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Sample records for protease inhibitor ritonavir

  1. Investigating and Targeting Chronic Lymphocytic Leukemia Metabolism with the HIV Protease Inhibitor Ritonavir and Metformin

    PubMed Central

    Adekola, Kehinde U.A.; Aydemir, Sevim D.; Ma, Shuo; Zhou, Zheng; Rosen, Steven T.; Shanmugam, Mala

    2016-01-01

    Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity. Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal. Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation. CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL. PMID:24828872

  2. 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

  3. HIV-1 subtype influences susceptibility and response to monotherapy with the protease inhibitor lopinavir/ritonavir

    PubMed Central

    Sutherland, K. A.; Ghosn, J.; Gregson, J.; Mbisa, J. L.; Chaix, M. L.; Cohen Codar, I.; Delfraissy, J. F.; Delaugerre, C.; Gupta, R. K.

    2015-01-01

    Objective PI susceptibility results from a complex interplay between protease and Gag proteins, with Gag showing wide variation across HIV-1 subtypes. We explored the impact of pre-treatment susceptibility on the outcome of lopinavir/ritonavir monotherapy. Methods Treatment-naive individuals who experienced lopinavir/ritonavir monotherapy failure from the MONARK study were matched (by subtype, viral load and baseline CD4 count) with those who achieved virological response (‘successes’). Successes were defined by viral load <400 copies/mL after week 24 and <50 copies/mL from week 48 to week 96. Full-length Gag–protease was amplified from patient samples for in vitro phenotypic susceptibility testing, with susceptibility expressed as fold change (FC) relative to a subtype B reference strain. Results Baseline lopinavir susceptibility was lower in viral failures compared with viral successes, but the differences were not statistically significant (median lopinavir susceptibility: 4.4 versus 8.5, respectively, P = 0.17). Among CRF02_AG/G patients, there was a significant difference in lopinavir susceptibility between the two groups (7.1 versus 10.4, P = 0.047), while in subtype B the difference was not significant (2.7 versus 3.4, P = 0.13). Subtype CRF02_AG/G viruses had a median lopinavir FC of 8.7 compared with 3.1 for subtype B (P = 0.001). Conclusions We report an association between reduced PI susceptibility (using full-length Gag–protease sequences) at baseline and subsequent virological failure on lopinavir/ritonavir monotherapy in antiretroviral-naive patients harbouring subtype CRF02_AG/G viruses. We speculate that this may be important in the context of suboptimal adherence in determining viral failure. PMID:25228587

  4. 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

  5. Resistance profiles observed in virological failures after 24 weeks of amprenavir/ritonavir containing regimen in protease inhibitor experienced patients.

    PubMed

    Marcelin, Anne-Geneviève; Affolabi, Dissou; Lamotte, Claire; Mohand, Hocine Ait; Delaugerre, Constance; Wirden, Marc; Voujon, Delphine; Bossi, Philippe; Ktorza, Nadine; Bricaire, François; Costagliola, Dominique; Katlama, Christine; Peytavin, Gilles; Calvez, Vincent

    2004-09-01

    Amprenavir (APV) is an HIV protease inhibitor (PI) used for the treatment of either naive or PI-experienced HIV-infected patients. Several genotypic resistance pathways in protease gene have been described to be associated to unboosted APV failure (I50V, V32I + I47V, I54L/M, or less commonly I84V, which may be accompanied by one ore more accessory mutations such as L10F, L33F, M46I/L). The aims of this study were to investigate the efficacy up to week 24 of an APV plus ritonavir containing regimen in PI experienced patients and to determine the genotypic resistance profiles emerging in patients failing to this therapy. Forty-nine, PI experienced but APV naïve patients were treated with APV (600 mg bid) plus ritonavir (100 mg bid). By intent-to-treat analysis, the median decrease in viral load (VL) was -1.32 log10 (min +0.6; max -2.8) and -1.46 log10 (min +0.5; max -2.8) 12 and 24 weeks after initiating APV plus ritonavir regimen, respectively. Twelve patients harboured a VL >200 copies/ml at week 24. Among these patients, the selection of mutations previously described with the use of APV as first PI (V32I, L33F, M46I/L, I50V, 54M/L, and I84V) was observed. However, in some cases, mutations classically described after the use of other PIs (V82F and L90M) were selected but always with APV-specific mutations. There was no relation between the resistance pathways selected with either APV or ritonavir plasma minimal concentration, but higher APV plasma minimal concentration were associated with a lower rate of resistance mutations selection. PMID:15258963

  6. Effects of NO-Hybridization on the Immunomodulatory Properties of the HIV Protease Inhibitors Lopinavir and Ritonavir.

    PubMed

    Fagone, Paolo; Mangano, Katia; Quattrocchi, Cinzia; Cavalli, Eugenio; Mammana, Santa; Lombardo, Giuseppe A G; Pennisi, Vincenzo; Zocca, Mai-Britt; He, Mingzhu; Al-Abed, Yousef; Nicoletti, Ferdinando

    2015-11-01

    HIV protease inhibitors (PIs) are antiretroviral agents, which have been found to also affect several cellular processes, such as inflammation and cell progression. In studies on non-steroidal, anti-inflammatory drugs, the addition of a nitric oxide (NO) moiety has been shown to both reduce their toxicity and enhance their pharmacological efficacy. Along this line of research, several derivatives of PIs have been synthesized by covalent attachment of NO moiety to the parental molecules. Previous work has indicated that NO-hybridization of the prototypical PI, Saquinavir leads to a derivative named Saquinavir-NO that while retaining the antiretroviral effect, acquires antitumoural and immunomodulatory properties along with reduced toxicity in vitro and in vivo. These data prompted us to evaluate the effects of NO-hybridization on two other PIs, Lopinavir and Ritonavir. The two NO-derivatives were compared head to head with their parental compounds on human primary peripheral blood mononuclear cells as well as on human primary macrophages. Lopinavir-NO and Lopinavir were also screened in an in vivo model of autoimmune hepatitis. Our results prove that Lopinavir-NO exerts markedly superior effects as compared to the parental compound both in vitro and in vivo. On the contrary, Ritonavir-NO effects overlapped those of Ritonavir. These data demonstrate that NO-hybridization of Lopinavir generates a derivative with significantly stronger immunomodulatory effects that are apparently related to an action of the compound on T-cell secretory capacity. Lopinavir-NO deserves additional studies for its possible use in T-cell-mediated autoimmune diseases including, but not limited to autoimmune hepatitis. PMID:25903922

  7. Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

    SciTech Connect

    Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.; Laurence, Jeffrey

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. Black-Right-Pointing-Pointer Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. Black-Right-Pointing-Pointer Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. Black-Right-Pointing-Pointer Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/{beta}-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of {beta}-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, {beta}-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in

  8. Sildenafil and bosentan plasma concentrations in a human immunodeficiency virus- infected patient with pulmonary arterial hypertension treated with ritonavir-boosted protease inhibitor.

    PubMed

    Chinello, Pierangelo; Cicalini, Stefania; Pichini, Simona; Pacifici, Roberta; Tempestilli, Massimo; Cicini, Maria P; Pucillo, Leopoldo P; Petrosillo, Nicola

    2015-02-24

    Sildenafil and bosentan are increasingly used for the treatment of pulmonary arterial hypertension (PAH) in HIV-infected patients. However, concerns exist about pharmacokinetic interactions among sildenafil, bosentan and antiretroviral drugs, including protease inhibitors (PI). We describe here the case of an HIV-infected patient with PAH, who was co-administered bosentan 125 mg twice daily and sildenafil 40 mg three times per day, together with a ritonavir-boosted PI-based antiretroviral therapy; plasma levels of bosentan, sildenafil, N-desmethylsildenafil, and PI were measured. The patient had a sildenafil Cthrough and Cmax of 276.94 ng/mL and 1733.19 ng/mL, respectively. The Cthrough and the Cmax of bosentan were 1546.53 ng/mL and 3365.99 ng/mL, respectively. The patient was able to tolerate as high sildenafil blood concentrations as 10 times those usually requested and did not report any significant adverse reaction to sildenafil during the follow-up period. Therapeutic drug monitoring should be considered during sildenafil therapy in patients concomitantly treated with ritonavir-boosted PI. PMID:25874072

  9. Sildenafil and Bosentan Plasma Concentrations in a Human Immunodeficiency Virus- Infected Patient with Pulmonary Arterial Hypertension Treated with Ritonavir-Boosted Protease Inhibitor

    PubMed Central

    Chinello, Pierangelo; Cicalini, Stefania; Pichini, Simona; Pacifici, Roberta; Tempestilli, Massimo; Cicini, Maria P.; Pucillo, Leopoldo P.; Petrosillo, Nicola

    2015-01-01

    Sildenafil and bosentan are increasingly used for the treatment of pulmonary arterial hypertension (PAH) in HIV-infected patients. However, concerns exist about pharmacokinetic interactions among sildenafil, bosentan and antiretroviral drugs, including protease inhibitors (PI). We describe here the case of an HIV-infected patient with PAH, who was co-administered bosentan 125 mg twice daily and sildenafil 40 mg three times per day, together with a ritonavir-boosted PI-based antiretroviral therapy; plasma levels of bosentan, sildenafil, N-desmethylsildenafil, and PI were measured. The patient had a sildenafil Cthrough and Cmax of 276.94 ng/mL and 1733.19 ng/mL, respectively. The Cthrough and the Cmax of bosentan were 1546.53 ng/mL and 3365.99 ng/mL, respectively. The patient was able to tolerate as high sildenafil blood concentrations as 10 times those usually requested and did not report any significant adverse reaction to sildenafil during the follow-up period. Therapeutic drug monitoring should be considered during sildenafil therapy in patients concomitantly treated with ritonavir-boosted PI. PMID:25874072

  10. Complex Drug Interactions of the HIV Protease Inhibitors 3: Effect of Simultaneous or Staggered Dosing of Digoxin and Ritonavir, Nelfinavir, Rifampin, or Bupropion

    PubMed Central

    Kirby, Brian J.; Collier, Ann C.; Kharasch, Evan D.; Whittington, Dale; Thummel, Kenneth E.

    2012-01-01

    As part of a larger clinical drug-drug interaction (DDI) study aimed at in vitro to in vivo prediction of HIV protease inhibitor metabolic and transporter-based DDIs, we measured the inductive (staggered administration) and inductive plus inhibitory (simultaneously administered) effect of multiple dose ritonavir (RTV), nelfinavir (NFV), or rifampin (RIF) on the pharmacokinetics of the P-glycoprotein probe, digoxin (DIG), when administered simultaneously or staggered with the protease inhibitors or RIF. In both cases, NFV did not significantly affect DIG disposition. RTV decreased DIG renal clearance (Clrenal) when administered simultaneously or staggered but significantly increased DIG area under the curve from time zero to 24 h (AUC0–24 h) only when administered simultaneously. RIF decreased DIG AUC0–24 h only when RIF and DIG administration was staggered. When RIF and DIG were administered simultaneously, DIG maximal observed plasma concentration and area under the curve from time zero to 4 h were significantly increased, and DIG Clrenal was decreased. An unexpected and potentially clinically significant DDI was observed between DIG and the CYP2B6 probe, bupropion, which decreased DIG AUC0–24 h 1.6-fold and increased Clrenal 1.8-fold. Because this was an unexpected DDI and our studies were not specifically designed to quantify this interaction, further studies are required to confirm the interaction and understand the mechanistic basis of the DDI. In summary, RTV or NFV do not induce P-glycoprotein activity measured with DIG, and RIF does so only under staggered administration. PMID:22190694

  11. Complex Drug Interactions of HIV Protease Inhibitors 1: Inactivation, Induction, and Inhibition of Cytochrome P450 3A by Ritonavir or Nelfinavir

    PubMed Central

    Kirby, Brian J.; Collier, Ann C.; Kharasch, Evan D.; Whittington, Dale; Thummel, Kenneth E.

    2011-01-01

    Conflicting drug-drug interaction (DDI) studies with the HIV protease inhibitors (PIs) suggest net induction or inhibition of intestinal or hepatic CYP3A. As part of a larger DDI study in healthy volunteers, we determined the effect of extended administration of two PIs, ritonavir (RTV) or nelfinavir (NFV), or the induction-positive control rifampin on intestinal and hepatic CYP3A activity as measured by midazolam (MDZ) disposition after a 14-day treatment with the PI in either staggered (MDZ ∼12 h after PI) or simultaneous (MDZ and PI coadministered) manner. Oral and intravenous MDZ areas under the plasma concentration-time curves were significantly increased by RTV or NFV and were decreased by rifampin. Irrespective of method of administration, RTV decreased net intestinal and hepatic CYP3A activity, whereas NFV decreased hepatic but not intestinal CYP3A activity. The magnitude of these DDIs was more accurately predicted using PI CYP3A inactivation parameters generated in sandwich-cultured human hepatocytes rather than human liver microsomes. PMID:21406602

  12. Genotypic Inhibitory Quotient as Predictor of Virological Response to Ritonavir-Amprenavir in Human Immunodeficiency Virus Type 1 Protease Inhibitor-Experienced Patients

    PubMed Central

    Marcelin, Anne-Geneviève; Lamotte, Claire; Delaugerre, Constance; Ktorza, Nadine; Ait Mohand, Hocine; Cacace, Raquel; Bonmarchand, Manuela; Wirden, Marc; Simon, Anne; Bossi, Philippe; Bricaire, François; Costagliola, Dominique; Katlama, Christine; Peytavin, Gilles; Calvez, Vincent

    2003-01-01

    Forty-nine protease inhibitor (PI)-experienced but amprenavir (APV)-naïve patients experiencing virological failure were treated with ritonavir (RTV) (100 mg twice a day [b.i.d.]) plus APV (600 mg b.i.d.). Patients responded to therapy with a median viral load decrease of −1.32 log10 by week 12. The addition of low-dose RTV enhanced the minimal APV concentration in plasma (APV Cmin) up to 10-fold compared with that obtained with APV (1,200 mg b.i.d.) without RTV. Baseline PI resistance mutations (L10F/I/V, K20M/R, E35D, R41K, I54V, L63P, V82A/F/T/S, I84V) identified by univariate analysis and included in a genotypic score and APV Cmin at week 8 were predictive of the virological response at week 12. The response to APV plus RTV was significantly reduced in patients with six or more of the resistance mutations among the ones defined above. The genotypic inhibitory quotient, calculated as the ratio of the APV Cmin to the number of human immunodeficiency virus type 1 protease mutations, was a better predictor than the virological or pharmacological variables used alone. This genotypic inhibitory quotient could be used in therapeutic drug monitoring to define the concentrations in plasma needed to control replication of viruses with different levels of PI resistance, as measured by the number of PI resistance mutations. PMID:12543665

  13. Low rate of virological failure and maintenance of susceptibility to HIV-1 protease inhibitors with first-line lopinavir/ritonavir-based antiretroviral treatment in clinical practice.

    PubMed

    Prosperi, Mattia C F; Zazzi, Maurizio; Punzi, Grazia; Monno, Laura; Colao, Grazia; Corsi, Paola; Di Giambenedetto, Simona; Meini, Genny; Ghisetti, Valeria; Bonora, Stefano; Pecorari, Monica; Gismondo, Maria Rita; Bagnarelli, Patrizia; Carli, Tiziana; De Luca, Andrea

    2010-12-01

    Protease inhibitor (PI)-resistant HIV-1 has hardly ever been detected at failed boosted PI-based first-line antiretroviral regimens in clinical trials. However, this phenomenon has not been investigated in clinical practice. To address this gap, data from patients starting a first-line lopinavir/ritonavir (LPV/rtv)-based therapy with available baseline HIV-1 RNA load, a viral genotype and follow-up viral load after 3 and 6 months of treatment were extracted from the Italian Antiretroviral Resistance Cohort Analysis (ARCA) observational database. Based on survival analysis, 39 (7.1%) and 43 (7.8%) of the 548 examined patient cases had an HIV-1 RNA >500 and >50 copies/ml, respectively, after 6 months of treatment. Cox proportional hazard models detected baseline HIV-1 RNA (RH 1.79, 95%CI 1.10-2.92 per 1-log(10) increase, P=0.02) and resistance to the nucleoside backbone (RH 1.04, 95%CI 1.02-1.06 per 10-point increase using the Stanford HIVdb algorithm, P<0.001) as independent predictors of HIV-1 RNA at >500 copies/ml, but not at the >50 copies/ml cutoff criteria. Higher baseline viral load, older patient age, heterosexual route of infection and use of tenofovir/emtricitabine were predictors of failure at month 3 using the 50-copy and/or 500-copy threshold. Resistance to LPV/rtv did not occur or increase in any of the available 36 follow-up HIV-1 genotypes. Resistance to the nucleoside backbone (M184V) developed in four cases. Despite the likely differences in patient population and adherence, both the low rate of virological failure and the lack of development of LPV/rtv resistance documented in clinical trials are thus confirmed in clinical practice. PMID:20981785

  14. Ombitasvir, Paritaprevir, and Ritonavir

    MedlinePlus

    ... from spreading inside the body. Paritaprevir is a protease inhibitor. It works by decreasing the amount of HCV in the body. Ritonavir is a protease inhibitor. It helps to increase the amount of ...

  15. Complex Drug Interactions of HIV Protease Inhibitors 2: In Vivo Induction and In Vitro to In Vivo Correlation of Induction of Cytochrome P450 1A2, 2B6, and 2C9 by Ritonavir or Nelfinavir

    PubMed Central

    Kirby, Brian J.; Collier, Ann C.; Kharasch, Evan D.; Dixit, Vaishali; Desai, Pankaj; Whittington, Dale; Thummel, Kenneth E.

    2011-01-01

    Drug-drug interactions (DDIs) with the HIV protease inhibitors (PIs) are complex, paradoxical (e.g., ritonavir/alprazolam), and involve multiple mechanisms. As part of a larger study to better understand these DDIs and to devise a framework for in vitro to in vivo prediction of these DDIs, we determined the inductive effect of ∼2 weeks of administration of two prototypic PIs, nelfinavir (NFV), ritonavir (RTV), and rifampin (RIF; induction positive control) on the cytochrome P450 enzymes CYP1A2, CYP2B6, CYP2C9, and CYP2D6 and the inductive or inductive plus inhibitory effect of NFV, RTV, or RIF on CYP3A and P-glycoprotein in healthy human volunteers. Statistically significant induction of CYP1A2 (2.1-, 2.9-, and 2.2-fold), CYP2B6 (1.8-, 2.4-, and 4-fold), and CYP2C9 (1.3-, 1.8-, and 2.6-fold) was observed after NFV, RTV, or RIF treatment, respectively (as expected, CYP2D6 was not induced). Moreover, we accurately predicted the in vivo induction of these enzymes by quantifying their induction by the PIs in human hepatocytes and by using RIF as an in vitro to in vivo scalar. On the basis of the modest in vivo induction of CYP1A2, CYP2B6, or CYP2C9, the in vivo paradoxical DDIs with the PIs are likely explained by mechanisms other than induction of these enzymes such as induction of other metabolic enzymes, transporters, or both. PMID:21930825

  16. Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

    SciTech Connect

    Liu, Zhigang; Yedidi, Ravikiran S.; Wang, Yong; Dewdney, Tamaria G.; Reiter, Samuel J.; Brunzelle, Joseph S.; Kovari, Iulia A.; Kovari, Ladislau C.

    2013-01-08

    Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTV and MDR HIV-1 protease.

  17. Estimated Glomerular Filtration Rate Trajectories in HIV-Infected Subjects Treated With Different Ritonavir-Boosted Protease Inhibitors and Tenofovir Disoproxil Fumarate or Abacavir.

    PubMed

    Gianotti, Nicola; Galli, Laura; Poli, Andrea; Salpietro, Stefania; Nozza, Silvia; Carbone, Alessia; Merli, Marco; Ripa, Marco; Lazzarin, Adriano; Castagna, Antonella

    2016-05-01

    The aim of the study was to evaluate in human immunodeficiency virus (HIV)-infected patients estimated glomerular filtration rate (eGFR) trajectories during treatment with different protease inhibitors (PIs) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) plus tenofovir (TDF) or abacavir (ABC) and lamivudine or emtricitabine (xTC).Retrospective study of patients followed at a single clinical center; all patients who started TDF or ABC for the first time with a NNRTI or lopinavir/r (LPV/r) or atazanavir/r (ATV/r) or darunavir/r (DRV/r), for whom at least 1 eGFR value before the start and during the studied treatment was known, were included in this analysis. eGFR was calculated by means of the CKD-EPI formula. Univariate and multivariate mixed linear model (MLM) was applied to estimate eGFR slope with the considered antiretroviral treatment.In the 1658 patients treated with TDF/xTC (aged 43 [37-48] years, with an eGFR of 105 [96; 113] mL/min/1.73 m, 80% males, 92% Caucasians, 10% coinfected with HCV, 4% with diabetes, 11% with hypertension, 38% naive for antiretroviral therapy (ART), 37% with HIV-RNA <50 copies/mL) the median follow-up was 2.5 (1.2-4.6) years. Their adjusted eGFR slopes (95% CI) were -1.26 (-1.58; -0.95), -0.43 (-1.20; +0.33), -0.86 (-1.28; -0.44), and -0.20 (-0.42; +0.02) mL/min/1.73 m per year in patients treated with ATV/r, DRV/r, LPV/r, and NNRTI, respectively. Patients receiving ATV/r or LPV/r had a greater adjusted decline in eGFR compared with those receiving NNRTIs (difference -1.06 [-1.44; -0.69] mL/min/1.73 m per year, P <0.001; and -0.66 [-1.13; -0.20] mL/min/1.73 m per year, P = 0.005, respectively); adjusted eGFR slopes were similar in patients receiving DRV/r and in those receiving NNRTIs. Patients receiving ATV/r had a greater adjusted eGFR decline than those treated with DRV/r (difference -0.83 [-1.65; -0.02] mL/min/1.73 m per year; P = 0.04), but not than those receiving LPV/r; no significant difference was

  18. Estimated Glomerular Filtration Rate Trajectories in HIV-Infected Subjects Treated With Different Ritonavir-Boosted Protease Inhibitors and Tenofovir Disoproxil Fumarate or Abacavir

    PubMed Central

    Gianotti, Nicola; Galli, Laura; Poli, Andrea; Salpietro, Stefania; Nozza, Silvia; Carbone, Alessia; Merli, Marco; Ripa, Marco; Lazzarin, Adriano; Castagna, Antonella

    2016-01-01

    Abstract The aim of the study was to evaluate in human immunodeficiency virus (HIV)-infected patients estimated glomerular filtration rate (eGFR) trajectories during treatment with different protease inhibitors (PIs) or a non-nucleoside reverse transcriptase inhibitor (NNRTI) plus tenofovir (TDF) or abacavir (ABC) and lamivudine or emtricitabine (xTC). Retrospective study of patients followed at a single clinical center; all patients who started TDF or ABC for the first time with a NNRTI or lopinavir/r (LPV/r) or atazanavir/r (ATV/r) or darunavir/r (DRV/r), for whom at least 1 eGFR value before the start and during the studied treatment was known, were included in this analysis. eGFR was calculated by means of the CKD-EPI formula. Univariate and multivariate mixed linear model (MLM) was applied to estimate eGFR slope with the considered antiretroviral treatment. In the 1658 patients treated with TDF/xTC (aged 43 [37–48] years, with an eGFR of 105 [96; 113] mL/min/1.73 m2, 80% males, 92% Caucasians, 10% coinfected with HCV, 4% with diabetes, 11% with hypertension, 38% naive for antiretroviral therapy (ART), 37% with HIV-RNA <50 copies/mL) the median follow-up was 2.5 (1.2–4.6) years. Their adjusted eGFR slopes (95% CI) were −1.26 (−1.58; −0.95), −0.43 (−1.20; +0.33), −0.86 (−1.28; −0.44), and −0.20 (−0.42; +0.02) mL/min/1.73 m2 per year in patients treated with ATV/r, DRV/r, LPV/r, and NNRTI, respectively. Patients receiving ATV/r or LPV/r had a greater adjusted decline in eGFR compared with those receiving NNRTIs (difference −1.06 [−1.44; −0.69] mL/min/1.73 m2 per year, P <0.001; and −0.66 [−1.13; −0.20] mL/min/1.73 m2 per year, P = 0.005, respectively); adjusted eGFR slopes were similar in patients receiving DRV/r and in those receiving NNRTIs. Patients receiving ATV/r had a greater adjusted eGFR decline than those treated with DRV/r (difference −0.83 [−1.65; −0.02] mL/min/1.73 m2 per year; P = 0.04), but not

  19. Effect of protease inhibitors on the sense of taste.

    PubMed

    Schiffman, S S; Zervakis, J; Heffron, S; Heald, A E

    1999-10-01

    The purpose of this study was to investigate the taste properties of protease inhibitors which are essential components of drug regimes used to treat human immunodeficiency virus (HIV) infection. In this study, the taste properties of four protease inhibitors (indinavir, ritonavir, saquinavir, and nelfinavir) were investigated in unmedicated HIV-infected patients and healthy controls. Three of the four protease inhibitors (indinavir, ritonavir, and saquinavir) were found to be predominantly bitter (with additional qualities of medicinal, metallic, astringent, sour, and burning). Nelfinavir was found to be relatively tasteless. HIV-infected and uninfected control subjects detected protease inhibitors at similar concentrations, but HIV-infected subjects perceived suprathreshold concentrations as more bitter than controls. Detection thresholds ranged from 0.0061 mM for saquinavir in HIV-infected patients to 0.0702 mM for ritonavir in uninfected control subjects. Suprathreshold studies indicated that protease inhibitors modified the taste perception of a variety of other taste compounds. These results are consistent with clinical findings that protease inhibitors produce taste complaints that can impact patient compliance. PMID:10501290

  20. Inhibitors of rhomboid proteases.

    PubMed

    Wolf, Eliane V; Verhelst, Steven H L

    2016-03-01

    Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes. Several assays have been developed that are able to monitor rhomboid activity. With the aid of these assays, different types of inhibitors have been found, all based on electrophiles that covalently react with the active site machinery. Although the currently available inhibitors have limited selectivity and moderate potency, they can function as research tools and as starting point for the development of activity-based probes, which are reagents that can specifically detect active rhomboid species. Structural studies on complexes of inhibitors with the Escherichia coli rhomboid GlpG have provided insight into how substrate recognition may occur. Future synthetic efforts, aided by high-throughput screening or structure-based design, may lead to more potent and selective inhibitors for this interesting family of proteases. PMID:26166068

  1. 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

  2. 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

  3. 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

  4. Elevated adiponectin prevents HIV protease inhibitor toxicity and preserves cerebrovascular homeostasis in mice.

    PubMed

    Dasuri, Kalavathi; Pepping, Jennifer K; Fernandez-Kim, Sun-Ok; Gupta, Sunita; Keller, Jeffrey N; Scherer, Philipp E; Bruce-Keller, Annadora J

    2016-06-01

    HIV protease inhibitors are key components of HIV antiretroviral therapies, which are fundamental in the treatment of HIV infection. However, the protease inhibitors are well-known to induce metabolic dysfunction which can in turn escalate the complications of HIV, including HIV associated neurocognitive disorders. As experimental and epidemiological data support a therapeutic role for adiponectin in both metabolic and neurologic homeostasis, this study was designed to determine if increased adiponectin could prevent the detrimental effects of protease inhibitors in mice. Adult male wild type (WT) and adiponectin-overexpressing (ADTg) mice were thus subjected to a 4-week regimen of lopinavir/ritonavir, followed by comprehensive metabolic, neurobehavioral, and neurochemical analyses. Data show that lopinavir/ritonavir-induced lipodystrophy, hypoadiponectinemia, hyperglycemia, hyperinsulinemia, and hypertriglyceridemia were attenuated in ADTg mice. Furthermore, cognitive function and blood-brain barrier integrity were preserved, while loss of cerebrovascular markers and white matter injury were prevented in ADTg mice. Finally, lopinavir/ritonavir caused significant increases in expression of markers of brain inflammation and decreases in synaptic markers in WT, but not in ADTg mice. Collectively, these data reinforce the pathophysiologic link from metabolic dysfunction to loss of cerebrovascular and cognitive homeostasis; and suggest that preservation and/or replacement of adiponectin could prevent these key aspects of HIV protease inhibitor-induced toxicity in clinical settings. PMID:26912411

  5. Monotherapy with lopinavir/ritonavir versus standard of care in HIV-infected patients virologically suppressed while on treatment with protease inhibitor-based regimens: results from the MoLo study.

