types of tumor cells. In this study, we examined the capability of BBI to enhance the cytotoxic effect of cisplatin in MM cells via the induction of Cx43. Human MM H28 cells were used. Cell viability was evaluated by a WST-1 assay and proteasomal activity was determined by fluorometric analysis. Protein and mRNA levels were determined by immunoblot analysis and real-time RT-PCR, respectively. GJ function mediated by Cx43 was evaluated using the scrape-loading method. BBI effectively inhibited H28 cell growth in a dose-dependent manner (200-400 μg/ml). In parallel with the growth inhibition, Cx43 levels (mRNA and protein) and GJ function were elevated by BBI treatment. Knockdown of BBI-induced Cx43 by an antisense nucleotide treatment almost cancelled the growth inhibition. BBI enhanced cisplatin-induced cytotoxicity in H28 cells, and down-regulation of Cx43 by the antisense nucleotide treatment abrogated the enhancing effect of BBI. The induction of Cx43 by BBI contributed to Src inactivation and subsequent induction of Bax. Furthermore, an Src inhibitor (SU6656) also enhanced cisplatin-induced cytotoxicity in H28 cells. These results suggest that BBI improves the cytotoxic efficacy of cisplatin in H28 cells via the inhibition of Src signaling.

  5. Association of Hypercholesterolemia Incidence With Antiretroviral Treatment, Including Protease Inhibitors, Among Perinatally HIV-Infected Children

    PubMed Central

    Tassiopoulos, Katherine; Williams, Paige L.; Seage, George R.; Crain, Marilyn; Oleske, James; Farley, John

    2011-01-01

    Context Antiretroviral therapy has been associated with hypercholesterolemia in HIV-infected children. Few longitudinal studies have been conducted to examine this association, however. Objective To evaluate the incidence of and risk factors for development of hypercholesterolemia in a large pediatric study. Design Prospective cohort study (Pediatric AIDS Clinical Trials Group 219C). Participants A total of 2122 perinatally HIV-infected children free of hypercholesterolemia at entry. Outcome Development of hypercholesterolemia (total cholesterol ≥220 mg/dL at 2 consecutive visits). Cox proportional hazards models were used to evaluate risk factors. Results Thirteen percent of children had hypercholesterolemia at entry, and an additional 13% developed hypercholesterolemia during follow-up for an incidence rate of 3.4 cases per 100 person-years (95% confidence interval [CI]: 3.0 to 3.9). After adjustment for age, boosted protease inhibitor (PI) use (hazard ratio [HR] = 13.9, 95% CI: 6.73 to 28.6), nonboosted PI use (HR = 8.65, 95% CI: 4.19 to 17.9), and nonnucleoside reverse transcriptase inhibitor use (HR = 1.33, 95% CI: 1.04 to 1.71) were associated with increased risk of hypercholesterolemia, and higher viral load was protective (>50,000 vs. ≤400 copies/mL; HR = 0.59, 95% CI: 0.39 to 0.90). Self-reported adherent subjects had higher risk. Conclusions PIs were significant risk factors for hypercholesterolemia. Higher viral load was protective and may reflect non-adherence. Further follow-up is critical to evaluate long-term consequences of chronic PI exposure and hypercholesterolemia. PMID:18209684

  6. Bowman-Birk protease inhibitor from soybeans enhances cisplatin-induced cytotoxicity in human mesothelioma cells

    PubMed Central

    KASHIWAGI, KOREHITO; VIRGONA, NANTIGA; YAMADA, JIN; SATO, AYAMI; OTA, MASAKO; YAZAWA, TAKUYA; YANO, TOMOHIRO

    2011-01-01

    Malignant mesothelioma (MM) is an aggressive cancer with no effective treatment options. Enforced expression of the gap junction (GJ) component connexin 43 (Cx43) increases the sensitivity of MM cells to cisplatin. Bowman-Birk protease inhibitor (BBI) induces the restoration of Cx43 in several types of tumor cells. In this study, we examined the capability of BBI to enhance the cytotoxic effect of cisplatin in MM cells via the induction of Cx43. Human MM H28 cells were used. Cell viability was evaluated by a WST-1 assay and proteasomal activity was determined by fluorometric analysis. Protein and mRNA levels were determined by immunoblot analysis and real-time RT-PCR, respectively. GJ function mediated by Cx43 was evaluated using the scrape-loading method. BBI effectively inhibited H28 cell growth in a dose-dependent manner (200–400 μg/ml). In parallel with the growth inhibition, Cx43 levels (mRNA and protein) and GJ function were elevated by BBI treatment. Knockdown of BBI-induced Cx43 by an antisense nucleotide treatment almost cancelled the growth inhibition. BBI enhanced cisplatin-induced cytotoxicity in H28 cells, and down-regulation of Cx43 by the antisense nucleotide treatment abrogated the enhancing effect of BBI. The induction of Cx43 by BBI contributed to Src inactivation and subsequent induction of Bax. Furthermore, an Src inhibitor (SU6656) also enhanced cisplatin-induced cytotoxicity in H28 cells. These results suggest that BBI improves the cytotoxic efficacy of cisplatin in H28 cells via the inhibition of Src signaling. PMID:22977565

  7. Resistance to amprenavir before and after treatment with lopinavir/ritonavir in highly protease inhibitor-experienced HIV patients.

    PubMed

    Hasson, Hamid; Gianotti, Nicola; Danise, Anna; Seminari, Elena; Boeri, Enzo; Nozza, Silvia; Castagna, Antonella; Lazzarin, Adriano

    2004-01-01

    Genotypes in nine highly protease inhibitor (PI)-experienced patients were studied before and after lopinavir/ritonavir (LPV/r) treatment. Resistance to amprenavir was the rule both before and after LPV/r treatment. Treatment with LPV/r can select for the 50 V mutation. In this setting, significant differences in the inference of the amprenavir phenotype from genotype were observed when using different algorithms.

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

    PubMed

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

    2015-10-01

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

  9. 3D-QSAR studies on chromone derivatives as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Ungwitayatorn, Jiraporn; Samee, Weerasak; Pimthon, Jutarat

    2004-02-01

    The three-dimensional quantitative structure-activity relationship (3D-QSAR) approach using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) was applied to a series of 30 chromone derivatives, a new class of HIV-1 protease inhibitors. The best predictive CoMFA model gives cross-validated r2 ( q2)=0.763, non-cross-validated r2=0.967, standard error of estimate ( S)=5.092, F=90.701. The best CoMSIA model has q2=0.707, non-cross-validated r2=0.943, S=7.018, F=51.734, included steric, electrostatic, hydrophobic, and hydrogen bond donor fields. The predictive ability of these models was validated by a set of five compounds that were not included in the training set. The calculated (predicted) and experimental inhibitory activities were well correlated. The contour maps obtained from CoMFA and CoMSIA models were in agreement with the previous docking study for this chromone series.

  10. Directed HIV-1 Evolution of Protease Inhibitor Resistance by Second-Generation Short Hairpin RNAs

    PubMed Central

    Schopman, Nick C. T.; Braun, Anja

    2012-01-01

    Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches. PMID:22064528

  11. Directed HIV-1 evolution of protease inhibitor resistance by second-generation short hairpin RNAs.

    PubMed

    Schopman, Nick C T; Braun, Anja; Berkhout, Ben

    2012-01-01

    Despite the success of antiretroviral drugs in decreasing AIDS-related mortality, a substantial fraction of HIV-infected patients experience therapy failure due to the emergence of drug-resistant virus variants. For durable inhibition of HIV-1 replication, the emergence of such escape viruses must be controlled. In addition to antiretroviral drugs, RNA interference (RNAi)-based gene therapy can be used to inhibit HIV-1 replication by targeting the viral RNA genome. RNAi is an evolutionary conserved gene silencing mechanism that mediates the sequence-specific breakdown of the targeted mRNA. Here we investigated an alternative strategy combining the activity of a protease inhibitor (PI) with second-generation short hairpin RNAs (shRNAs) designed to specifically block the emergence of PI-resistant HIV-1 variants. We demonstrate that dominant viral escape routes can be effectively blocked by second-generation shRNAs and that virus evolution can be redirected toward less-fit variants. These results are of importance for a deeper understanding of HIV-1 evolution under combined drug and RNAi pressure and may be used to design future therapeutic approaches. PMID:22064528

  12. Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors.

    PubMed

    Spaeny-Dekking, E H; Kamp, A M; Froelich, C J; Hack, C E

    2000-02-15

    Granzyme A (GrA) and B (GrB) together with perforin are the main constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytotoxic proteins are released to deliver a lethal hit during contact between the CTL or NK cell and target cell. With the use of an enzyme-linked immunosorbent assay for antigenic levels, we showed in a recent study that plasma of patients with activated CTLs and NK cells contain elevated levels of extracellular GrA. In this study, we determined the form and proteolytic capacity of this extracellular GrA detected in plasma. With the use of various assays, we show that part of the extracellular GrA circulates in the mature conformation and is bound to proteoglycans that protect it against inactivation by protease inhibitors, such as antithrombin III and alpha-2-macroglobulin, whereas another part of GrA circulates as a complex with antithrombin III. Finally, with the use of a novel assay for active GrA, we demonstrate that some plasma samples with high levels of extracellular GrA contain active GrA. These results suggest that various forms of extracellular GrA occur in vivo and that the regulation of GrA activity may be modified by proteoglycans. These data support the notion that granzymes may exert extracellular functions distant from the site of CTL or NK cell interaction with their target cells. (Blood. 2000;95:1465-1472)

  13. Function of Secretory Leukocyte Protease Inhibitor (SLPI) in Odontoblast During Mouse Tooth Development.

    PubMed

    Jeong, Je-O; Wang, Guanlin; Jeong, Soon-Jeong; Choi, Baik-Dong; Lee, Hye-Yon; Jeong, Moon-Jin

    2015-01-01

    Secretory leuckocyte protease inhibitor (SLPI) is thought as a regulating protein on the synthesis and degradation of matrix proteins. But there was no report of expression and function of SLPI on the tooth development, especially on the odontoblasts. As observed by in-situ hybridization and immunohistochemical analysis, SLPI was expressed in odontoblasts and predentin on post-natal day 4 (PN4). On PN10, SLPI was observed under the dentin and apical region including odontoblasts processes. Further, on PN15, expression of SLPI was the same pattern compared to PN10. SLPI was expressed under layer of the odontoblasts and in odontoblasts on PN20. Matrix metalloproteinase-2 (MMP-2) and -9 levels in SLPI/MDPC-23 cells were higher than that of the MDPC-23 cells. The gene expression of SLPI, bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I (Col I) was higher in SLPI/MDPC-23 than that of MDPC-23 cells and the expression of dentin sialophosphoprotein (DSPP) was lower in SLPI/MDPC-23. Taken together, our results suggest that SLPI may be a MMP-2 and -9 regulating molecule in odontoblasts during dentin matrix formation and acts as a signaling molecule for dentin matrix related proteins during odontoblasts differentiation and mineralization.

  14. Serine protease inhibitor 6 is required to protect dendritic cells from the kiss of death.

    PubMed

    Lovo, Elena; Zhang, Manling; Wang, Lihui; Ashton-Rickardt, Philip G

    2012-02-01

    How dendritic cells (DC) present Ag to cytotoxic T cells (CTL) without themselves being killed through contact-mediated cytotoxicity (so-called kiss of death) has proved to be controversial. Using mice deficient in serine protease inhibitor 6 (Spi6), we show that Spi6 protects DC from the kiss of death by inhibiting granzyme B (GrB) delivered by CTL. Infection of Spi6 knockout mice with lymphocytic choriomeningitis virus revealed impaired survival of CD8α DC. The impaired survival of Spi6 knockout CD8α DC resulted in impaired priming and expansion of both primary and memory lymphocytic choriomeningitis virus-specific CTL, which could be corrected by GrB deficiency. The rescue in the clonal burst obtained by GrB elimination demonstrated that GrB was the physiological target through which Spi6 protected DC from CTL. We conclude that the negative regulation of DC priming of CD8 T lymphocyte immunity by CTL killing is mitigated by the physiological inhibition of GrB by Spi6.

  15. Enhancement of cellular uptake, transport and oral absorption of protease inhibitor saquinavir by nanocrystal formulation

    PubMed Central

    He, Yuan; Xia, Deng-ning; Li, Qiu-xia; Tao, Jin-song; Gan, Yong; Wang, Chi

    2015-01-01

    Aim: Saquinavir (SQV) is the first protease inhibitor for the treatment of HIV infection, but with poor solubility. The aim of this study was to prepare a colloidal nanocrystal suspension for improving the oral absorption of SQV. Methods: SQV nanocrystals were prepared using anti-solvent precipitation–high pressure homogenization method. The nanocrystals were characterized by a Zetasizer and transmission electron microscopy (TEM). Their dissolution, cellular uptake and transport across the human colorectal adenocarcinoma cell line (Caco-2) monolayer were investigated. Bioimaging of ex vivo intestinal sections of rats was conducted with confocal laser scanning microscopy. Pharmacokinetic analysis was performed in rats administered nanocrystal SQV suspension (50 mg/kg, ig), and the plasma SQV concentrations were measured with HPLC. Results: The SQV nanocrystals were approximately 200 nm in diameter, with a uniform size distribution. The nanocrystals had a rod-like shape under TEM. The dissolution, cellular uptake, and transport across a Caco-2 monolayer of the nanocrystal formulation were significantly improved compared to those of the coarse crystals. The ex vivo intestinal section study revealed that the fluorescently labeled nanocrystals were located in the lamina propria and the epithelium of the duodenum and jejunum. Pharmacokinetic study showed that the maximal plasma concentration (Cmax) was 2.16-fold of that for coarse crystalline SQV suspension, whereas the area under the curve (AUC) of nanocrystal SQV suspension was 1.95-fold of that for coarse crystalline SQV suspension. Conclusion: The nanocrystal drug delivery system significantly improves the oral absorption of saquinavir. PMID:26256404

  16. Pregnane X Receptor Mediates Dyslipidemia Induced by the HIV Protease Inhibitor Amprenavir in Mice

    PubMed Central

    Helsley, Robert N.; Sui, Yipeng; Ai, Ni; Park, Se-Hyung; Welsh, William J.

    2013-01-01

    Human immunodeficiency virus (HIV) protease inhibitors (PIs) have been used successfully in extending the life span of people infected with HIV. The use of PIs has also been associated with dyslipidemia and an increased risk of cardiovascular disease, but the underlying mechanisms remain elusive. Several PIs have been implicated in activating the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. Recent studies indicate that PXR may also play an important role in the regulation of lipid homeostasis. In the present study, we identified amprenavir, a widely used HIV PI, as a potent PXR-selective agonist. Computational docking studies combined with site-direct mutagenesis identified several key residues within the ligand-binding pocket of PXR that constitute points of interaction with amprenavir. Amprenavir efficiently activated PXR and induced PXR target gene expression in vitro and in vivo. Short-term exposure to amprenavir significantly increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. Amprenavir-mediated PXR activation stimulated the expression of several key intestinal genes involved in lipid homeostasis. These findings provide critical mechanistic insight for understanding the impact of PIs on cardiovascular disease and demonstrate a potential role of PXR in mediating the adverse effects of HIV PIs in humans. PMID:23519392

  17. Novel Dengue Virus NS2B/NS3 Protease Inhibitors

    PubMed Central

    Wu, Hongmei; Bock, Stefanie; Snitko, Mariya; Berger, Thilo; Weidner, Thomas; Holloway, Steven; Kanitz, Manuel; Diederich, Wibke E.; Steuber, Holger; Walter, Christof; Hofmann, Daniela; Weißbrich, Benedikt; Spannaus, Ralf; Acosta, Eliana G.; Bartenschlager, Ralf; Engels, Bernd; Schirmeister, Tanja

    2014-01-01

    Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. PMID:25487800

  18. The circadian Clock gene regulates acrosin activity of sperm through serine protease inhibitor A3K

    PubMed Central

    Cheng, Shuting; Liang, Xin; Wang, Yuhui; Jiang, Zhou; Liu, Yanyou; Hou, Wang; Li, Shiping; Zhang, Jing

    2015-01-01

    Our previous study found that CLOCK knockdown in the testes of male mice led to a reduced fertility, which might be associated with the lower acrosin activity. In this present study, we examined the differential expression in proteins of CLOCK knockdown sperm. Clock gene expression was knocked down in cells to confirm those differentially expressions and serine protease inhibitor SERPINA3K was identified as a potential target. The up-regulated SERPINA3K revealed an inverse relationship with Clock knockdown. Direct treatment of normal sperm with recombinant SERPINA3K protein inhibited the acrosin activity and reduced in vitro fertilization rate. The luciferase reporter gene assay showed that the down-regulated of Clock gene could activate the Serpina3k promoter, but this activation was not affected by the mutation of E-box core sequence. Co-IP demonstrated a natural interaction between SERPIAN3K and RORs (α and β). Taken together, these results demonstrated that SERPINA3K is involved in the Clock gene-mediated male fertility by regulating acrosin activity and provide the first evidence that SERPINA3K could be regulated by Clock gene via retinoic acid-related orphan receptor response elements. PMID:26264441

  19. Ethical assessment of hepatitis C virus treatment: The lesson from first generation protease inhibitors.

    PubMed

    Sacchini, Dario; Craxì, Lucia; Refolo, Pietro; Minacori, Roberta; Cicchetti, Americo; Gasbarrini, Antonio; Cammà, Calogero; Spagnolo, Antonio G

    2015-05-01

    Since chronic hepatitis C has mostly become curable, issues concerning choice and allocation of treatment are of major concern. We assessed the foremost ethical issues in hepatitis C virus therapy with 1st generation protease inhibitors using the personalist ethical framework within the health technology assessment methodology. Our aim was to identify values at stake/in conflict and to support both the physicians' choices in hepatitis C therapy and social (macro-) allocation decision-making. The ethical assessment indicates that: (1) safety/effectiveness profile of treatment is guaranteed if its use is restricted to the patients subgroups who may benefit from it; (2) patients should be carefully informed, particularly on treatment deferral, and widespread information on these therapies should be implemented; (3) since treatment was proven to be cost-effective, its use is acceptable respecting resource macro-allocation. Concerning individual (micro-) location criteria: (a) criteria for eligibility to treatment should be clearly identified and updated periodically; (b) information on criteria for eligibility/deferral to treatment for specific patients' subgroups should be made widely known. Interferon-based regimens will disappear from use within the next year, with the introduction of highly effective/tolerable combination regimens of direct-acting antivirals, thus profoundly changing social choices. Nonetheless, our model could support future ethical assessment since the evaluation pertaining ethical domains remains generally applicable.

  20. Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors.

    PubMed

    Geitmann, Matthis; Dahl, Göran; Danielson, U Helena

    2011-01-01

    The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization. PMID:21194118

  1. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

    NASA Astrophysics Data System (ADS)

    Becker, Daniel; Kaczmarska, Zuzanna; Arkona, Christoph; Schulz, Robert; Tauber, Carolin; Wolber, Gerhard; Hilgenfeld, Rolf; Coll, Miquel; Rademann, Jörg

    2016-09-01

    Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme-inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.

  2. 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.

  3. Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer.

    PubMed

    Han, Zhenfu; Harris, Peter K W; Jones, Darin E; Chugani, Ryan; Kim, Tommy; Agarwal, Manjula; Shen, Wei; Wildman, Scott A; Janetka, James W

    2014-11-13

    Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with K is = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON. PMID:25408834

  4. Inhibitors of HGFA, Matriptase, and Hepsin Serine Proteases: A Nonkinase Strategy to Block Cell Signaling in Cancer

    PubMed Central

    2014-01-01

    Hepatocyte growth factor activators (HGFA), matriptase, and hepsin are S1 family trypsin-like serine proteases. These proteases proteolytically cleave the single-chain zymogen precursors, pro-HGF (hepatocyte growth factor), and pro-MSP (macrophage stimulating protein) into active heterodimeric forms. HGF and MSP are activating ligands for the oncogenic receptor tyrosine kinases (RTKs), c-MET and RON, respectively. We have discovered the first substrate-based ketothiazole inhibitors of HGFA, matriptase and hepsin. The compounds were synthesized using a combination of solution and solid-phase peptide synthesis (SPPS). Compounds were tested for protease inhibition using a kinetic enzyme assay employing fluorogenic peptide substrates. Highlighted HGFA inhibitors are Ac-KRLR-kt (5g), Ac-SKFR-kt (6c), and Ac-SWLR-kt (6g) with Kis = 12, 57, and 63 nM, respectively. We demonstrated that inhibitors block the conversion of native pro-HGF and pro-MSP by HGFA with equivalent potency. Finally, we show that inhibition causes a dose-dependent decrease of c-MET signaling in MDA-MB-231 breast cancer cells. This preliminary investigation provides evidence that HGFA is a promising therapeutic target in breast cancer and other tumor types driven by c-MET and RON. PMID:25408834

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

    SciTech Connect

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

    2012-09-11

    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 the 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 Asp29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies.

  6. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

    PubMed Central

    Becker, Daniel; Kaczmarska, Zuzanna; Arkona, Christoph; Schulz, Robert; Tauber, Carolin; Wolber, Gerhard; Hilgenfeld, Rolf; Coll, Miquel; Rademann, Jörg

    2016-01-01

    Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme–inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases. PMID:27677239

  7. Design, Synthesis, Evaluation and Thermodynamics of 1-Substituted Pyridylimidazo[1,5-a]Pyridine Derivatives as Cysteine Protease Inhibitors

    PubMed Central

    Khan, Mohd Sajid; Baig, Mohd Hassan; Ahmad, Saheem; Siddiqui, Shapi Ahmad; Srivastava, Ashwini Kumar; Srinivasan, Kumar Venkatraman; Ansari, Irfan A.

    2013-01-01

    Targeting papain family cysteine proteases is one of the novel strategies in the development of chemotherapy for a number of diseases. Novel cysteine protease inhibitors derived from 1-pyridylimidazo[1,5-a]pyridine representing pharmacologically important class of compounds are being reported here for the first time. The derivatives were initially designed and screened in silico by molecular docking studies against papain to explore the possible mode of action. The molecular interaction between the compounds and cysteine protease (papain) was found to be very similar to the interactions observed with the respective epoxide inhibitor (E-64c) of papain. Subsequently, compounds were synthesized to validate their efficacy in wet lab experiments. When characterized kinetically, these compounds show their Ki and IC50 values in the range of 13.75 to 99.30 µM and 13.40 to 96.50 µM, respectively. The thermodynamics studies suggest their binding with papain hydrophobically and entropically driven. These inhibitors also inhibit the growth of clinically important different types of Gram positive and Gram negative bacteria having MIC50 values in the range of 0.6–1.4 µg/ml. Based on Lipinski’s rule of Five, we also propose these compounds as potent antibacterial prodrugs. The most active antibacterial compound was found to be 1-(2-pyridyl)-3-(2-hydroxyphenyl)imidazo[1,5-a]pyridine (3a). PMID:23940536

  8. Complementary Antiviral Efficacy of Hydroxyurea and Protease Inhibitors in Human Immunodeficiency Virus-Infected Dendritic Cells and Lymphocytes

    PubMed Central

    Piccinini, Giampiero; Foli, Andrea; Comolli, Giuditta; Lisziewicz, Julianna; Lori, Franco

    2002-01-01

    Dendritic cells are susceptible to human immunodeficiency virus (HIV) infection and may transmit the virus to T cells in vivo. Scarce information is available about drug efficacy in dendritic cells because preclinical testing of antiretroviral drugs has been limited predominantly to T cells and macrophages. We compared the antiviral activities of hydroxyurea and two protease inhibitors (indinavir and ritonavir) in monocyte-derived dendritic cells and in lymphocytes. At therapeutic concentrations (50 to 100 μM), hydroxyurea inhibited supernatant virus production from monocyte-derived dendritic cells in vitro but the drug was ineffective in activated lymphocytes. Concentrations of hydroxyurea insufficient to be effective in activated lymphocytes cultured alone strongly inhibited supernatant virus production from cocultures of uninfected, activated lymphocytes with previously infected monocyte-derived dendritic cells in vitro. In contrast, protease inhibitors were up to 30-fold less efficient in dendritic cells than in activated lymphocytes. Our data support the rationale for testing of the combination of hydroxyurea and protease inhibitors, since these drugs may have complementary antiviral efficacies in different cell compartments. A new criterion for combining drugs for the treatment of HIV infection could be to include at least one drug that selectively targets HIV in viral reservoirs. PMID:11836405

  9. Effect of synthesized inhibitors on babesipain-1, a new cysteine protease from the bovine piroplasm Babesia bigemina.

    PubMed

    Martins, T M; Gonçalves, L M D; Capela, R; Moreira, R; do Rosário, V E; Domingos, A

    2010-04-01

    Papain-like cysteine proteases (CP) have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. One gene was identified by sequence similarity search to be homologous to the CP family in the ongoing Babesia bigemina genome sequencing project database. The newly identified CP gene, called babesipain-1, was cloned and expressed as a fusion protein, and the effect of different inhibitors on proteolytic activity was tested. A series of new artemisinin-vinyl sulfone hybrid molecules were tested as inhibitors being effective on the range of 0.3-30 microm, depending on the core-containing molecule. PMID:20537109

  10. Synthesis and structure-activity relationship of α-keto amides as enterovirus 71 3C protease inhibitors.

    PubMed

    Zeng, Debin; Ma, Yuying; Zhang, Rui; Nie, Quandeng; Cui, Zhengjie; Wang, Yaxin; Shang, Luqing; Yin, Zheng

    2016-04-01

    α-Keto amide derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. structure-activity relationship (SAR) study indicated that small moieties were primarily tolerated at P1' and the introduction of para-fluoro benzyl at P2 notably improved the potency of inhibitor. Inhibitors 8v, 8w and 8x exhibited satisfactory activity (IC50=1.32±0.26μM, 1.88±0.35μM and 1.52±0.31μM, respectively) and favorable CC50 values (CC50>100μM). α-Keto amide may represent a good choice as a warhead for EV71 3C(pro) inhibitor.

  11. Synthesis and herbicidal evaluation of novel benzothiazole derivatives as potential inhibitors of D1 protease.

    PubMed

    Huang, Tonghui; Sun, Jie; An, Lin; Zhang, Lixian; Han, Cuiping

    2016-04-01

    D1 protease is a C-terminal processing protease that has been predicted to be an ideal herbicidal target. Three novel series of benzothiazole derivatives were synthesized and evaluated for their herbicidal activities against Brassica napus (rape) and Echinochloa crusgalli (barnyard grass). The preliminary bioassay indicated that most of the synthesized compounds possess promising D1 protease inhibitory activities and considerable herbicidal activities. Molecular docking was performed to position representative compounds into the active site of D1 protease to determine a probable binding model. PMID:26905829

  12. INTERACTIONS OF DIFFERENT INHIBITORS WITH ACTIVE-SITE ASPARTYL RESIDUES OF HIV-1 PROTEASE AND POSSIBLE RELEVANCE TO PEPSINS

    PubMed Central

    Sayer, Jane M.; Louis, John M.

    2008-01-01

    The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors (symmetric cyclic urea inhibitor DMP323, non-hydrolysable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)) was assessed by differential scanning calorimetry. ΔTm values, defined as the difference in Tm for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 °C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by ΔTm ratios [ΔTm(PR)/ΔTm(PRD25N)] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (ΔTm ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (ΔTm(PR)/ΔTm(PRD29N) ≥ 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (ΔTm ratios of 1.4-2.2). Of the 9 FDA approved clinical HIV-1 protease inhibitors screened, APV, RTV and DRV competitively inhibit porcine pepsin with Ki values of 0.3, 0.6 and 2.14 μM, respectively. DSC results were consistent with this relatively weak binding of APV (ΔTm 2.7 °C) compared with the tight binding of AcPEP (ΔTm ≥17 °C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32 and Ser219 in pepsin, equivalent to Asp25, Asp25′ and Asp29 in PR, in the binding and stabilization of the pepsin/APV complex. PMID:18951411

  13. Impact of oilseed rape expressing the insecticidal cysteine protease inhibitor oryzacystatin on the beneficial predator Harmonia axyridis (multicoloured Asian ladybeetle).

    PubMed

    Ferry, N; Raemaekers, R J M; Majerus, M E N; Jouanin, L; Port, G; Gatehouse, J A; Gatehouse, A M R

    2003-02-01

    Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) on the predatory ladybird Harmonia axyridis were investigated using diamondback moth Plutella xylostella as the pest species. As expected, oilseed rape expressing OC-1 had no effects on either development or survival of the pest, which utilizes serine digestive proteases. Immunoassays confirmed accumulation of the transgene product in pest larval tissues at levels of up to 3 ng per gut. Characterization of proteolytic digestive enzymes of H. axyridis demonstrated that larvae and adults utilize cysteine and aspartic proteases; the former activity was completely inhibited by oryzacystatin in vitro. However, when H. axyridis larvae consumed prey reared on OC-1 expressing plants over their entire life cycle, no significant effects upon survival or overall development were observed. The inhibitor initially stimulated development, with a shortening of the developmental period of the second instar by 27% (P < 0.0001) accompanied by a 36% increase in weight of second instar larvae (P = 0.007). OC-1 had no detrimental effects on reproductive fitness of adult H. axyridis. Interestingly there was a significant increase in consumption of OC-1 dosed prey. The results show that prey reared on transgenic plants expressing a protein which inhibited ladybird digestive enzymes in vitro had no effects in vivo; the ladybird was able to up-regulate digestive proteases in response to the inhibitor.

  14. Identification of a novel inhibitor of dengue virus protease through use of a virtual screening drug discovery Web portal.

    PubMed

    Viswanathan, Usha; Tomlinson, Suzanne M; Fonner, John M; Mock, Stephen A; Watowich, Stanley J

    2014-10-27

    We report the discovery of a novel small-molecule inhibitor of the dengue virus (DENV) protease (NS2B-NS3pro) using a newly constructed Web-based portal (DrugDiscovery@TACC) for structure-based virtual screening. Our drug discovery portal, an extension of virtual screening studies performed using IBM's World Community Grid, facilitated access to supercomputer resources managed by the Texas Advanced Computing Center (TACC) and enabled druglike commercially available small-molecule libraries to be rapidly screened against several high-resolution DENV NS2B-NS3pro crystallographic structures. Detailed analysis of virtual screening docking scores and hydrogen-bonding interactions between each docked ligand and the NS2B-NS3pro Ser135 side chain were used to select molecules for experimental validation. Compounds were ordered from established chemical companies, and compounds with established aqueous solubility were tested for their ability to inhibit DENV NS2B-NS3pro cleavage of a model substrate in kinetic studies. As a proof-of-concept, we validated a small-molecule dihydronaphthalenone hit as a single-digit-micromolar mixed noncompetitive inhibitor of the DENV protease. Since the dihydronaphthalenone was predicted to interact with NS2B-NS3pro residues that are largely conserved between DENV and the related West Nile virus (WNV), we tested this inhibitor against WNV NS2B-NS3pro and observed a similar mixed noncompetitive inhibition mechanism. However, the inhibition constants were ∼10-fold larger against the WNV protease relative to the DENV protease. This novel validated lead had no chemical features or pharmacophores associated with adverse toxicity, carcinogenicity, or mutagenicity risks and thus is attractive for additional characterization and optimization. PMID:25263519

  15. Turnover of mitochondrial steroidogenic acute regulatory (StAR) protein by Lon protease: the unexpected effect of proteasome inhibitors.

    PubMed

    Granot, Zvi; Kobiler, Oren; Melamed-Book, Naomi; Eimerl, Sarah; Bahat, Assaf; Lu, Bin; Braun, Sergei; Maurizi, Michael R; Suzuki, Carolyn K; Oppenheim, Amos B; Orly, Joseph

    2007-09-01

    Steroidogenic acute regulatory protein (StAR) is a vital mitochondrial protein promoting transfer of cholesterol into steroid making mitochondria in specialized cells of the adrenal cortex and gonads. Our previous work has demonstrated that StAR is rapidly degraded upon import into the mitochondrial matrix. To identify the protease(s) responsible for this rapid turnover, murine StAR was expressed in wild-type Escherichia coli or in mutant strains lacking one of the four ATP-dependent proteolytic systems, three of which are conserved in mammalian mitochondria-ClpP, FtsH, and Lon. StAR was rapidly degraded in wild-type bacteria and stabilized only in lon (-)mutants; in such cells, StAR turnover was fully restored upon coexpression of human mitochondrial Lon. In mammalian cells, the rate of StAR turnover was proportional to the cell content of Lon protease after expression of a Lon-targeted small interfering RNA, or overexpression of the protein. In vitro assays using purified proteins showed that Lon-mediated degradation of StAR was ATP-dependent and blocked by the proteasome inhibitors MG132 (IC(50) = 20 microm) and clasto-lactacystin beta-lactone (cLbetaL, IC(50) = 3 microm); by contrast, epoxomicin, representing a different class of proteasome inhibitors, had no effect. Such inhibition is consistent with results in cultured rat ovarian granulosa cells demonstrating that degradation of StAR in the mitochondrial matrix is blocked by MG132 and cLbetaL but not by epoxomicin. Both inhibitors also blocked Lon-mediated cleavage of the model substrate fluorescein isothiocyanate-casein. Taken together, our former studies and the present results suggest that Lon is the primary ATP-dependent protease responsible for StAR turnover in mitochondria of steroidogenic cells.