    PubMed

    Gianotti, Nicola; Poli, Andrea; Galli, Massimo; Pan, Angelo; Rizzardini, Giuliano; Soria, Alessandro; Viale, Pierluigi; Di Biagio, Antonio; Quirino, Tiziana; Viganò, Paolo; Bonfanti, Paolo; d'Arminio Monforte, Antonella; Fortino, Ida; Lazzarin, Adriano

    2014-10-01

    This study compared the cost-efficacy ratios of lopinavir/ritonavir monotherapy (LPV/r-MT) and of standard of care in virologically suppressed HIV-infected patients. The results of the efficacy and safety analyses are presented. We conducted a multicentre, randomised, open-label trial of HIV-infected adults on stable treatment, with HIV- RNA <50 copies/mL, randomised to continue the ongoing regimen (cART-arm) or to switch to LPV/r (400/100 mg BID) MT (MT-arm). Time to virological rebound (VR = confirmed HIV-RNA ?50 copies/mL) was estimated by Ka- plan-Meier method and changes in laboratory values during follow-up were evaluated by univariate mixed-linear models. Ninety-four patients were randomised and analysed (43 in the MT-arm and 51 in the cART-arm). Five (four in the MT and 1 in the cART-arm; p=0.175) had VR, but time to VR did not statistically differ between the two arms (p=0.143). Major PI mutations were not detected at VR. Patients on MT had significant increases in total choles- terol [difference in mean change between MT and cART arm: 0.77 (±0.30) mg/dL per month; p=0.012] and eGFR [difference in mean change between MT and cART arm: 0.24 (±0.11) mL/min/1.73 m2 per month; p=0.029]. LPV/r-MT seems safe in most patients and should be considered in patients who have developed kidney toxicity from tenofovir. PMID:25387282

  6. Lopinavir and Ritonavir

    MedlinePlus

    ... with other medications to treat human immunodeficiency virus (HIV) infection. Lopinavir and ritonavir are in a class ... inhibitors. They work by decreasing the amount of HIV in the blood. When lopinavir and ritonavir are ...

  7. Protein protease inhibitors in insects and comparison with mammalian inhibitors.

    PubMed

    Eguchi, M

    1993-01-01

    1. Studies on insect protein protease inhibitors are summarized. Biochemical, genetic and physiological investigations of the silkworm are performed. 2. In addition, the properties and characteristics of fungal protease inhibitors from the silkworm (Bombyx mori) are described and their importance as defensive functions is emphasized. 3. This review also concerns comparative and evolutionary studies of protease inhibitors from various sources. 4. The biological significance of inhibitors is discussed in view of the extensive experimental results. PMID:8365101

  8. Solubility profiling of HIV protease inhibitors in human intestinal fluids.

    PubMed

    Wuyts, Benjamin; Brouwers, Joachim; Mols, Raf; Tack, Jan; Annaert, Pieter; Augustijns, Patrick

    2013-10-01

    The present study pursued to profile the intestinal solubility of nine HIV protease inhibitors (PIs) in fasted- and fed-state human intestinal fluids (FaHIF, FeHIF) aspirated from four volunteers. In addition, the ability of fasted- and fed-state simulated intestinal fluids (FaSSIF, FeSSIF) to predict the intestinal solubility was evaluated. All PIs were poorly soluble in FaHIF (from 7 μM for ritonavir to 327 μM for darunavir) and FeHIF (from 15 μM for atazanavir to 409μM for darunavir). For four of nine PIs, food intake significantly enhanced the solubilizing capacity of intestinal fluids (up to 18.4-fold increase for ritonavir). The intersubject variability (average coefficient of variance CVfed = 60.6%, CVfasted = 40.4%) was higher as compared with the intrasubject variability (CVfed = 41.3%, CVfasted = 20.5%). PI solubilities correlated reasonably well between FaSSIF and FaHIF (R = 0.817), but not between FeSSIF and FeHIF (R = 0.617). To conclude, postprandial conditions increased the inter- and intrasubject variability of the PIs. The inability of FeSSIF to accurately predict the FeHIF solubility emphasizes the need for a multivariate approach to determine solubility profiles, taking into account solid-state characteristics, pH, mixed bile acid/phospholipid micelles, and digestive products. PMID:23939880

  9. 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

  10. Progress and prospects on DENV protease inhibitors.

    PubMed

    Timiri, Ajay Kumar; Sinha, Barij Nayan; Jayaprakash, Venkatesan

    2016-07-19

    New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B-NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective. PMID:27092412

  11. 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

  12. Synthesis of amino heterocycle aspartyl protease inhibitors.

    PubMed

    Chambers, Rachel K; Khan, Tanweer A; Olsen, David B; Sleebs, Brad E

    2016-06-14

    Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds. PMID:27143279

  13. Metabolomic screening and identification of bioactivation pathways of ritonavir

    PubMed Central

    Li, Feng; Lu, Jie; Ma, Xiaochao

    2011-01-01

    Ritonavir-boosted protease inhibitor regimens are widely used for HIV chemotherapy. However, ritonavir causes multiple side effects, and the mechanisms are not fully understood. The current study was designed to explore the metabolic pathways of ritonavir that may be related to its toxicity. Metabolomic analysis screened out 26 ritonavir metabolites in mice, and half of them are novel. These novel ritonavir metabolites include two glycine conjugated, two N-acetylcysteine conjugated and three ring-open products. Accompanied with the generation of ritonavir ring-open metabolites, the formation of methanethioamide and 2-methylpropanethioamide were expected. Based upon the structures of these novel metabolites, five bioactivation pathways are proposed, which may be associated with sulfation and epoxidation. By using Cyp3a-null mice, we confirmed that CYP3A is involved in four pathways of RTV bioactivation. In addition, all these five bioactivation pathways were recapitulated in the incubation of ritonavir in human liver microsomes. Further studies are suggested to determine the role of CYP3A and these bioactivation pathways in ritonavir toxicity. PMID:22040299

  14. Phenotypic characterization of virological failure following lopinavir/ritonavir monotherapy using full-length gag–protease genes

    PubMed Central

    Sutherland, Katherine A.; Mbisa, Jean L.; Ghosn, Jade; Chaix, Marie-Laure; Cohen-Codar, Isabelle; Hue, Stephane; Delfraissy, Jean-Francois; Delaugerre, Constance; Gupta, Ravindra K.

    2014-01-01

    Objectives Major protease mutations are rarely observed following first-line failure with PIs and interpretation of genotyping results in this context may be difficult. We performed extensive phenotyping of viruses from five patients failing lopinavir/ritonavir monotherapy in the MONARK study without major PI mutations by standard genotyping. Methods Phenotypic susceptibility testing and viral infectivity assessments were performed using a single-cycle assay and fold changes (FC) relative to a lopinavir-susceptible reference strain were calculated. Results >10-fold reduced baseline susceptibility to lopinavir occurred in two of five patients and >5-fold in another two. Four of five patients exhibited phylogenetic evidence of a limited viral evolution between baseline and failure, with amino acid changes at drug resistance-associated positions in one: T81A emerged in Gag with M36I in the protease gene, correlating with a reduction in lopinavir susceptibility from FC 7 (95% CI 6–8.35) to FC 13 (95% CI 8.11–17.8). Reductions in darunavir susceptibility (>5 FC) occurred in three individuals. Discussion This study suggests both baseline reduced susceptibility and evolution of resistance could be contributing factors to PI failure, despite the absence of classical PI resistance mutations by standard testing methods. Use of phenotyping also reveals lower darunavir susceptibility, warranting further study as this agent is commonly used following lopinavir failure. PMID:25096075

  15. Concentration-dependent effects and intracellular accumulation of HIV protease inhibitors in cultured CD4 T cells and primary human lymphocytes

    PubMed Central

    Janneh, Omar; Bray, Patrick G.; Jones, Elizabeth; Wyen, Christoph; Chiba, Peter; Back, David J.; Khoo, Saye H.

    2010-01-01

    Background The intracellular and plasma concentrations of HIV protease inhibitors (HPIs) vary widely in vivo. It is unclear whether there is a concentration-dependent effect of HPIs such that at increasing concentration they may either block their own efflux (leading to ‘autoboosting’) or influx (leading to saturability/decreased intracellular accumulation). Method The effects of various concentrations (0–30 µM) of lopinavir, saquinavir, ritonavir and atazanavir on the accumulation of [14C]lopinavir, [3H]saquinavir, [3H]ritonavir and [3H]atazanavir, respectively, were investigated in CEMparental, CEMVBL [P-glycoprotein (ABCB1) overexpressing], CEME1000 (MRP1 overexpressing) and in peripheral blood mononuclear cells (PBMCs). We also investigated the effects of inhibitors of ABCB1/ABCG2 (tariquidar), ABCC (MK571) and ABCC1/2 (frusemide), singly and in combination with HPIs, on cellular accumulation. Results In all the cell lines, with increasing concentration of lopinavir, saquinavir and ritonavir, there was a significant increase in the cellular accumulation of [14C]lopinavir, [3H]saquinavir and [3H]ritonavir. Tariquidar, MK571 and frusemide (alone and in combination with lopinavir, saquinavir and ritonavir) significantly increased the accumulation of [14C]lopinavir, [3H]saquinavir and [3H]ritonavir. Ritonavir (alone or in combination with tariquidar) decreased the intracellular accumulation of [3H]ritonavir in PBMCs. Atazanavir decreased the accumulation of [3H]atazanavir in a concentration-dependent manner in all of the cells tested. Conclusions There are complex and variable drug-specific rather than class-specific effects of the HPIs on their own accumulation. PMID:20237075

  16. Modulation of the LDL receptor and LRP levels by HIV protease inhibitors.

    PubMed

    Tran, Huan; Robinson, Susan; Mikhailenko, Irina; Strickland, Dudley K

    2003-10-01

    Inhibitors of the human immunodeficiency virus (HIV)-1 protease have proven to be effective antiretroviral drugs. However, patients receiving these drugs develop serious metabolic abnormalities, including hypercholesterolemia. The objective of the present study was to identify mechanisms by which HIV protease inhibitors increase plasma cholesterol levels. We hypothesized that HIV protease inhibitors may affect gene regulation of certain LDL receptor (LDLR) family members, thereby altering the catabolism of cholesterol-containing lipoproteins. In this present study we investigated the effect of several HIV protease inhibitors (ABT-378, Amprenavir, Indinavir, Nelfinavir, Ritonavir, and Saquinavir) on mRNA, protein, and functional levels of LDLR family members. Our results demonstrate that one of these drugs, Nelfinavir, significantly decreases LDLR and LDLR-related protein (LRP) mRNA and protein levels, resulting in the reduced functional activity of these two receptors. Nelfinavir exerts its effect by reducing levels of active SREBP1 in the nucleus. The finding that Nelfinavir reduces the levels of two key receptors (LRP and LDLR) involved in lipoprotein catabolism and maintenance of vessel wall integrity identifies a mechanism that causes hypercholesterolemia complications in HIV patients treated with this drug and raises concerns about the atherogenic nature of Nelfinavir. PMID:12837856

  17. Protease inhibitors targeting coronavirus and filovirus entry.

    PubMed

    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

    2015-04-01

    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

  18. 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

  19. Comparative study on the protease inhibitors from fish eggs

    NASA Astrophysics Data System (ADS)

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

    2005-07-01

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

  20. Neuroserpin, an axonally secreted serine protease inhibitor.

    PubMed Central

    Osterwalder, T; Contartese, J; Stoeckli, E T; Kuhn, T B; Sonderegger, P

    1996-01-01

    We have identified and chromatographically purified an axonally secreted glycoprotein of CNS and PNS neurons. Several peptides derived from it were microsequenced. Based on these sequences, a fragment of the corresponding cDNA was amplified and used as a probe to isolate a full length cDNA from a chicken brain cDNA library. Because the deduced amino acid sequence qualified the protein as a novel member of the serpin family of serine protease inhibitors, we called it neuroserpin. Analysis of the primary structural features further characterized neuroserpin as a heparin-independent, functional inhibitor of a trypsin-like serine protease. In situ hybridization revealed a predominantly neuronal expression during the late stages of neurogenesis and in the adult brain in regions which exhibit synaptic plasticity. Thus, neuroserpin might function as an axonally secreted regulator of the local extracellular proteolysis involved in the reorganization of the synaptic connectivity during development and synapse plasticity in the adult. Images PMID:8670795

  1. Impact of protease inhibitors on the evolution of urinary markers: Subanalyses from an observational cross-sectional study.

    PubMed

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

    2016-08-01

    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 renal

  2. Fosamprenavir calcium plus ritonavir for HIV infection.

    PubMed

    Torres, Harrys A; Arduino, Roberto C

    2007-06-01

    Fosamprenavir is a protease inhibitor (PI) approved for the treatment of HIV-1 infection. Fosamprenavir is a prodrug of amprenavir developed to reduce the pill burden yet maintain the unique resistance pattern and efficacy associated with amprenavir. In a head-to-head, noninferiority trial in antiretroviral treatment-naive HIV-infected patients, the antiviral efficacy and tolerability of ritonavir-boosted fosamprenavir was not inferior to ritonavir-boosted lopinavir, when the PIs were combined with two other nucleoside reverse transcriptase inhibitors. There are fewer studies published about fosamprenavir use in antiretroviral treatment-experienced HIV-infected patients. The high genetic barrier to the development of resistance to fosamprenavir and the low level of cross-resistance between ritonavir-boosted fosamprenavir and other PI regimens are notable. As with amprenavir, gastrointestinal disturbance and rash are the most frequent short-term treatment-limiting events with fosamprenavir. Treatment with ritonavir-boosted fosamprenavir can produce a durable response. To date, fosamprenavir is one of the recommended preferred PI components for the treatment of antiretroviral-naive HIV-infected patients. PMID:17547501

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

    PubMed Central

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

    2015-01-01

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

  4. Effect of fosamprenavir/ritonavir on the pharmacokinetics of single-dose olanzapine in healthy volunteers.

    PubMed

    Jacobs, Birgit S; Colbers, Angela P H; Velthoven-Graafland, Kirsten; Schouwenberg, Bas J J W; Burger, David M

    2014-08-01

    Psychosis and other mental illnesses are common in HIV-infected patients. Olanzapine is one of the preferred antipsychotic agents for the treatment of schizophrenia. Olanzapine is primarily metabolised by CYP1A2 and uridine diphosphate glucuronosyltransferase (UGT). High-dose ritonavir has been shown to increase olanzapine elimination through induction of CYP1A2 and/or UGT, but the effect of low-dose ritonavir on olanzapine pharmacokinetics is unknown. Fosamprenavir is an HIV protease inhibitor that is boosted by low-dose ritonavir. To compensate for the induction of olanzapine metabolism by fosamprenavir/ritonavir, we hypothesised that a dose increase of olanzapine to 15 mg with fosamprenavir/ritonavir would lead to a similar area under the concentration-time curve (AUC) compared with olanzapine 10 mg alone. An open-label, randomised, two-period, cross-over, single-centre trial was conducted in 24 healthy volunteers. Subjects were randomised to one of the following treatments: (A) fosamprenavir/ritonavir 700/100 mg twice daily (b.i.d.) for 16 days with a single dose of olanzapine 15 mg on Day 13, a wash-out period of 31 days and a single dose of olanzapine 10 mg on Day 48; or (B) the same medication in reverse order. Twenty subjects completed the trial. The geometric mean ratios (90% CI) of olanzapine AUClast, maximum drug concentration (C(max)) and apparent elimination half-life (t(1/2)) when taken with fosamprenavir/ritonavir versus olanzapine alone were 1.00 (0.93-1.08), 1.32 (1.18-1.47) and 0.68 (0.63-0.74), respectively. Fosamprenavir/ritonavir 700/100 mg b.i.d. appeared to induce olanzapine metabolism. We therefore propose a 50% dosage increase of olanzapine when combining with a ritonavir-boosted protease inhibitor. PMID:24929949

  5. HIV-1 protease mutations and protease inhibitor cross-resistance.

    PubMed

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W

    2010-10-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  6. 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

  7. 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

  8. A human immunodeficiency virus protease inhibitor is a novel functional inhibitor of human pregnane X receptor.

    PubMed

    Healan-Greenberg, Christine; Waring, Jeffrey F; Kempf, Dale J; Blomme, Eric A G; Tirona, Rommel G; Kim, Richard B

    2008-03-01

    Drug-drug interactions involving induction of cytochrome P450 enzymes (P450s) can lead to loss of drug efficacy. Certain drugs, particularly those used to treat mycobacterial and human immunodeficiency virus (HIV) infections, are especially prone to induce P450s. During studies to examine drug-interaction potential of compounds in cultured human hepatocytes, exposure with (S)-1-[(1S,3S,4S)-4-[(S)-2-(3-benzyl-2-oxo-imidazolidin-1-yl)-3,3-dimethyl-butyrylamino]-3-hydroxy-5-phenyl-1-(4-pyridin-2-yl-benzyl)-pentylcarbamoyl]-2,2-dimethyl-propyl-carbamic acid methyl ester (A-792611), a novel HIV protease inhibitor (PI) previously under investigation for the treatment of HIV infection, resulted in significant down-regulation of constitutive CYP3A4 expression. Furthermore, coadministration of A-792611 was found to attenuate CYP3A4 induction mediated by known inducers rifampin and efavirenz. A-792611 also attenuated the rifampin and ritonavir-mediated activation of the human pregnane X receptor (PXR) in luciferase reporter assays. Microarray analysis on cultured human hepatocytes revealed that A-792611 treatment down-regulated the expression of PXR target genes CYP3A4, CYP2B6, CYP2C8, and CYP2C9, whereas there was a lack of inductive effect observed in treated rat hepatocytes. A-792611 did not interact with other ligand-activated nuclear receptors that regulate P450 expression such as constitutive androstane receptor, farnesoid X receptor, vitamin D receptor, and peroxisome proliferator-activated receptor alpha. These data suggest that A-792611 is a functional and effective human PXR inhibitor. Among the class of HIV-PIs, which are typically PXR activators, A-792611 seems to have a unique property for PXR antagonism and could be a useful tool for studying nuclear receptor pathway regulation. PMID:18096673

  9. Switching to a Protease Inhibitor–Containing, Nucleoside-Sparing Regimen (Lopinavir/Ritonavir Plus Efavirenz) Increases Limb Fat But Raises Serum Lipid Levels

    PubMed Central

    Tebas, Pablo; Zhang, Jiameng; Yarasheski, Kevin; Evans, Scott; Fischl, Margaret A.; Shevitz, Abby; Feinberg, Judith; Collier, Ann C.; Shikuma, Cecilia; Brizz, Barbara; Sattler, Fred

    2015-01-01

    Background Subcutaneous limb fat loss continues to be one the most troubling side effects of long-term antiretroviral regimens. Nucleoside analogues and protease inhibitors (PIs) have been linked to the development of this complication. Methods We evaluated the effects of nucleoside-sparing and PI-sparing regimens on fat distribution, bone mineral density, and metabolic parameters in 62 subjects, who were not selected for lipoatrophy, with advanced HIV (nadir CD4 count ≤200 cells/mm3 or HIV RNA level ≥80,000 copies/mL) and an undetectable HIV viral load. Participants were randomized to switch their initial successful antiretroviral regimen to open-label lopinavir/ritonavir (LPV/r) at a dose of 533/133 mg twice a day and efavirenz (EFV) at a dose of 600 mg/d (the nucleoside-sparing arm) versus EFV and 2 nucleoside analogues (the PI-sparing arm). Findings At week 48, the median change in limb fat in the nucleoside-sparing arm was 562 g (6%, interquartile range [IQR]: −218–1186 g) versus a loss of −242 g (−4%, IQR: −539–452 g) in the nucleoside-containing PI-sparing arm (P = 0.086). At the time of last observation (median = 102 weeks, IQR: 73–152 weeks), a median gain of 782 g (10%, IQR: −380–1168 g) of limb fat was noted in the nonnucleoside arm (n = 22) versus a loss of 850 g (−15%, IQR: −1270 to −526 g) in the nucleoside-containing arm (n = 25; P = 0.002). Interpretation The switch to a nucleoside-sparing combination antiretroviral regimen (LPV/r + EFV) was associated with significant improvement in limb fat. These results provide additional evidence that nucleoside analogues are important in the progressive limb fat loss that characterizes antiretroviral treatment and that switching medications can significantly improve this complication. This option has to be carefully balanced with the potential to increase serum lipid levels and the trend to increase virologic failure. PMID:17527093

  10. Susceptibility of the Porcine Endogenous Retrovirus to Reverse Transcriptase and Protease Inhibitors

    PubMed Central

    Qari, Shoukat H.; Magre, Saema; García-Lerma, J. Gerardo; Hussain, Althaf I.; Takeuchi, Yasuhiro; Patience, Clive; Weiss, Robin A.; Heneine, Walid

    2001-01-01

    Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC50s and IC90s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC50s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culture-based, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC50s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine. PMID:11134319

  11. Structure and mechanism of the complex between cytochrome P4503A4 and ritonavir

    PubMed Central

    Sevrioukova, Irina F.; Poulos, Thomas L.