  16. Genetic correlates of in vivo viral resistance to indinavir, a human immunodeficiency virus type 1 protease inhibitor.

    PubMed Central

    Condra, J H; Holder, D J; Schleif, W A; Blahy, O M; Danovich, R M; Gabryelski, L J; Graham, D J; Laird, D; Quintero, J C; Rhodes, A; Robbins, H L; Roth, E; Shivaprakash, M; Yang, T; Chodakewitz, J A; Deutsch, P J; Leavitt, R Y; Massari, F E; Mellors, J W; Squires, K E; Steigbigel, R T; Teppler, H; Emini, E A

    1996-01-01

    Indinavir (IDV) (also called CRIXIVAN, MK-639, or L-735,524) is a potent and selective inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease. During early clinical trials, in which patients initiated therapy with suboptimal dosages of IDV, we monitored the emergence of viral resistance to the inhibitor by genotypic and phenotypic characterization of primary HIV-1 isolates. Development of resistance coincided with variable patterns of multiple substitutions among at least 11 protease amino acid residues. No single substitution was present in all resistant isolates, indicating that resistance evolves through multiple genetic pathways. Despite this complexity, all of 29 resistant isolates tested exhibited alteration of residues M-46 (to I or L) and/or V-82 (to A, F, or T), suggesting that screening of these residues may be useful in predicting the emergence of resistance. We also extended our previous finding that IDV-resistant viral variants exhibit various patterns of cross-resistance to a diverse panel of HIV-1 protease inhibitors. Finally, we noted an association between the number of protease amino acid substitutions and the observed level of IDV resistance. No single substitution or pair of substitutions tested gave rise to measurable viral resistance to IDV. The evolution of this resistance was found to be cumulative, indicating the need for ongoing viral replication in this process. These observations strongly suggest that therapy should be initiated with the most efficacious regimen available, both to suppress viral spread and to inhibit the replication that is required for the evolution of resistance. PMID:8970946

  17. A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A.

    PubMed

    Zaidi, Nousheen; Burster, Timo; Sommandas, Vinod; Herrmann, Timo; Boehm, Bernhard O; Driessen, Christoph; Voelter, Wolfgang; Kalbacher, Hubert

    2007-12-14

    Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp(43-58) (penetratin), Tat(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.

  18. Identification of a novel inhibitor of dengue virus protease through use of a virtual screening drug discovery Web portal.

    PubMed

    Viswanathan, Usha; Tomlinson, Suzanne M; Fonner, John M; Mock, Stephen A; Watowich, Stanley J

    2014-10-27

    We report the discovery of a novel small-molecule inhibitor of the dengue virus (DENV) protease (NS2B-NS3pro) using a newly constructed Web-based portal (DrugDiscovery@TACC) for structure-based virtual screening. Our drug discovery portal, an extension of virtual screening studies performed using IBM's World Community Grid, facilitated access to supercomputer resources managed by the Texas Advanced Computing Center (TACC) and enabled druglike commercially available small-molecule libraries to be rapidly screened against several high-resolution DENV NS2B-NS3pro crystallographic structures. Detailed analysis of virtual screening docking scores and hydrogen-bonding interactions between each docked ligand and the NS2B-NS3pro Ser135 side chain were used to select molecules for experimental validation. Compounds were ordered from established chemical companies, and compounds with established aqueous solubility were tested for their ability to inhibit DENV NS2B-NS3pro cleavage of a model substrate in kinetic studies. As a proof-of-concept, we validated a small-molecule dihydronaphthalenone hit as a single-digit-micromolar mixed noncompetitive inhibitor of the DENV protease. Since the dihydronaphthalenone was predicted to interact with NS2B-NS3pro residues that are largely conserved between DENV and the related West Nile virus (WNV), we tested this inhibitor against WNV NS2B-NS3pro and observed a similar mixed noncompetitive inhibition mechanism. However, the inhibition constants were ∼10-fold larger against the WNV protease relative to the DENV protease. This novel validated lead had no chemical features or pharmacophores associated with adverse toxicity, carcinogenicity, or mutagenicity risks and thus is attractive for additional characterization and optimization.

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

    PubMed

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

    2013-08-01

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

  20. Simple in vitro translation assay to analyze inhibitors of rhinovirus proteases.

    PubMed Central

    Heinz, B A; Tang, J; Labus, J M; Chadwell, F W; Kaldor, S W; Hammond, M

    1996-01-01

    We have developed a simple in vitro translation method to analyze compounds that inhibit the rhinovirus 3C protease in peptide substrate assays but demonstrate no antiviral activity. This complementary assay, which provides both qualitative and quantitative results, detects the inhibition of the 3CD protease in the native polyprotein form. PMID:8787922

  1. In vitro effects of cysteine protease inhibitors on Trichomonas foetus-induced cytopathic changes in porcine intestinal epithelial cells.

    PubMed

    Tolbert, M Katherine; Brand, Mabre D; Gould, Emily N

    2016-08-01

    OBJECTIVE To investigate the effects of specific cysteine protease (CP) inhibitors on cytopathic changes to porcine intestinal epithelial cells induced by Tritrichomonas foetus isolated from naturally infected cats. SAMPLE T foetus isolates from 4 naturally infected cats and nontransformed porcine intestinal epithelial cells. PROCEDURES T foetus isolates were treated with or without 0.1 to 1.0mM of the CP inhibitors antipain, cystatin, leupeptin, and chymostatin and the vinyl sulfone inhibitors WRR-483 and K11777. In-gel gelatin zymography was performed to evaluate the effects of these inhibitors on CP activity of T foetus isolates. Each treated or untreated isolate was also cocultured with monolayers of porcine intestinal epithelial cells for 24 hours, and cytopathic effects of T foetus were evaluated by light microscopy and crystal violet spectrophotometry. RESULTS Results of in-gel gelatin zymography suggested an ability of WRR-483, K11777, and cystatin to target specific zones of CP activity of the T foetus isolates. These inhibitors had no effect on T foetus growth, and the cytopathic changes to the intestinal epithelium induced by all 4 T foetus isolates were significantly inhibited. CONCLUSIONS AND CLINICAL RELEVANCE This study revealed that certain protease inhibitors were capable of inhibiting regions of CP activity (which has been suggested to cause intestinal cell damage in cats) in T foetus organisms and of ameliorating T foetus-induced cytopathic changes to porcine intestinal epithelium in vitro. Although additional research is needed, these inhibitors might be useful in the treatment of cats with trichomonosis. PMID:27463553

  2. Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily, Cause Nagashima-type Palmoplantar Keratosis

    PubMed Central

    Kubo, Akiharu; Shiohama, Aiko; Sasaki, Takashi; Nakabayashi, Kazuhiko; Kawasaki, Hiroshi; Atsugi, Toru; Sato, Showbu; Shimizu, Atsushi; Mikami, Shuji; Tanizaki, Hideaki; Uchiyama, Masaki; Maeda, Tatsuo; Ito, Taisuke; Sakabe, Jun-ichi; Heike, Toshio; Okuyama, Torayuki; Kosaki, Rika; Kosaki, Kenjiro; Kudoh, Jun; Hata, Kenichiro; Umezawa, Akihiro; Tokura, Yoshiki; Ishiko, Akira; Niizeki, Hironori; Kabashima, Kenji; Mitsuhashi, Yoshihiko; Amagai, Masayuki

    2013-01-01

    “Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK. PMID:24207119

  3. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    SciTech Connect

    Cooper, Jon; Coates, Leighton; Hussey, Robert

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  4. Protease inhibitors partially overcome the interferon nonresponse phenotype in patients with chronic hepatitis C.

    PubMed

    Duarte-Rojo, A; Fischer, S E; Adeyi, O; Zita, D; Deneke, M G; Selzner, N; Chen, L; Malespin, M; Cotler, S J; McGilvray, I D; Feld, J J

    2016-05-01

    The outcome of triple therapy with protease inhibitors (PI) depends on the intrinsic response to interferon. Interferon-stimulated gene (ISG) expression differs by cell type in the liver and is a strong predictor of interferon responsiveness. Patients who respond well to interferon have low/absent ISG expression in hepatocytes but significant ISG expression in macrophages. Nonresponders (NRs) show the opposite pattern. We aimed to determine the association between cell-type-specific ISG staining and treatment outcome with PI-based triple therapy. Liver biopsy tissue from consecutive patients treated with boceprevir or telaprevir with peginterferon and ribavirin was stained for myxovirus A (MxA). Staining was scored 0-3 in macrophages (M-MxA) and hepatocytes (H-MxA), and IL28B genotyping was performed. Of 56 patients included 41 achieved SVR (73%) (sustained virological response), 2 (4%) relapsed, 10 (18%) were NRs, and 3 (5%) were lost to follow-up. Median M-MxA staining was stronger and H-MxA staining was weaker in patients who achieved SVR. MxA staining correlated with IL28B genotype and with the HCV RNA decline during lead-in phase. However, unlike with dual therapy, the negative predictive value (NPV) of absent or weak M-MxA staining was poor (42%), while the positive predictive value improved (93%). Although by multivariable logistic regression M-MxA staining was significantly associated with SVR (OR 4.35, 1.32-14.28, P = 0.012), the predictive ability was inadequate to withhold therapy. The interaction between macrophages and hepatocytes plays a critical role in interferon responsiveness; however, the addition of a PI at least partially overcomes the interferon nonresponse phenotype making the predictive ability of ISG staining less clinically useful. PMID:26710754

  5. Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection.

    PubMed

    Skeate, Joseph G; Porras, Tania B; Woodham, Andrew W; Jang, Julie K; Taylor, Julia R; Brand, Heike E; Kelly, Thomas J; Jung, Jae U; Da Silva, Diane M; Yuan, Weiming; Kast, W Martin

    2016-02-01

    Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.

  6. Species-Specific Serological Detection for Schistosomiasis by Serine Protease Inhibitor (SERPIN) in Multiplex Assay

    PubMed Central

    Tanigawa, Chihiro; Fujii, Yoshito; Miura, Masashi; Nzou, Samson Muuo; Mwangi, Anne Wanjiru; Nagi, Sachiyo; Hamano, Shinjiro; Njenga, Sammy M.; Mbanefo, Evaristus Chibunna; Hirayama, Kenji; Mwau, Matilu; Kaneko, Satoshi

    2015-01-01

    Background Both Schistosoma mansoni and Schistosoma haematobium cause schistosomiasis in sub-Saharan Africa. We assessed the diagnostic value of selected Schistosoma antigens for the development of a multiplex serological immunoassay for sero-epidemiological surveillance. Methodology/Principal Findings Diagnostic ability of recombinant antigens from S. mansoni and S. haematobium was assessed by Luminex multiplex immunoassay using plasma from school children in two areas of Kenya, endemic for different species of schistosomiasis. S. mansoni serine protease inhibitor (SERPIN) and Sm-RP26 showed significantly higher reactivity to patient plasma as compared to the control group. Sm-Filamin, Sm-GAPDH, Sm-GST, Sm-LAP1, Sm-LAP2, Sm-Sm31, Sm-Sm32 and Sm-Tropomyosin did not show difference in reactivity between S. mansoni infected and uninfected pupils. Sm-RP26 was cross-reactive to plasma from S. haematobium patients, whereas Sm-SERPIN was species-specific. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. ROC analysis for Sm-RP26, Sm-SERPIN and Sh-SERPIN showed AUC values of 0.833, 0.888 and 0.947, respectively. Using Spearman’s rank correlation coefficient analysis, we also found significant positive correlation between the number of excreted eggs and median fluorescence intensity (MFI) from the multiplex immunoassays for Sm-SERPIN (ρ = 0.430, p-value = 0.003) and Sh-SERPIN (ρ = 0.433, p-value = 0.006). Conclusions/Significance Sm-SERPIN is a promising species-specific diagnostic antigen. Sh-SEPRIN was partially cross-reactive to S. mansoni infected patients. SERPINs showed correlation with the number of excreted eggs. These indicate prospects for inclusion of SERPINs in the multiplex serological immunoassay system. PMID:26291988

  7. Structural and electronic properties of new fullerene derivatives and their possible application as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Ibrahim, Medhat; Saleh, Noha A.; Hameed, Ali Jameel; Elshemey, Wael M.; Elsayed, Anwar A.

    2010-02-01

    Density functional theory (DFT) calculations have been carried out at the hybrid Becke 3-Lee-Yang-Parr; B3LYP/3-21G** level of theory to study two series of hydroxy-chalca-acetic acid-(4-pyrrolidin-1-yl-phenyl) ester [C 60-C 2H 4N-(4-XCOCH 2OH)C 6H 4] and hydroxy-chalcoacetic acid-[2-(2-hydroxy-acetylchalcanyl)-4-pyrrolidin-1-yl-phenyl] ester[C 60-C 2H 4N-(3,4-XCOCH 2OH)C 6H 4]. The X atom is O, S or Se for the two series. The vibrational spectra, physical, chemical, thermodynamics and Quantitative Structure Activity Relationship (QSAR) properties of the studied molecules are calculated and discussed. We have evaluated these molecules as HIV-1 protease inhibitors based on the hydrogenation interaction between the hydroxymethylcarbonyl (HMC) groups and the two aspartic acid of the HIV-1 protease active site. Results show that some of the investigated fullerene-based derivatives can be considered promising as HIV-1 protease inhibitors.

  8. Transcript profiling reveals that cysteine protease inhibitors are up-regulated in tuber sprouts after extended darkness.

    PubMed

    Grandellis, Carolina; Giammaria, Veronica; Fantino, Elisa; Cerrudo, Ignacio; Bachmann, Sandra; Santin, Franco; Ulloa, Rita M

    2016-07-01

    Potato (Solanum tuberosum L.) tubers are an excellent staple food due to its high nutritional value. When the tuber reaches physiological competence, sprouting proceeds accompanied by changes at mRNA and protein levels. Potato tubers become a source of carbon and energy until sprouts are capable of independent growth. Transcript profiling of sprouts grown under continuous light or dark conditions was performed using the TIGR 10K EST Solanaceae microarray. The profiles analyzed show a core of highly expressed transcripts that are associated to the reactivation of growth. Under light conditions, the photosynthetic machinery was fully activated; the highest up-regulation was observed for the Rubisco activase (RCA), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the Photosystem II 22 kDa protein (CP22) genes, among others. On the other hand, sprouts exposed to continuous darkness elongate longer, and after extended darkness, synthesis of chloroplast components was repressed, the expression of proteases was reduced while genes encoding cysteine protease inhibitors (CPIs) and metallocarboxypeptidase inhibitors (MPIs) were strongly induced. Northern blot and RT-PCR analysis confirmed that MPI levels correlated with the length of the dark period; however, CPI expression was strong only after longer periods of darkness, suggesting a feedback loop (regulation mechanism) in response to dark-induced senescence. Prevention of cysteine protease activity in etiolated sprouts exposed to extended darkness could delay senescence until they emerge to light.

  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. PMID:26456256

  10. Crystallization of a Nonclassical Kazal-type Carcinoscorpius Rotundicauda Serine Protease Inhibitor, CrSPI-1, Complexed with Subtilisin

    SciTech Connect

    Tulsidas, S.; Thangamani, S; Ho, B; Sivaraman, J; Ding, J

    2009-01-01

    Serine proteases play a major role in host-pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 {angstrom}resolution and belonged to space group P2{sub 1}, with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 {angstrom}, {beta} = 95.4. The Matthews coefficient (VM = 2.64 {angstrom}3 Da-1, corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.