    2010-01-01

    Ritonavir is a HIV protease inhibitor routinely prescribed to HIV patients that also potently inactivates cytochrome P4503A4 (CYP3A4), the major human drug-metabolizing enzyme. By inhibiting CYP3A4, ritonavir increases plasma concentrations of other anti-HIV drugs oxidized by CYP3A4 thereby improving clinical efficacy. Despite the importance and wide use of ritonavir in anti-HIV therapy, the precise mechanism of CYP3A4 inhibition remains unclear. The available data are inconsistent and suggest that ritonavir acts as a mechanism-based, competitive or mixed competitive-noncompetitive CYP3A4 inactivator. To resolve this controversy and gain functional and structural insights into the mechanism of CYP3A4 inhibition, we investigated the ritonavir binding reaction by kinetic and equilibrium analysis, elucidated how the drug affects redox properties of the hemoprotein, and determined the 2.0 Å X-ray structure of the CYP3A4-ritonavir complex. Our results show that ritonavir is a type II ligand that perfectly fits into the CYP3A4 active site cavity and irreversibly binds to the heme iron via the thiazole nitrogen, which decreases the redox potential of the protein and precludes its reduction with the redox partner, cytochrome P450 reductase. PMID:20937904

  12. A symmetric inhibitor binds HIV-1 protease asymmetrically.

    PubMed

    Dreyer, G B; Boehm, J C; Chenera, B; DesJarlais, R L; Hassell, A M; Meek, T D; Tomaszek, T A; Lewis, M

    1993-01-26

    Potential advantages of C2-symmetric inhibitors designed for the symmetric HIV-1 protease include high selectivity, potency, stability, and bioavailability. Pseudo-C2-symmetric monools and C2-symmetric diols, containing central hydroxymethylene and (R,R)-dihydroxyethylene moieties flanked by a variety of hydrophobic P1/P1' side chains, were studied as HIV-1 protease inhibitors. The monools and diols were synthesized in 8-10 steps from D-(+)-arabitol and D-(+)-mannitol, respectively. Monools with ethyl or isobutyl P1/P1' side chains were weak inhibitors of recombinant HIV-1 protease (Ki > 10 microM), while benzyl P1/P1' side chains afforded a moderately potent inhibitor (apparent Ki = 230 nM). Diols were 100-10,000x more potent than analogous monools, and a wider range of P1/P1' side chains led to potent inhibition. Both classes of compounds exhibited lower apparent Ki values under high-salt conditions. Surprisingly, monool and diol HIV-1 protease inhibitors were potent inhibitors of porcine pepsin, a prototypical asymmetric monomeric aspartic protease. These results were evaluated in the context of the pseudosymmetric structure of monomeric aspartic proteases and their evolutionary kinship with the retroviral proteases. The X-ray crystal structure of HIV-1 protease complexed with a symmetric diol was determined at 2.6 A. Contrary to expectations, the diol binds the protease asymmetrically and exhibits 2-fold disorder in the electron density map. Molecular dynamics simulations were conducted beginning with asymmetric and symmetric HIV-1 protease/inhibitor model complexes. A more stable trajectory resulted from the asymmetric complex, in agreement with the observed asymmetric binding mode. A simple four-point model was used to argue more generally that van der Waals and electrostatic force fields can commonly lead to an asymmetric association between symmetric molecules. PMID:8422397

  13. Protease inhibitor-induced nausea and vomiting is attenuated by a peripherally acting, opioid-receptor antagonist in a rat model

    PubMed Central

    Yuan, Chun-Su; Wang, Chong-Zhi; Mehendale, Sangeeta R; Aung, Han H; Foo, Adela; Israel, Robert J

    2009-01-01

    Background Protease inhibitors such as ritonavir can cause nausea and vomiting which is the most common reason for discontinuation. Rats react to nauseous and emetic stimuli by increasing their oral intake of non-nutritive substances like kaolin, known as pica behavior. In this study, we evaluated the effects of methylnaltrexone, a peripherally acting mu-opioid receptor antagonist that does not affect analgesia, on ritonavir-induced nausea and vomiting in a rat pica model. Results We observed that 24 to 48 hr after administration of oral ritonavir 20 mg/kg, kaolin consumption increased significantly in rats (P < 0.01). This increase was attenuated by pretreatment with an intraperitoneal injection of methylnaltrexone (0.3–3.0 mg/kg) in a dose dependent manner (P < 0.01) and also with naloxone (0.1–0.3 mg/kg) (P < 0.01). The areas under the curve for kaolin intake from time 0 to 120 hr were significantly reduced after administration of the opioid antagonists. Food intake was not significantly affected. Plasma naltrexone levels were measured after methylnaltrexone injection, and no detectable levels were found, indicating that methylnaltrexone was not demethylated in our experimental paradigm. Conclusion These results suggest that methylnaltrexone may have potential clinical utility in reducing nausea and vomiting in HIV patients who take ritonavir. PMID:19698111

  14. A NOVEL APPROACH TO REGULATE NITROGEN MINERALIZATION USING PROTEASE INHIBITORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mineralization of organic N sources by extracellular proteases affects both the availability of inorganic N to plants and losses of N to the environment. We hypothesized that (i) application of purified protease inhibitors would slow down soil N mineralization, and (ii) elevated concentrations of pr...

  15. 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

  16. 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.

  17. Delay of Iris flower senescence by protease inhibitors.

    PubMed

    Pak, Caroline; van Doorn, Wouter G

    2005-02-01

    Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than half to serine proteases, with a minor role of metalloproteases. Treatment of isolated tepals with the purported serine protease inhibitors AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride] or DFP (diisopropyl-fluorophosphate) prevented the increase in endoprotease activity and considerably delayed or prevented the normal senescence symptoms. The specific cysteine protease-specific E-64d reduced maximum endoprotease activity by 30%, but had no effect on the time to visible senescence. Zinc chloride and aprotinin reduced maximum endoprotease activity by c. 50 and 40%, respectively, and slightly delayed visible senescence. A proteasome inhibitor (Z-leu-leu-Nva-H) slightly delayed tepal senescence, which indicates that protein degradation in the proteasome may play a role in induction of the visible senescence symptoms. It is concluded that visible senescence is preceded by large-scale protein degradation, which is apparently mainly due to cysteine- and serine protease activity, and that two (unspecific) inhibitors of serine proteases considerably delay the senescence symptoms. PMID:15720658

  18. Protease inhibitors decrease the resistance of Vitaceae to Plasmopara viticola.

    PubMed

    Gindro, Katia; Berger, Valentine; Godard, Sophie; Voinesco, Francine; Schnee, Sylvain; Viret, Olivier; Alonso-Villaverde, Virginia

    2012-11-01

    Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll). PMID:22906813

  19. Fighting an enemy within: cytoplasmic inhibitors of bacterial cysteine proteases.

    PubMed

    Potempa, Jan; Golonka, Ewa; Filipek, Renata; Shaw, Lindsey N

    2005-08-01

    The genes encoding secreted, broad-spectrum activity cysteine proteases of Staphylococcus spp. (staphopains) and Streptococcus pyogenes (streptopain, SpeB) are genetically linked to genes encoding cytoplasmic inhibitors. While staphopain inhibitors have lipocalin-like folds, streptopain is inhibited by a protein bearing the scaffold of the enzyme profragment. Bioinformatic analysis of other prokaryotic genomes has revealed that two more species may utilize this same genetic arrangement to control streptopain-like proteases with lipocalin-like inhibitors, while three other species may employ a C-terminally located domain that resembles the profragment. This apparently represents a novel system that bacteria use to control the intracellular activity of their proteases. PMID:16045606

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

    PubMed

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

    2013-02-01

    Telaprevir and boceprevir are the first direct-acting antiviral agents approved for use in HCV treatment and represent a significant advance in HCV therapy. However, these first-generation drugs also have significant limitations related to thrice-daily dosing, clinically challenging side-effect profiles, low barriers to resistance and a lack of pan-genotype activity. A second wave of protease inhibitors are in phase II and III trials and promise to provide a drug regimen with a better dosing schedule and improved tolerance. These second-wave protease inhibitors will probably be approved in combination with PEG-IFN and Ribavirin (RBV), as well as future all-oral regimens. The true second-generation protease inhibitors are in earlier stages of development and efficacy data are anxiously awaited as they may provide pan-genotypic antiviral activity and a high genetic barrier to resistance. PMID:23286850

  1. 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

  2. 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…

  3. Protease inhibitor expression in soybean roots exhibiting susceptible and resistance reactions to soybean cyst nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protease inhibitors play a role in regulating proteases during cellular development and in plant defense against insects and nematodes. We identified, cloned and sequenced cDNAs encoding six protease inhibitors expressed in soybean roots infected with soybean cyst nematode. Four of these protease in...

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

    PubMed Central

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

    2002-01-01

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

  5. 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

  6. Nutritional status changes in HIV-infected children receiving combined antiretroviral therapy including protease inhibitors.

    PubMed

    Fiore, P; Donelli, E; Boni, S; Pontali, E; Tramalloni, R; Bassetti, D

    2000-11-01

    Maintaining linear growth and weight gain in HIV-infected children is often difficult. Nutritional evaluation and support are recognised as important factors to improve their quality of life. Combination antiretroviral therapy including protease inhibitors (HAART) reduces HIV-viral load and improves survival, quality of life and nutritional status. Our study aimed to determine changes in nutrional status based on body weight, height and nutritional habits, of HIV-infected children receiving HAART. Possible side effects of lipid metabolism were also studied. Twenty five children, 13 treated with HAART (group B) were followed up for 12 months. We did not observe statistically significant differences in nutritional status over that time or between groups A and B. Inadequate energy intake was more common in patients with advanced HIV-disease. Hyperlipidemia was found in 70% of children receiving ritonavir and in approximately 50% of children receiving nelfinavir. We observed an important although not statistically significative modification in the height of those in group B. PMID:11091066

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

    PubMed

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

    2016-06-01

    HIV(+) subjects on optimal antiretroviral therapy have persistently impaired antibody responses to pneumococcal vaccination. We explored the possibility that this effect may be due to HIV protease inhibitors (PIs). We found that in humans and mice, PIs do not affect antibody production in response to pneumococcal vaccination. PMID:27074938

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

    PubMed Central

    Troll, W

    1989-01-01

    Many tumor promoters are inflammatory agents that stimulate the formation of oxygen radicals (.O2-) and hydrogen peroxide (H2O2) in phagocytic neutrophils. The neutrophils use the oxygen radicals to kill bacteria, which are recognized by the cell membrane of phagocytic cells causing a signal to mount the oxygen response. The tumor promoter isolated from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimics the signal, causing an oxygen radical release that is intended to kill bacteria; instead, it injures cells in the host. Oxygen radicals cause single strand breaks in DNA and modify DNA bases. These damaging reactions appear to be related to tumor promotion, as three types of chemopreventive agents, retinoids, onion oil, and protease inhibitors, suppress the induction of oxygen radicals in phagocytic neutrophils and suppress tumor promotion in skin cancer in mice. Protease inhibitors also suppress breast and colon cancers in mice. Protease inhibitors capable of inhibiting chymotrypsin show a greater suppression of the oxygen effect and are better suppressors of tumor promotion. In addition, oxygen radicals may be one of the many agents that cause activation of oncogenes. Since retinoids and protease inhibitors suppress the expression of the ras oncogene in NIH 3T3 cells, NIH 3T3 cells may serve as a relatively facile model for finding and measuring chemopreventive agents that interfere with the carcinogenic process. PMID:2667986

  9. Computational Prediction of HIV-1 Resistance to Protease Inhibitors.

    PubMed

    Hosseini, Ali; Alibés, Andreu; Noguera-Julian, Marc; Gil, Victor; Paredes, Roger; Soliva, Robert; Orozco, Modesto; Guallar, Victor

    2016-05-23

    The development of mutations in HIV-1 protease (PR) hinders the activity of antiretroviral drugs, forcing changes in drug prescription. Most resistance assessments used to date rely on expert-based rules on predefined sets of stereotypical mutations; such an information-driven approach cannot capture new polymorphisms or be applied for new drugs. Computational modeling could provide a more general assessment of drug resistance and could be made available to clinicians through the Internet. We have created a protocol involving sequence comparison and all-atom protein-ligand induced fit simulations to predict resistance at the molecular level. We first compared our predictions with the experimentally determined IC50 values of darunavir, amprenavir, ritonavir, and indinavir from reference PR mutants displaying different resistance levels. We then performed analyses on a large set of variants harboring more than 10 mutations. Finally, several sequences from real patients were analyzed for amprenavir and darunavir. Our computational approach detected all of the genotype changes triggering high-level resistance, even those involving a large number of mutations. PMID:27082876

  10. Computing Clinically Relevant Binding Free Energies of HIV-1 Protease Inhibitors

    PubMed Central

    2014-01-01

    The use of molecular simulation to estimate the strength of macromolecular binding free energies is becoming increasingly widespread, with goals ranging from lead optimization and enrichment in drug discovery to personalizing or stratifying treatment regimes. In order to realize the potential of such approaches to predict new results, not merely to explain previous experimental findings, it is necessary that the methods used are reliable and accurate, and that their limitations are thoroughly understood. However, the computational cost of atomistic simulation techniques such as molecular dynamics (MD) has meant that until recently little work has focused on validating and verifying the available free energy methodologies, with the consequence that many of the results published in the literature are not reproducible. Here, we present a detailed analysis of two of the most popular approximate methods for calculating binding free energies from molecular simulations, molecular mechanics Poisson–Boltzmann surface area (MMPBSA) and molecular mechanics generalized Born surface area (MMGBSA), applied to the nine FDA-approved HIV-1 protease inhibitors. Our results show that the values obtained from replica simulations of the same protease–drug complex, differing only in initially assigned atom velocities, can vary by as much as 10 kcal mol–1, which is greater than the difference between the best and worst binding inhibitors under investigation. Despite this, analysis of ensembles of simulations producing 50 trajectories of 4 ns duration leads to well converged free energy estimates. For seven inhibitors, we find that with correctly converged normal mode estimates of the configurational entropy, we can correctly distinguish inhibitors in agreement with experimental data for both the MMPBSA and MMGBSA methods and thus have the ability to rank the efficacy of binding of this selection of drugs to the protease (no account is made for free energy penalties associated with

  11. HIV Protease Inhibitors Act as Competitive Inhibitors of the Cytoplasmic Glucose Binding Site of GLUTs with Differing Affinities for GLUT1 and GLUT4

    PubMed Central

    Hresko, Richard C.; Hruz, Paul W.

    2011-01-01

    The clinical use of several first generation HIV protease inhibitors (PIs) is associated with the development of insulin resistance. Indinavir has been shown to act as a potent reversible noncompetitive inhibitor of zero-trans glucose influx via direct interaction with the insulin responsive facilitative glucose transporter GLUT4. Newer drugs within this class have differing effects on insulin sensitivity in treated patients. GLUTs are known to contain two distinct glucose-binding sites that are located on opposite sides of the lipid bilayer. To determine whether interference with the cytoplasmic glucose binding site is responsible for differential effects of PIs on glucose transport, intact intracellular membrane vesicles containing GLUT1 and GLUT4, which have an inverted transporter orientation relative to the plasma membrane, were isolated from 3T3-L1 adipocytes. The binding of biotinylated ATB-BMPA, a membrane impermeable bis-mannose containing photolabel, was determined in the presence of indinavir, ritonavir, atazanavir, tipranavir, and cytochalasin b. Zero-trans 2-deoxyglucose transport was measured in both 3T3-L1 fibroblasts and primary rat adipocytes acutely exposed to these compounds. PI inhibition of glucose transport correlated strongly with the PI inhibition of ATB-BMPA/transporter binding. At therapeutically relevant concentrations, ritonavir was not selective for GLUT4 over GLUT1. Indinavir was found to act as a competitive inhibitor of the cytoplasmic glucose binding site of GLUT4 with a KI of 8.2 µM. These data establish biotinylated ATB-BMPA as an effective probe to quantify accessibility of the endofacial glucose-binding site in GLUTs and reveal that the ability of PIs to block this site differs among drugs within this class. This provides mechanistic insight into the basis for the clinical variation in drug-related metabolic toxicity. PMID:21966466

  12. Natural products from Garcinia brasiliensis as Leishmania protease inhibitors.

    PubMed

    Pereira, Ivan O; Assis, Diego M; Juliano, Maria A; Cunha, Rodrigo L O R; Barbieri, Clara L; do Sacramento, Luis V S; Marques, Marcos J; dos Santos, Marcelo H

    2011-06-01

    The infections by protozoans of the genus Leishmania are a major worldwide health problem, with high endemicity in developing countries. The drugs of choice for the treatment of leishmaniasis are the pentavalent antimonials, which cause renal and cardiac toxicity. As part of a search for new drugs against leishmaniasis, we evaluated the in vitro Leishmania protease inhibition activity of extracts (hexanic, ethyl-acetate, and ethanolic) and fukugetin, a bioflavonoid purified from the ethyl-acetate extract of the pericarp of the fruit of Garcinia brasiliensis, a tree native to Brazilian forests. The isolated compound was characterized by using spectral analyses with nuclear magnetic resonance, mass spectroscopy, ultraviolet, and infrared techniques. The ethyl-acetate extract and the compound fukugetin showed significant activity as inhibitors of Leishmania's proteases, with mean (±SD) IC(50) (50% inhibition concentration of protease activity) values of 15.0±1.3 μg/mL and 3.2±0.5 μM/mL, respectively, characterizing a bioguided assay. In addition, this isolated compound showed no activity against promastigote and amastigote forms of L. (L.) amazonensis and mammalian cells. These results suggest that fukugetin is a potent protease inhibitor of L. (L.) amazonensis and does not cause toxicity in mammalian or Leishmania cells in vitro. This study provides new perspectives on the development of novel drugs that have leishmanicidal activity obtained from natural products and that target the parasite's proteases. PMID:21554130

  13. 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

  14. 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

  15. Protease Inhibitors in View of Peptide Substrate Databases.

    PubMed

    Waldner, Birgit J; Fuchs, Julian E; Schauperl, Michael; Kramer, Christian; Liedl, Klaus R

    2016-06-27

    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

  16. Hepatitis C Virus NS3/4A Protease Inhibitors.

    PubMed

    López-Labrador, Francesc-Xavier

    2008-11-01

    Chronic hepatitis C virus infection is a global problem worldwide due to the lack of an effective therapy (the current standard of care treatment is effective in about 40-50% of the cases), and the difficulties in developing a protective vaccine. Chronic infection progresses to end-stage liver disease and liver failure in a considerable number of infected individuals. Once liver function is compromised, the only reliable therapeutic intervention is liver transplantation. Unfortunately, re-infection of the graft is unavoidable, and a new chronic hepatitis is early established in transplant recipients, that can result in graft loss. Thus, there is an urgent need for new, specifically targeted therapies for the treatment of HCV chronic infection. Among the viral proteins, the NS3/4A protease and the NS5b RNA-dependent RNA-polymerase, essential for the virus life cycle, have concentrated the efforts in the development of new antivirals, and some promising ones have already entered clinical trials. In particular, inhibitors of the HCV NS3/4A protease are the most advanced in clinical development. This review summarizes the available data for the most important HCV NS3/4A protease inhibitors in development, the most recent patents of these type of compounds, the envisioned options for future HCV therapies, and the eventual impact of HCV genetic variability on resistance to new NS3/4A protease inhibitors. PMID:18991798

  17. Schistosome serine protease inhibitors: parasite defense or homeostasis?

    PubMed Central

    Lopez Quezada, Landys A.; McKerrow, James H.

    2016-01-01

    Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL). Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases. PMID:21670886

  18. The effect of HIV protease inhibitors on amyloid-β peptide degradation and synthesis in human cells and Alzheimer's disease animal model.

    PubMed

    Lan, Xiqian; Kiyota, Tomomi; Hanamsagar, Richa; Huang, Yunlong; Andrews, Scott; Peng, Hui; Zheng, Jialin C; Swindells, Susan; Carlson, George A; Ikezu, Tsuneya

    2012-06-01

    Combined antiretroviral therapy (ART) tremendously improved the lifespan and symptoms associated with AIDS-defining illness in affected individuals. However, chronic ART-treated patients frequently develop age-dependent complications, including dementia, diabetes, and hyperlipidemia: all risk factors of Alzheimer's disease. Importantly, the effect of ART compounds on amyloid generation and clearance has never been systematically examined. Nine prescribed HIV protease inhibitors were tested for their effect on amyloid-β peptide (Aβ) clearance in primary cultured human monocyte-derived macrophages. Atazanavir, ritonavir, and saquinavir modestly inhibited of Aβ degradation, while lopinavir, nelfinavir, and ritonavir enhanced secretion of undigested Aβ after phagocytosis. Lopinavir, nelfinavir, ritonavir, and saquinavir inhibited endogenous Aβ40 production from primary cultured human cortical neurons, which were associated with reduction in Beta-site APP Converting Enzyme 1 (BACE1) and γ-secretase enzyme activities. However, ART compounds showed little inhibition of purified BACE1 activity in vitro, suggesting the indirect effect of ART compounds on BACE1 activity in neurons. Finally, nefinavir or lopinavir/ritonavir (Kaletra) were orally administered for 30 days into APP SCID mice expressing a double mutant form of APP 695 (KM670/671NL + V717F) in homozygosity for the scid allele of Prkdc. There was no difference in beta-amyloidosis by ART drug administration as determined by both immunohistochemistry and ELISA measurements although the therapeutic doses of the ART compounds was present in the brain. These data demonstrated that ART drugs can inhibit Aβ clearance in macrophages and Aβ production in neurons, but these effects did not significantly alter Aβ accumulation in the mouse brain. PMID:21826404

  19. Characterization of resistance mutations against HCV ketoamide protease inhibitors.

    PubMed

    Tong, Xiao; Bogen, Stephane; Chase, Robert; Girijavallabhan, V; Guo, Zhuyan; Njoroge, F George; Prongay, Andrew; Saksena, Anil; Skelton, Angela; Xia, Ellen; Ralston, Robert

    2008-03-01

    An issue of clinical importance in the development of new antivirals for HCV is emergence of resistance. Several resistance loci to ketoamide inhibitors of the NS3/4A protease have been identified (residues V36, T54, R155, A156, and V170) by replicon and clinical studies. Using SCH 567312, a more potent protease inhibitor derived from SCH 503034 (boceprevir) series, we identified two new positions (Q41 and F43) that confer resistance to the ketoamide class. The catalytic efficiency of protease enzymes was not affected by most resistance mutations, whereas replicon fitness varied with specific mutations. SCH 503034 and another ketoamide inhibitor, VX-950 (telaprevir), showed moderate losses of activity against most resistance mutations (< or =10-fold); the highest resistance level was conferred by mutations at A156 locus. Although SCH 503034 and VX-950 bind similarly to the active site, differences in resistance level were observed with specific mutations. Changes at V36 and R155 had more severe impact on VX-950, whereas mutations at Q41, F43 and V170 conferred higher resistance to SCH 503034. Structural analysis of resistance mutations on inhibitor binding is discussed. PMID:18201776

  20. HIV Protease Inhibitors: Effect on the Opportunistic Protozoan Parasites

    PubMed Central

    Alfonso, Yenisey; Monzote, Lianet

    2011-01-01

    The impact of highly active antiretroviral therapy (HAART) in the natural history of AIDS disease has been allowed to prolong the survival of people with HIV infection, particularly whose with increased HIV viral load. Additionally, the antiretroviral therapy could exert a certain degree of protection against parasitic diseases. A number of studies have been evidenced a decrease in the incidence of opportunistic parasitic infections in the era of HAART. Although these changes have been attributed to the restoration of cell-mediated immunity, induced by either non-nucleoside reverse transcriptase inhibitors or HIV protease inhibitors, in combination with at least two nucleoside reverse transcriptase inhibitors included in HAART, there are evidence that the control of these parasitic infections in HIV-positive persons under HAART, is also induced by the inhibition of the proteases of the parasites. This review focuses on the principal available data related with therapeutic HIV-protease inhibitors and their in vitro and in vivo effects on the opportunistic protozoan parasites. PMID:21629510

  1. 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

  2. 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

  3. Ritonavir-Mediated Induction of Apoptosis in Pancreatic Cancer Occurs via the RB/E2F-1 and AKT Pathways

    PubMed Central

    Batchu, Ramesh B.; Gruzdyn, Oksana V.; Bryant, Christopher S.; Qazi, Aamer M.; Kumar, Sanjeev; Chamala, Sreedhar; Kung, Shu T.; Sanka, Ramana S.; Puttagunta, Udaya S.; Weaver, Donald W.; Gruber, Scott A.

    2014-01-01

    Recent observations suggest a lower incidence of malignancies in patients infected with HIV during treatment with Highly Active Anti-Retroviral Therapy (HAART) utilizing protease inhibitors. We investigated the effects of ritonavir, a FDA approved HIV protease inhibitor, on proliferation of pancreatic ductal adeno-carcinoma (PDAC) cell lines. Human PDAC cell lines BxPC-3, MIA PaCa-2, and PANC-1 were propagated under standard conditions and treated with serial dilutions of ritonavir. Ritonavir inhibited cell growth in a dose-dependent manner as well as activated the intrinsic apoptotic pathway in human pancreatic ductal adenocarcinoma (PDAC) cell lines. We observed down-modulation of cell-cycle promoting and up-regulation of cell-cycle inhibitory genes; enhanced interaction of retinoblastoma protein (RB) with E2F-1 transcription factor; inhibition of phosphorylation of RB, resulting in sequestration of E2F-1 and subsequent down-regulation of S phase genes; decreased interaction of E2F-1 with its consensus binding sites; inhibition of cell motility and invasiveness; and inhibition of the AKT pathway. Our results demonstrate a potential use of ritonavir as part of combination chemotherapy for PDAC. Since ritonavir is FDA approved for HIV, drug repositioning for PDAC would limit the costs and reduce risks. PMID:24451403

  4. 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

  5. Metabolism and Disposition of the Hepatitis C Protease Inhibitor Paritaprevir in Humans.

    PubMed

    Shen, Jianwei; Serby, Michael; Reed, Aimee; Lee, Anthony J; Zhang, Xiaomei; Marsh, Kennan; Khatri, Amit; Menon, Rajeev; Kavetskaia, Olga; Fischer, Volker

    2016-08-01

    Paritaprevir (also known as ABT-450), a potent NS3-4A serine protease inhibitor [identified by AbbVie (North Chicago, IL) and Enanta Pharmaceuticals (Watertown, MA)] of the hepatitis C virus (HCV), has been developed in combination with ombitasvir and dasabuvir in a three-direct-acting antiviral agent (DAA) oral regimen for the treatment of patients infected with HCV genotype 1. This article describes the mass balance, metabolism, and disposition of paritaprevir in humans. After the administration of a single 200-mg oral dose of [(14)C]paritaprevir coadministered with 100 mg of ritonavir to four male healthy volunteers, the mean total percentage of the administered radioactive dose recovered was 96.5%, with recovery in individual subjects ranging from 96.0% to 96.9%. Radioactivity derived from [(14)C]paritaprevir was primarily eliminated in feces (87.8% of the dose). Radioactivity recovered in urine accounted for 8.8% of the dose. The biotransformation of paritaprevir in humans involves: 1) P450-mediated oxidation on the olefinic linker, the phenanthridine group, the methylpyrazinyl group, or combinations thereof; and 2) amide hydrolysis at the acyl cyclopropane-sulfonamide moiety and the pyrazine-2-carboxamide moiety. Paritaprevir was the major component in plasma [90.1% of total radioactivity in plasma, AUC from time 0 to 12 hours (AUC0-12hours) pool]. Five minor metabolites were identified in plasma, including the metabolites M2, M29, M3, M13, and M6; none of the metabolites accounted for greater than 10% of the total radioactivity. Paritaprevir was primarily eliminated through the biliary-fecal route followed by microflora-mediated sulfonamide hydrolysis to M29 as a major component in feces (approximately 60% of dose). In summary, the biotransformation and clearance pathways of paritaprevir were characterized, and the structures of metabolites in circulation and excreta were elucidated. PMID:27179127

  6. Heme oxygenase-1-derived bilirubin counteracts HIV protease inhibitor-mediated endothelial cell dysfunction.

    PubMed

    Liu, Xiao-Ming; Durante, Zane E; Peyton, Kelly J; Durante, William

    2016-05-01

    The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-l-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS-Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy. PMID:26968795

  7. 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

  8. Hepatitis C protease and polymerase inhibitors in development.

    PubMed

    Liu-Young, Gustine; Kozal, Michael J

    2008-06-01

    Hepatitis C infection (HCV) remains a global problem and the current anti-HCV therapies available in the clinic have sustained virologic response rates (SVR) of only about 50%, especially in HCV genotype 1-infected subjects. The SVR is even lower in HIV-HCV co-infected patients, estimated at only about 30-40%. However, exciting new research is under way to find new anti-HCV therapies. Presently, efforts to develop new anti-HCV agents for HCV-infected persons who fail pegylated interferon and ribavirin-based therapies have focused on inhibitors of key HCV enzymes such as the HCV NS3 protease and the NS5B polymerase. There are two protease inhibitors, telaprevir (VX-950, Vertex) and boceprevir (SCH 503034, Schering-Plough); and three polymerase inhibitors, valopicitabine (NM283, Idenix), R1626 (Roche), and HCV-796 (Viropharma) that have advanced to late-stage clinical trials. Of these aforementioned agents, telaprevir is the most advanced in clinical development. Early trial results on efficacy, safety, and HCV drug-resistance profiles of these novel agents will be discussed in this review paper. PMID:18479202

  9. Review: Hepatitis C Protease and Polymerase Inhibitors in Development

    PubMed Central

    Kozal, Michael J.

    2008-01-01

    Abstract Hepatitis C infection (HCV) remains a global problem and the current anti-HCV therapies available in the clinic have sustained virologic response rates (SVR) of only about 50%, especially in HCV genotype 1–infected subjects. The SVR is even lower in HIV-HCV co-infected patients, estimated at only about 30–40%. However, exciting new research is under way to find new anti-HCV therapies. Presently, efforts to develop new anti-HCV agents for HCV-infected persons who fail pegylated interferon and ribavirin-based therapies have focused on inhibitors of key HCV enzymes such as the HCV NS3 protease and the NS5B polymerase. There are two protease inhibitors, telaprevir (VX-950, Vertex) and boceprevir (SCH 503034, Schering-Plough); and three polymerase inhibitors, valopicitabine (NM283, Idenix), R1626 (Roche), and HCV-796 (Viropharma) that have advanced to late-stage clinical trials. Of these aforementioned agents, telaprevir is the most advanced in clinical development. Early trial results on efficacy, safety, and HCV drug-resistance profiles of these novel agents will be discussed in this review paper. PMID:18479202

  10. 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.

  11. [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

  12. A controlled Phase II trial assessing three doses of enfuvirtide (T-20) in combination with abacavir, amprenavir, ritonavir and efavirenz in non-nucleoside reverse transcriptase inhibitor-naive HIV-infected adults.

    PubMed

    Lalezari, Jacob P; DeJesus, Edwin; Northfelt, Donald W; Richmond, Gary; Wolfe, Peter; Haubrich, Richard; Henry, David; Powderly, William; Becker, Stephen; Thompson, Melanie; Valentine, Fred; Wright, David; Carlson, Margrit; Riddler, Sharon; Haas, Frances F; DeMasi, Ralph; Sista, Prokash R; Salgo, Miklos; Delehanty, John

    2003-08-01

    Enfuvirtide is a novel antiretroviral that blocks HIV-1 cell fusion and viral entry. This Phase II, controlled, open-label, randomized, multicentre dose-ranging trial explored the safety, antiviral activity and pharmacokinetics of enfuvirtide, administered by subcutaneous (s.c.) injection, in 71 HIV-1-infected, protease inhibitor-experienced, non-nucleoside reverse transcriptase inhibitor (NNRTI)-naive adults for 48 weeks. Study participants were randomized to receive enfuvirtide at a deliverable dose of 45, 67.5 or 90 mg twice daily; the 45 mg twice daily dose required 2 injections/day, while the higher doses required 4 injections/day. A background oral antiretroviral (ARV) regimen of abacavir (300 mg twice daily), amprenavir (1200 mg twice daily), ritonavir (200 mg twice daily) and efavirenz (600 mg once daily) was provided with enfuvirtide. A control group received the background ARV regimen alone. All potential participants underwent an HIV genotype at screen to ensure a homogenous population and to exclude patients with evidence of genotypic resistance to NNRTIs. Overall, the tolerability of the combination of abacavir, amprenavir, ritonavir, efavirenz and enfuvirtide was generally comparable to control through 48 weeks. No enfuvirtide dose-dependent adverse events (AEs) were observed across treatment groups. Injection site reactions (ISRs) occurred at least once in 68.5% of the enfuvirtide-treated population, and most ISRs were mild to moderate in severity, with no apparent dose relationship. Excluding ISRs, the most common treatment-emergent AEs were nausea, diarrhoea, dizziness and fatigue; with no clinically significant differences in the incidence of AEs observed between the control and enfuvirtide groups. Each treatment group benefited from ARV therapy, with a trend of increasing antiviral and immunological activity associated with increasing enfuvirtide dose. At 48 weeks, the median HIV-1 RNA change from baseline for the ITT population was -2.24 log10

  13. Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters.

    PubMed

    Vermeer, Lydia M M; Isringhausen, Caleb D; Ogilvie, Brian W; Buckley, David B

    2016-03-01

    Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporter-expressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics. PMID:26668209

  14. Antiretroviral drugs saquinavir and ritonavir reduce inhibitory concentration values of itraconazole against Histoplasma capsulatum strains in vitro.

    PubMed

    Brilhante, Raimunda Sâmia Nogueira; Caetano, Érica Pacheco; Riello, Giovanna Barbosa; Guedes, Glaucia Morgana de Melo; Castelo-Branco, Débora de Souza Collares Maia; Fechine, Maria Auxiliadora Bezerra; Oliveira, Jonathas Sales de; Camargo, Zoilo Pires de; Mesquita, Jacó Ricarte Lima de; Monteiro, André Jalles; Cordeiro, Rossana de Aguiar; Rocha, Marcos Fábio Gadelha; Sidrim, José Júlio Costa

    2016-01-01

    Recent studies have shown that some drugs that are not routinely used to treat fungal infections have antifungal activity, such as protease inhibitor antiretroviral drugs. This study investigated the in vitro susceptibility of Histoplasma capsulatum var. capsulatum to saquinavir and ritonavir, and its combination with the antifungal itraconazole. The susceptibility assay was performed according to Clinical and Laboratory Standards Institute guidelines. All strains were inhibited by the protease inhibitor antiretroviral drugs. Saquinavir showed minimum inhibitory concentrations ranging from 0.125 to 1μgmL(-1) for both phases, and ritonavir presented minimum inhibitory concentrations ranging from 0.0312 to 4μgmL(-1)and from 0.0625 to 1μgmL(-1) for filamentous and yeast phase, respectively. Concerning the antifungal itraconazole, the minimum inhibitory concentration values ranged from 0.0019 to 0.125μgmL(-1) and from 0.0039 to 0.0312μgmL(-1) for the filamentous and yeast phase, respectively. The combination of saquinavir or ritonavir with itraconazole was synergistic against H. capsulatum, with a significant reduction in the minimum inhibitory concentrations of both drugs against the strains (p<0.05). These data show an important in vitro synergy between protease inhibitors and itraconazole against the fungus H. capsulatum. PMID:26748233

  15. Action of anti-HIV drugs and resistance: reverse transcriptase inhibitors and protease inhibitors.

    PubMed

    Imamichi, Tomozumi

    2004-01-01

    Currently, 20 drugs have been approved for Human Immunodeficiency Virus type-1 (HIV-1) clinical therapy. These drugs inhibit HIV-1 reverse transcriptase, protease, or virus entry. Introduction of a combination therapy with reverse transcriptase inhibitors and protease inhibitors has resulted in a drastic decrease in HIV-1 related mortality. Although the combination therapy can suppress viral replication below detection levels in current available assays, low levels of on-going viral replication still persist in some patients. Long-term administration of the combination therapy may increase selective pressure against viruses, and subsequently induce emergence of multiple drug-resistant HIV-1 variants. Attempts have been made to design novel antiretroviral drugs that would be able to suppress replication of the resistant variants. At present, several investigational drugs are being tested in clinical trials. These drugs target not only the resistant variants, but also improvement in oral bioavilability or other viral proteins such as HIV-1 integrase, ribonuclease H, and HIV-1 entry (CD4 attachment inhibitors, chemokine receptors antagonists, and fusion inhibitors). Understanding mechanism(s) of action of the drugs and mechanisms of drug resistance is necessary for successful designs in the next generation of anti-HIV-1 drugs. In this review, the mechanisms of action of reverse transcriptase- and protease-inhibitors, and the mechanism of resistance to these inhibitors, are described. PMID:15579086

  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. Epidermal differentiation: the role of proteases and their inhibitors.

    PubMed

    Zeeuwen, Patrick L J M

    2004-12-01

    Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent

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

    PubMed

    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

  19. Serine protease inhibitor A3 in atherosclerosis and aneurysm disease.

    PubMed

    Wågsäter, Dick; Johansson, Daniel; Fontaine, Vincent; Vorkapic, Emina; Bäcklund, Alexandra; Razuvaev, Anton; Mäyränpää, Mikko I; Hjerpe, Charlotta; Caidahl, Kenneth; Hamsten, Anders; Franco-Cereceda, Anders; Wilbertz, Johannes; Swedenborg, Jesper; Zhou, Xinghua; Eriksson, Per

    2012-08-01

    Remodeling of extracellular matrix (ECM) plays an important role in both atherosclerosis and aneurysm disease. Serine protease inhibitor A3 (serpinA3) is an inhibitor of several proteases such as elastase, cathepsin G and chymase derived from mast cells and neutrophils. In this study, we investigated the putative role of serpinA3 in atherosclerosis and aneurysm formation. SerpinA3 was expressed in endothelial cells and medial smooth muscle cells in human atherosclerotic lesions and a 14-fold increased expression of serpinA3n mRNA was found in lesions from Apoe-/- mice compared to lesion-free vessels. In contrast, decreased mRNA expression (-80%) of serpinA3 was found in biopsies of human abdominal aortic aneurysm (AAA) compared to non-dilated aortas. Overexpression of serpinA3n in transgenic mice did not influence the development of atherosclerosis or CaCl2-induced aneurysm formation. In situ zymography analysis showed that the transgenic mice had lower cathepsin G and elastase activity, and more elastin in the aortas compared to wild-type mice, which could indicate a more stable aortic phenotype. Differential vascular expression of serpinA3 is clearly associated with human atherosclerosis and AAA but serpinA3 had no major effect on experimentally induced atherosclerosis or AAA development in mouse. However, serpinA3 may be involved in a phenotypic stabilization of the aorta. PMID:22580763

  20. 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

  1. Design of mutation-resistant HIV protease inhibitors with the substrate envelope hypothesis.

    PubMed

    Chellappan, Sripriya; Kiran Kumar Reddy, G S; Ali, Akbar; Nalam, Madhavi N L; Anjum, Saima Ghafoor; Cao, Hong; Kairys, Visvaldas; Fernandes, Miguel X; Altman, Michael D; Tidor, Bruce; Rana, Tariq M; Schiffer, Celia A; Gilson, Michael K

    2007-05-01

    There is a clinical need for HIV protease inhibitors that can evade resistance mutations. One possible approach to designing such inhibitors relies upon the crystallographic observation that the substrates of HIV protease occupy a rather constant region within the binding site. In particular, it has been hypothesized that inhibitors which lie within this region will tend to resist clinically relevant mutations. The present study offers the first prospective evaluation of this hypothesis, via computational design of inhibitors predicted to conform to the substrate envelope, followed by synthesis and evaluation against wild-type and mutant proteases, as well as structural studies of complexes of the designed inhibitors with HIV protease. The results support the utility of the substrate envelope hypothesis as a guide to the design of robust protease inhibitors. PMID:17539822

  2. Ombitasvir, Paritaprevir, and Ritonavir

    MedlinePlus

    ... ritonavir comes as a tablet to take by mouth. Take ombitasvir, paritaprevir, and ritonavir every morning with ... in Advair); tacrolimus (Astagraf XL, Prograf, Protopic); and voriconazole (Vfend). Your doctor may need to change the ...

  3. Severe synergistic toxicity from docetaxel in a patient treated concurrently with protease inhibitors as part of HIV post-exposure prophylaxis: a case report

    PubMed Central

    2009-01-01

    Introduction Docetaxel is a semisynthetic taxane commonly used in solid tumour oncology. Its pharmacokinetics has been widely studied, and it is well established that it is metabolized to pharmacologically inactive products by the cytochrome P450 3A iso-enzymes. However, there have been few reports of the consequences of drug interactions between taxanes and other drugs metabolized by the cytochrome P450 pathway. To the best of our knowledge, this is the first case report of the potentially life-threatening interaction that can occur between docetaxel and the protease inhibitors lopinavir and ritonavir. Case presentation A 30-year-old Caucasian woman presented with symptoms suggestive of severe docetaxel toxicity, that is, prolonged myelosuppression, grade 4 mucositis and desquamating rash, following the commencement of post-exposure prophylaxis for a needlestick injury. She had previously received docetaxel chemotherapy with minimal side effects. Conclusion This case report highlights a probable and novel drug interaction between docetaxel and lopinavir and/or ritonavir, which is largely unreported in the medical literature. Even though these interactions may be more relevant in the field of HIV medicine, knowledge of these interactions is also beneficial to oncologists and dermatologists, as well as those providing acute medical care.

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

    PubMed

    Dabhade, Arvind; Patel, Priti; Pati, Ulhas

    2013-10-01

    A thermo-stable, proteinaceous protease inhibitor (LPI) from Lawsonia inermis is reported. The LPI was purified from Lawsonia inermis seeds by subsequent ammonium sulfate precipitation, ion exchange chromatography (DEAE-Cellulose) and gel permeation chromatography (Sephadex-50). The purified protease inhibitor is effective against a wide range of proteases viz. papain, trypsin, pepsin and metallo-protease. The apparent molecular weight of the protease inhibitor is 19 kDa, determined by SDS-PAGE electrophoresis. The protease inhibitor was found to be stable at 70 degrees C for 30 min. It was also examined for antibacterial activity against Pseudomonas aeruginosa MTCC 7926 and Staphylococcus aureus NCIM 2079; the IC50 values of the purified LPI were 11.4 microg/mL and 16.6 microg/mL respectively. PMID:24354203

  5. 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

  6. Iatrogenic Cushing Syndrome Secondary to Ritonavir-Epidural Triamcinolone Interaction: An Illustrative Case and Review

    PubMed Central

    Berg, Melody L.

    2014-01-01

    HIV positive patients on ritonavir-containing antiretroviral therapy (ART) can develop iatrogenic Cushing syndrome (IACS) and adrenal insufficiency as a result of drug-drug interactions with inhaled or intranasal glucocorticoid therapy. Reports related to epidural triamcinolone injections are relatively uncommon but increasingly reported. We describe a 48-year-old woman with immunologically and virologically well-controlled HIV on ritonavir-based ART, who developed headache, dizziness, and candida and herpes simplex virus (HSV) ulcerative esophagitis 7 days after receiving an epidural triamcinolone injection for cervical radicular pain. Iatrogenic Cushing syndrome and relative adrenal insufficiency were suspected and proven. The patient's ART was changed to a non-HIV protease inhibitor- (PI-) containing program, her symptoms improved, and she did not require hydrocortisone replacement. In this paper, we review the literature on IACS and relative secondary adrenal insufficiency from epidural triamcinolone injections in HIV patients on ritonavir-containing ART regimens. A high index of clinical suspicion is needed for diagnosis. Prevention of drug-drug interactions by taking a thorough medication history for patients on ritonavir-containing ART regimens before prescribing any form of corticosteroid is crucial and effective and sustained interdisciplinary communication in the care of such patients. PMID:24895495

  7. Beneficial effects of secretory leukocyte protease inhibitor after spinal cord injury

    PubMed Central

    Ghasemlou, Nader; Bouhy, Delphine; Yang, Jingxuan; López-Vales, Rubèn; Haber, Michael; Thuraisingam, Thusanth; He, Guoan; Radzioch, Danuta; Ding, Aihao

    2010-01-01

    Secretory leukocyte protease inhibitor is a serine protease inhibitor produced by various cell types, including neutrophils and activated macrophages, and has anti-inflammatory properties. It has been shown to promote wound healing in the skin and other non-neural tissues, however, its role in central nervous system injury was not known. We now report a beneficial role for secretory leukocyte protease inhibitor after spinal cord injury. After spinal cord contusion injury in mice, secretory leukocyte protease inhibitor is expressed primarily by astrocytes and neutrophils but not macrophages. We show, using transgenic mice over-expressing secretory leukocyte protease inhibitor, that this molecule has an early protective effect after spinal cord contusion injury. Furthermore, wild-type mice treated for the first week after spinal cord contusion injury with recombinant secretory leukocyte protease inhibitor exhibit sustained improvement in locomotor control and reduced secondary tissue damage. Recombinant secretory leukocyte protease inhibitor injected intraperitoneally localizes to the nucleus of circulating leukocytes, is detected in the injured spinal cord, reduces activation of nuclear factor-κB and expression of tumour necrosis factor-α. Administration of recombinant secretory leukocyte protease inhibitor might therefore be useful for the treatment of acute spinal cord injury. PMID:20047904

  8. Microarray analysis reveals strategies of Tribolium castaneum larvae to compensate for cysteine and serine protease inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microarrays containing Tribolium castaneum whole-genome sequences were developed to study the transcriptome response of T. castaneum larvae to dietary protease inhibitors. In larvae fed diets containing 0.1% of the cysteine protease inhibitor E-64 alone or in combination with 5.0% of the serine pro...

  9. 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

  10. Antiretroviral Therapy and Efficacy After Virologic Failure on First-line Boosted Protease Inhibitor Regimens

    PubMed Central

    Zheng, Yu; Hughes, Michael D.; Lockman, Shahin; Benson, Constance A.; Hosseinipour, Mina C.; Campbell, Thomas B.; Gulick, Roy M.; Daar, Eric S.; Sax, Paul E.; Riddler, Sharon A.; Haubrich, Richard; Salata, Robert A.; Currier, Judith S.

    2014-01-01

    Background. Virologic failure (VF) on a first-line ritonavir-boosted protease inhibitor (PI/r) regimen is associated with low rates of resistance, but optimal management after failure is unknown. Methods. The analysis included participants in randomized trials who experienced VF on a first-line regimen of PI/r plus 2 nucleoside reverse transcriptase inhibitors (NRTIs) and had at least 24 weeks of follow-up after VF. Antiretroviral management and virologic suppression (human immunodeficiency virus type 1 [HIV-1] RNA <400 copies/mL) after VF were assessed. Results. Of 209 participants, only 1 participant had major PI-associated treatment-emergent mutations at first-line VF. The most common treatment approach after VF (66%) was to continue the same regimen. The virologic suppression rate 24 weeks after VF was 64% for these participants, compared with 72% for those who changed regimens (P = .19). Participants remaining on the same regimen had lower NRTI resistance rates (11% vs 30%; P = .003) and higher CD4+ cell counts (median, 275 vs 213 cells/µL; P = .005) at VF than those who changed. Among participants remaining on their first-line regimen, factors at or before VF significantly associated with subsequent virologic suppression were achieving HIV-1 RNA <400 copies/mL before VF (odds ratio [OR], 3.39 [95% confidence interval {CI}, 1.32–8.73]) and lower HIV-1 RNA at VF (OR for <10 000 vs ≥10 000 copies/mL, 3.35 [95% CI, 1.40–8.01]). Better adherence after VF was also associated with subsequent suppression (OR for <100% vs 100%, 0.38 [95% CI, .15–.97]). For participants who changed regimens, achieving HIV-1 RNA <400 copies/mL before VF also predicted subsequent suppression. Conclusions. For participants failing first-line PI/r with no or limited drug resistance, remaining on the same regimen is a reasonable approach. Improving adherence is important to subsequent treatment success. PMID:24842909

  11. Randomised Pharmacokinetic Trial of Rifabutin with Lopinavir/Ritonavir-Antiretroviral Therapy in Patients with HIV-Associated Tuberculosis in Vietnam

    PubMed Central

    Lan, Nguyen Thi Ngoc; Thu, Nguyen Thi Nguyet; Barrail-Tran, Aurélie; Duc, Nguyen Hong; Lan, Nguyen Ngoc; Laureillard, Didier; Thi Xuan Lien, Truong; Borand, Laurence; Quillet, Catherine; Connolly, Catherine; Lagarde, Dominique; Pym, Alexander; Lienhardt, Christian; Dung, Nguyen Huy; Taburet, Anne-Marie; Harries, Anthony D

    2014-01-01

    Background Rifampicin and protease inhibitors are difficult to use concomitantly in patients with HIV-associated tuberculosis because of drug-drug interactions. Rifabutin has been proposed as an alternative rifamycin, but there is concern that the current recommended dose is suboptimal. The principal aim of this study was to compare bioavailability of two doses of rifabutin (150 mg three times per week and 150 mg daily) in patients with HIV-associated tuberculosis who initiated lopinavir/ritonavir-based antiretroviral therapy in Vietnam. Concentrations of lopinavir/ritonavir were also measured. Methods This was a randomized, open-label, multi-dose, two-arm, cross-over trial, conducted in Vietnamese adults with HIV-associated tuberculosis in Ho Chi Minh City (Clinical trial registry number NCT00651066). Rifabutin pharmacokinetics were evaluated before and after the introduction of lopinavir/ritonavir -based antiretroviral therapy using patient randomization lists. Serial rifabutin and 25-O-desacetyl rifabutin concentrations were measured during a dose interval after 2 weeks of rifabutin 300 mg daily, after 3 weeks of rifabutin 150 mg daily with lopinavir/ritonavir and after 3 weeks of rifabutin 150 mg three times per week with lopinavir/ritonavir. Results Sixteen and seventeen patients were respectively randomized to the two arms, and pharmacokinetic analysis carried out in 12 and 13 respectively. Rifabutin 150 mg daily with lopinavir/ritonavir was associated with a 32% mean increase in rifabutin average steady state concentration compared with rifabutin 300 mg alone. In contrast, the rifabutin average steady state concentration decreased by 44% when rifabutin was given at 150 mg three times per week with lopinavir/ritonavir. With both dosing regimens, 2 – 5 fold increases of the 25-O-desacetyl- rifabutin metabolite were observed when rifabutin was given with lopinavir/ritonavir compared with rifabutin alone. The different doses of rifabutin had no significant

  12. Protease inhibitor monotherapy for long-term management of HIV infection: a randomised, controlled, open-label, non-inferiority trial

    PubMed Central

    Paton, Nicholas I; Stöhr, Wolfgang; Arenas-Pinto, Alejandro; Fisher, Martin; Williams, Ian; Johnson, Margaret; Orkin, Chloe; Chen, Fabian; Lee, Vincent; Winston, Alan; Gompels, Mark; Fox, Julie; Scott, Karen; Dunn, David T

    2015-01-01

    Summary Background Standard-of-care antiretroviral therapy (ART) uses a combination of drugs deemed essential to minimise treatment failure and drug resistance. Protease inhibitors are potent, with a high genetic barrier to resistance, and have potential use as monotherapy after viral load suppression is achieved with combination treatment. We aimed to assess clinical risks and benefits of protease inhibitor monotherapy in long-term clinical use: in particular, the effect on drug resistance and future treatment options. Methods In this pragmatic, parallel-group, randomised, controlled, open-label, non-inferiority trial, we enrolled adults (≥18 years of age) positive for HIV attending 43 public sector treatment centres in the UK who had suppressed viral load (<50 copies per mL) for at least 24 weeks on combination ART with no change in the previous 12 weeks and a CD4 count of more than 100 cells per μL. Participants were randomly allocated (1:1) to maintain ongoing triple therapy (OT) or to switch to a strategy of physician-selected ritonavir-boosted protease inhibitor monotherapy (PI-mono); we recommended ritonavir (100 mg)-boosted darunavir (800 mg) once daily or ritonavir (100 mg)-boosted lopinavir (400 mg) twice daily, with prompt return to combination treatment if viral load rebounded. All treatments were oral. Randomisation was with permuted blocks of varying size and stratified by centre and baseline ART; we used a computer-generated, sequentially numbered randomisation list. The primary outcome was loss of future drug options, defined as new intermediate-level or high-level resistance to one or more drugs to which the patient's virus was deemed sensitive at trial entry (assessed at 3 years; non-inferiority margin of 10%). We estimated probability of rebound and resistance with Kaplan-Meier analysis. Analyses were by intention to treat. This trial is registered with the International Standard Randomised Controlled Trial Number registry, number ISRCTN

  13. Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?

    PubMed

    Casares-Crespo, L; Vicente, J S; Talaván, A M; Viudes-de-Castro, M P

    2016-03-15

    The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of aminopeptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 ± 0.26 and 9.3 ± 0.23 vs. 8.2 ± 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size. PMID:26639641

  14. Streptomyces serine protease (SAM-P20): recombinant production, characterization, and interaction with endogenous protease inhibitor.