  11. Abacavir/Lamivudine Versus Tenofovir/Emtricitabine in Virologically Suppressed Patients Switching from Ritonavir-Boosted Protease Inhibitors to Raltegravir

    PubMed Central

    d'Albuquerque, Polyana M.; Pérez, Ignacio; Pich, Judit; Gatell, José M.

    2013-01-01

    Abstract There are few clinical data on the combination abacavir/lamivudine plus raltegravir. We compared the outcomes of patients from the SPIRAL trial receiving either abacavir/lamivudine or tenofovir/emtricitabine at baseline who had taken at least one dose of either raltegravir or ritonavir-boosted protease inhibitors. For the purpose of this analysis, treatment failure was defined as virological failure (confirmed HIV-1 RNA ≥50 copies/ml) or discontinuation of abacavir/lamivudine or tenofovir/emtricitabine because of adverse events, consent withdrawal, or lost to follow-up. There were 143 (72.59%) patients with tenofovir/emtricitabine and 54 (27.41%) with abacavir/lamivudine. In the raltegravir group, there were three (11.11%) treatment failures with abacavir/lamivudine and eight (10.96%) with tenofovir/emtricitabine (estimated difference 0.15%; 95% CI −17.90 to 11.6). In the ritonavir-boosted protease inhibitor group, there were four (14.81%) treatment failures with abacavir/lamivudine and 12 (17.14%) with tenofovir/emtricitabine (estimated difference −2.33%; 95% CI −16.10 to 16.70). Triglycerides decreased and HDL cholesterol increased through the study more pronouncedly with abacavir/lamivudine than with tenofovir/emtricitabine and differences in the total-to-HDL cholesterol ratio between both combinations of nucleoside reverse transcriptase inhibitors (NRTIs) tended to be higher in the raltegravir group, although differences at 48 weeks were not significant. While no patient discontinued abacavir/lamivudine due to adverse events, four (2.80%) patients (all in the ritonavir-boosted protease inhibitor group) discontinued tenofovir/emtricitabine because of adverse events (p=0.2744). The results of this analysis do not suggest that outcomes of abacavir/lamivudine are worse than those of tenofovir/emtricitabine when combined with raltegravir in virologically suppressed HIV-infected adults. PMID:22916715

  12. Abacavir/lamivudine versus tenofovir/emtricitabine in virologically suppressed patients switching from ritonavir-boosted protease inhibitors to raltegravir.

    PubMed

    Martínez, Esteban; d'Albuquerque, Polyana M; Pérez, Ignacio; Pich, Judit; Gatell, José M

    2013-02-01

    There are few clinical data on the combination abacavir/lamivudine plus raltegravir. We compared the outcomes of patients from the SPIRAL trial receiving either abacavir/lamivudine or tenofovir/emtricitabine at baseline who had taken at least one dose of either raltegravir or ritonavir-boosted protease inhibitors. For the purpose of this analysis, treatment failure was defined as virological failure (confirmed HIV-1 RNA ≥50 copies/ml) or discontinuation of abacavir/lamivudine or tenofovir/emtricitabine because of adverse events, consent withdrawal, or lost to follow-up. There were 143 (72.59%) patients with tenofovir/emtricitabine and 54 (27.41%) with abacavir/lamivudine. In the raltegravir group, there were three (11.11%) treatment failures with abacavir/lamivudine and eight (10.96%) with tenofovir/emtricitabine (estimated difference 0.15%; 95% CI -17.90 to 11.6). In the ritonavir-boosted protease inhibitor group, there were four (14.81%) treatment failures with abacavir/lamivudine and 12 (17.14%) with tenofovir/emtricitabine (estimated difference -2.33%; 95% CI -16.10 to 16.70). Triglycerides decreased and HDL cholesterol increased through the study more pronouncedly with abacavir/lamivudine than with tenofovir/emtricitabine and differences in the total-to-HDL cholesterol ratio between both combinations of nucleoside reverse transcriptase inhibitors (NRTIs) tended to be higher in the raltegravir group, although differences at 48 weeks were not significant. While no patient discontinued abacavir/lamivudine due to adverse events, four (2.80%) patients (all in the ritonavir-boosted protease inhibitor group) discontinued tenofovir/emtricitabine because of adverse events (p=0.2744). The results of this analysis do not suggest that outcomes of abacavir/lamivudine are worse than those of tenofovir/emtricitabine when combined with raltegravir in virologically suppressed HIV-infected adults. PMID:22916715

  13. Molecular Modeling and Docking Study to Elucidate Novel Chikungunya Virus nsP2 Protease Inhibitors

    PubMed Central

    Agarwal, T.; Asthana, Somya; Bissoyi, A.

    2015-01-01

    Chikungunya is one of the tropical viral infections that severely affect the Asian and African countries. Absence of any suitable drugs or vaccines against Chikungunya virus till date makes it essential to identify and develop novel leads for the same. Recently, nsP2 cysteine protease has been classified as a crucial drug target to combat infections caused by Alphaviruses including Chikungunya virus due to its involvement viral replication. Here in, we investigated the structural aspects of the nsP2 protease through homology modeling based on nsP2 protease from Venezuelan equine encephalitis virus. Further, the ligands were virtually screened based on various pharmacological, ADME/Tox filters and subjected to docking with the modeled Chikungunya nsP2 protease using AutoDock4.2. The interaction profiling of ligand with the protein was carried out using LigPlot+. The results demonstrated that the ligand with PubChem Id (CID_5808891) possessed highest binding affinity towards Chikungunya nsP2 protease with a good interaction profile with the active site residues. We hereby propose that these compounds could inhibit the nsP2 protease by binding to its active site. Moreover, they may provide structural scaffold for the design of novel leads with better efficacy and specificity for the nsP2 protease. PMID:26664062

  14. Characterization of HIV drug resistance mutations among patients failing first-line antiretroviral therapy from a tertiary referral center in Lusaka, Zambia.

    PubMed

    Seu, Lillian; Mulenga, Lloyd B; Siwingwa, Mpanji; Sikazwe, Izukanji; Lambwe, Nason; Guffey, M Bradford; Chi, Benjamin H

    2015-07-01

    In settings of resource constraint, an understanding of HIV drug resistance can guide antiretroviral therapy (ART) at switch to second-line therapy. To determine the prevalence of such HIV drug resistance mutations (HIV DRM), we used an in-house sequencing assay in the pol gene (protease and partial reverse transcriptase) in a cohort of patients suspected of failing a first-line regimen, which in Zambia comprises two nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI). Our analysis cohort (n = 68) was referred to the University Teaching Hospital in Lusaka from November 2009 to October 2012. Median duration on first-line ART to suspected treatment failure was 3.2 years (IQR 1.7-4.7 years). The majority of patients (95%) harbored HIV-1 subtype C virus. Analysis of reverse transcriptase revealed M184V (88%), K103N/S (32%), and Y181C/I/V (41%) DRMs, with the latter conferring reduced susceptibility to the salvage therapy candidates etravirine and rilpivirine. Three patients (5%) had major protease inhibitor (PI) resistance mutations: all three had the V82A mutation, and one patient (Clade J virus) had a concurrent M46I, Q58E, and L76V DRM. HIV-1 genotyping revealed major and minor DRMs as well as high levels of polymorphisms in subtype C isolates from patients failing first-line antiretroviral therapy. Closer monitoring of DRM mutations at first-line failure can inform clinicians about future options for salvage therapy.

  15. Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.

    PubMed

    Franco, Francisco M; Jones, Darin E; Harris, Peter K W; Han, Zhenfu; Wildman, Scott A; Jarvis, Cassie M; Janetka, James W

    2015-05-15

    Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.

  16. Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.

    PubMed

    Franco, Francisco M; Jones, Darin E; Harris, Peter K W; Han, Zhenfu; Wildman, Scott A; Jarvis, Cassie M; Janetka, James W

    2015-05-15

    Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA. PMID:25882520

  17. Protease Inhibitor-Resistant Hepatitis C Virus Mutants with Reduced Fitness from Impaired Production of Infectious Virus

    PubMed Central

    Shimakami, Tetsuro; Welsch, Christoph; Yamane, Daisuke; McGivern, David; Yi, MinKyung; Zeuzem, Stefan; Lemon, Stanley M.

    2010-01-01

    Background & Aims Several small molecule inhibitors of the hepatitis C virus (HCV) NS3/4A protease have advanced successfully to clinical trials. However, the selection of drug-resistant mutants is a significant issue with protease inhibitors (PIs). A variety of amino acid substitutions in the protease domain of NS3 can lead to PI resistance. Many of these significantly impair the replication fitness of HCV RNA replicons. However, it is not known whether these mutations also adversely affect infectious virus assembly and release, processes in which NS3 also participates. Methods We studied the impact of 25 previously identified PI-resistance mutations on the capacity of genotype 1a H77S RNA to replicate in cell culture and produce infectious virus. Results Most PI-resistance mutations resulted in moderate loss of replication competence, although several (V36A/L/M, R109K, and D168E) demonstrated fitness comparable to wild type, whereas others (S138T and A156V) were severely impaired both in RNA replication and infectious virus production. Although reductions in RNA replication capacity correlated well with decreased yields of infectious virus for most mutations, a subset of mutants (Q41R, F43S, R155T, A156S, and I170A/T) reproducibly demonstrated greater impairment in their ability to produce virus than predicted from reductions in RNA replication capacity. More detailed examination of the I170A mutant demonstrated no defect in release of virus from cells, and no significant difference in specific infectivity of extracellular virus particles. Conclusions Replicon-based assays might underestimate the loss of fitness caused by PI-resistance mutations, as some mutations in the NS3 protease domain specifically impair late steps in the viral lifecycle that involve intracellular assembly of infectious virus. PMID:21056040

  18. 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.

  19. 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

    2014-01-01

    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. PMID:25361421

  20. Purification of a cysteine protease inhibitor from larval hemolymph of the Tobacco Hornworm (Manduca sexta) and functional expression of the recombinant protein.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5 kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration of Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, ...

  1. Comparison of efavirenz and protease inhibitor based combination antiretroviral therapy regimens in treatment-naïve people living with HIV with baseline resistance.

    PubMed

    Lim, Charlotte; McFaul, Katie; Kabagambe, Samuel; Sonecha, Sonali; Jones, Rachael; Asboe, David; Pozniak, Anton; Nwokolo, Nneka; Boffito, Marta

    2016-07-17

    A retrospective cohort analysis comparing the efficacy of boosted protease inhibitor-based and efavirenz-based combination antiretroviral therapy in treatment-naïve people living with HIV with baseline resistance found that efavirenz-based treatment led to a shorter mean time to undetectable viral load. A higher proportion of patients with nonnucleoside reverse transcriptase inhibitor related baseline resistance mutations in the efavirenz-treatment group achieved an undetectable viral load at both 6 and 12 months post-treatment initiation, compared with the boosted protease-inhibitor-treatment group.Supplementary content: http://links.lww.com/QAD/A930. PMID:27139315

  2. A selective cysteine protease inhibitor is non-toxic and cerebroprotective in rats undergoing transient middle cerebral artery ischemia.

    PubMed

    Seyfried, D M; Veyna, R; Han, Y; Li, K; Tang, N; Betts, R L; Weinsheimer, S; Chopp, M; Anagli, J

    2001-05-18

    Ischemic neuronal injury mediated by cysteine proteases such as calpains and caspases has been demonstrated in various experimental models. Cathepsins B and L are also cysteine proteases which may contribute to neuronal death after ischemia. The authors measured in vitro and in vivo toxicity and post-ischemic cytoprotective effects of a cysteine protease inhibitor which does not block calpain or caspase but, rather, is relatively selective for cathepsins B and L. The compound belongs to the peptidyl-diazomethane family (cysteine protease inhibitor 1, termed CP-1). In vitro toxicity was measured using an assay of cell viability, and in vivo toxicity was measured by histological tissue analysis after infusion of CP-1 in rats. Two hours of middle cerebral artery (MCA) occlusion in rats was performed by the intravascular suture method. Immediately following reperfusion, intravenous infusion of CP-1 or vehicle was performed for 4 h at 0.9 ml/h. After a 7-day survival, the infarct volumes were measured. CP-1 was non-toxic to cultured glial cells to a local concentration of 200 microM, and relatively non-toxic to cultured endothelial cells at concentrations of 100-200 microM. No animal exhibited toxic effects at any of the doses used. Histologic comparisons revealed no signs of tissue toxicity. CP-1 significantly reduced hemispheric infarct volume compared to control (37+/-8.2%) at concentrations of 10, 50, and 250 microM [22+/-15%, P=0.008; 20+/-13%, P=0.002; 23+/-15%, P=0.022, respectively (mean+/-standard deviation; N=7-10 per group)]. CP-1, at the concentration of 50 microM, improved the functional score of the animals, but did not significantly alter cerebral blood flow. This study supports the hypothesis that the lysosomal cathepsins B and/or L contribute to cerebral injury after focal ischemia with reperfusion. Cysteine protease inhibitors which are relatively selective for cathepsins B and L, but not the calpains or caspases, are effective at reducing infarct

  3. Clinical pharmacology profile of boceprevir, a hepatitis C virus NS3 protease inhibitor: focus on drug-drug interactions.

    PubMed

    Khalilieh, Sauzanne; Feng, Hwa-Ping; Hulskotte, Ellen G J; Wenning, Larissa A; Butterton, Joan R

    2015-06-01

    Boceprevir is a potent, orally administered ketoamide inhibitor that targets the active site of the hepatitis C virus (HCV) non-structural (NS) 3 protease. The addition of boceprevir to peginterferon plus ribavirin resulted in higher rates of sustained virologic response (SVR) than for peginterferon plus ribavirin alone in phase III studies in both previously treated and untreated patients with HCV infection. Because boceprevir is metabolized by metabolic routes common to many other drugs, and is an inhibitor of cytochrome P450 (CYP) 3A4/5, there is a high potential for drug-drug interactions when boceprevir is administered with other therapies, particularly when treating patients with chronic HCV infection who are often receiving other medications concomitantly. Boceprevir is no longer widely used in the US or EU due to the introduction of second-generation treatments for HCV infection. However, in many other geographic regions, first-generation protease inhibitors such as boceprevir continue to form an important treatment option for patients with HCV infection. This review summarizes the interactions between boceprevir and other therapeutic agents commonly used in this patient population, indicating dose adjustment requirements where needed. Most drug interactions do not affect boceprevir plasma concentrations to a clinically meaningful extent, and thus efficacy is likely to be maintained when boceprevir is coadministered with the majority of other therapeutics. Overall, the drug-drug interaction profile of boceprevir suggests that this agent is suitable for use in a wide range of HCV-infected patients receiving concomitant therapies. PMID:25787025

  4. The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

    PubMed

    Lung, Oliver; Tram, Uyen; Finnerty, Casey M; Eipper-Mains, Marcie A; Kalb, John M; Wolfner, Mariana F

    2002-01-01

    Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating. PMID:11805057

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

    PubMed

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

    2016-08-19

    Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to "shotgun" clone the cDNAs encoding two novel but structurally-related peptides from the lyophilised skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin. PMID:27311856

  6. Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: lessons learned from HIV-1 protease inhibition.

    PubMed

    Shen, Yang; Radhakrishnan, Mala L; Tidor, Bruce

    2015-02-01

    Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV-1 protease inhibition and the molecular designs targeted a panel of wild-type and drug-resistant mutant HIV-1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders.

  7. Characterisation of a secretory serine protease inhibitor (SjB6) from Schistosoma japonicum

    PubMed Central

    2014-01-01

    Background Proteins belonging to the serine protease inhibitor (serpin) superfamily play essential physiological roles in many organisms. In pathogens, serpins are thought to have evolved specifically to limit host immune responses by interfering with the host immune-stimulatory signals. Serpins are less well characterised in parasitic helminths, although some are thought to be involved in mechanisms associated with host immune modulation. In this study, we cloned and partially characterised a secretory serpin from Schistosoma japonicum termed SjB6, these findings provide the basis for possible functional roles. Methods SjB6 gene was identified through database mining of our previously published microarray data, cloned and detailed sequence and structural analysis and comparative modelling carried out using various bioinformatics and proteomics tools. Gene transcriptional profiling was determined by real-time PCR and the expression of native protein determined by immunoblotting. An immunological profile of the recombinant protein produced in insect cells was determined by ELISA. Results SjB6 contains an open reading frame of 1160 base pairs that encodes a protein of 387 amino acid residues. Detailed sequence analysis, comparative modelling and structural-based alignment revealed that SjB6 contains the essential structural motifs and consensus secondary structures typical of inhibitory serpins. The presence of an N-terminal signal sequence indicated that SjB6 is a secretory protein. Real-time data indicated that SjB6 is expressed exclusively in the intra-mammalian stage of the parasite life cycle with its highest expression levels in the egg stage (p < 0.0001). The native protein is approximately 60 kDa in size and recombinant SjB6 (rSjB6) was recognised strongly by sera from rats experimentally infected with S. japonicum. Conclusions The significantly high expression of SjB6 in schistosome eggs, when compared to other life cycle stages, suggests a possible

  8. Cost-Effectiveness of Tenofovir Instead of Zidovudine for Use in First-Line Antiretroviral Therapy in Settings without Virological Monitoring

    PubMed Central

    von Wyl, Viktor; Cambiano, Valentina; Jordan, Michael R.; Bertagnolio, Silvia; Miners, Alec; Pillay, Deenan; Lundgren, Jens; Phillips, Andrew N.