    PubMed Central

    Taguchi, S; Suzuki, M; Kojima, S; Miura, K; Momose, H

    1995-01-01

    Previously, we isolated a candidate for an endogenous target enzyme(s) of the Streptomyces subtilisin inhibitor (SSI), termed SAM-P20, from a non-SSI-producing mutant strain (S. Taguchi, A. Odaka, Y. Watanabe, and H. Momose, Appl. Environ. Microbiol. 61:180-186, 1995). In this study, in order to investigate the detailed enzymatic properties of this protease, an overproduction system of recombinant SAM-P20 was established in Streptomyces coelicolor with the SSI gene promoter. The recombinant SAM-P20 was purified by salting out and by two successive ion-exchange chromatographies to give a homogeneous band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial peptide mapping and amino acid composition analysis revealed that the recombinant SAM-P20 was identical to natural SAM-P20. From the results for substrate specificity and inhibitor sensitivity, SAM-P20 could be categorized as a chymotrypsin-like protease with an arginine-cleavable activity, i.e., a serine protease with broad substrate specificity. For proteolytic activity, the optimal pH was 10.0 and the optimal temperature was shifted from 50 to 80 degrees C by the addition of 10 mM calcium ion. The strong stoichiometric inhibition of SAM-P20 activity by SSI dimer protein occurred in a subunit molar ratio of these two proteins of about 1, and an inhibitor constant of SSI toward SAM-P20 was estimated to be 8.0 x 10(-10) M. The complex formation of SAM-P20 and SSI was monitored by analytical gel filtration, and a complex composed of two molecules of SAM-P20 and one dimer molecule of SSI was detected, in addition to a complex of one molecule of SAM-P20 bound to one dimer molecule of SSI. The reactive site of SSI toward SAM-P20 was identified as Met-73-Val-74 by sequence analysis of the modified form of SSI, which was produced by the acidification of the complex of SSI and SAM-P20. This reactive site is the same that toward an exogenous target enzyme, subtilisin BPN'. PMID:7592444

  15. 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

  16. In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV.

    PubMed

    Hazen, Richard; Harvey, Robert; Ferris, Robert; Craig, Charles; Yates, Phillip; Griffin, Philip; Miller, John; Kaldor, Istvan; Ray, John; Samano, Vincente; Furfine, Eric; Spaltenstein, Andrew; Hale, Michael; Tung, Roger; St Clair, Marty; Hanlon, Mary; Boone, Lawrence

    2007-09-01

    Brecanavir, a novel tyrosyl-based arylsulfonamide, high-affinity, human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), has been evaluated for anti-HIV activity in several in vitro assays. Preclinical assessment of brecanavir indicated that this compound potently inhibited HIV-1 in cell culture assays with 50% effective concentrations (EC(50)s) of 0.2 to 0.53 nM and was equally active against HIV strains utilizing either the CXCR4 or CCR5 coreceptor, as was found with other PIs. The presence of up to 40% human serum decreased the anti-HIV-1 activity of brecanavir by 5.2-fold, but under these conditions the compound retained single-digit nanomolar EC(50)s. When brecanavir was tested in combination with nucleoside reverse transcriptase inhibitors, the antiviral activity of brecanavir was synergistic with the effects of stavudine and additive to the effects of zidovudine, tenofovir, dideoxycytidine, didanosine, adefovir, abacavir, lamivudine, and emtricitabine. Brecanavir was synergistic with the nonnucleoside reverse transcriptase inhibitor nevirapine or delavirdine and was additive to the effects of efavirenz. In combination with other PIs, brecanavir was additive to the activities of indinavir, lopinavir, nelfinavir, ritonavir, amprenavir, saquinavir, and atazanavir. Clinical HIV isolates from PI-experienced patients were evaluated for sensitivity to brecanavir and other PIs in a recombinant virus assay. Brecanavir had a <5-fold increase in EC(50)s against 80% of patient isolates tested and had a greater mean in vitro potency than amprenavir, indinavir, lopinavir, atazanavir, tipranavir, and darunavir. Brecanavir is by a substantial margin the most potent and broadly active antiviral agent among the PIs tested in vitro. PMID:17620375

  17. Boceprevir, an NS3 protease inhibitor of HCV.

    PubMed

    Berman, Kenneth; Kwo, Paul Y

    2009-08-01

    Hepatitis C virus (HCV) is a major cause of chronic liver disease leading to death from liver failure or hepatocellular carcinoma. Hepatitis C is the most common indication for liver transplantation worldwide and is a major cause of the increased incidence of hepatocellular cancer in the United States. The current paradigm for HCV treatment relies on pegylated interferon and ribavirin as agents that enhance endogenous mechanisms for viral clearance and are dependent on host factors. In patients with genotype 1 HCV infection, sustained viral response (SVR) rates remain suboptimal, with less than half of genotype 1-infected individuals going on to achieve SVR. This has led to a shift in the investigational focus for treatment of HCV toward specifically targeted antiviral therapy for HCV agents. This review focuses on boceprevir, a protease inhibitor, and discusses its mechanism of action, effects on HCV, and viral resistance. PMID:19628159

  18. 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.

  19. HIV protease inhibitors in pulmonary hypertension: rationale and design of a pilot trial in idiopathic pulmonary arterial hypertension

    PubMed Central

    Li, Ying; Li, Xiao-hui; Yu, Zai-xin; Cai, Jing-jing; Billiar, Timothy R.; Chen, Alex F.; Lv, Ben; Chen, Zi-ying; Huang, Zhi-jun; Yang, Guo-ping; Song, Jie; Liu, Bin

    2015-01-01

    Abstract We propose an exploratory clinical study, the first of its kind to our knowledge, to determine the safety and potential clinical benefit of the combination of the HIV protease inhibitors (HIV-PIs) saquinavir and ritonavir (SQV+RIT) in patients with idiopathic pulmonary arterial hypertension (IPAH). This study is based on evidence that (1) HIV-PIs can improve pulmonary hemodynamics in experimental models; (2) both Toll-like receptor 4 and high-mobility group box 1 (HMGB1) participate in the pathogenesis of experimental pulmonary hypertension; and (3) a high-throughput screen for inhibitors of HMGB1-induced macrophage activation yielded HIV-PIs as potent inhibitors of HMGB1-induced cytokine production. In this proposed open-label, pre-post study, micro, low, and standard doses of SQV+RIT will be given to IPAH patients for 14 days. Patients will receive follow-up for the next 14 days. The primary outcome to be evaluated is change in HMGB1 level from baseline at 14 days. The secondary outcome is changes in tumor necrosis factor α, interleukin 1β, interleukin 6, C-reactive protein, pulmonary arterial pressure based on echocardiography parameters and New York Heart Association/World Health Organization functional class, and Brog dyspnea scale index from baseline at 14 days. Other secondary measurements will include N-terminal pro-brain natriuretic peptide, atrial natriuretic peptide, and 6-minute walk distance. We propose that SQV+RIT treatment will improve inflammatory disorders and pulmonary hemodynamics in IPAH patients. If the data support a potentially useful therapeutic effect and suggest that SQV+RIT is safe in IPAH patients, the study will warrant further investigation. (ClinicalTrials.gov identifier: NCT02023450.) PMID:26401255

  20. 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

  1. The effect of lopinavir/ritonavir on the renal clearance of tenofovir in HIV-infected patients.

    PubMed

    Kiser, J J; Carten, M L; Aquilante, C L; Anderson, P L; Wolfe, P; King, T M; Delahunty, T; Bushman, L R; Fletcher, C V

    2008-02-01

    We determined the effects of lopinavir/ritonavir on tenofovir renal clearance. Human immunodeficiency virus-infected subjects taking tenofovir disoproxil fumarate (TDF) were matched on age, race, and gender and were enrolled into one of the following two groups: group 1: subjects taking TDF plus lopinavir/ritonavir plus other nucleoside reverse transcriptase inhibitors (NRTIs); group 2: subjects taking TDF plus NRTIs and/or non-NRTIs but no protease inhibitors. Twenty-four-hour blood and urine collections were carried out in subjects for tenofovir quantification. Drug transporter genotype associations with tenofovir pharmacokinetics were examined. In 30 subjects, median (range) tenofovir apparent oral clearance, renal clearance, and fraction excreted in urine were 34.6 l/h (20.6-89.5), 11.3 l/h (6.2-22.6), and 0.33 (0.23-0.5), respectively. After adjusting for renal function, tenofovir renal clearance was 17.5% slower (P=0.04) in subjects taking lopinavir/ritonavir versus those not taking a protease inhibitor, consistent with a renal interaction between these drugs. Future studies should clarify the exact mechanism and whether there is an increased risk of nephrotoxicity. PMID:17597712

  2. 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

  3. 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

  4. 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

  5. Cyanohydrin as an Anchoring Group for Potent and Selective Inhibitors of Enterovirus 71 3C Protease.

    PubMed

    Zhai, Yangyang; Zhao, Xiangshuai; Cui, Zhengjie; Wang, Man; Wang, Yaxin; Li, Linfeng; Sun, Qi; Yang, Xi; Zeng, Debin; Liu, Ying; Sun, Yuna; Lou, Zhiyong; Shang, Luqing; Yin, Zheng

    2015-12-10

    Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2'S,5S)-16 and (1R,2S,2'S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C(pro). Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors. PMID:26571192

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

    PubMed

    Mourão, Caroline B F; Schwartz, Elisabeth F

    2013-06-01

    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

  7. 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

  8. HIV-1 Protease Mutations and Protease Inhibitor Cross-Resistance▿ † ‡

    PubMed Central

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W. Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W.

    2010-01-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  9. X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid. beta. -protein precursor

    SciTech Connect

    Hynes, T.R.; Randal, M.; Kennedy, L.A.; Eigenbrot, C.; Kossiakoff, A.A. Univ. of California, San Francisco )

    1990-10-01

    Alzheimer's amyloid {beta}-protein precursor contains a Kunitz protease inhibitor domain (APPI) potentially involved in proteolytic events leading to cerebral amyloid deposition. To facilitate the identification of the physiological target of the inhibitor, the crystal structure of APPI has been determined and refined to 1.5-{Angstrom} resolution. Sequences in the inhibitor-protease interface of the correct protease target will reflect the molecular details of the APPI structure. While the overall tertiary fold of APPI is very similar to that of the Kunitz inhibitor BPTI, a significant rearrangement occurs in the backbone conformation of one of the two protease binding loops. A number of Kunitz inhibitors have similar loop sequences, indicating the structural alteration is conserved and potentially an important determinant of inhibitor specificity. In a separate region of the protease binding loops, APPI side chains Met-17 and Phe-34 create an exposed hydrophobic surface in place of Arg-17 and Val-34 in BPTI. The restriction this change places on protease target sequences is seen when the structure of APPI is superimposed on BPTI complexed to serine proteases, where the hydrophobic surface of APPI faces a complementary group of nonpolar side chains on kallikrein A versus polar side chains on trypsin.

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

    PubMed

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

    2014-01-01

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

  11. α-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB

    PubMed Central

    Steert, Koen; Berg, Maya; Mottram, Jeremy C.; Westrop, Gareth D.; Coombs, Graham H.; Cos, Paul; Maes, Louis; Joossens, Jurgen; Van der Veken, Pieter; Haemers, Achiel; Augustyns, Koen

    2011-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of α-ketoheterocycles 1-15 has been developed as reversible inhibitors of a recombinant L. mexicana cysteine protease CPB2.8. The isoxazoles 1-3 and especially the oxadiazole 15 are potent reversible inhibitors of CPB2.8, however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease. PMID:20799311

  12. 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

  13. De novo design and discovery of potent, nonpeptidal HIV-1 protease inhibitors

    SciTech Connect

    Lam, P.Y.S.; Eyermann, C.J.; Hodge, C.N.; Jadhav, P.K.; Ru, Yu; Bacheler, L.T.; Meek, J.L.; Otto, M.J.; Rayner, M.M.; Wong, N.Y.; Chang, C.H.; Weber, P.C.; Jackson, D.A.; Sharpe, T.R.; Erickson-Viitanen, S.K.

    1993-12-31

    Intense worldwide research in HIV-1 protease inhibition has resulted in many inhibitors with nanomolar Ki. However, they are mostly pseudopeptides (containing amide bonds) and substrate-like. In this work the authors report that using 3-D database searching, computer modeling and x-ray structures of the HIV-1 protease/inhibitor complex, a completely novel class of potent nonpeptides has been designed and synthesized. The Ki is in the subnanomolar range and the IC90 for the cell assays in the submicromolar range. Confirmation of the mode of binding was achieved by a high resolution x-ray structure of a HIV-1 protease/inhibitor complex. Molecular recognition studies between HIV-1 protease and these inhibitors will also be discussed.

  14. Evaluation of the substrate envelope hypothesis for inhibitors of HIV-1 protease.

    PubMed

    Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X; Schiffer, Celia; Gilson, Michael K

    2007-08-01

    Crystallographic data show that various substrates of HIV protease occupy a remarkably uniform region within the binding site; this region has been termed the substrate envelope. It has been suggested that an inhibitor that fits within the substrate envelope should tend to evade viral resistance because a protease mutation that reduces the affinity of the inhibitor will also tend to reduce the affinity of substrate, and will hence decrease the activity of the enzyme. Accordingly, inhibitors that fit the substrate envelope better should be less susceptible to clinically observed resistant mutations, since these must also allow substrates to bind. The present study describes a quantitative measure of the volume of a bound inhibitor falling outside the substrate envelope, and observes that this quantity correlates with the inhibitor's losses in affinity to clinically relevant mutants. This measure may thus be useful as a penalty function in the design of robust HIV protease inhibitors. PMID:17474129

  15. Impact of protease inhibitors on dentin matrix degradation by collagenase.

    PubMed

    Kato, M T; Leite, A L; Hannas, A R; Calabria, M P; Magalhães, A C; Pereira, J C; Buzalaf, M A R

    2012-12-01

    This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 µM), chlorhexidine (CHX, 0.012%), FeSO(4) (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37°C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey's test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO(4) led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM. PMID:23023765

  16. 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

  17. Enantioselective Synthesis of Dioxatriquinane Structural Motifs for HIV-1 Protease Inhibitors Using a Cascade Radical Cyclization†

    PubMed Central

    Ghosh, Arun K.; Xu, Chun-Xiao; Osswald, Heather L.

    2015-01-01

    Synthesis of novel HIV-1 protease inhibitors incorporating dioxatriquinane-derived P2-ligands is described. The tricyclic ligand alcohol contains five contiguous chiral centers. The ligand alcohols were prepared in optically active form by an enzymatic asymmetrization of mesodiacetate, cascade radical cyclization, and Lewis acid catalyzed reduction as the key steps. Inhibitors with dioxatriquinane-derived P2-ligands exhibited low nanomolar HIV-1 protease activity. PMID:26185337

  18. Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

    PubMed

    Terelius, Ylva; Figler, Robert A; Marukian, Svetlana; Collado, Maria S; Lawson, Mark J; Mackey, Aaron J; Manka, David; Qualls, Charles W; Blackman, Brett R; Wamhoff, Brian R; Dash, Ajit

    2016-08-01

    Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 μM) and Ritonavir (3.5 and 62.4 μM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain

  19. 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

  20. Protein structure-based design of potent orally bioavailable, nonpeptide inhibitors of human immunodeficiency virus protease.

    PubMed Central

    Reich, S H; Melnick, M; Davies, J F; Appelt, K; Lewis, K K; Fuhry, M A; Pino, M; Trippe, A J; Nguyen, D; Dawson, H

    1995-01-01

    A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species. Images Fig. 2 PMID:7724556

  1. Coadministration of ticagrelor and ritonavir: Toward prospective dose adjustment to maintain an optimal platelet inhibition using the PBPK approach.

    PubMed

    Marsousi, N; Samer, C F; Fontana, P; Reny, J L; Rudaz, S; Desmeules, J A; Daali, Y

    2016-09-01

    Ticagrelor is a potent antiplatelet drug metabolized by cytochrome (CYP)3A. It is contraindicated in patients with human immunodeficiency virus (HIV) because of the expected CYP3A inhibition by most protease inhibitors, such as ritonavir and an increased bleeding risk. In this study, a physiologically based pharmacokinetic (PBPK) model was created for ticagrelor and its active metabolite (AM). Based on the simulated interaction between ticagrelor 180 mg and ritonavir 100 mg, a lower dose of ticagrelor was calculated to obtain, when coadministered with ritonavir, the same pharmacokinetic (PK) and platelet inhibition as ticagrelor administered alone. A clinical study was thereafter conducted in healthy volunteers. Observed PK profiles of ticagrelor and its AM were successfully predicted with the model. Platelet inhibition was nearly complete in both sessions despite administration of a fourfold lower dose of ticagrelor in the second session. This PBPK model could be prospectively used to broaden the usage of ticagrelor in patients with ritonavir-treated HIV regardless of the CYP3A inhibition. PMID:27264793

  2. A novel HCV NS3 protease mutation selected by combination treatment of the protease inhibitor boceprevir and NS5B polymerase inhibitors.

    PubMed

    Chase, Robert; Skelton, Angela; Xia, Ellen; Curry, Stephanie; Liu, Shaotang; McMonagle, Patricia; Huang, H-C; Tong, Xiao

    2009-11-01

    Boceprevir (SCH 503034) is an orally active novel inhibitor of the hepatitis C virus (HCV) NS3 protease currently in clinical development for the treatment of hepatitis C. In this in vitro study, we demonstrate that combination of boceprevir with a nucleoside analog or a non-nucleoside HCV NS5B polymerase inhibitor was superior to treatment by single agents in inhibiting viral RNA replication in replicon cells. In the presence of boceprevir (at 5xEC(90)), the addition of 2'-C-methyl-adenosine or an indole-N-acetamide targeting the polymerase finger-loop site (at 1xEC(90)) significantly reduced the emergence of resistant replicon colonies. A higher dose (5xEC(90)) of either of the polymerase inhibitors in combination with boceprevir suppressed replicon resistance further to below detectable levels. Sequencing analysis of replicon cells selected by the combination treatment revealed known resistance mutations to the two polymerase inhibitors but no previously reported resistance mutations to boceprevir. Interestingly, a novel mutation (M175L) in the protease domain was identified. The dually resistant replicon cells were monitored for over 30 passages and sensitivity to polymerase inhibitors was found to decrease over time in a manner that correlated with the increasing prevalence of specific resistance mutations. Importantly, these cells remained sensitive to interferon-alpha and different classes of polymerase inhibitors. These findings support the rationale for clinical evaluation of combination treatment of HCV protease and polymerase inhibitors. PMID:19747948

  3. Treatment of chronic hepatitis C: anticipated impact of resistance in patients treated with protease inhibitors.

    PubMed

    Kronenberger, Bernd; Zeuzem, Stefan

    2009-02-01

    A main target of specifically targeted antiviral therapy for hepatitis C (STAT-C) is the NS3-protease, which has key functions in the hepatitis C virus (HCV) replication cycle. HCV/NS3-protease inhibitors have shown high antiviral activity in vitro and in patients with chronic hepatitis C. Protease-resistant HCV variants occurred rapidly in patients receiving protease-inhibitor monotherapy. The development of resistance can be best explained by selection of preexisting resistant variants, which grow out under selective pressure. Numerous mutations associated with resistance were identified. Clinical trials showed that protease-resistant strains are sensitive to interferon and that a triple combination of protease inhibitors, peginterferon, and ribavirin may improve the sustained virologic response rate compared with standard peginterferon/ribavirin combination therapy. Overall, it can be anticipated that successful treatment with protease inhibitors will require either combination therapy with peginterferon/ribavirin or a combination of STAT-C compounds with distinct modes of action and resistance patterns. PMID:19166654

  4. HIV Protease Inhibitors Sensitize Human Head and Neck Squamous Carcinoma Cells to Radiation by Activating Endoplasmic Reticulum Stress

    PubMed Central

    Liu, Runping; Zhang, Luyong; Yang, Jing; Zhang, Xiaoxuan; Mikkelsen, Ross; Song, Shiyu; Zhou, Huiping

    2015-01-01

    Background Human head and neck squamous cell carcinoma (HNSCC) is the sixth most malignant cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, there is no significant improvement of mortality rates for this disease in the past decades. Radiotherapy is the core component of the clinical combinational therapies for HNSCC. However, the tumor cells have a tendency to develop radiation resistance, which is a major barrier to effective treatment. HIV protease inhibitors (HIV PIs) have been reported with radiosensitizing activities in HNSCC cells, but the underlying cellular/molecular mechanisms remain unclear. Our previous study has shown that HIV PIs induce cell apoptosis via activation of endoplasmic reticulum (ER) stress. The aim of this study was to examine the role of ER stress in HIV PI-induced radiosensitivity in human HNSCC. Methodology and Principal Findings HNSCC cell lines, SQ20B and FaDu, and the most commonly used HIV PIs, lopinavir and ritonavir (L/R), were used in this study. Clonogenic assay was used to assess the radiosensitivity. Cell viability, apoptosis and cell cycle were analyzed using Cellometer Vision CBA. The mRNA and protein levels of ER stress-related genes (eIF2α, CHOP, ATF-4, and XBP-1), as well as cell cycle related protein, cyclin D1, were detected by real time RT-PCR and Western blot analysis, respectively. The results demonstrated that L/R dose-dependently sensitized HNSCC cells to irradiation and inhibited cell growth. L/R-induced activation of ER stress was correlated to down-regulation of cyclin D1 expression and cell cycle arrest under G0/G1 phase. Conclusion and Significance HIV PIs sensitize HNSCC cells to radiotherapy by activation of ER stress and induction of cell cycle arrest. Our results provided evidence that HIV PIs can be potentially used in combination with radiation in the treatment of HNSCC. PMID:25933118

  5. 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.

  6. 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

  7. 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. PMID:26342866

  8. 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

  9. Maternal Lopinavir/Ritonavir Is Associated with Fewer Adverse Events in Infants than Nelfinavir or Atazanavir

    PubMed Central

    Weinberg, Adriana; Forster, Jeri E.; Levin, Myron J.; Davies, Jill; Pappas, Jennifer; Kinzie, Kay; Barr, Emily; Paul, Suzanne; McFarland, Elizabeth J.

    2016-01-01

    Combination antiretroviral therapy (cART) is successfully used for prevention of perinatal HIV transmission. To investigate safety, we compared adverse events (AE) among infants exposed to different maternal cART regimens. We reviewed 158 HIV-uninfected infants born between 1997 and 2009, using logistic regression to model grade ≥1 AE and grade ≥3 AE as a function of maternal cART and confounding variables (preterm, C-section, illicit drug use, race, ethnicity, infant antiretrovirals, and maternal viremia). Frequently used cART regimens included zidovudine (63%), lamivudine (80%), ritonavir-boosted lopinavir (37%), nelfinavir (26%), and atazanavir (10%). At birth, anemia occurred in 13/140 infants (9%), neutropenia in 27/107 (25%), thrombocytopenia in 5/133 (4%), and liver enzyme elevation in 21/130 (16%). Corresponding rates of AE at 4 weeks were 59/141 (42%), 54/130 (42%), 3/137 (2%), and 3/104 (3%), respectively. Serious AE (grade ≥ 3) exceeded 2% only for neutropenia (13% at birth; 9% at 4 weeks). Compared with infants exposed to maternal lopinavir/ritonavir, infants exposed to nelfinavir and atazanavir had a 5-fold and 4-fold higher incidence of AE at birth, respectively. In conclusion, hematologic and hepatic AE were frequent, but rarely serious. In this predominantly protease inhibitor-treated population, lopinavir/ritonavir was associated with the lowest rate of infant AE. PMID:27127401

  10. 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.

  11. 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

  12. 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

  13. Plasminogen activator and serine protease inhibitor-E2 (protease nexin-1) expression by bovine granulosa cells in vitro.

    PubMed

    Cao, Mingju; Sahmi, Malha; Lussier, Jacques G; Price, Christopher A

    2004-09-01

    Remodeling of the extracellular matrix (ECM) occurs during antral follicle growth, and the plasminogen activators (PA) have been implicated in this process in rodents. In the present study, we measured the expression and secretion of PA and the PA inhibitor protease nexin-1 (SerpinE2) in antral and basal bovine granulosa cells from small (<6 mm), medium (6-8 mm), and large follicles (>8 mm) during 6 days of culture in serum-free medium. Casein zymography revealed that the cells secreted predominantly tissue-type PA (tPA) with urokinase (uPA) being associated mainly with cell lysates, and Western blot demonstrated that the cells secreted SerpinE2. Overall, secreted tPA activity was higher in cultures of cells from small follicles compared with large follicles, and secreted SerpinE2 levels were higher in cultures of cells from large follicles. In cultures of cells from small follicles, secreted tPA levels increased with time of culture for antral but not basal cells, and SerpinE2 levels increased with time for basal but not antral cells. In cultures of granulosa cells from large follicles, tPA activity increased significantly with time of culture, whereas SerpinE2 levels decreased. Cell-associated uPA activity decreased with time in cells from medium and large follicles. Reverse-transcription polymerase chain reaction and Northern blot analysis showed that SerpinE2 secretion was regulated largely at the transcriptional level, whereas tPA secretion was not. The data suggest stage-dependent regulation of granulosa cell PA and SerpinE2 production, consistent with a role in ECM remodeling during follicle growth. PMID:15128599

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

    PubMed

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

    2016-03-15

    Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. PMID:26879854

  15. 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.

  16. 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.

  17. Protease inhibitors decrease IgG shedding from Staphylococcus aureus, increasing complement activation and phagocytosis efficiency.

    PubMed

    Fernandez Falcon, Maria F; Echague, Charlene G; Hair, Pamela S; Nyalwidhe, Julius O; Cunnion, Kenji M

    2011-10-01

    Staphylococcus aureus is a major pathogen for immunologically intact humans and its pathogenesis is a model system for evasion of host defences. Antibodies and complement are essential elements of the humoral immune system for prevention and control of S. aureus infections. The specific hypothesis for the proposed research is that S. aureus modifies humoral host defences by cleaving IgG that has bound to the bacterial surface, thereby inhibiting opsonophagocytosis. S. aureus was coated with pooled, purified human IgG and assayed for the shedding of cleaved IgG fragments using ELISA and Western blot analysis. Surface-bound IgG was shed efficiently from S. aureus in the absence of host blood proteins. Broad-spectrum protease inhibitors prevented cleavage of IgG from the S. aureus surface, suggesting that staphylococcal proteases are responsible for IgG cleavage. Serine protease inhibitors and cysteine protease inhibitors decreased the cleavage of surface-bound IgG; however, a metalloprotease inhibitor had no effect. Using protease inhibitors to prevent the cleavage of surface-bound IgG increased the binding of complement C3 fragments on the surface of S. aureus, increased the association with human neutrophils and increased phagocytosis by human neutrophils. PMID:21636671

  18. 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.