    2012-01-01

    Background The most recent World Health Organization (WHO) antiretroviral treatment guidelines recommend the inclusion of zidovudine (ZDV) or tenofovir (TDF) in first-line therapy. We conducted a cost-effectiveness analysis with emphasis on emerging patterns of drug resistance upon treatment failure and their impact on second-line therapy. Methods We used a stochastic simulation of a generalized HIV-1 epidemic in sub-Saharan Africa to compare two strategies for first-line combination antiretroviral treatment including lamivudine, nevirapine and either ZDV or TDF. Model input parameters were derived from literature and, for the simulation of resistance pathways, estimated from drug resistance data obtained after first-line treatment failure in settings without virological monitoring. Treatment failure and cost effectiveness were determined based on WHO definitions. Two scenarios with optimistic (no emergence; base) and pessimistic (extensive emergence) assumptions regarding occurrence of multidrug resistance patterns were tested. Results In the base scenario, cumulative proportions of treatment failure according to WHO criteria were higher among first-line ZDV users (median after six years 36% [95% simulation interval 32%; 39%]) compared with first-line TDF users (31% [29%; 33%]). Consequently, a higher proportion initiated second-line therapy (including lamivudine, boosted protease inhibitors and either ZDV or TDF) in the first-line ZDV user group 34% [31%; 37%] relative to first-line TDF users (30% [27%; 32%]). At the time of second-line initiation, a higher proportion (16%) of first-line ZDV users harboured TDF-resistant HIV compared with ZDV-resistant viruses among first-line TDF users (0% and 6% in base and pessimistic scenarios, respectively). In the base scenario, the incremental cost effectiveness ratio with respect to quality adjusted life years (QALY) was US$83 when TDF instead of ZDV was used in first-line therapy (pessimistic scenario: US$ 315), which

  9. Design, Synthesis, and X-ray Structure of Substituted Bis-tetrahydrofuran (Bis-THF)-Derived Potent HIV-1 Protease Inhibitors

    SciTech Connect

    Ghosh, Arun K.; Martyr, Cuthbert D.; Steffey, Melinda; Wang, Yuan-Fang; Agniswamy, Johnson; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2012-06-18

    We investigated substituted bis-THF-derived HIV-1 protease inhibitors in order to enhance ligand-binding site interactions in the HIV-1 protease active site. In this context, we have carried out convenient syntheses of optically active bis-THF and C4-substituted bis-THF ligands using a [2,3]-sigmatropic rearrangement as the key step. The synthesis provided convenient access to a number of substituted bis-THF derivatives. Incorporation of these ligands led to a series of potent HIV-1 protease inhibitors. Inhibitor 23c turned out to be the most potent (K{sub i} = 2.9 pM; IC{sub 50} = 2.4 nM) among the inhibitors. An X-ray structure of 23c-bound HIV-1 protease showed extensive interactions of the inhibitor with the protease active site, including a unique water-mediated hydrogen bond to the Gly-48 amide NH in the S2 site.

  10. Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies

    SciTech Connect

    Ghosh, Arun K.; Leshchenko-Yashchuk, Sofiya; Anderson, David D.; Baldridge, Abigail; Noetzel, Marcus; Miller, Heather B.; Tie, Yunfeng; Wang, Yuan-Fang; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki

    2009-09-02

    Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1{prime}-ligands. These ligands are designed to interact with Gly-27{prime} carbonyl and Arg-8 side chain in the S1{prime}-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1{prime}-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1{prime} and S2 subsites of HIV-1 protease.

  11. HIV-protease inhibitors block the replication of both vesicular stomatitis and influenza viruses at an early post-entry replication step

    SciTech Connect

    Federico, Maurizio

    2011-08-15

    The inhibitors of HIV-1 protease (PIs) have been designed to block the activity of the viral aspartyl-protease. However, it is now accepted that this family of inhibitors can also affect the activity of cell proteases. Since the replication of many virus species requires the activity of host cell proteases, investigating the effects of PIs on the life cycle of viruses other than HIV would be of interest. Here, the potent inhibition induced by saquinavir and nelfinavir on the replication of both vesicular stomatitis and influenza viruses is described. These are unrelated enveloped RNA viruses infecting target cells upon endocytosis and intracellular fusion. The PI-induced inhibition was apparently a consequence of a block at the level of the fusion between viral envelope and endosomal membranes. These findings would open the way towards the therapeutic use of PIs against enveloped RNA viruses other than HIV.

  12. Characterization of 8-hydroxyquinoline derivatives containing aminobenzothiazole as inhibitors of dengue virus type 2 protease in vitro

    PubMed Central

    Lai, Huiguo; Prasad, G. Sridhar; Padmanabhan, Radhakrishnan

    2013-01-01

    Four serotypes of Dengue virus (DENV1–4), mosquito-borne members of Flaviviridae family cause frequent epidemics causing considerable morbidity and mortality in humans throughout tropical regions of the world. There is no vaccine or antiviral therapeutics available for human use. In a previous study, we reported that compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of West Nile virus serine protease. In this study, we analyzed potencies of some compounds with (8-HQ)-aminobenzothiazole derivatives for inhibition of DENV2 protease in vitro. We identified analogs 1–4 with 2-aminothiazole or 2-aminobenzothiazole scaffold with submicromolar potencies (IC50) in the in vitro protease assays. The kinetic constant (Ki) for the most potent 8-HQ-aminobenzothiazoleinhibitor (compound 1) with an IC50 value of 0.91 ± 0.05 µM was determined to be 2.36 ± 0.13 µM. This compound inhibits the DENV2 NS2B/NS3pro by a competitive mode of inhibition. PMID:23127365

  13. The QSAR and docking calculations of fullerene derivatives as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Saleh, Noha A.

    2015-02-01

    The inhibition of HIV-1 protease is considered as one of the most important targets for drug design and the deactivation of HIV-1. In the present work, the fullerene surface (C60) is modified by adding oxygen atoms as well as hydroxymethylcarbonyl (HMC) groups to form 6 investigated fullerene derivative compounds. These compounds have one, two, three, four or five O atoms + HMC groups at different positions on phenyl ring. The effect of the repeating of these groups on the ability of suggested compounds to inhibit the HIV protease is studied by calculating both Quantitative Structure Activity Relationship (QSAR) properties and docking simulation. Based on the QSAR descriptors, the solubility and the hydrophilicity of studied fullerene derivatives increased with increasing the number of oxygen atoms + HMC groups in the compound. While docking calculations indicate that, the compound with two oxygen atoms + HMC groups could interact and binds with HIV-1 protease active site. This is could be attributed to the active site residues of HIV-1 protease are hydrophobic except the two aspartic acids. So that, the increase in the hydrophilicity and polarity of the compound is preventing and/or decreasing the hydrophobic interaction between the compound and HIV-1 protease active site.

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

    PubMed

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

    2008-10-01

    Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities. PMID:18582511

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

    PubMed

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

    2008-10-01

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

  16. Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis

    PubMed Central

    Habgood, Anthony N; Tatler, Amanda L; Porte, Joanne; Wahl, Sharon M; Laurent, Geoffrey J; John, Alison E; Johnson, Simon R; Jenkins, Gisli

    2016-01-01

    Idiopathic Pulmonary Fibrosis (IPF) is a progressive, fatal disease with limited treatment options. Protease mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi−/− mice following injury. Slpi−/−, and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase-2 (MMP-2) and MMP-9 were measured. Lung fibrosis was determined by collagen subtype specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi−/− animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post-bleomycin. In contrast Slpi−/− mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi−/− mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1 and Col4α1 gene

  17. Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis.

    PubMed

    Habgood, Anthony N; Tatler, Amanda L; Porte, Joanne; Wahl, Sharon M; Laurent, Geoffrey J; John, Alison E; Johnson, Simon R; Jenkins, Gisli

    2016-06-01

    Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi(-/-) mice following injury. Slpi(-/-), and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi(-/-) animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi(-/-) mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi(-/-) mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression

  18. Crystal structure of chemically synthesized HIV-1 protease and a ketomethylene isostere inhibitor based on the p2/NC cleavage site

    SciTech Connect

    Torbeev, Vladimir Yu.; Mandal, Kalyaneswar; Terechko, Valentina A.; Kent, Stephen B.H.

    2009-09-02

    Here we report the X-ray structures of chemically synthesized HIV-1 protease and the inactive [D25N]HIV-1 protease complexed with the ketomethylene isostere inhibitor Ac-Thr-Ile-Nle{psi}[CO-CH{sub 2}]Nle-Gln-Arg.amide at 1.4 and 1.8 {angstrom} resolution, respectively. In complex with the active enzyme, the keto-group was found to be converted into the hydrated gem-diol, while the structure of the complex with the inactive D25N enzyme revealed an intact keto-group. These data support the general acid-general base mechanism for HIV-1 protease catalysis.

  19. Highly Potent HIV-1 Protease Inhibitors with Novel Tricyclic P2 Ligands: Design, Synthesis, and Protein-Ligand X-ray Studies

    SciTech Connect

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

    2013-10-08

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

  20. Design of HIV-1 Protease Inhibitors with C3-Substituted Hexahydrocyclopentafuranyl Urethanes as P2-Ligands: Synthesis, Biological Evaluation, and Protein-Ligand X-ray Crystal Structure

    SciTech Connect

    Ghosh, Arun K; Chapsal, Bruno D; Parham, Garth L; Steffey, Melinda; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T; Mitsuya, Hiroaki

    2011-11-07

    We report the design, synthesis, biological evaluation, and the X-ray crystal structure of a novel inhibitor bound to the HIV-1 protease. Various C3-functionalized cyclopentanyltetrahydrofurans (Cp-THF) were designed to interact with the flap Gly48 carbonyl or amide NH in the S2-subsite of the HIV-1 protease. We investigated the potential of those functionalized ligands in combination with hydroxyethylsulfonamide isosteres. Inhibitor 26 containing a 3-(R)-hydroxyl group on the Cp-THF core displayed the most potent enzyme inhibitory and antiviral activity. Our studies revealed a preference for the 3-(R)-configuration over the corresponding 3-(S)-derivative. Inhibitor 26 exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray structure of 26-bound HIV-1 protease revealed important molecular insight into the ligand-binding site interactions.

  1. A Treatment with a Protease Inhibitor Recombinant from the Cattle Tick (Rhipicephalus Boophilus microplus) Ameliorates Emphysema in Mice

    PubMed Central

    Lourenço, Juliana D.; Neves, Luana P.; Olivo, Clarice R.; Duran, Adriana; Almeida, Francine M.; Arantes, Petra M. M.; Prado, Carla M.; Leick, Edna Aparecida; Tanaka, Aparecida S.; Martins, Mílton A.; Sasaki, Sergio D.; Lopes, Fernanda D. T. Q. S.

    2014-01-01

    Aims To determine whether a serine protease inhibitor treatment can prevent or minimize emphysema in mice. Methods C57BL/6 mice were subjected to porcine pancreatic elastase (PPE) nasal instillation to induce emphysema and were treated with a serine protease inhibitor (rBmTI-A) before (Protocol 1) and after (Protocol 2) emphysema development. In both protocols, we evaluated lung function to evaluate the airway resistance (Raw), tissue damping (Gtis) and tissue elastance (Htis). The inflammatory profile was analyzed in the bronchoalveolar lavage (BALF) and through the use of morphometry; we measured the mean linear intercept (Lm) (to verify alveolar enlargement), the volume proportion of collagen and elastic fibers, and the numbers of macrophages and metalloprotease 12 (MMP-12) positive cells in the parenchyma. We showed that at both time points, even after the emphysema was established, the rBmTI-A treatment was sufficient to reverse the loss of elastic recoil measured by Htis, the alveolar enlargement and the increase in the total number of cells in the BALF, with a primary decrease in the number of macrophages. Although, the treatment did not control the increase in macrophages in the lung parenchyma, it was sufficient to decrease the number of positive cells for MMP-12 and reduce the volume of collagen fibers, which was increased in PPE groups. These findings attest to the importance of MMP-12 in PPE-induced emphysema and suggest that this metalloprotease could be an effective therapeutic target. PMID:24886716

  2. Evaluation of novel Saquinavir analogs for resistance mutation compatibility and potential as an HIV-Protease inhibitor drug

    PubMed Central

    Jayaswal, Amit; Mishra, Ankita; Mishra, Hirdyesh; Shah, Kavita

    2014-01-01

    A fundamental issue related to therapy of HIV-1 infection is the emergence of viral mutations which severely limits the long term efficiency of the HIV-protease (HIV-PR) inhibitors. Development of new drugs is therefore continuously needed. Chemoinformatics enables to design and discover novel molecules analogous to established drugs using computational tools and databases. Saquinavir, an anti-HIV Protease drug is administered for HIV therapy. In this work chemoinformatics tools were used to design structural analogs of Saquinavir as ligand and molecular dockings at AutoDock were performed to identify potential HIV-PR inhibitors. The analogs S1 and S2 when docked with HIV-PR had binding energies of -4.08 and -3.07 kcal/mol respectively which were similar to that for Saquinavir. The molecular docking studies revealed that the changes at N2 of Saquinavir to obtain newly designed analogs S1 (having N2 benzoyl group at N1) and S2 (having 3-oxo-3phenyl propanyl group at N2) were able to dock with HIV-PR with similar affinity as that of Saquinavir. Docking studies and computationally derived pharmacodynamic and pharmacokinetic properties׳ comparisons at ACD/I-lab establish that analog S2 has more potential to evade the problem of drug resistance mutation against HIV-1 PR subtype-A. S2 can be further developed and tested clinically as a real alternative drug for HIV-1 PR across the clades in future. PMID:24966525

  3. P1-Substituted Symmetry-Based Human Immunodeficiency Virus Protease Inhibitors with Potent Antiviral Activity against Drug-Resistant Viruses

    SciTech Connect

    DeGoey, David A.; Grampovnik, David J.; Chen, Hui-Ju; Flosi, William J.; Klein, Larry L.; Dekhtyar, Tatyana; Stoll, Vincent; Mamo, Mulugeta; Molla, Akhteruzzaman; Kempf, Dale J.

    2013-03-07

    Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistant mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.

  4. Clonidine displacement from type 1 imidazoline receptor by p-aminobenzamidine, the prototype of trypsin-like serine protease inhibitors.