  19. 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

  20. A Novel Bis-Tetrahydrofuranylurethane-Containing Nonpeptidic Protease Inhibitor (PI), GRL-98065, Is Potent Against Mulutiple-PI-Resistant Human Immunodeficiency Virus in Vitro

    SciTech Connect

    Amano,M.; Koh, Y.; Das, D.; Li, J.; Leschenko, S.; Wang, Y.; Boross, P.; Weber, I.; Ghosh, A.; Mitsuya, H.

    2007-01-01

    We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC{sub 50}], 0.0002 to 0.0005 {mu}M) with minimal cytotoxicity (50% cytotoxicity, 35.7 {mu}M in CD4{sup +} MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1{sub NL4-3} variants exposed to and selected by up to a 5 {mu}M concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 {mu}M concentration of lopinavir or atazanavir (EC{sub 50}, 0.0015 to 0.0075 {mu}M), although it was less active against HIV-1{sub NLV4-3} selected by amprenavir (EC{sub 50}, 0.032 {mu}M). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.

  1. Ombitasvir/Paritaprevir/Ritonavir Plus Dasabuvir: A Review in Chronic HCV Genotype 1 Infection.

    PubMed

    Deeks, Emma D

    2015-06-01

    A fixed-dose tablet comprising ombitasvir (an NS5A replication complex inhibitor), paritaprevir (an NS3/4A protease inhibitor) and ritonavir (a cytochrome P450 inhibitor) taken in combination with dasabuvir (an NS5B polymerase inhibitor) is indicated for the treatment of chronic hepatitis C virus (HCV) genotype 1 infection in several countries, including the USA (copackaged as Viekira Pak(™)) and those of the EU (Viekirax(®) and Exviera(®)). In phase II and III trials, this interferon-free regimen, taken ± ribavirin, provided high rates of sustained virological response 12 weeks post-treatment in adults with chronic HCV genotype 1a or 1b infection, including those with compensated cirrhosis, liver transplants or HIV-1 co-infection. The regimen was generally well tolerated, with nausea, insomnia, asthenia, pruritus, other skin reactions and fatigue being among the most common tolerability issues. Thus, ombitasvir/paritaprevir/ritonavir plus dasabuvir is an effective interferon-free, direct-acting antiviral regimen for use ± ribavirin in a broad range of adults chronically infected with HCV genotype 1. PMID:26059288

  2. Lopinavir and Ritonavir

    MedlinePlus

    ... Although lopinavir and ritonavir will not cure HIV, these medications may decrease your chance of developing acquired ... illnesses such as serious infections or cancer. Taking these medications along with practicing safer sex and making ...

  3. Inhibition of soybean seeds in warm water results in the release of copious amounts of Bowman-Birk protease inhibitor, a putative anticarcinogenic agent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protease inhibitors play a protective role against pathogenic microorganisms and herbivorous insects. The two predominant protease inhibitors of soybean seeds are the Kunitz trypsin inhibitor (KTI) and Bowman-Birk protease inhibitor (BBI). In this study we report that soybean seeds incubated in war...

  4. 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.

  5. 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

  6. Expedient screening for HIV-1 protease inhibitors using a simplified immunochromatographic assay.

    PubMed

    Kitidee, Kuntida; Khamaikawin, Wannisa; Thongkum, Weeraya; Tawon, Yardpiroon; Cressey, Tim R; Jevprasesphant, Rachaneekorn; Kasinrerk, Watchara; Tayapiwatana, Chatchai

    2016-05-15

    A colloidal gold-based immunochromatographic (IC) strip test was developed and validated for the detection of HIV-1 protease (HIV-PR) activity and inhibitory effect of HIV-PR inhibitors (PIs). It is a unique 'two-step' process requiring the combination of proteolysis of HIV-PR and an immunochromatographic reaction. Monoclonal antibodies to the free C-terminus of HIV matrix protein (HIV-MA) conjugated to gold particles and a monoclonal antibody against intact and cleaved forms of the HIV-MA are immobilized on the 'Test'-line of the IC strip. Using lopinavir, a potent HIV protease inhibitor, the IC-strip was optimized to detect inhibitory activity against HIV-protease. At a lopinavir concentration of 1000ng/mL (its suggested minimum effective concentration), a HIV-PRH6 concentration of 6mg/mL and incubation period of 60min were the optimal conditions. A preliminary comparison between a validated high-performance liquid chromatography assay and the IC-strip to semi-quantify HIV protease inhibitor concentrations (lopinavir and atazanavir) demonstrated good agreement. This simplified method is suitable for the rapid screening of novel protease inhibitors for future therapeutic use. Moreover, the IC strip could also be optimized to semi-quantify PIs concentrations in plasma samples. PMID:26490422

  7. Modifying the substrate specificity of Carcinoscorpius rotundicauda serine protease inhibitor domain 1 to target thrombin.

    PubMed

    Giri, Pankaj Kumar; Tang, Xuhua; Thangamani, Saravanan; Shenoy, Rajesh T; Ding, Jeak Ling; Swaminathan, Kunchithapadam; Sivaraman, J

    2010-01-01

    Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC(50) of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. PMID:21188150

  8. Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

    PubMed Central

    Giri, Pankaj Kumar; Tang, Xuhua; Thangamani, Saravanan; Shenoy, Rajesh T.; Ding, Jeak Ling; Swaminathan, Kunchithapadam; Sivaraman, J.

    2010-01-01

    Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases. PMID:21188150

  9. In Vitro Characterization of GS-8374, a Novel Phosphonate-Containing Inhibitor of HIV-1 Protease with a Favorable Resistance Profile ▿ †

    PubMed Central

    Callebaut, Christian; Stray, Kirsten; Tsai, Luong; Williams, Matt; Yang, Zheng-Yu; Cannizzaro, Carina; Leavitt, Stephanie A.; Liu, Xiaohong; Wang, Kelly; Murray, Bernard P.; Mulato, Andrew; Hatada, Marcos; Priskich, Tina; Parkin, Neil; Swaminathan, Swami; Lee, William; He, Gong-Xin; Xu, Lianhong; Cihlar, Tomas

    2011-01-01

    GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+ T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients. PMID:21245449

  10. Interactions of Streptomyces serine-protease inhibitors with Streptomyces griseus metalloendopeptidase II.

    PubMed

    Kajiwara, K; Fujita, A; Tsuyuki, H; Kumazaki, T; Ishii, S

    1991-09-01

    Streptomyces griseus metalloendopeptidase II (SGMPII) was shown to form tight complexes with several Streptomyces protein inhibitors which had been believed to be specific to serine proteases, such as Streptomyces subtilisin inhibitor (SSI), plasminostreptin (PS), and alkaline protease inhibitor-2c' (API-2c'), as well as with Streptomyces metalloprotease inhibitor (SMPI). The dissociation constants of complexes between SGMPII and these inhibitors were successfully determined by using a novel fluorogenic bimane-peptide substrate. The values ranged from nM to pM. The results of studies by gel chromatographic and enzymatic analyses indicated that SGMPII is liberated from the complex with SSI by the addition of subtilisin BPN'. SGMPII and subtilisin BPN' proved, therefore, to interact with SSI in a competitive manner, despite the difference in the chemical nature of their active sites. PMID:1769961

  11. 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. PMID:25460544

  12. 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

  13. 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.

  14. Ombitasvir, Paritaprevir, Ritonavir, and Dasabuvir

    MedlinePlus

    Viekira Pak® (as a combination product containing Ombitasvir, Paritaprevir, Ritonavir, Dasabuvir) ... combination of ombitasvir, paritaprevir, ritonavir, and dasabuvir comes as tablets to take by mouth. This medication comes ...

  15. Echinacea Purpurea Significantly Induces Cytochrome P450 3A (CYP3A) but does not alter Lopinavir-Ritonavir Exposure in Healthy Subjects

    PubMed Central

    Penzak, Scott R.; Robertson, Sarah M.; Hunt, Jennifer D.; Chairez, Cheryl; Malati, Christine Y.; Alfaro, Raul M.; Stevenson, James M.; Candidate, Pharm.D.; Kovacs, Joseph A.

    2012-01-01

    the presence of the potent CYP3A inhibitor, ritonavir. Echinacea purpurea is unlikely to alter the pharmacokinetics of ritonavir-boosted protease inhibitors but may cause modest decreases in plasma concentrations of other CYP3A substrates. PMID:20653355

  16. 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

  17. Second-line protease inhibitor-based highly active antiretroviral therapy after failing non-nucleoside reverse transcriptase inhibitors-based regimens in Asian HIV-infected children

    PubMed Central

    Bunupuradah, Torsak; Puthanakit, Thanyawee; Fahey, Paul; Kariminia, Azar; Yusoff, Nik Khairulddin Nik; Khanh, Truong Huu; Sohn, Annette H.; Chokephaibulkit, Kulkanya; Lumbiganon, Pagakrong; Hansudewechakul, Rawiwan; Razali, Kamarul; Kurniati, Nia; Huy, Bui Vu; Sudjaritruk, Tavitiya; Kumarasamy, Nagalingeswaran; Fong, Siew Moy; Saphonn, Vonthanak; Ananworanich, Jintanat

    2013-01-01

    Background The WHO recommends boosted protease inhibitor (bPI)-based highly active antiretroviral therapy (HAART) after failing non-nucleoside reverse transcriptase inhibitor (NNRTI) treatment. We examined outcomes of this regimen in Asian HIV-infected children. Methods Children from five Asian countries in the TREAT Asia Pediatric HIV Observational Database (TApHOD) with ≥24 weeks of NNRTI-based HAART followed by ≥24 weeks of bPI-based HAART were eligible. Primary outcomes were the proportions with virologic suppression (HIV-RNA <400 copies/ml) and immune recovery (CD4% ≥25% if age <5 years and CD4 count ≥500 cells/mm3 if age ≥5 years) at 48 and 96 weeks. Results Of 3422 children, 153 were eligible; 52% were female. At switch, median age was 10 years, 26% were in WHO stage 4. Median weight-for-age z-score (WAZ) was −1.9 (n=121), CD4% was 12.5% (n=106), CD4 count was 237 (n=112) cells/mm3, and HIV-RNA was 4.6 log10copies/ml (n=61). The most common PI was lopinavir/ritonavir (83%). At 48 weeks, 61% (79/129) had immune recovery, 60% (26/43) had undetectable HIV-RNA and 73% (58/79) had fasting triglycerides ≥130mg/dl. By 96 weeks, 70% (57/82) achieved immune recovery, 65% (17/26) virologic suppression, and hypertriglyceridemia occurred in 66% (33/50). Predictors for virologic suppression at week 48 were longer duration of NNRTI-based HAART (p=0.006), younger age (p=0.007), higher WAZ (p=0.020), and HIV-RNA at switch <10,000 copies/ml (p=0.049). Conclusion In this regional cohort of Asian children on bPI-based second-line HAART, 60% of children tested had immune recovery by one year, and two-thirds had hyperlipidemia, highlighting difficulties in optimizing second-line HAART with limited drug options. PMID:23296119

  18. Atazanavir/ritonavir-based combination antiretroviral therapy for treatment of HIV-1 infection in adults.

    PubMed

    Achenbach, Chad J; Darin, Kristin M; Murphy, Robert L; Katlama, Christine

    2011-02-01

    In the past 15 years, improvements in the management of HIV infection have dramatically reduced morbidity and mortality. Similarly, rapid advances in antiretroviral medications have resulted in the possibility of life-long therapy with simple and tolerable regimens. Protease inhibitors have been important medications in regimens of combination antiretroviral therapy for the treatment of HIV. One of the recommended and commonly used therapies in this class is once-daily-administered atazanavir, pharmacologically boosted with ritonavir (atazanavir/r). Clinical studies and practice have shown these drugs, in combination with other antiretroviral agents, to be potent, safe and easy to use in a variety of settings. Atazanavir/r has minimal short-term toxicity, including benign bilirubin elevation, and has less potential for long-term complications of hyperlipidemia and insulin resistance compared with other protease inhibitors. A high genetic barrier to resistance and a favorable resistance profile make it an excellent option for initial HIV treatment or as the first drug utilized in the protease inhibitors class. Atazanavir/r is also currently being studied in novel treatment strategies, including combinations with new classes of antiretrovirals to assess nucleoside reverse transcriptase inhibitor-sparing regimens. In this article we review atazanavir/r as a treatment for HIV infection and discuss the latest information on its pharmacology, efficacy and toxicity. PMID:21731578

  19. Atazanavir/ritonavir-based combination antiretroviral therapy for treatment of HIV-1 infection in adults

    PubMed Central

    Achenbach, Chad J; Darin, Kristin M; Murphy, Robert L; Katlama, Christine

    2011-01-01

    In the past 15 years, improvements in the management of HIV infection have dramatically reduced morbidity and mortality. Similarly, rapid advances in antiretroviral medications have resulted in the possibility of life-long therapy with simple and tolerable regimens. Protease inhibitors have been important medications in regimens of combination antiretroviral therapy for the treatment of HIV. One of the recommended and commonly used therapies in this class is once-daily-administered atazanavir, pharmacologically boosted with ritonavir (atazanavir/r). Clinical studies and practice have shown these drugs, in combination with other antiretroviral agents, to be potent, safe and easy to use in a variety of settings. Atazanavir/r has minimal short-term toxicity, including benign bilirubin elevation, and has less potential for long-term complications of hyperlipidemia and insulin resistance compared with other protease inhibitors. A high genetic barrier to resistance and a favorable resistance profile make it an excellent option for initial HIV treatment or as the first drug utilized in the protease inhibitors class. Atazanavir/r is also currently being studied in novel treatment strategies, including combinations with new classes of antiretrovirals to assess nucleoside reverse transcriptase inhibitor-sparing regimens. In this article we review atazanavir/r as a treatment for HIV infection and discuss the latest information on its pharmacology, efficacy and toxicity. PMID:21731578

  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. 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

  2. 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

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

    PubMed Central

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

    1996-01-01

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

  4. Binding modes of a new epoxysuccinyl-peptide inhibitor of cysteine proteases. Where and how do cysteine proteases express their selectivity?

    PubMed

    Czaplewski, C; Grzonka, Z; Jaskólski, M; Kasprzykowski, F; Kozak, M; Politowska, E; Ciarkowski, J

    1999-05-18

    Papain from Carica papaya, an easily available cysteine protease, is the best-studied representative of this family of enzymes. The three dimensional structure of papain is very similar to that of other cysteine proteases of either plant (actinidin, caricain, papaya protease IV) or animal (cathepsins B, K, L, H) origin. As abnormalities in the activities of mammalian cysteine proteases accompany a variety of diseases, there has been a long-lasting interest in the development of potent and selective inhibitors for these enzymes. A covalent inhibitor of cysteine proteases, designed as a combination of epoxysuccinyl and peptide moieties, has been modeled in the catalytic pocket of papain. A number of its configurations have been generated and relaxed by constrained simulated annealing-molecular dynamics in water. A clear conformational variability of this inhibitor is discussed in the context of a conspicuous conformational diversity observed earlier in several solid-state structures of other complexes between cysteine proteases and covalent inhibitors. The catalytic pockets S2 and even more so S3, as defined by the pioneering studies on the papain-ZPACK, papain-E64c and papain-leupeptin complexes, appear elusive in view of the evident flexibility of the present inhibitor and in confrontation with the obvious conformational scatter seen in other examples. This predicts limited chances for the development of selective structure-based inhibitors of thiol proteases, designed to exploit the minute differences in the catalytic pockets of various members of this family. A simultaneous comparison of the three published proenzyme structures suggests the enzyme's prosegment binding loop-prosegment interface as a new potential target for selective inhibitors of papain-related thiol proteases. PMID:10350606

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

    NASA Astrophysics Data System (ADS)

    Windsor, Ian W.; Raines, Ronald T.

    2015-08-01

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

  6. Parallel ultra high pressure liquid chromatography-mass spectrometry for the quantification of HIV protease inhibitors using dried spot sample collection format.

    PubMed

    Watanabe, Kyoko; Varesio, Emmanuel; Hopfgartner, Gérard

    2014-08-15

    An assay was developed and validated for the quantification of eight protease inhibitors (indinavir (IDV), ritonavir (RTV), lopinavir (LPV), saquinavir (SQV), amprenavir (APV), nelfinavir (NFV), atazanavir (AZV) and darunavir (DRV)) in dried plasma spots using parallel ultra-high performance liquid chromatography and mass spectrometry detection in the multiple reaction monitoring mode. For each analyte an isotopically labeled internal standard was used and the assay based on liquid-solid extraction the area response ratio (analyte/IS) was found to be linear; from 0.025 μg/ml to 20 μg/ml for IDV, SQV, DRV, AZV, LPV, from 0.025 μg/ml to 10 μg/ml for NFV, APV and from 0.025 μg/ml to 5 μg/ml for RTV using 15 μl of plasma spotted on filter paper placed in a sample tube. The total analysis time was of 4 min and inter-assay accuracies and precisions were in the range of 87.7-109% and 2.5-11.8%, respectively. On dried plasma spots all analytes were found to be stable for at least 7 days. Practicability of the assay to blood was also demonstrated. The sample drying process could be reduced to 5 min using a commercial microwave system without any analyte degradation. Together with quantification, confirmatory analysis was performed on representative clinical samples. PMID:25049214

  7. Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants.

    PubMed

    Robert, Stéphanie; Jutras, Philippe V; Khalf, Moustafa; D'Aoust, Marc-André; Goulet, Marie-Claire; Sainsbury, Frank; Michaud, Dominique

    2016-01-01

    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts. PMID:26614285

  8. Functional domains of the human epididymal protease inhibitor, eppin.

    PubMed

    McCrudden, Maelíosa T C; Dafforn, Tim R; Houston, David F; Turkington, Philip T; Timson, David J

    2008-04-01

    Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin's domains are involved in the protein's antibacterial activity, only the Kunitz domain is required for selective protease inhibition. PMID:18331357

  9. Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling.

    PubMed

    Cohen, F E; Gregoret, L M; Amiri, P; Aldape, K; Railey, J; McKerrow, J H

    1991-11-26

    Computer modeling of the three-dimensional structure of an enzyme, based upon its primary sequence alone, is a potentially powerful tool to elucidate the function of enzymes as well as design specific inhibitors. The cercarial (larval) protease from the blood fluke Schistosoma mansoni is a serine protease hypothesized to assist the schistosome parasite in invading the human circulatory system via the skin. A three-dimensional model of the protease was built, taking advantage of the similarity of the sequence of the cercarial enzyme to the trypsin-like class of serine proteases. A large hydrophobic S-1 binding pocket, suspected from previous kinetic studies, was located in the model and confirmed by new kinetic studies with both synthetic peptide substrates and inhibitors. Unexpected structural characteristics of the enzyme were also predicted by the model, including a large S-4 binding pocket, again confirmed by assays with synthetic peptides. The model was then used to design a peptide inhibitor with 4-fold increased solubility, and a series of synthetic inhibitors were tested against live cercariae invading human skin to confirm that predictions of the model were also applicable in a biologic assay. PMID:1958659

  10. 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…

  11. 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

  12. Effect of Semen on Vaginal Fluid Cytokines and Secretory Leukocyte Protease Inhibitor

    PubMed Central

    Agnew, Kathy J.; Aura, Jan; Nunez, Norma; Lee, Zandra; Lawler, Rick; Richardson, Carol E.; Culhane, Jennifer; Hitti, Jane

    2008-01-01

    The presence of semen in vaginal fluid, as identified by an acid phosphatase spot test, does not influence vaginal proinflammatory cytokine concentrations. Objective: determine whether semen, as detected by acid phosphatase, influences vaginal cytokines or secretory leukocyte protease inhibitor concentrations. Methods: 138 pregnant women had vaginal fluid collected for Gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-Beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. Results for women with and without acid phosphatase were compared by Mann-Whitney test. Results: of 138 subjects, 28 (20%) had acid phosphatase detected; of these, only 19 (68%) reported recent intercourse and 3 (11%) had sperm seen on Gram stain. There were no significant differences in proinflammatory cytokine concentrations; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase. Conclusions: proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. Detection of semen by acid phosphatase was associated with higher vaginal SLPI concentrations, however, the presence of semen did not appear to influence vaginal proinflammatory cytokine concentrations. PMID:18615190

  13. Allosteric Inhibitors of the NS3 Protease from the Hepatitis C Virus

    PubMed Central

    Abian, Olga; Vega, Sonia; Sancho, Javier; Velazquez-Campoy, Adrian

    2013-01-01

    The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn+2-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn+2, the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn+2-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease. PMID:23936097

  14. 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

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

    PubMed

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

    2015-07-01

    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides. PMID:26071808

  16. Real-time fluorometric turn-on assay for protease activity and inhibitor screening with a benzoperylene probe.

    PubMed

    Zhou, Chuibei; Li, Wenying; Chen, Jian; Yang, Meiding; Li, Yang; Zhu, Jintao; Yu, Cong

    2014-03-01

    A real-time fluorescence turn-on strategy for protease activity and inhibitor screening has been developed. A negatively charged benzo[ghi]perylene derivative (probe 1) was employed. Protamine is a cationic protein which can induce aggregation of probe 1 via strong electrostatic and hydrophobic interactions. The fluorescence of probe 1 was efficiently quenched. In the presence of a protease, protamine was enzymatically hydrolyzed and probe 1 de-aggregated. The recovery of the probe 1 monomer fluorescence could be detected. The protease activity could be monitored in real-time. In addition, upon addition of a protease inhibitor, the protease-catalyzed hydrolysis was inhibited, which led to a decreased fluorescence recovery. The fluorometric assay thus could also be employed for screening protease inhibitors. PMID:24427771

  17. The serine protease inhibitor protease nexin-1 controls mammary cancer metastasis through LRP-1-mediated MMP-9 expression.

    PubMed

    Fayard, Bérengère; Bianchi, Fabrizio; Dey, Julien; Moreno, Eliza; Djaffer, Sabrina; Hynes, Nancy E; Monard, Denis

    2009-07-15

    Through their ability to degrade the extracellular matrix, proteases mediate cancer cell invasion and metastasis. Paradoxically, some serine protease inhibitors (serpins) are often overexpressed in human tumors. Using computational analysis, we found that the RNA level of protease nexin-1 (PN-1), a serpin that blocks numerous proteases activity, is significantly elevated in estrogen receptor-alpha-negative and in high-grade breast cancer. The in silico approach was complemented by mechanistic studies on two mammary cancer cell lines, the PN-1-negative 168FARN cells and the PN-1-positive 4T1 cells, both of which form primary mammary tumors, but only 4T1 tumors are able to metastasize to the lungs. We show that treatment of 168FARN cells with PN-1 stimulates extracellular signal-regulated kinase activation via low-density lipoprotein receptor-related protein-1 (LRP-1) binding, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity. PN-1-silenced 4T1 cells express low MMP-9 levels. Moreover, injection of PN-1-silenced cells into mice did not affect 4T1 primary mammary tumor outgrowth; however, the tumors had impaired metastatic potential, which could be restored by reexpressing soluble MMP-9 in the PN-1-silenced 4T1 cells. Thus, using mammary tumor models, we describe a novel pathway whereby the serpin PN-1 by binding LRP-1 stimulates extracellular signal-regulated kinase signaling, MMP-9 expression, and metastatic spread of mammary tumors. Importantly, an analysis of 126 breast cancer patients revealed that those whose breast tumors had elevated PN-1 levels had a significantly higher probability to develop lung metastasis, but not metastasis to other sites, on relapse. These results suggest that PN-1 might become a prognostic marker in breast cancer. PMID:19584287

  18. 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. PMID:26645974

  19. 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

  20. Protease Inhibitor Profile of Black Americans With and Without Chronic Cardiopulmonary Disease

    PubMed Central

    Young, Roscoe C.; Headings, Verle E.; Henderson, Anita L.; Bose, Sikta; Hackney, Robert L.

    1978-01-01

    An epidemiologic study of protease inhibitor (alpha1-antitrypsin) was undertaken among 599 ambulatory and hospitalized black American patients with chronic cardiopulmonary disease referred for pulmonary function testing, and 115 ethnically matched, healthy control subjects. Clinical evaluation consisted of respiratory questionnaire completion, physical examination, chest radiograph, and spirography. Protease inhibitor evaluation consisted of measurement of serum trypsin inhibitory capacity in all subjects corrected by comparison with control sera, while 200 of these subjects were phenotyped for alpha1-antitrypsin electrophoretic variants. Results showed mean serum trypsin inhibitory capacity for all subjects was 1.56, SD ± 0.47 mg/ml, while corrected values were 111.2, SD ± 30.5 percent of control. Acute phase reactivity was present for patients with heart disease, pulmonary malignancy, p<0.01 for both, and pulmonary fibrosis, p<0.05, when compared with controls. Prevalence of protease inhibitor variants in 29 controls was two heterozygotes for the Z variant (seven percent), and one homozygote for the S variant. Among 94 patients with chronic obstructive pulmonary disease, prevalence was 1.1 percent each for ZZ and SZ phenotypes, and 2.1 percent for MZ. Suprprisingly, the sole ZZ patient had asthmatic bronchitis rather than emphysema. Computed allele frequencies for Pi M and Z were comparable to those for a random sample of black Americans in St. Louis, but differed from a sample of black infants in Brooklyn, NY. These results indicate that protease inhibitor deficiency variants are not as uncommon among black Americans as the literature suggests. Furthermore, the heterozygous state is not necessarily a risk factor in development of chronic obstructive pulmonary disease. Protease inhibitor deficiency states therefore appear to play less important a role in etiology of chronic cardiopulmonary disease in black Americans than among their Caucasian counterparts

  1. 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. PMID:26546840

  2. 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

  3. 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

  4. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  5. 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.

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

    PubMed Central

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

    2013-01-01

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

  7. Cysteine protease and its inhibitor in experimentally produced squamous cell carcinomas in hairless mouse skin.

    PubMed

    Alidina, R; Kikuchi, M; Kashima, M; Epstein, J H; Fukuyama, K

    1988-08-01

    Squamous cell carcinomas (SCC) were experimentally produced in hairless mouse skin, and cysteine protease and its inhibitor were simultaneously purified from extracts of 1 g of tissue of SCC and normal skin. Activity of cysteine proteinases, Mr greater than 50,000 and Mr 28,000, increased in SCC compared to those in normal skin. SCC also showed elevation of cysteine proteinase inhibitor activity and Mr 13,000 and Mr 82,000 inhibitors were purified. Mr 13,000 inhibitor was found to have biochemical properties which were the same as those of the inhibitor present in normal skin. Mr 82,000 inhibitor was not detectable in normal skin and it differed from a serum inhibitor with a similar Mr in terms of activity and stability at acidic pH. The findings suggest that the increased activity of both cysteine proteases and endogenous inhibitors may be involved in the regulatory mechanisms of malignant cell metabolism and tissue remodeling associated with SCC development. PMID:3396664

  8. Synergistic inhibitor binding to the papain-like protease of human SARS coronavirus: mechanistic and inhibitor design implications.

    PubMed

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

    2013-08-01

    We previously developed two potent chemical classes that inhibit the essential papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus. 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 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 on a computational fragment-merging approach. This approach provides an alternative strategy for lead optimization for cases in which direct co-crystallization is difficult. PMID:23788528

  9. 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

  10. Interaction of trypsin-like protease from Streptomyces griseus with an immobilized inhibitor from kidney bean.

    PubMed

    Mosolov, V V; Fedurkina, N V; Valueva, T A

    1978-01-12

    An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties. PMID:413581

  11. 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.

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

    PubMed Central

    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-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 findings 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 we have called ADR may be a protease itself. PMID:3540956

  13. The Genetic Basis of HIV-1 Resistance to Reverse Transcriptase and Protease Inhibitors

    PubMed Central

    Shafer, Robert W.; Kantor, Rami; Gonzales, Matthew J.

    2008-01-01

    HIV-1 drug resistance is caused by mutations in the reverse transcriptase (RT) and protease enzymes, the molecular targets of antiretroviral therapy. At the beginning of the year 2000, two expert panels recommended that HIV-1 RT and protease susceptibility testing be used to help select antiretroviral drugs for HIV-1-infected patients. Genotypic assays have been developed to detect HIV-1 mutations known to confer antiretroviral drug resistance. Genotypic assays using dideoxynucleoside sequencing provide extensive insight into the presence of drug-resistant variants in the population of viruses within an individual. However, the interpretation of these assays in clinical settings is formidable because of the large numbers of drug resistance mutations and because these mutations interact with one another and emerge in complex patterns. In addition, cross-resistance between antiretroviral drugs is greater than that anticipated from initial in vitro studies. This review summarises the published data linking HIV-1 RT and protease mutations to in vitro and clinical resistance to the currently available nucleoside RT inhibitors, non-nucleoside RT inhibitors, and protease inhibitors. PMID:19096725

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

    PubMed

    Steverding, Dietmar

    2015-01-01

    Chemotherapy of human African trypanosomiasis (HAT) is unsatisfactory because only a few drugs, with serious side effects and poor efficacy, are available. As drug combination regimes often achieve greater therapeutic efficacy than monotherapies, here the trypanocidal activity of the cysteine protease inhibitor K11777 in combination with current anti-HAT drugs using bloodstream forms of Trypanosoma brucei was investigated. Isobolographic analysis was used to determine the interaction between cysteine protease inhibitors (K11777, CA-074Me and CAA0225) and anti-HAT drugs (suramin, pentamidine, melarsoprol and eflornithine). Bloodstream forms of T. brucei were incubated in culture medium containing cysteine protease inhibitors or anti-HAT drugs alone or in combination at a 1:1 fixed-dose ratio. After 48 h incubation, live cells were counted, the 50% growth inhibition values determined and combination indices calculated. The general cytotoxicity of drug combinations was evaluated with human leukaemia HL-60 cells. Combinations of K11777 with suramin, pentamidine and melarsoprol showed antagonistic effects while with eflornithine a synergistic effect was observed. Whereas eflornithine antagonises with CA-074Me, an inhibitor inactivating the targeted TbCATL only under reducing conditions, it synergises with CAA0255, an inhibitor structurally related to CA-074Me which inactivates TbCATL independently of thiols. These findings indicate an essential role of thiols for the synergistic interaction between K11777 and eflornithine. Encouragingly, the K11777/eflornithine combination displayed higher trypanocidal than cytotoxic activity. The results of this study suggest that the combination of the cysteine protease inhibitor K11777 and eflornithine display promising synergistic trypanocidal activity that warrants further investigation of the drug combination as possible alternative treatment of HAT. PMID:25662707

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

    PubMed

    Kumaresan, Venkatesh; Harikrishnan, Ramaswamy; Arockiaraj, Jesu

    2015-02-01

    Kazal-type serine protease inhibitor (KSPI) is a pancreatic secretary trypsin inhibitor which involves in various cellular component regulations including development and defense process. In this study, we have characterized a KSPI cDNA sequence of freshwater striped murrel fish Channa striatus (Cs) at molecular level. Cellular location analysis predicted that the CsKSPI was an extracellular protein. The domain analysis showed that the CsKSPI contains a Kazal domain at 47-103 along with its family signature between 61 and 83. Phylogenetically, CsKSPI is closely related to KSPI from Maylandia zebra and formed a sister group with mammals. The 2D structure of CsKSPI showed three α-helical regions which are connected with random coils, one helix at signal sequence and two at the Kazal domain region. The relative gene expression showed that the CsKSPI was highly expressed in gills and its expression was induced upon fungus (Aphanomyces invadans), bacteria (Aeromonas hydrophila) and poly I:C (a viral analogue) challenge. The CsKSPI recombinant protein was produced to characterize and study the CsKSPI gene specific functions. The recombinant CsKSPI strongly inhibited trypsin compared to other tested proteases. The results of the kinetic activity of CsKSPI against trypsin was V(max)s = 1.62 nmol/min, K(M)s = 0.21 mM and K(i)s = 15.37 nM. Moreover, the recombinant CsKSPI inhibited the growth of Gram-negative bacteria A. hydrophila at 20 μM and Gram-positive bacteria Bacillus subtilis at the MIC50 of 15 μM. Overall, the study indicated that the CsKSPI was a potential trypsin inhibitor which involves in antimicrobial activity. PMID:25433138

  16. 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

  17. 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

  18. Deep Sequencing of Protease Inhibitor Resistant HIV Patient Isolates Reveals Patterns of Correlated Mutations in Gag and Protease

    PubMed Central

    Tan, Zhiqiang; Oliveira, Glenn; Yuan, Jinyun; Okulicz, Jason F.; Torbett, Bruce E.; Levy, Ronald M.

    2015-01-01

    While the role of drug resistance mutations in HIV protease has been studied comprehensively, mutations in its substrate, Gag, have not been extensively cataloged. Using deep sequencing, we analyzed a unique collection of longitudinal viral samples from 93 patients who have been treated with therapies containing protease inhibitors (PIs). Due to the high sequence coverage within each sample, the frequencies of mutations at individual positions were calculated with high precision. We used this information to characterize the variability in the Gag polyprotein and its effects on PI-therapy outcomes. To examine covariation of mutations between two different sites using deep sequencing data, we developed an approach to estimate the tight bounds on the two-site bivariate probabilities in each viral sample, and the mutual information between pairs of positions based on all the bounds. Utilizing the new methodology we found that mutations in the matrix and p6 proteins contribute to continued therapy failure and have a major role in the network of strongly correlated mutations in the Gag polyprotein, as well as between Gag and protease. Although covariation is not direct evidence of structural propensities, we found the strongest correlations between residues on capsid and matrix of the same Gag protein were often due to structural proximity. This suggests that some of the strongest inter-protein Gag correlations are the result of structural proximity. Moreover, the strong covariation between residues in matrix and capsid at the N-terminus with p1 and p6 at the C-terminus is consistent with residue-residue contacts between these proteins at some point in the viral life cycle. PMID:25894830

  19. 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.

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

    PubMed

    Harvey, A L; Karlsson, E

    1982-09-01

    1 Five polypeptides, which were isolated from elapid snake venoms and which are structurally related to protease inhibitors, were tested for action on isolated biventer cervicis nerve-muscle preparations of the chick. 2 Dendrotoxin from the Eastern green mamba (Dendroaspis angusticeps) and toxins K and I from the black mamba (Dendroaspis polylepis polylepis) increased to indirect stimulation without affecting responses to exogenous acetylcholine, carbachol of KCl. 3 The two other protease inhibitor homologues, HHV-II from Ringhals cobra (Hemachatus haemachatus) and NNV-II from Cape cobra (Naja nivea) did not increase responses to nerve stimulation. Trypsin inhibitor from bovine pancreas also had no facilitatory effects on neuromuscular transmission. 4 The facilitatory toxins from mamba venoms interacted with the prejunctional blocking toxins, beta-bungarotoxin, crotoxin and notexin, but not with taipoxin. The blocking effects of beta-bungarotoxin were reduced by pretreatment with the mamba toxins, whereas the blocking actions of crotoxin and notexin were enhanced. 5 The results indicate that protease inhibitor homologues from mamba venoms form a new class of neurotoxin, which acts to increase the release of acetylcholine in response to motor nerve stimulation. 6 From the interaction studies it is concluded that the facilitatory toxins bind to motor nerve terminals at sites related to those occupied by the prejunctional blocking toxins. However, differences in interactions with individual toxins suggest that there must be several related binding sites on the nerve terminals. PMID:6751453

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

    PubMed

    Börner, Stefan; Ragg, Hermann

    2008-05-31

    The mutually exclusive use of alternative reactive site loop (RSL) cassettes due to alternative splicing of serpin (serine protease inhibitor) gene transcripts is a widespread strategy to create target-selective protease inhibitors in the animal kingdom. Since molecular basis and evolution of serpin RSL cassette exon amplification and diversification are unexplored, the exon-intron organization of the serpin gene spn4 from 12 species of the genus Drosophila was studied. The analysis of the gene structures shows that both number and target enzyme specificities of Spn4 RSL cassettes are highly variable in fruit flies and includes inhibitor variants with novel antiproteolytic activities in some species, indicating that RSL diversity is the result of adaptive evolution. Comparative genomics suggests that interallelic gene conversion and/or recombination events contribute to RSL cassette exon amplification. Due to an intron that is located at the most suitable position within the RSL region, multiple inhibitors can be formed in an economic manner that are both efficient and target-selective, allowing fruit flies to control an astonishing variety of proteases with different cleavage chemistry and evolutionary ancestry. PMID:18395367

  2. Structural Basis for the Immunomodulatory Function of Cysteine Protease Inhibitor from Human Roundworm Ascaris lumbricoides

    PubMed Central

    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. PMID:24781326

  3. 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

  4. 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.

  5. 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.

  6. 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

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

    PubMed Central

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

    1994-01-01

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

  8. Design of Specific Serine Protease Inhibitors Based on a Versatile Peptide Scaffold: Conversion of a Urokinase Inhibitor to a Plasma Kallikrein Inhibitor.

    PubMed

    Xu, Peng; Xu, Mingming; Jiang, Longguang; Yang, Qinglan; Luo, Zhipu; Dauter, Zbigniew; Huang, Mingdong; Andreasen, Peter A

    2015-11-25

    All serine proteases hydrolyze peptide bonds by the same basic mechanism and have very similar active sites, in spite of the fact that individual proteases have different physiological functions. We here report a strategy for designing high-affinity and high-specificity serine protease inhibitors using a versatile peptide scaffold, a 10-mer peptide, mupain-1 (CPAYSRYLDC). Mupain-1 was previously reported as a specific inhibitor of murine urokinase-type plasminogen activator (Ki = 0.55 μM) without measurable affinity to plasma kallikrein (Ki > 1000 μM). On the basis of a structure-based rational design, we substituted five residues of mupain-1 and converted it to a potent plasma kallikrein inhibitor (Ki = 0.014 μM). X-ray crystal structure analysis showed that the new peptide was able to adapt a new set of enzyme surface interactions by a slightly changed backbone conformation. Thus, with an appropriate re-engineering, mupain-1 can be redesigned to specific inhibitors of other serine proteases. PMID:26536069

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

    PubMed

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

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields. PMID:25453359

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

    PubMed

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

    2015-04-01

    The nsP2 protease of chikungunya virus (CHIKV) is one of the essential components of viral replication and it plays a crucial role in the cleavage of polyprotein precursors for the viral replication process. Therefore, it is gaining attention as a potential drug design target against CHIKV. Based on the recently determined crystal structure of the nsP2 protease of CHIKV, this study identified potential inhibitors of the virus using structure-based approaches with a combination of molecular docking, virtual screening and molecular dynamics (MD) simulations. The top hit compounds from database searching, using the NCI Diversity Set II, with targeting at five potential binding sites of the nsP2 protease, were identified by blind dockings and focused dockings. These complexes were then subjected to MD simulations to investigate the stability and flexibility of the complexes and to gain a more detailed insight into the interactions between the compounds and the enzyme. The hydrogen bonds and hydrophobic contacts were characterized for the complexes. Through structural alignment, the catalytic residues Cys1013 and His1083 were identified in the N-terminal region of the nsP2 protease. The absolute binding free energies were estimated by the linear interaction energy approach and compared with the binding affinities predicted with docking. The results provide valuable information for the development of inhibitors for CHIKV. PMID:25622129

  11. 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.

  12. 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

  13. Secretory Leukocyte Protease Inhibitor (SLPI): Emerging Roles in CNS Trauma and Repair.

    PubMed

    Hannila, Sari S

    2015-12-01

    At first glance, secretory leukocyte protease inhibitor (SLPI) would appear to have little relevance to the central nervous system (CNS). This serine protease inhibitor is most commonly found in mucosal fluids such as saliva and is best known for its anti-inflammatory and antimicrobial properties. It has been shown to promote wound healing by reducing expression of pro-inflammatory cytokines, and it can also inhibit bacterial growth and block HIV infection of macrophages. In the past 10 years, however, several studies have reported that SLPI is strongly up-regulated in response to CNS injury and that exogenous administration of SLPI is neuroprotective. It has also been shown that SLPI can overcome inhibition by CNS myelin and promote axonal regeneration. In this review, we will discuss these studies, examine the molecular mechanisms underlying SLPI's effects, and consider SLPI's potential for therapeutic use in cerebral ischemia, spinal cord injury, and multiple sclerosis. PMID:25118190

  14. Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.

    PubMed

    Kantyka, Tomasz; Fischer, Jan; Wu, Zhihong; Declercq, Wim; Reiss, Karina; Schröder, Jens-Michael; Meyer-Hoffert, Ulf

    2011-06-01

    Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K(i) around 1nM, KLK4 with K(i)=27.3nM, KLK6 with K(i)=140nM, caspase-14 with a K(i) approximating 1μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members. PMID:21439340

  15. 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. PMID:25724418

  16. Secretory leukocyte protease inhibitor (SLPI), a multifunctional protein in the host defense response.

    PubMed

    Majchrzak-Gorecka, Monika; Majewski, Pawel; Grygier, Beata; Murzyn, Krzysztof; Cichy, Joanna

    2016-04-01

    Secretory leukocyte protease inhibitor (SLPI), a ∼12kDa nonglycosylated cationic protein, is emerging as an important regulator of innate and adaptive immunity and as a component of tissue regenerative programs. First described as an inhibitor of serine proteases such as neutrophil elastase, this protein is increasingly recognized as a molecule that benefits the host via its anti-proteolytic, anti-microbial and immunomodulatory activities. Here, we discuss the diverse functions of SLPI. Moreover, we review several novel layers of SLPI-mediated control that protect the host from excessive/dysregulated inflammation typical of infectious, allergic and autoinflammatory diseases and that support healing responses through affecting cell proliferation, differentiation and apoptosis. PMID:26718149

  17. Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI).

    PubMed

    Schechter, Norman M; Choi, Eun-Jung; Wang, Zhe-Mei; Hanakawa, Yasushi; Stanley, John R; Kang, Ya'an; Clayman, Gary L; Jayakumar, Arumugam

    2005-11-01

    LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin. PMID:16307483

  18. Enhanced Secretory Leukocyte Protease Inhibitor in Human Immunodeficiency Virus Type 1-Infected Patients

    PubMed Central

    Baqui, A. A. M. A.; Meiller, Timothy F.; Falkler, William A.

    1999-01-01

    Secretory leukocyte protease inhibitor (SLPI) has been found to possess activity against the human immunodeficiency virus type 1 (HIV-1) in vitro at physiological concentrations. A study was undertaken to evaluate SLPI levels in human saliva and plasma among HIV-positive (HIV+) patients with various HIV-1 viral loads in comparison to uninfected controls. Whole blood in EDTA and unstimulated saliva samples were collected from 37 HIV+ patients, of whom 20 had a history of intravenous drug abuse (IVDA). Control samples were collected from 20 appropriate age- and sex-matched HIV-1-negative individuals. SLPI was estimated from both saliva and serum samples by an enzyme-linked immunosorbent assay. HIV viral load was determined using a quantitative reverse transcription-PCR. SLPI levels were increased 16.7% in plasma and 10.3% in saliva among HIV+ patients in comparison to uninfected controls. SLPI levels were increased 5.9% in saliva and 3.9% in plasma among HIV+ patients with a high viral load (>10,000 copies/ml) as compared to patients with a low viral load (<400 copies/ml). Only 23% of patients with a high viral load used combination therapy with protease inhibitor drugs, whereas 92.9% of HIV+ patients with a low viral load used protease inhibitors. SLPI levels did not differ significantly among the IVDA patients, patients with different viral loads, or patients using protease inhibitor drugs. There was a statistically significant increase in SLPI levels in saliva among HIV patients in comparison to non-HIV-infected controls. An increase in SLPI levels among HIV+ patients may be a natural consequence of HIV pathogenesis and an important factor in preventing oral transmission of HIV, but this increase may not be evident during plasma viremia in patients with a high viral load. PMID:10548568

  19. Protease inhibitors suppress the survival increase mediated by uncouplers in X-irradiated mammalian cells.

    PubMed

    Michel, S; Laval, F

    1982-01-01

    When mammalian cells are incubated with an uncoupler of oxidative phosphorylation prior to and during X-irradiation, the survival and the mutation frequency are markedly increased. This process requires protein synthesis and is inhibited when the cells are plated in the presence of a protease inhibitor (antipain or leupeptin). These results suggest the existence of an error-prone DNA repair process in X-irradiated mammalian cells. PMID:6814524

  20. PLANT-PIs: a database for plant protease inhibitors and their genes

    PubMed Central

    De Leo, F.; Volpicella, M.; Licciulli, F.; Liuni, S.; Gallerani, R.; Ceci, L. R.

    2002-01-01

    PLANT-PIs is a database developed to facilitate retrieval of information on plant protease inhibitors (PIs) and related genes. For each PI, links to sequence databases are reported together with a summary of the functional properties of the molecule (and its mutants) as deduced from literature. PLANT-PIs contains information for 351 plant PIs, plus several isoinhibitors. The database is accessible at http://bighost.area.ba.cnr.it/PLANT-PIs. PMID:11752333

  1. Protease inhibitors decrease rabbit cartilage degradation after meniscectomy

    SciTech Connect

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

    1988-01-01

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

  2. 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

  3. A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

    PubMed Central

    Nijhuis, Monique; van Maarseveen, Noortje M; Lastere, Stephane; Schipper, Pauline; Coakley, Eoin; Glass, Bärbel; Rovenska, Mirka; de Jong, Dorien; Chappey, Colombe; Goedegebuure, Irma W; Heilek-Snyder, Gabrielle; Dulude, Dominic; Cammack, Nick; Brakier-Gingras, Lea; Konvalinka, Jan; Parkin, Neil; Kräusslich, Hans-Georg; Brun-Vezinet, Francoise; Boucher, Charles A. B

    2007-01-01

    Background HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. Methods and Findings We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with

  4. 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

  5. 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

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

    PubMed

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

    2013-11-01

    Marine alkaline protease (MP,(2) accession no. ACY25898) is produced by a marine bacterium strain isolated from Yellow Sea sediment in China. Previous research has shown that this protease is a cold-adapted enzyme with antioxidant activity that could be used as a detergent additive. Owing to its instability in the liquid state, MP's application in liquid detergents was limited. Therefore, the discovery of reversible MP inhibitors to stabilize the protease was imperative. Here, we used the X-ray structure of MP and recompiled AutoDock 4.2 with refined Zn(2+) characters to screen the free chemical database ZINC. After completing the docking procedure, we applied strategies including the "initial filter", consensus scoring and pharmocophore model to accelerate the process and improve the virtual screening success rate. The "initial filter" was built based on the docking results of boronic acid derivatives validated as reversible inhibitors of MP by our previous studies. Finally, ten compounds were purchased or synthetized to test their binding affinity for MP. Three of the compounds could reversibly inhibit MP with apparent Ki values of 0.8-1.2 mmol. These active compounds and their binding modes provide useful information for understanding the molecular mechanism of reversible MP inhibition. The results may also serve as the foundation for further screening and design of reversible MP inhibitors. PMID:24200527

  7. 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. PMID:15643975

  8. Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor.

    PubMed

    García-Fernández, Rossana; Pons, Tirso; Perbandt, Markus; Valiente, Pedro A; Talavera, Ariel; González-González, Yamile; Rehders, Dirk; Chávez, María A; Betzel, Christian; Redecke, Lars

    2012-11-01

    Proteins isolated from marine invertebrates are frequently characterized by exceptional structural and functional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactyla helianthus, displays activity not only against serine-, but also against cysteine-, and aspartate proteases. As an initial step to evaluate the molecular basis of its activities, we describe the crystallographic structure of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7Å resolution. The overall structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-like Kunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitz inhibitor complexes. However, a prominent stabilizing role within the interface was attributed to arginine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinity against trypsin, if the P3 residue of ShPI-1 is mutated to alanine. Together with the increased role of Arg(11) at P3 position, slightly reduced interactions at the prime side (Pn') of the primary binding loop and at the secondary binding loop of ShPI-1 were detected. In addition, the structure provides important information for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnological applications. PMID:22975140

  9. 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.

  10. 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

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

    PubMed Central

    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

  12. 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

  13. Joint X-ray/neutron crystallographic study of HIV-1 protease with clinical inhibitor amprenavir – insights for drug design

    PubMed Central

    Weber, Irene T.; Waltman, Mary Jo; Mustyakimov, Marat; Blakeley, Matthew P.; Keen, David A.; Ghosh, Arun K.; Langan, Paul; Kovalevsky, Andrey Y.

    2013-01-01

    HIV-1 protease is an important target for the development of antiviral inhibitors to treat AIDS. A room-temperature joint X-ray/neutron structure of the protease in complex with clinical drug amprenavir has been determined at 2.0 Å resolution. The structure provides direct determination of hydrogen atom positions in the enzyme active site. Analysis of the enzyme-drug interactions suggests that some hydrogen bonds may be weaker than deduced from the non-hydrogen interatomic distances. This information may be valuable for the design of improved protease inhibitors. PMID:23772563

  14. Secretory Leukocyte Protease Inhibitor Suppresses the Inflammation and Joint Damage of Bacterial Cell Wall–Induced Arthritis

    PubMed Central

    Song, Xiao-yu; Zeng, Li; Jin, Wenwen; Thompson, John; Mizel, Diane E.; Lei, Ke-jian; Billinghurst, R.C.; Poole, A. Robin; Wahl, Sharon M.

    1999-01-01

    Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor α and nuclear factor κB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases. PMID:10449524

  15. 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

  16. 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.

  17. Complementary approaches to understanding the role of proteases and their natural inhibitors in neoplastic development: retrospect and prospect.

    PubMed

    Rubin, Harry

    2003-05-01

    A great deal of evidence has accumulated in recent years for an important but complex role for proteases in tumor development. However, attempts to treat cancer in humans with anti-proteases have been disappointing, and it has been suggested that more basic groundwork is needed before anti-proteases can be effectively applied. Considerable basic information comes from the recognition that earlier results on transformation of chicken embryo fibroblasts (CEF) by the Bryan strain of Rous sarcoma virus (B-RSV) can be explained in terms of proteases and their inhibitors. In particular, the full but reversible normalization of discrete transformed foci by appropriate concentrations of fetal bovine or of calf serum implies a causal role for multiple proteases in transformation, and the efficacy of treatment with a physiological balance of their natural inhibitors. Addition of certain proteases to contact-inhibited normal cultures was then found to stimulate their proliferation. The toxicity of medium produced by CEF heavily transformed with B-RSV suggests that cachexia and other systemic effects of human cancer may result from vascular dissemination of peptides from pericellular proteolysis within tumors. Comprehensive studies revealed significant increases of plasminogen activator and matrix metalloproteinases (MMPs) after infection of CEF with other strains of RSV, and correlation of the proteases with aspects of transformation. A similar role for proteases is indicated in the transformation of mammalian cells by chemical and physical agents. The information gained from functional experiments on cell transformation in culture is complementary to that obtained from the molecular identification of proteases and their inhibitors in all stages of tumor development. The speed, quantification and easy manipulation of the RSV-CEF transformation assay can be combined with current methods of characterizing proteases and anti-proteases to further enrich our basic knowledge of

  18. Selection and analysis of human immunodeficiency virus type 1 variants with increased resistance to ABT-538, a novel protease inhibitor.