    PubMed

    Pallottini, Valentina; Marino, Maria; Ascenzi, Paolo

    2002-11-01

    p-Aminobenzamidine inhibits competitively the catalytic activity of enzymes that recognize preferentially the L-arginyl side chain and related structures. Notably, p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors. Furthermore, p-aminobenzamidine inhibits the catalytic activity of nitric oxide synthase type I and type II as well as copper amine oxidase. Taking into account the structural similarity between p-aminobenzamidine, agmatine (the putative endogenous ligand of the membrane type 1 imidazoline receptor (I1-R)), and N-amidino-2-hydroxypyrrolidine (the product of agmatine oxidation by copper amine oxidase), the [3H]clonidine displacement from I1-R in rat heart membranes by p-aminobenzamidine was investigated. p-Aminobenzamidine is as effective as agmatine and N-amidino-2-hydroxypyrrolidine and more effective than the antihypertensive drug clonidine to displace [3H]clonidine from I1-R. Therefore, trypsin-like serine protease inhibitors structurally related to p-aminobenzamidine should be administrated under careful control. PMID:12587981

  5. Insights from atomic-resolution X-ray structures of chemically synthesized HIV-1 protease in complex with inhibitors.

    PubMed

    Johnson, Erik C B; Malito, Enrico; Shen, Yuequan; Pentelute, Brad; Rich, Dan; Florián, Jan; Tang, Wei-Jen; Kent, Stephen B H

    2007-10-26

    The human immunodeficiency virus 1 (HIV-1) protease (PR) is an aspartyl protease essential for HIV-1 viral infectivity. HIV-1 PR has one catalytic site formed by the homodimeric enzyme. We chemically synthesized fully active HIV-1 PR using modern ligation methods. When complexed with the classic substrate-derived inhibitors JG-365 and MVT-101, the synthetic HIV-1 PR formed crystals that diffracted to 1.04- and 1.2-A resolution, respectively. These atomic-resolution structures revealed additional structural details of the HIV-1 PR's interactions with its active site ligands. Heptapeptide inhibitor JG-365, which has a hydroxyethylamine moiety in place of the scissile bond, binds in two equivalent antiparallel orientations within the catalytic groove, whereas the reduced isostere hexapeptide MVT-101 binds in a single orientation. When JG-365 was converted into the natural peptide substrate for molecular dynamic simulations, we found putative catalytically competent reactant states for both lytic water and direct nucleophilic attack mechanisms. Moreover, free energy perturbation calculations indicated that the insertion of catalytic water into the catalytic site is an energetically favorable process.

  6. PL-100, a novel HIV-1 protease inhibitor displaying a high genetic barrier to resistance: an in vitro selection study.

    PubMed

    Dandache, Serge; Coburn, Craig A; Oliveira, Maureen; Allison, Timothy J; Holloway, M Katharine; Wu, Jinzi J; Stranix, Brent R; Panchal, Chandra; Wainberg, Mark A; Vacca, Joseph P

    2008-12-01

    The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations. PMID:19040279

  7. Staphylococcal proteases aid in evasion of the human complement system.

    PubMed

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz; Bielecka, Ewa; Miller, Halie K; Kalinska, Magdalena; Dubin, Grzegorz; Garred, Peter; Shaw, Lindsey N; Blom, Anna M

    2014-01-01

    Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system.

  8. Strength of hydrogen bond network takes crucial roles in the dissociation process of inhibitors from the HIV-1 protease binding pocket.

    PubMed

    Li, Dechang; Ji, Baohua; Hwang, Keh-Chih; Huang, Yonggang

    2011-01-01

    To understand the underlying mechanisms of significant differences in dissociation rate constant among different inhibitors for HIV-1 protease, we performed steered molecular dynamics (SMD) simulations to analyze the entire dissociation processes of inhibitors from the binding pocket of protease at atomistic details. We found that the strength of hydrogen bond network between inhibitor and the protease takes crucial roles in the dissociation process. We showed that the hydrogen bond network in the cyclic urea inhibitors AHA001/XK263 is less stable than that of the approved inhibitor ABT538 because of their large differences in the structures of the networks. In the cyclic urea inhibitor bound complex, the hydrogen bonds often distribute at the flap tips and the active site. In contrast, there are additional accessorial hydrogen bonds formed at the lateral sides of the flaps and the active site in the ABT538 bound complex, which take crucial roles in stabilizing the hydrogen bond network. In addition, the water molecule W301 also plays important roles in stabilizing the hydrogen bond network through its flexible movement by acting as a collision buffer and helping the rebinding of hydrogen bonds at the flap tips. Because of its high stability, the hydrogen bond network of ABT538 complex can work together with the hydrophobic clusters to resist the dissociation, resulting in much lower dissociation rate constant than those of cyclic urea inhibitor complexes. This study may provide useful guidelines for design of novel potent inhibitors with optimized interactions.

  9. Crystal Structure of An FIV/HIV Chimeric Protease Complexed With the Broad-Based Inhibitor, TL-3

    SciTech Connect

    Heaslet, H.; Lin, Y.-C.; Tam, K.; Torbett, B.E.; Elder, J.E.; Stout, C.D.; /Pfizer Global Res. Devel. /Scripps Res. Inst.

    2007-07-09

    We have obtained the 1.7 angstrom crystal structure of FIV protease (PR) in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR). The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants [1-4]. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively [2-4]. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed the formation of additional van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

  10. Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

    PubMed Central

    Wu, Deng-wei; Mao, Fei; Ye, Yan; Li, Jian; Xu, Chuan-lian; Luo, Xiao-min; Chen, Jing; Shen, Xu

    2015-01-01

    Aim: Dengue is a severe epidemic disease caused by dengue virus (DENV) infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 (NS3) and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENV drugs. Here, we report a novel inhibitor of DENV2 NS2B/NS3 protease and its antiviral action. Methods: An enzymatic inhibition assay was used for screening DENV2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis. Results: In our in-house library of old drugs (∼1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC50 of 4.99 μg/mL, whereas its IC50 for cytotoxicity to BHK-21 cells was 459.45 μg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with site-directed mutagenesis, we demonstrated that the residues Gln106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding. Conclusion: Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease. PMID:26279156

  11. Carotid artery intima–media thickness and HIV infection: traditional risk factors overshadow impact of protease inhibitor exposure

    PubMed Central

    Currier, Judith S.; Kendall, Michelle A.; Zackin, Robert; Henry, W. Keith; Alston-Smith, Beverly; Torriani, Francesca J.; Schouten, Jeff; Mickelberg, Keith; Li, Yanjie; Hodis, Howard N.

    2005-01-01

    Context The impact of HIV infection and exposure to antiretroviral therapy on the development of subclinical atherosclerosis is incompletely understood. Objective To compare intima–media thickness (IMT) of the carotid artery between HIV-infected subjects receiving protease inhibitor-containing regimens and subjects not receiving these regimens and to compare differences in the IMT of the carotid artery between HIV-infected subjects and HIV-uninfected subjects. Methods A prospective matched cohort study in university-based outpatient clinics. Groups of three individuals (triads) matched on the following characteristics were enrolled: age, sex, race/ethnicity, smoking status, blood pressure and menopausal status. Group 1, HIV-infected subjects with continuous use of protease inhibitor (PI) therapy for ≥ 2 years; group 2, HIV-infected subjects without prior PI use; and group 3: HIV-uninfected. Ultrasonographers at six sites sent standardized ultrasound images to a central reading site for carotid IMT measurements. Carotid IMT was compared within the HIV-infected groups (1 and 2) and between the HIV-infected and uninfected groups in a matched analysis. Results One hundred and thirty-four individuals were enrolled in 45 triads. The median IMT in groups 1, 2 and 3 was 0.690, 0.712 and 0.698 mm, respectively. There were no statistically significant differences in IMT between groups 1 and 2, or in the combined HIV groups compared with the HIV uninfected group. Significant predictors of carotid IMT in a multivariate model included high-density lipoprotein (HDL) cholesterol, the interaction of HDL cholesterol and triglycerides, age and body mass index. Conclusions We found no association between PI inhibitor exposure or HIV infection and carotid IMT. PMID:15905673

  12. Long-term changes in bone mineral density after switching to a protease inhibitor monotherapy in HIV-infected subject.

    PubMed

    Negredo, Eugènia; Bonjoch, Anna; Puig, Jordi; Echeverría, Patricia; Estany, Carla; Santos, José R; Moltó, José; Pérez-Álvarez, Nuria; Ornelas, Arelly; Clotet, Bonaventura

    2015-04-01

    Although some clinical trials have studied the impact of treatments on bone mineral density (BMD), scarce data are available about the impact of protease inhibitor (PI) monotherapies on BMD. The aim of this study was to evaluate changes in BMD in patients after one, two, or three years of a PI monotherapy. This study included 46 HIV-infected patients who switched from a conventional triple antiretroviral strategy to a monotherapy with lopinavir/ritonavir (LPV/r) or darunavir/ritonavir (DRV/r) for one (one-year group, n=16), two (two-year group, n=20), and three (three-year group, n=10) years. BMD was assessed by dual-energy X-ray absorptiometry (DXA). The median percentage of change in total femur BMD was 0.20% after one, 0.79% after two, and -0.31% after three years. The change in lumbar spine was -0.08%, -0.14%, and 0.50% % after the same years. No significant differences were found when patients were classified regarding the type of PI and whether or not had previously received PI or tenofovir. However, patients who interrupted tenofovir or those who started with DRV/r had a higher BMD increment. Patients who had taken non-nucleoside reverse transcriptase inhibitors previously decreased BMD when started PIs. Monotherapy treatment with ritonavir-boosted protease inhibitors (both LPV/r and DRV/r) during one, two, or three years leads to the stabilization of BMD in HIV-infected patients with long-term virological suppression. Larger studies are necessary to compare the effect of starting or withdrawing PIs on BMD. PMID:25938744

  13. Discordances between Interpretation Algorithms for Genotypic Resistance to Protease and Reverse Transcriptase Inhibitors of Human Immunodeficiency Virus Are Subtype Dependent

    PubMed Central

    Snoeck, Joke; Kantor, Rami; Shafer, Robert W.; Van Laethem, Kristel; Deforche, Koen; Carvalho, Ana Patricia; Wynhoven, Brian; Soares, Marcelo A.; Cane, Patricia; Clarke, John; Pillay, Candice; Sirivichayakul, Sunee; Ariyoshi, Koya; Holguin, Africa; Rudich, Hagit; Rodrigues, Rosangela; Bouzas, Maria Belen; Brun-Vézinet, Françoise; Reid, Caroline; Cahn, Pedro; Brigido, Luis Fernando; Grossman, Zehava; Soriano, Vincent; Sugiura, Wataru; Phanuphak, Praphan; Morris, Lynn; Weber, Jonathan; Pillay, Deenan; Tanuri, Amilcar; Harrigan, Richard P.; Camacho, Ricardo; Schapiro, Jonathan M.; Katzenstein, David; Vandamme, Anne-Mieke

    2006-01-01

    The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype. PMID:16436728

  14. Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF-κB signaling pathway.

    PubMed

    Mikami, Yoshikazu; Iwase, Takashi; Komiyama, Yusuke; Matsumoto, Naoyuki; Oki, Hidero; Komiyama, Kazuo

    2015-10-01

    Polymeric immunoglobulin receptor (pIgR) plays an important role in mucosal immune systems. Secretory immunoglobulin A, composed of secretory component of pIgR and a dimeric form of immunoglobulin A, is secreted on mucosal surfaces and serves as a biological defense factor. pIgR gene expression is reportedly induced by activation of the transcription factor nuclear factor (NF)-κB. On the other hand, secretory leukocyte protease inhibitor (SLPI) is a glycoprotein that functions as a serine protease inhibitor. In alveolar epithelial cells, SLPI increases the level of IκBβ, which indicates that it is an inhibitor of NF-κB at the protein level. Taken together, SLPI may regulate pIgR expression; however, the specific mechanism by which this occurs is unclear. Therefore, the aim of this study was to elucidatethe influence of SLPI on pIgR expression.SLPI and pIgR localized in goblet cells and ciliated epithelial cells of the gastrointestinal tract, respectively. No cells were detected in which SLPI and pIgR were co-expressed. In addition, recombinant human SLPI stimulation of an epithelial cell line (HT-29) decreased the pIgR expression. The pIgR expression was also higher in SLPI-deficient Ca9-22 cells than in wild-type Ca9-22 cells. Furthermore, a luciferase assay using a NF-κB reporter plasmid and real-time RT-PCR analysis indicated that when SLPI was present, the transcriptional activity of NF-κB protein was suppressed, which was accompanied by anincrease in the protein, but not the mRNA,expression of IκBβ. These results demonstrate that SLPI down-regulates pIgR expression through the NF-κB signaling pathway by inhibiting degradation of IκBβ protein. PMID:26239418

  15. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

  16. Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins.

    PubMed

    Lee, Young Ah; Saito-Nakano, Yumiko; Kim, Kyeong Ah; Min, Arim; Nozaki, Tomoyoshi; Shin, Myeong Heon

    2015-02-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1(+)) or repressed by antisense small RNA-mediated gene silencing (ICP1(-)). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1(+) strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1(-) strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1(+) amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1(-) amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis. PMID:25500214

  17. Molecular dynamics study of the connection between flap closing and binding of fullerene-based inhibitors of the HIV-1 protease.

    PubMed

    Zhu, Zhongwei; Schuster, David I; Tuckerman, Mark E

    2003-02-11

    The complementary spatial relationship between fullerene C(60) and the hydrophobic cavity region of the human immunodeficiency virus (HIV) protease, which houses the active site of the enzyme, has led to the suggestion that fullerene-based derivatives could have potential use as effective HIV protease inhibitors. The ability of such compounds to desolvate the cavity region leads to a strong hydrophobic interaction between the C(60) moiety and residues in the cavity region. In this study, the connection between the motion of the so-called flexible flaps of the cavity and favorable binding of a fullerene-based protease inhibitor is explored using multiple-time scale molecular dynamics simulations and free energy techniques. In addition, the effect of the interaction between the C(60) moiety and the residues in the cavity region on the water content of the cavity is also investigated. Conformational free energy profiles along a suitably chosen flap opening coordinate show a considerable barrier to flap opening in the presence of the inhibitor, while no such barrier exists for the protease alone. This result is interpreted in terms of a strong hydrophobic interaction between the C(60) moiety and the flexible flaps, which cause the latter to close tightly around the inhibitor, thereby expelling water from the cavity and leading to a favorable binding interaction. This interpretation is rationalized by direct analysis of the water content in the cavity in the presence and absence of the inhibitor.

  18. The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase.

    PubMed Central

    Eggers, Christopher T; Murray, Iain A; Delmar, Valerie A; Day, Anthony G; Craik, Charles S

    2004-01-01

    Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane. PMID:14705961

  19. Herbivore damage-induced production and specific anti-digestive function of serine and cysteine protease inhibitors in tall goldenrod, Solidago altissima L. (Asteraceae).

    PubMed

    Bode, Robert F; Halitschke, Rayko; Kessler, André

    2013-05-01

    Plant protease inhibitors (PIs) are among the most well-studied and widely distributed resistance traits that plants use against their herbivore attackers. There are different types of plant PIs which putatively function against the different types of proteases expressed in insect guts. Serine protease inhibitors (SPIs) and cysteine protease inhibitors (CPIs) are hypothesized to differentially function against the predominant gut proteases in lepidopteran and coleopteran herbivores, respectively. Here, we test the hypothesis that tall goldenrod, Solidago altissima, can specifically respond to damage by different herbivores and differentially induce SPIs and CPIs in response to damage by lepidopteran and coleopteran herbivores. Moreover, we ask if the concerted induction of different types of PIs accounts for variation in induced resistance to herbivory. We altered and optimized a rapid and effective existing methodology to quantitatively analyze both SPI and CPI activity simultaneously from a single tissue sample and to use the same plant extracts directly for characterization of inhibitory effects on insect gut protease activity. We found that both SPIs and CPIs are induced in S. altissima in response to damage, regardless of the damaging herbivore species. However, only SPIs were effective against Spodoptera exigua gut proteases. Our data suggest that plant PI responses are not necessarily specific to the identity of the attacking organism but that different components of generally induced defense traits can specifically affect different herbivore species. While providing an efficient and broadly applicable methodology to analyze multiple PIs extracted from the same tissue, this study furthers our understanding of specificity in induced plant resistance. PMID:23371287

  20. Structure-guided Discovery of a Novel Non-peptide Inhibitor of Dengue Virus NS2B-NS3 Protease.

    PubMed

    Li, Linfeng; Basavannacharya, Chandrakala; Chan, Kitti Wing Ki; Shang, Luqing; Vasudevan, Subhash G; Yin, Zheng

    2015-09-01

    Dengue fever is a fast emerging epidemic-prone viral disease caused by dengue virus serotypes 1-4. NS2B-NS3 protease of dengue virus is a validated target to develop antiviral agents. A major limitation in developing dengue virus protease inhibitors has been the lack of or poor cellular activity. In this work, we extracted and refined a pharmacophore model based on X-ray crystal structure and predicted binding patterns, followed by a three-dimensional flexible database filtration. These output molecules were screened according to a docking-based protocol, leading to the discovery of a compound with novel scaffold and good cell-based bioactivity that has potential to be further optimized. The discovery of this novel scaffold by combination of in silico methods suggests that structure-guided drug discovery can lead to the development of potent dengue virus protease inhibitors.

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

    PubMed

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

    2016-07-01

    The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity. PMID:26993255

  2. Nafamostat mesilate, a broad spectrum protease inhibitor, modulates platelet, neutrophil and contact activation in simulated extracorporeal circulation.

    PubMed

    Sundaram, S; Gikakis, N; Hack, C E; Niewiarowski, S; Edmunds, L H; Koneti Rao, A; Sun, L; Cooper, S L; Colman, R W

    1996-01-01

    Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet beta-thromboglobulin (beta TG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-alpha 1-antitrypsin complex is 0.16 micrograms/ml in the NM group and 1.24 micrograms/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB.

  3. Molecular cloning, sequencing analysis, and chromosomal localization of the human protease inhibitor 4 (Kallistatin) gene (P14)

    SciTech Connect

    Chai, K.X.; Chao, J.; Chao, L.; Ward, D.C.

    1994-09-15

    The gene encoding human protease inhibitor 4 (kallistatin; gene symbol PI4), a novel serine proteinase inhibitor (serpin), has been isolated and completely sequenced. The kallistatin gene is 9618 bp in length and contains five exons and four introns. The structure and organization of the kallistatin gene are similar to those of the genes encoding {alpha}{sub 1}-antichymotrypsin. The kallistatin gene is also similar to the genes encoding rat and mouse kallikrein-binding proteins. The first exon of the kallistatin gene is a noncoding 89-bp fragment, as determined by primer extension. The fifth exon, which contains 308 bp of noncoding sequence, encodes the reactive center of kallistatin. In the 5`-flanking region of the kallistatin gene, 1125 bp have been sequenced and a consensus promoter segment with potential transcription regulatory sites, including CAAT and TATA boxes, an AP-2 binding site, a GC-rich region, a cAMP response element, and an AP-1 binding site, has been identified within this region. The kallistatin gene was localized by in situ hybridization to human chromosome 14q31-132.1, close to the serpin genes encoding {alpha}{sub 1}-antichymotrypsin, protein C inhibitor, {alpha}{sub 1}-antitrypsin, and corticosteroid-binding globulin. In a genomic DNA Southern blot, kallistatin-related genes were identified in monkey, mouse, rat, bovine, dog, cat, and a ground mole. The patterns of hybridization revealed clues of human serpin evolution. 34 refs., 6 figs.

  4. Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction.

    PubMed

    Beek, J; Nauwynck, H; Appeltant, R; Maes, D; Van Soom, A

    2015-11-01

    Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 10(5) spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF.

  5. Inhibition of human preadipocyte proteasomal activity by HIV protease inhibitors or specific inhibitor lactacystin leads to a defect in adipogenesis, which involves matrix metalloproteinase-9.

    PubMed

    De Barros, Sandra; Zakaroff-Girard, Alexia; Lafontan, Max; Galitzky, Jean; Bourlier, Virginie

    2007-01-01

    In a previous publication, we reported that human immunodeficiency virus (HIV) protease inhibitors (PIs) inhibited the differentiation of human preadipocytes in primary culture, reducing the expression and secretion of matrix metalloproteinase 9 (MMP-9). The present work was performed to clarify this mechanism. Interestingly, HIV-PIs have been reported to be inhibitors of the proteasome complex, which is known to regulate nuclear factor (NF)-kappaB activation and transcription of its target genes, among them MMP-9. We thus investigated the potential involvement of the proteasome in the antiadipogenic effects of HIV-PIs. The effect of four HIV-PIs was tested on preadipocyte proteasomal activity, and chronic treatment with the specific proteasome inhibitor lactacystin was performed to evaluate alterations of adipogenesis and MMP-9 expression/secretion. Finally, modifications of the NF-kappaB pathway induced by either HIV-PIs or lactacystin were studied. We demonstrated that preadipocyte proteasomal activity was decreased by several HIV-PIs and that chronic treatment with lactacystin mimicked the effects of HIV-PIs by reducing adipogenesis and MMP-9 expression/secretion. Furthermore, we observed an intracellular accumulation of the NF-kappaB inhibitor, IkappaBbeta, with chronic treatment with HIV-PIs or lactacystin as well as a decrease in MMP-9 expression induced by acute tumor necrosis factor-alpha stimulation. These results indicate that inhibition of the proteasome by specific (lactacystin) or nonspecific (HIV-PIs) inhibitors leads to a reduction of human adipogenesis, and they therefore implicate deregulation of the NF-kappaB pathway and the related decrease of the key adipogenic factor, MMP-9. This study adds significantly to recent reports that have linked HIV-PI-related lipodystrophic syndrome with altered proteasome function, endoplasmic reticulum stress, and metabolic disorders.

  6. Docking and 3-D QSAR studies on the binding of tetrahydropyrimid-2-one HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Rao, Shashidhar N.; Balaji, Govardhan A.; Balaji, Vitukudi N.

    2013-06-01

    We present molecular docking and 3-D QSAR studies on a series of tetrahydropyrimid-2-one HIV-1 protease inhibitors whose binding affinities to the enzyme span nearly 6 orders of magnitude. The docking investigations have been carried out with Surflex (GEOM, GEOMX) and Glide (SP and XP) methodologies available through Tripos and Schrodinger suite of tools in the context of Sybyl-X and Maestro interfaces, respectively. The alignments for 3-D QSAR studies were obtained by using the automated Surflex-SIM methodology in Sybyl-X and the analyses were performed using the CoMFA and CoMSIA methods. Additionally, the top-ranked poses obtained from various docking protocols were also employed to generate CoMFA and CoMSIA models to evaluate the qualitative consistency of the docked models with experimental data. Our studies demonstrate that while there are a number of common features in the docked models obtained from Surflex-dock and Glide methodologies, the former sets of models are generally better correlated with deduced experimental binding modes based on the X-ray structures of known HIV-1 protease complexes with cyclic ureas. The urea moiety common to all the ligands are much more tightly aligned in Surflex docked structures than in the models obtained from Glide SP and XP dockings. The 3-D QSAR models are qualitatively and quantitatively similar to those previously reported, suggesting the utility of automatically generated alignments from Surflex-SIM methodology.

  7. Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease.

    PubMed Central

    Shahabuddin, M; Toyoshima, T; Aikawa, M; Kaslow, D C

    1993-01-01

    During development in the mosquito midgut, malarial parasites must traverse a chitin-containing peritrophic matrix (PM) that forms around the food bolus. Previously Huber et al. [Huber, M., Cabib, E. & Miller, L. H. (1991) Proc. Natl. Acad. Sci. USA 88, 2807-2810] reported that the parasite secretes a protein with chitinase activity, and they suggested that parasite chitinase (EC 3.2.1.14) plays an important role in the parasite's egress from the blood meal. We found that allosamidin, a specific inhibitor of chitinase, completely blocked oocyst development in vivo and thus blocked malaria parasite transmission. Addition of exogenous chitinase to the blood meal prevented the PM from forming and reversed the transmission-blocking activity of allosamidin. Using exogenous chitinase, we also found that the PM does not limit the number of parasites that develop into oocysts, suggesting that the parasite produces sufficient quantities of chitinase to penetrate this potential barrier. In addition, we found that treatment of parasite chitinase with a diisopropyl fluorophosphate-sensitive trypsinlike protease from the mosquito midgut or endoproteinase Lys-C increased its enzymatic activity. These results suggest that malaria parasite has evolved an intricate mechanism to adapt to the PM and the protease-rich environment of the mosquito midgut. Images Fig. 2 PMID:8483942

  8. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation.

    PubMed

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-02-01

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent. PMID:26875984

  9. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation

    PubMed Central

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L.; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-01-01

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent. PMID:26875984

  10. Suppressive Effects of the Site 1 Protease (S1P) Inhibitor, PF-429242, on Dengue Virus Propagation.

    PubMed

    Uchida, Leo; Urata, Shuzo; Ulanday, Gianne Eduard L; Takamatsu, Yuki; Yasuda, Jiro; Morita, Kouichi; Hayasaka, Daisuke

    2016-02-10

    Dengue virus (DENV) infection causes one of the most widespread mosquito-borne diseases in the world. Despite the great need, effective vaccines and practical antiviral therapies are still under development. Intracellular lipid levels are regulated by sterol regulatory elements-binding proteins (SREBPs), which are activated by serine protease, site 1 protease (S1P). Small compound PF-429242 is known as a S1P inhibitor and the antivirus effects have been reported in some viruses. In this study, we examined the anti-DENV effects of PF-429242 using all four serotypes of DENV by several primate-derived cell lines. Moreover, emergence of drug-resistant DENV mutants was assessed by sequential passages with the drug. DENV dependency on intracellular lipids during their infection was also evaluated by adding extracellular lipids. The addition of PF-429242 showed suppression of viral propagation in all DENV serotypes. We showed that drug-resistant DENV mutants are unlikely to emerge after five times sequential passages through treatment with PF-429242. Although the levels of intracellular cholesterol and lipid droplets were reduced by PF-429242, viral propagations were not recovered by addition of exogenous cholesterol or fatty acids, indicating that the reduction of LD and cholesterol caused by PF-429242 treatment is not related to its mechanism of action against DENV propagation. Our results suggest that PF-429242 is a promising candidate for an anti-DENV agent.

  11. Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

    PubMed

    Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

    2016-10-01

    Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. PMID:27353494

  12. Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

    PubMed

    Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

    2016-10-01

    Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions.

  13. Antiviral activities of 15 dengue NS2B-NS3 protease inhibitors using a human cell-based viral quantification assay.

    PubMed

    Chu, Justin Jang Hann; Lee, Regina Ching Hua; Ang, Melgious Jin Yan; Wang, Wei-Ling; Lim, Huichang Annie; Wee, John Liang Kuan; Joy, Joma; Hill, Jeffrey; Brian Chia, C S

    2015-06-01

    The dengue virus is a mosquito-borne pathogen responsible for an estimated 50-100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor's ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 μM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 μM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay.

  14. Structural Basis for Dual-Inhibition Mechanism of a Non-Classical Kazal-Type Serine Protease Inhibitor from Horseshoe Crab in Complex with Subtilisin

    SciTech Connect

    Shenoy, Rajesh T.; Thangamani, Saravanan; Velazquez-Campoy, Adrian; Ho, Bow; Ding, Jeak Ling; Sivaraman, J.; Kursula, Petri

    2011-04-26

    Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki=1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1:2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.

  15. Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin.

    PubMed

    Shenoy, Rajesh T; Thangamani, Saravanan; Velazquez-Campoy, Adrian; Ho, Bow; Ding, Jeak Ling; Sivaraman, J

    2011-01-01

    Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.

  16. Adaptive mechanisms of insect pests against plant protease inhibitors and future prospects related to crop protection: a review.

    PubMed

    Macedo, Maria L R; de Oliveira, Caio F R; Costa, Poliene M; Castelhano, Elaine C; Silva-Filho, Marcio C

    2015-01-01

    The overwhelming demand for food requires the application of technology on field. An important issue that limits the productivity of crops is related to insect attacks. Hence, several studies have evaluated the application of different compounds to reduce the field losses, especially insecticide compounds from plant sources. Among them, plant protease inhibitors (PIs) have been studied in both basic and applied researches, displaying positive results in control of some insects. However, certain species are able to bypass the insecticide effects exerted by PIs. In this review, we disclosed the adaptive mechanisms showed by lepidopteran and coleopteran insects, the most expressive insect orders related to crop predation. The structural aspects involved in adaptation mechanisms are presented as well as the newest alternatives for pest control. The application of biotechnological tools in crop protection will be mandatory in agriculture, and it will be up to researchers to find the best candidates for effective control in long-term. PMID:25329404

  17. Flexible Cyclic Ethers/Polyethers as Novel P2-Ligands for HIV-1 Protease Inhibitors: Design, Synthesis, Biological Evaluation, and Protein-Ligand X-Ray Studies

    SciTech Connect

    Ghosh, Arun; Gemma, Sandra; Baldridge, Abigal; Wang, Yuan-Fang; Kovalevsky, Andrey; Koh, Yashiro; Weber, Irene; Mitsuya, Hiroaki

    2008-12-05

    We report the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors. The inhibitors incorporate stereochemically defined flexible cyclic ethers/polyethers as high affinity P2-ligands. Inhibitors containing small ring 1,3-dioxacycloalkanes have shown potent enzyme inhibitory and antiviral activity. Inhibitors 3d and 3h are the most active inhibitors. Inhibitor 3d maintains excellent potency against a variety of multi-PI-resistant clinical strains. Our structure-activity studies indicate that the ring size, stereochemistry, and position of oxygens are important for the observed activity. Optically active synthesis of 1,3-dioxepan-5-ol along with the syntheses of various cyclic ether and polyether ligands have been described. A protein-ligand X-ray crystal structure of 3d-bound HIV-1 protease was determined. The structure revealed that the P2-ligand makes extensive interactions including hydrogen bonding with the protease backbone in the S2-site. In addition, the P2-ligand in 3d forms a unique water-mediated interaction with the NH of Gly-48.

  18. A combined 3D-QSAR and docking studies for the In-silico prediction of HIV-protease inhibitors

    PubMed Central

    2013-01-01

    Background Tremendous research from last twenty years has been pursued to cure human life against HIV virus. A large number of HIV protease inhibitors are in clinical trials but still it is an interesting target for researchers due to the viral ability to get mutated. Mutated viral strains led the drug ineffective but still used to increase the life span of HIV patients. Results In the present work, 3D-QSAR and docking studies were performed on a series of Danuravir derivatives, the most potent HIV- protease inhibitor known so far. Combined study of 3D-QSAR was applied for Danuravir derivatives using ligand-based and receptor-based protocols and generated models were compared. The results were in good agreement with the experimental results. Additionally, docking analysis of most active 32 and least active 46 compounds into wild type and mutated protein structures further verified our results. The 3D-QSAR and docking results revealed that compound 32 bind efficiently to the wild and mutated protein whereas, sufficient interactions were lost in compound 46. Conclusion The combination of two computational techniques would helped to make a clear decision that compound 32 with well inhibitory activity bind more efficiently within the binding pocket even in case of mutant virus whereas compound 46 lost its interactions on mutation and marked as least active compound of the series. This is all due to the presence or absence of substituents on core structure, evaluated by 3D-QSAR studies. This set of information could be used to design highly potent drug candidates for both wild and mutated form of viruses. PMID:23683267

  19. Molecular Cloning and Functional Studies of Two Kazal-Type Serine Protease Inhibitors Specifically Expressed by Nasonia vitripennis Venom Apparatus

    PubMed Central

    Qian, Cen; Fang, Qi; Wang, Lei; Ye, Gong-Yin

    2015-01-01

    Two cDNA sequences of Kazal-type serine protease inhibitors (KSPIs) in Nasonia vitripennis, NvKSPI-1 and NvKSPI-2, were characterized and their open reading frames (ORFs) were 198 and 264 bp, respectively. Both NvKSPI-1 and NvKSPI-2 contained a typical Kazal-type domain. Real-time quantitative PCR (RT-qPCR) results revealed that NvKSPI-1 and NvKSPI-2 mRNAs were mostly detected specifically in the venom apparatus, while they were expressed at lower levels in the ovary and much lower levels in other tissues tested. In the venom apparatus, both NvKSPI-1 and NvKSPI-2 transcripts were highly expressed on the fourth day post eclosion and then declined gradually. The NvKSPI-1 and NvKSPI-2 genes were recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvKSPI-1 and GST-NvKSPI-2 to three serine protease inhibitors (trypsin, chymotrypsin, and proteinase K) were tested and results showed that only NvKSPI-1 could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvKSPI-1 and GST-NvKSPI-2 on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by both recombinant proteins; however, there was no significant inhibition on the PO activity. Our results suggested that NvKSPI-1 and NvKSPI-2 could inhibit PPO activation in host hemolymph and trypsin activity in vitro. PMID:26248077

  20. Secretory leukocyte protease inhibitor promotes differentiation and mineralization of MC3T3-E1 preosteoblasts on a titanium surface.

    PubMed

    Choi, Baik-Dong; Lee, Seung-Yeon; Jeong, Soon-Jeong; Lim, Do-Seon; Cha, Hee-Jae; Chung, Won-Gyun; Jeong, Moon-Jin

    2016-08-01

    Mineralized bone matrix constituted with collagenous and non-collagenous proteins was synthesized by osteoblasts differentiated from mesenchymal stem cells. Secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor, promotes cell migration and proliferation, and suppresses the inflammatory response. Recent studies reported that SLPI regulates the formation of dentin and mineralization by odontoblasts and increases the adhesion and viability of preosteoblasts on a titanium (Ti) surface. Ti and its alloys are widely used implant materials in artificial joints and dental implants owing to their biocompatibility with bone. Therefore, this study aimed to examine whether SLPI can be an effective molecule in promoting differentiation and mineralization of osteoblasts on a Ti surface. In order to investigate the effects of SLPI on osteoblasts, an MTT assay, PCR, western blotting and Alizarin Red S staining were performed. The results demonstrated that SLPI increased the viability of osteoblasts during differentiation on Ti discs compared with that of the control. The expression levels of SLPI mRNA and protein were higher than that of the control after treatment of osteoblasts with SLPI on Ti discs during differentiation. SLPI increased the formation of mineralized nodules and mRNA expression of alkaline phosphatase, dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and collagen I in osteoblasts on Ti discs compared with that of the control. In conclusion, SLPI increases the viability and promotes the differentiation and mineralization of osteoblasts on Ti surfaces, suggesting that SLPI is an effective molecule for achieving successful osseointegration between osteoblasts and a Ti surface. PMID:27279420

  1. Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease.

    PubMed Central

    Ullrich, B.; Laberge, M.; Tölgyesi, F.; Szeltner, Z.; Polgár, L.; Fidy, J.

    2000-01-01

    The Q7K/L331/L631 HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with k(q1) = 6.85 x 10(9) M(-1) s(-1) and k(q2) = 1.88 x 10(9) M(-1) s(-1), that merge into one in the complex with k(q) = 1.78 x 10(9) M(-1) s(-1). 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity. PMID:11152134

  2. Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases in silico docked to different inhibitors

    PubMed Central

    2010-01-01

    Background Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes. Results We propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues (IFR) for serine proteases and their inhibitors. Currently, the Protein Data Bank (PDB) does not contain all the data that our analysis would require. Therefore, an in silico approach was designed for building corresponding complexes The IFRs are obtained by "rigid body docking" among 70 structurally aligned, sequence wise non-redundant, serine protease structures with 3 inhibitors: bovine pancreatic trypsin inhibitor (BPTI), ecotine and ovomucoid third domain inhibitor. The table (matrix) of all amino acid positions at the interface and their respective occupancy is created. We also developed a new computational protocol for predicting IFRs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least 0.97. Conclusions The serine proteases interfaces prefer polar (including glycine) residues (with some exceptions). Charged residues were found to be uniquely prevalent at the interfaces between the

  3. Synthesis, in vitro evaluation, and docking studies of novel chromone derivatives as HIV-1 protease inhibitor

    NASA Astrophysics Data System (ADS)

    Ungwitayatorn, Jiraporn; Wiwat, Chanpen; Samee, Weerasak; Nunthanavanit, Patcharawee; Phosrithong, Narumol

    2011-08-01

    Novel chromone derivatives with a benzopyran-4-one scaffold have been prepared by the one-pot cyclization reaction. The in vitro inhibitory activity of these new compounds towards HIV-1 protease have been evaluated using stop time HPLC method as the preliminary screening. The most potent compound, 7,8-dihydroxy-2-(3'-trifluoromethyl phenyl)-3-(3″-trifluoromethylbenzoyl)chromone ( 32), showed IC 50 = 0.34 μM. The molecular docking study supported results from experimental activity testing and also provided structure-activity relationship of this series.

  4. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    SciTech Connect

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  5. Intraspecies differences in hemostatic venom activities of the South American rattlesnakes, Crotalus durissus cumanensis, as revealed by a range of protease inhibitors.

    PubMed

    Salazar, Ana M; Aguilar, Irma; Guerrero, Belsy; Girón, María E; Lucena, Sara; Sánchez, Elda E; Rodríguez-Acosta, Alexis

    2008-09-01

    Crotalus durissus cumanensis is an endemic rattlesnake found in Venezuela and Colombia. In this study, a comparative analysis of hemorrhagic, coagulation and fibrino(geno)lytic activities in the presence or absence of protease inhibitors was performed with venoms of the same species Crotalus durissus cumanensis, from seven geographical regions of Venezuela (Lagunetica, Santa Teresa, Carrizales, Guarenas, Anzoátegui, Margarita and Maracay). Lagunetica, Carrizales and Anzoátegui venoms induced hemorrhagic activity. All venoms, except that of snakes from the Carrizales region presented thrombin-like activity, which was inhibited completely by phenylmethanesulfonyl fluoride and ethylene glycol-bis-N, N,N',N'-tetraacetic acid. This effect of the latter could be explained by the high chelant calcium effect, which is a cofactor for the fibrin polymerization process. Soybean trypsin inhibitor was effective on Santa Teresa venom. Antithrombin III/Hep complex and phenantroline partially inhibited this activity in all venoms except Margarita and Anzoátegui, respectively, which were not inhibited. Serine protease inhibitors were more effective against thrombin, kallikrein and plasmin-like amidolytic activities. Additionally, metalloprotease inhibitors significantly inhibited the t-PA-like amidolytic activity and completely the hemorrhagic and fibrino(geno)lytic activities. In conclusion, the thrombin-like activity observed in these venoms was partially reduced by serine protease inhibitors, indicating the possible presence of catalytic domains in those enzymes that do not interact with these inhibitors. Using protease inhibitors on venom hemostatic activities could contribute to our understanding of the active components of snake venom on the hemostatic system, and further reveal the intraspecies variation of venoms, which is important in the treatment of envenomation, and in addition represents an interesting model for the study of venom in hemostasis.

  6. Response to First-Line Ritonavir-Boosted Protease Inhibitors (PI/r)-Based Regimens in HIV Positive Patients Presenting to Care with Low CD4 Counts: Data from the Icona Foundation Cohort

    PubMed Central

    d’Arminio Monforte, Antonella; Cozzi-Lepri, Alessandro; Maggiolo, Franco; Rizzardini, Giuliano; Manconi, Paolo Emilio; Gianotti, Nicola; Quirino, Tiziana; Pinnetti, Carmela; Rusconi, Stefano; De Luca, Andrea; Antinori, Andrea

    2016-01-01

    Background There are no data comparing the response to PI/r-based regimens in people presenting for care with low CD4 counts or AIDS (LC). Aim To compare the response to LPV/r-, DRV/r- or ATV/r-based cART regimens in LC initiating cART from ART-naive. Methods We included people enrolled in Icona with either CD4 counts ≤350 cells/mm3 (low CD4-LC) or CD4 counts ≤200 cells/mm3 (very low CD4-VLC) and/or AIDS, starting their first PI/r-based regimen after 2008. Initial regimens were compared by intention-to-treat: i) time to viral failure (VF) (first of 2 consecutive VL>200 copies/mL after≥6 months); II) time to PI/r discontinuation/switching for any cause (TD) and for toxicity (TDT); III) treatment failure (TF) (VF or TD). Kaplan-Meier and Cox analyses were used. Results 1,362 LC patients were included (DRV/r 607; ATV/r 552; LPV/r 203); 813 VLC. In a median of 18 months (IQR:7–35), the 1-year probability of VF and TF were 2.8% (1.9–3.8) and 21.1% (18.7–23.4). In the adjusted analysis, patients initiating ATV/r had a 53% lower chance, and those initiating DRV/r a 61% lower chance of TD, as compared to LPV/r; the risk of TF was more likely in people starting LPV/r. Results were similar among VLC; in this subgroup LPV/r including regimens demonstrated a lower chance of VF. Conclusions We confirmed in LC a low chance of virological failure by 1 year, with small differences according to PI/r. However, larger differences were observed when comparing longer-term endpoints such as treatment failure. These results are important for people presenting late for care. PMID:27348592

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

    PubMed Central

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

    2013-01-01

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

  8. Structural and thermodynamic basis for the binding of TMC114, a next-generation human immunodeficiency virus type 1 protease inhibitor.

    PubMed

    King, Nancy M; Prabu-Jeyabalan, Moses; Nalivaika, Ellen A; Wigerinck, Piet; de Béthune, Marie-Pierre; Schiffer, Celia A

    2004-11-01

    TMC114, a newly designed human immunodeficiency virus type 1 (HIV-1) protease inhibitor, is extremely potent against both wild-type (wt) and multidrug-resistant (MDR) viruses in vitro as well as in vivo. Although chemically similar to amprenavir (APV), the potency of TMC114 is substantially greater. To examine the basis for this potency, we solved crystal structures of TMC114 complexed with wt HIV-1 protease and TMC114 and APV complexed with an MDR (L63P, V82T, and I84V) protease variant. In addition, we determined the corresponding binding thermodynamics by isothermal titration calorimetry. TMC114 binds approximately 2 orders of magnitude more tightly to the wt enzyme (K(d) = 4.5 x 10(-12) M) than APV (K(d) = 3.9 x 10(-10) M). Our X-ray data (resolution ranging from 2.2 to 1.2 A) reveal strong interactions between the bis-tetrahydrofuranyl urethane moiety of TMC114 and main-chain atoms of D29 and D30. These interactions appear largely responsible for TMC114's very favorable binding enthalpy to the wt protease (-12.1 kcal/mol). However, TMC114 binding to the MDR HIV-1 protease is reduced by a factor of 13.3, whereas the APV binding constant is reduced only by a factor of 5.1. However, even with the reduction in binding affinity to the MDR HIV protease, TMC114 still binds with an affinity that is more than 1.5 orders of magnitude tighter than the first-generation inhibitors. Both APV and TMC114 fit predominantly within the substrate envelope, a property that may be associated with decreased susceptibility to drug-resistant mutations relative to that of first-generation inhibitors. Overall, TMC114's potency against MDR viruses is likely a combination of its extremely high affinity and close fit within the substrate envelope.

  9. Structure-based design of a novel series of azetidine inhibitors of the hepatitis C virus NS3/4A serine protease.

    PubMed

    Parsy, Christophe; Alexandre, François-René; Brandt, Guillaume; Caillet, Catherine; Cappelle, Sylvie; Chaves, Dominique; Convard, Thierry; Derock, Michel; Gloux, Damien; Griffon, Yann; Lallos, Lisa; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna-Giulia; Moulat, Laure; Musiu, Chiara; Rahali, Houcine; Roques, Virginie; Seifer, Maria; Standring, David; Surleraux, Dominique

    2014-09-15

    Structural homology between thrombin inhibitors and the early tetrapeptide HCV protease inhibitor led to the bioisosteric replacement of the P2 proline by a 2,4-disubstituted azetidine within the macrocyclic β-strand mimic. Molecular modeling guided the design of the series. This approach was validated by the excellent activity and selectivity in biochemical and cell based assays of this novel series and confirmed by the co-crystal structure of the inhibitor with the NS3/4A protein (PDB code: 4TYD).

  10. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. PMID:25910649

  11. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress.

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

    SciTech Connect

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

    2006-12-01

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

  13. Boosted protease inhibitor monotherapy in HIV-infected adults: outputs from a pan-European expert panel meeting

    PubMed Central

    2013-01-01

    While the introduction of combination highly active antiretroviral therapy (HAART) regimens represents an important advance in the management of human immunodeficiency virus (HIV)-infected patients, tolerability can be an issue and the use of several different agents may produce problems. The switch of combination HAART to ritonavir-boosted protease inhibitor (PI) monotherapy may offer the opportunity to maintain antiviral efficacy while reducing treatment complexity and the risks of toxicity. Current European AIDS Clinical Society (EACS) guidelines recognise ritonavir-boosted PI monotherapy with twice-daily lopinavir/ritonavir or once-daily darunavir/ritonavir as a possible option in patients who have intolerance to nucleoside reverse transcriptase inhibitors, or for treatment simplification. Clinical trials data for PI boosted monotherapy are encouraging, showing substantial efficacy in the majority of patients; however, further data are required before this approach can be recommended as a routine treatment. Available data indicate that the most suitable candidates for the use of boosted PI monotherapy are long-term virologically suppressed patients who have demonstrated good adherence to antiretroviral therapy, who do not have chronic hepatitis B, have no history of treatment failure on PIs and are able to tolerate low-dose ritonavir. PMID:23347595

  14. Crystallization and preliminary X-ray diffraction analysis of a protease inhibitor from the haemolymph of the Indian tasar silkworm Antheraea mylitta

    SciTech Connect

    Roy, Sobhan; Aravind, Penmatsa; Madhurantakam, Chaithanya; Ghosh, Ananta Kumar; Sankaranarayanan, Rajan; Das, Amit Kumar

    2006-07-01

    The crystallization and preliminary X-ray crystallographic analysis of a protease inhibitor from the haemolymph of the Indian tasar silk worm A. mylitta is reported. A protein with inhibitory activity against fungal proteases was purified from the haemolymph of the Indian tasar silkworm Antheraea mylitta and was crystallized using the hanging-drop vapour-diffusion method. Polyethylene glycol 3350 was used as a precipitant. Crystals belonged to space group P6{sub 3}22, with unit-cell parameters a = b = 60.6, c = 85.1 Å. X-ray diffraction data were collected and processed to a maximum resolution of 2.1 Å.

  15. Synthesis and antiviral evaluation of a novel series of homoserine-based inhibitors of the hepatitis C virus NS3/4A serine protease.

    PubMed

    Alexandre, François-René; Brandt, Guillaume; Caillet, Catherine; Chaves, Dominique; Convard, Thierry; Derock, Michel; Gloux, Damien; Griffon, Yann; Lallos, Lisa; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna-Giulia; Moulat, Laure; Musiu, Chiara; Parsy, Christophe; Rahali, Houcine; Roques, Virginie; Seifer, Maria; Standring, David; Surleraux, Dominique

    2015-09-15

    We disclose here the synthesis of a series of macrocyclic HCV protease inhibitors, where the homoserine linked together the quinoline P2' motif and the macrocyclic moiety. These compounds exhibit potent inhibitory activity against HCV NS3/4A protease and replicon cell based assay. Their enzymatic and antiviral activities are modulated by substitutions on the quinoline P2' at position 8 by methyl and halogens and by small heterocycles at position 2. The in vitro structure activity relationship (SAR) studies and in vivo pharmacokinetic (PK) evaluations of selected compounds are described herein.

  16. A pharmacologic inhibitor of the protease Taspase1 effectively inhibits breast and brain tumor growth.

    PubMed

    Chen, David Y; Lee, Yishan; Van Tine, Brian A; Searleman, Adam C; Westergard, Todd D; Liu, Han; Tu, Ho-Chou; Takeda, Shugaku; Dong, Yiyu; Piwnica-Worms, David R; Oh, Kyoung J; Korsmeyer, Stanley J; Hermone, Ann; Gussio, Richard; Shoemaker, Robert H; Cheng, Emily H-Y; Hsieh, James J-D

    2012-02-01

    The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 μmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.

  17. Potential of protease inhibitor in 3-nitropropionic acid induced Huntington's disease like symptoms: mitochondrial dysfunction and neurodegeneration.

    PubMed

    Hariharan, Ashwini; Shetty, Shruthi; Shirole, Trupti; Jagtap, Aarti G

    2014-12-01

    Huntington's disease (HD) is a genetic, neurodegenerative disorder mainly characterized by motor dysfunction, cognitive decline and psychiatric disturbances. 3-Nitropropionic acid (3-NP) is an inhibitor of succinate dehydrogenase (Complex II) of the mitochondrial respiratory chain, which thereby reduces production of ATP. It induces neurotoxicity by causing striatal degeneration, energy deficit and oxidative stress. Angiotensin converting enzyme (ACE) is an important protease in the renin angiotensin system (RAS) responsible for the conversion of Angiotensin I to Angiotensin II. Angiotensin-II stimulates mitochondrial oxidant release leading to depression of energy metabolism. ACE inhibitors have shown promise in disorders like stress, anxiety, and depression in addition to showing beneficial effects in cognitive disorders like Alzheimer's. Angiotensin-II inhibition enhances energy production by lowering mitochondrial oxidant production, and hence protects mitochondrial structure. Trandolapril is a centrally active ACE inhibitor. 3-NP administered systematically (20mg/kg, i.p) for 4 days consecutively induced HD like symptoms - loss of body weight, neurobehavioral alterations like memory dysfunction (elevated plus maze, Morris water maze performance), Hind-limb impairment (Narrow beam test), motor incoordination (locomotor activity). Biochemical studies on brain tis