    PubMed Central

    Markowitz, M; Mo, H; Kempf, D J; Norbeck, D W; Bhat, T N; Erickson, J W; Ho, D D

    1995-01-01

    Inhibitors of the human immunodeficiency virus protease represent a promising new class of antiretroviral drugs for the treatment of AIDS. We now report the in vitro selection of viral variants with decreased sensitivity to a symmetry-based protease inhibitor, ABT-538, currently being tested in clinical trials. Molecular characterization of the variants shows that an isoleucine-to-valine substitution at position 84 results in a substantial decrease in sensitivity to the drug. Moreover, an additional mutation at position 82, valine to phenylalanine, further decreases viral susceptibility to ABT-538. Three-dimensional analysis of the protease-drug complex provides a structural explanation for the relative drug resistance induced by these two mutations. These findings emphasize the importance of closely monitoring patients receiving ABT-538 for the emergence of viral resistance and provide information that may prove useful in designing the next generation of protease inhibitors. PMID:7815532

  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. PMID:27386922

  20. 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

  1. 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

  2. Small Molecule Pan-dengue and West Nile Virus NS3 Protease Inhibitors

    PubMed Central

    Cregar-Hernandez, Lynne; Jiao, Guan-Sheng; Johnson, Alan T.; Lehrer, Axel T.; Wong, Teri Ann S.; Margosiak, Stephen A.

    2011-01-01

    Background Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome are caused by infections with any of the four serotypes of the dengue virus (DENV) and are an increasing global health risk. The related West Nile Virus (WNV) causes significant morbidity and mortality as well and continues to be a threat in endemic areas. Currently no FDA approved vaccines or therapeutics are available to prevent or treat any of these infections. Like the other members of the Flaviviridae family, DENV and WNV encode a protease (NS3) which is essential for viral replication and therefore is a promising target for developing therapies to treat dengue and West Nile infections. Methods Flaviviral protease inhibitors were identified and biologically characterized for mechanism of inhibition and DENV anti-viral activity. Results A guanidinylated 2, 5-dideoxystreptamine class of compounds was identified that competitively inhibited the NS3 protease from DENV(1-4) and WNV with IC50 values in the 1-70 μM range. Cytotoxicity was low; however, antiviral activity versus DENV-2 on VERO cells was not detectable. Conclusions This class of compounds is the first to demonstrate competitive pan-dengue and WNV NS3 protease inhibition and, given the sequence conservation among flavivirus NS3 proteins, suggests that developing a pan-dengue or possibly pan-flavivirus therapeutic is feasible. PMID:21566267

  3. 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. PMID:27226628

  4. Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

    PubMed

    Manetta, J V; Lai, M H; Osborne, H E; Dee, A; Margolin, N; Sportsman, J R; Vlahos, C J; Yan, S B; Heath, W F

    1992-04-01

    A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible. PMID:1621970

  5. 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

  6. Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts.

    PubMed

    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

    2016-02-17

    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 of 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

  7. The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin*

    PubMed Central

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

    2014-01-01

    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. PMID:24962579

  8. 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

  9. Identification of HCV protease inhibitor resistance mutations by selection pressure-based method

    PubMed Central

    Qiu, Ping; Sanfiorenzo, Vincent; Curry, Stephanie; Guo, Zhuyan; Liu, Shaotang; Skelton, Angela; Xia, Ellen; Cullen, Constance; Ralston, Robert; Greene, Jonathan; Tong, Xiao

    2009-01-01

    A major challenge to successful antiviral therapy is the emergence of drug-resistant viruses. Recent studies have developed several automated analyses of HIV sequence polymorphism based on calculations of selection pressure (Ka/Ks) to predict drug resistance mutations. Similar resistance analysis programs for HCV inhibitors are not currently available. Taking advantage of the recently available sequence data of patient HCV samples from a Phase II clinical study of protease inhibitor boceprevir, we calculated the selection pressure for all codons in the HCV protease region (amino acid 1–181) to identify potential resistance mutations. The correlation between mutations was also calculated to evaluate linkage between any two mutations. Using this approach, we identified previously known major resistant mutations, including a recently reported mutation V55A. In addition, a novel mutation V158I was identified, and we further confirmed its resistance to boceprevir in protease enzyme and replicon assay. We also extended the approach to analyze potential interactions between individual mutations and identified three pairs of correlated changes. Our data suggests that selection pressure-based analysis and correlation mapping could provide useful tools to analyze large amount of sequencing data from clinical samples and to identify new drug resistance mutations as well as their linkage and correlations. PMID:19395595

  10. Identification of HCV protease inhibitor resistance mutations by selection pressure-based method.

    PubMed

    Qiu, Ping; Sanfiorenzo, Vincent; Curry, Stephanie; Guo, Zhuyan; Liu, Shaotang; Skelton, Angela; Xia, Ellen; Cullen, Constance; Ralston, Robert; Greene, Jonathan; Tong, Xiao

    2009-06-01

    A major challenge to successful antiviral therapy is the emergence of drug-resistant viruses. Recent studies have developed several automated analyses of HIV sequence polymorphism based on calculations of selection pressure (K(a)/K(s)) to predict drug resistance mutations. Similar resistance analysis programs for HCV inhibitors are not currently available. Taking advantage of the recently available sequence data of patient HCV samples from a Phase II clinical study of protease inhibitor boceprevir, we calculated the selection pressure for all codons in the HCV protease region (amino acid 1-181) to identify potential resistance mutations. The correlation between mutations was also calculated to evaluate linkage between any two mutations. Using this approach, we identified previously known major resistant mutations, including a recently reported mutation V55A. In addition, a novel mutation V158I was identified, and we further confirmed its resistance to boceprevir in protease enzyme and replicon assay. We also extended the approach to analyze potential interactions between individual mutations and identified three pairs of correlated changes. Our data suggests that selection pressure-based analysis and correlation mapping could provide useful tools to analyze large amount of sequencing data from clinical samples and to identify new drug resistance mutations as well as their linkage and correlations. PMID:19395595

  11. [Positioning of lopinavir/ritonavir in antiretroviral treatment schemes].

    PubMed

    Camacho, Ángela; Rivero, Antonio

    2014-11-01

    Lopinavir/ritonavir (LPV/r) was approved for use in the treatment of human immunodeficiency virus (HIV) infection in 2001 and is the protease inhibitor that has been most widely studied in clinical trials. Despite the time interval since its approval, all the evidence accumulated in the last 14 years indicates that LPV/r continues to occupy an important position among antiretroviral drugs. Firstly, LPV/r plus 2 nucleoside/nucleotide analogs is still considered a good option for initial antiretroviral therapy (ART). Secondly, numerous studies have evaluated the efficacy and safety of new initial ART strategies based on LPV/r in dual therapy. The results obtained suggest that LPV/r plus lamivudine (3TC) or raltegravir can be as effective in initial ART as standard triple therapy and justify their consideration as alternative regimens in this scenario. Thirdly, LPV/r is a pioneer drug, as well as being the agent with the largest amount of evidence from clinical trials on simplification to monotherapy (LPV/r) or dual therapy (LPV/r + 3TC). Lastly, LPV/r is highly useful is special situations. It has a low risk of liver toxicity in patients with chronic liver disease, its use is preferred in the treatment of patients with HIV-2, and it is safe and effective in preventing vertical HIV transmission. PMID:25542874

  12. Antiviral activity of human immunodeficiency virus type 1 protease inhibitors in a single cycle of infection: evidence for a role of protease in the early phase.

    PubMed Central

    Nagy, K; Young, M; Baboonian, C; Merson, J; Whittle, P; Oroszlan, S

    1994-01-01

    The antiviral activities of two substrate-based inhibitors of human immunodeficiency virus type 1 (HIV-1) protease, UK-88,947 and Ro 31-8959, were studied in acute infections. H9 and HeLaCD4-LTR/beta-gal cells were infected either with HIV-1IIIB or a replication-defective virus, HIV-gpt(HXB-2). Both inhibitors were capable of blocking early steps of HIV-1 replication if added to cells prior to infection. Partial inhibition was also obtained by addition of inhibitor at the time of or as late as 15 min after infection. The inhibitors were ineffective if added 30 min postinfection. The inhibitory effects were studied by cDNA analysis with PCR followed by Southern blot hybridization and by infectivity assays allowing quantitation of HIV-1 in a single cycle of replication. When UK-88,947-treated H9 cells were coinfected with HIV-1 and human T-cell leukemia virus type I only the replication of HIV-1 was inhibited, demonstrating viral specificity. Pretreating the infectious virus stocks with the inhibitors also prevented replication, indicating that the inhibitors block the action of the viral protease and not a cellular protease. A panel of primer sets was used to analyze cDNA from cell lysates by PCR amplification at 4 and 18 h postinfection. Four hours after infection, viral specific cDNA was detected with all of the four primer pairs used: R/U5, nef/U3, 5' gag, and long terminal repeat (LTR)/gag. However, after 18 h, only the R/U5 and nef/U3 primer pairs and not the 5' gag or LTR/gag primer pair were able to allow amplification of cDNA. The results suggest a crucial role of HIV-1 protease in the early phase of viral replication. Although it is not clear what early steps are affected by the protease, it is likely that the target is the NC protein, as referred from our previous reports of the in situ cleavage of the nucleocapsid (NC) protein by the viral protease inside lentiviral capsids. The results suggest that it is not the inhibition of initiation and progression

  13. Increased Risk of Preterm Delivery Among HIV-Infected Women Randomized to Protease Versus Nucleoside Reverse Transcriptase Inhibitor-Based HAART During Pregnancy

    PubMed Central

    Kitch, Douglas; Ogwu, Anthony; Hughes, Michael D.; Lockman, Shahin; Leidner, Jean; van Widenfelt, Erik; Moffat, Claire; Moyo, Sikhulile; Makhema, Joseph; Essex, Max; Shapiro, Roger L.

    2011-01-01

    (See the editorial commentary by Kourtis, on pages 493–4.) Background. Protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) use in pregnancy has been associated with preterm deliveries in some observational studies. Methods. HIV-infected, HAART-naive pregnant women with CD4+ counts ≥200 cells/mm3 were randomized between 26 and 34 weeks gestation to lopinavir/ritonavir/zidovudine/lamivudine (PI group) or abacavir/zidovudine/lamivudine (NRTI group) in a clinical trial to prevent mother-to-child HIV transmission. Risk factors for preterm delivery (<37 weeks) and differences by randomization arm were evaluated for live infants by logistic regression. Results. Preterm delivery rates were higher among 267 women in the PI group than 263 women in the NRTI group (21.4% vs 11.8%, P = .003). PI-based HAART was the most significant risk factor for preterm delivery [odds ratio = 2.03, 95% confidence interval 1.26–3.27, P = .004]. Mean change in maternal body mass index (BMI) 1 month after HAART initiation was lower in the PI group (P < .001); however, this was not significantly associated with preterm delivery. Neither infant hospitalizations nor mortality through 6 months of life differed by maternal regimen. Conclusions. PI-based HAART was associated with increased preterm delivery but not increased infant hospitalizations or mortality in a clinical trial setting. The association between PI use and lower increase in BMI in late pregnancy warrants further study. PMID:21791651

  14. Potent ketoamide inhibitors of HCV NS3 protease derived from quaternized P1 groups.

    PubMed

    Venkatraman, Srikanth; Velazquez, Francisco; Wu, Wanli; Blackman, Melissa; Madison, Vincent; Njoroge, F George

    2010-04-01

    Blood borne hepatitis C infections are the primary cause for liver cirrhosis and hepatocellular carcinoma. HCV NS3 protease, a pivotal enzyme in the replication cycle of HCV virus has been the primary target for development of new drug candidates. Boceprevir and telaprevir are two novel ketoamide derived inhibitors that are currently undergoing phase-III clinical trials. These inhibitors include ketoamide functionality as serine trap and have an acidic alpha-ketoamide center that undergoes epimerization under physiological conditions. Our initial attempts to arrest this epimerization by introducing quaternary amino acids at P(1) had resulted in significantly diminished activity. In this manuscript we describe alpha quaternized P(1) group that result in potent inhibitors in the enzyme assay and demonstrate cellular activity comparable to boceprevir. PMID:20226659

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

    PubMed Central

    Yanuar, Arry; Suhartanto, Heru; Mun׳im, Abdul; Anugraha, Bram Hik; Syahdi, Rezi Riadhi

    2014-01-01

    HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4'-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside. PMID:24616554

  16. Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

    PubMed Central

    Kleina, L G; Grubman, M J

    1992-01-01

    The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed. Images PMID:1331517

  17. HIV protease inhibitor nelfinavir inhibits growth of human melanoma cells by induction of cell cycle arrest.

    PubMed

    Jiang, Wei; Mikochik, Peter J; Ra, Jin H; Lei, Hanqin; Flaherty, Keith T; Winkler, Jeffrey D; Spitz, Francis R

    2007-02-01

    HIV protease inhibitors (HIV PI) are a class of antiretroviral drugs that are designed to target the viral protease. Unexpectedly, this class of drugs is also reported to have antitumor activity. In this study, we have evaluated the in vitro activity of nelfinavir, a HIV PI, against human melanoma cells. Nelfinavir inhibits the growth of melanoma cell lines at low micromolar concentrations that are clinically attainable. Nelfinavir promotes apoptosis and arrests cell cycle at G(1) phase. Cell cycle arrest is attributed to inhibition of cyclin-dependent kinase 2 (CDK2) and concomitant dephosphorylation of retinoblastoma tumor suppressor. We further show that nelfinavir inhibits CDK2 through proteasome-dependent degradation of Cdc25A phosphatase. Our results suggest that nelfinavir is a promising candidate chemotherapeutic agent for advanced melanoma, for which novel and effective therapies are urgently needed. PMID:17283158

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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...

  3. Comparative molecular field analysis of a series of inhibitors of HIV-1 protease.

    PubMed

    Ferreira, Leonardo G; Leitão, Andrei; Montanari, Carlos A; Andricopulo, Adriano D

    2011-03-01

    Several protease inhibitors have reached the world market in the last fifteen years, dramatically improving the quality of life and life expectancy of millions of HIV-infected patients. In spite of the tremendous research efforts in this area, resistant HIV-1 variants are constantly decreasing the ability of the drugs to efficiently inhibit the enzyme. As a consequence, inhibitors with novel frameworks are necessary to circumvent resistance to chemotherapy. In the present work, we have created 3D QSAR models for a series of 82 HIV-1 protease inhibitors employing the comparative molecular field analysis (CoMFA) method. Significant correlation coefficients were obtained (q(2) = 0.82 and r(2) = 0.97), indicating the internal consistency of the best model, which was then used to evaluate an external test set containing 17 compounds. The predicted values were in good agreement with the experimental results, showing the robustness of the model and its substantial predictive power for untested compounds. The final QSAR model and the information gathered from the CoMFA contour maps should be useful for the design of novel anti-HIV agents with improved potency. PMID:21222610

  4. 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.

  5. 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

  6. 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

  7. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions.

    PubMed

    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

  8. Evaluating the substrate-envelope hypothesis: structural analysis of novel HIV-1 protease inhibitors designed to be robust against drug resistance.

    PubMed

    Nalam, Madhavi N L; Ali, Akbar; Altman, Michael D; Reddy, G S Kiran Kumar; Chellappan, Sripriya; Kairys, Visvaldas; Ozen, Aysegül; Cao, Hong; Gilson, Michael K; Tidor, Bruce; Rana, Tariq M; Schiffer, Celia A

    2010-05-01

    Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to "lock" into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors. PMID:20237088

  9. Evaluating the Substrate-Envelope Hypothesis: Structural Analysis of Novel HIV-1 Protease Inhibitors Designed To Be Robust against Drug Resistance ▿

    PubMed Central

    Nalam, Madhavi N. L.; Ali, Akbar; Altman, Michael D.; Reddy, G. S. Kiran Kumar; Chellappan, Sripriya; Kairys, Visvaldas; Özen, Ayşegül; Cao, Hong; Gilson, Michael K.; Tidor, Bruce; Rana, Tariq M.; Schiffer, Celia A.

    2010-01-01

    Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to “lock” into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors. PMID:20237088

  10. 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

  11. 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

  12. Attenuation of the Virulence of Porphyromonas gingivalis by Using a Specific Synthetic Kgp Protease Inhibitor

    PubMed Central

    Curtis, M. A.; Aduse Opoku, J.; Rangarajan, M.; Gallagher, A.; Sterne, J. A. C.; Reid, C. R.; Evans, H. E. A.; Samuelsson, B.

    2002-01-01

    The Arg- and Lys-gingipains of Porphyromonas gingivalis are important virulence determinants in periodontal disease and may correspond to targets for immune- or drug-based treatment strategies. In this investigation we aimed to determine which of these enzymes represents the most promising molecular target for protease inhibitor-based therapy and to examine the effectiveness of the resultant compound in a murine virulence assay. Isogenic mutants with mutations in rgpA and rgpB (encoding Arg-gingipains) and in kgp (encoding Lys-gingipain) and a double mutant with mutations in rgpA and rgpB were prepared by using P. gingivalis W50. The virulence of these mutants indicated that Kgp is a promising drug target. Combinatorial chemistry was used to define the optimal substrate of Kgp, and from this information a specific slowly reversible inhibitor with a nanomolar Ki was designed and synthesized. Growth of P. gingivalis W50 in the presence of this compound resembled the phenotype of the kgp isogenic mutant; in both instances bacterial colonies failed to form pigment on blood agar, and only poor growth was obtained in a defined medium containing albumin as the sole protein source. Furthermore, pretreatment of the wild-type organism with the Kgp inhibitor led to a significant reduction in virulence in the murine assay. These data emphasize the conclusion that Kgp is an important factor for both nutrition and virulence of P. gingivalis and that inhibitors of this enzyme may have therapeutic potential for the control of P. gingivalis infections. Protease inhibitors may be a potentially novel class of antimicrobial agents with relevance to the control of other bacterial pathogens. PMID:12438376

  13. 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.

  14. 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

  15. A novel locust (Schistocerca gregaria) serine protease inhibitor with a high affinity for neutrophil elastase

    PubMed Central

    Brillard-Bourdet, Michèle; Hamdaoui, Ahmed; Hajjar, Eric; Boudier, Christian; Reuter, Nathalie; Ehret-Sabatier, Laurence; Bieth, Joseph G.; Gauthier, Francis

    2006-01-01

    We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (kass=1.2×107 M−1·s−1, Ki=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM≤Ki≤153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 °C), low or high pH (2.5–11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases. PMID:16839309

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Emergence of Resistance to Protease Inhibitor Amprenavir in Human Immunodeficiency Virus Type 1-Infected Patients: Selection of Four Alternative Viral Protease Genotypes and Influence of Viral Susceptibility to Coadministered Reverse Transcriptase Nucleoside Inhibitors

    PubMed Central

    Maguire, Michael; Shortino, Denise; Klein, Astrid; Harris, Wendy; Manohitharajah, Varsha; Tisdale, Margaret; Elston, Robert; Yeo, Jane; Randall, Sharon; Xu, Fan; Parker, Hayley; May, Jackie; Snowden, Wendy

    2002-01-01

    Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (P = 0.014 and P = 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure. PMID:11850255

  1. Secretory leukocyte protease inhibitor and elafin/trappin-2: versatile mucosal antimicrobials and regulators of immunity.

    PubMed

    Sallenave, Jean-Michel

    2010-06-01

    Elafin and secretory leukocyte protease inhibitor (SLPI) are pleiotropic molecules chiefly synthesized at the mucosal surface that have a fundamental role in the surveillance against microbial infections. Their initial discovery as anti-proteases present in the inflammatory milieu in chronic pathologies such as those of the lung suggested that they may play a role in keeping in check extracellular proteases released during the excessive activation of innate immune cells such as neutrophils. This soon proved to be a simplistic explanation, as other functions were also soon ascribed to these molecules (antimicrobial, modulation of innate and adaptive immunity, regulation of tissue repair). Data emanating from patients with chronic pathologies (in the lung and elsewhere) have shown that SLPI and elafin are often inactivated in inflammatory secretions, either through the action of host or microbial products, justifying attempts at antiprotease supplementation in clinical protocols. Although these have been sparse, proof of principle has been demonstrated, and future challenges will undoubtedly rest with improvements in methods of delivery in the context of tissue inflammation and in careful selection of patients more likely to benefit from SLPI/elafin augmentation. PMID:20395631

  2. Protease inhibitor reduces airway response and underlying inflammation in cockroach allergen-induced murine model.

    PubMed

    Saw, Sanjay; Arora, Naveen

    2015-04-01

    Protease(s) enhances airway inflammation and allergic cascade. In the present study, effect of a serine protease inhibitor was evaluated in mouse model of airway disease. Mice were sensitized with cockroach extract (CE) or Per a 10 and treated with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) 1 h before or after challenge to measure airway response. Mice were euthanized to collect bronchoalveolar lavage fluid (BALF), blood, and lung to evaluate inflammation. AEBSF treatment significantly reduced the AHR in allergen-challenged mice in dose-dependent manner (p≤ 0.01). IgE (p≤0.05) and Th2 cytokines (p≤0.05) were significantly reduced in treated mice. AEBSF treatment lowered total cell (p≤0.05), eosinophil (p≤0.05), and neutrophil (p≤0.05) in BALF and lung tissue. Oxidative stress parameters were impaired on treatment in allergen-challenged mice (p≤0.05). AEBSF had therapeutic effect in allergen-induced airway resistance and underling inflammation and had potential for combination or as add-on therapy for respiratory diseases. PMID:25052477

  3. Sequence-specific alterations of epitope production by HIV Protease Inhibitors

    PubMed Central

    Kourjian, Georgio; Xu, Yang; Mondesire-Crump, Ijah; Shimada, Mariko; Gourdain, Pauline; Le Gall, Sylvie

    2014-01-01

    Antigen processing by intracellular proteases and peptidases and epitope presentation are critical for recognition of pathogen-infected cells by CD8+ T lymphocytes. First generation HIV protease inhibitors (PIs) alter proteasome activity, but the effect of first or second generation PIs on other cellular peptidases, the underlying mechanism and impact on antigen processing and epitope presentation to CTL are still unknown. Here we demonstrate that several HIV PIs altered not only proteasome but also aminopeptidase activities in PBMC. Using an in vitro degradation assay involving PBMC cytosolic extracts we showed that PIs altered the degradation patterns of oligopeptides and peptide production in a sequence-specific manner, enhancing the cleavage of certain residues and reducing others’. PIs affected the sensitivity of peptides to intracellular degradation, altered the kinetics and amount of HIV epitopes produced intracellularly. Accordingly the endogenous degradation of incoming virions in the presence of PIs led to variations in CTL-mediated killing of HIV-infected cells. By altering host protease activities and the degradation patterns of proteins in a sequence-specific manner, HIV PIs may diversify peptides available for MHC-I presentation to CTL, alter the patters of CTL responses, and may provide a complementary approach to current therapies for the CTL-mediated clearance of abnormal cells in infection, cancer or other immune disease. PMID:24616479

  4. Staphylococcus aureus secretes a unique class of neutrophil serine protease inhibitors.

    PubMed

    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

    2014-09-01

    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

  5. 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

  6. 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

  7. 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

  8. 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...

  9. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    PubMed

    Weiss, André; Joerss, Hanna; Brockmeyer, Jens

    2014-01-01

    EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

  10. Structural and Functional Characterization of Cleavage and Inactivation of Human Serine Protease Inhibitors by the Bacterial SPATE Protease EspPα from Enterohemorrhagic E. coli

    PubMed Central

    Weiss, André; Joerss, Hanna; Brockmeyer, Jens

    2014-01-01

    EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition. PMID:25347319

  11. Modulation of the Bacillus anthracis secretome by the immune inhibitor A1 protease.

    PubMed

    Pflughoeft, Kathryn J; Swick, Michelle C; Engler, David A; Yeo, Hye-Jeong; Koehler, Theresa M

    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

  12. Serum albumin as a probe for testing the selectivity of irreversible cysteine protease inhibitors: The case of vinyl sulfones.

    PubMed

    Regazzoni, Luca; Colombo, Simone; Mazzolari, Angelica; Vistoli, Giulio; Carini, Marina

    2016-05-30

    Vinyl sulfones are used for drug design of irreversible inhibitors of cysteine proteases since they are able to alkylate cysteine thiols inside the catalytic pocket of this class of enzymes. Some authors have reported the lack of reactivity towards glutathione as sufficient evidence of the selectivity of such a mechanism. Herein, we demonstrate that some simple molecules containing a vinyl sulfone moiety are not thiol-specific alkylants since they react with some albumin nucleophiles including side chains of Cys34 and His146. Such side-reactions are not desirable for any drug candidate since they limit serum stability, bioavailability and they possibly trigger toxicity mechanisms. In silico predictions, indicate that the compounds tested share similar structural features with reported inhibitors of cysteine proteases, as well as similar poses around the main albumin nucleophiles. Altogether, the data suggest that albumin is better than glutathione for the setup of early in vitro tests probing the selectivity of cysteine protease inhibitors. PMID:26970985

  13. Mapping Inhibitor Binding Modes on an Active Cysteine Protease via NMR Spectroscopy

    PubMed Central

    Lee, Gregory M.; Balouch, Eaman; Goetz, David H.; Lazic, Ana; McKerrow, James H.; Craik, Charles S.

    2013-01-01

    Cruzain is a member of the papain/cathepsin-L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. We report an auto-induction methodology that provides soluble-cruzain at high yields (> 30 mg per liter in minimal media). These increased yields provide sufficient quantities of active enzyme for use in NMR-based ligand mapping. Using CD and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective 15N-Cys, 15N-His, and 13C-Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verifies that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely covalent, non-covalent, and non-interacting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions in order to facilitate lead compound identification and subsequent structural studies. PMID:23181936

  14. 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.

  15. 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

  16. 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.

  17. Proresolving Actions of Synthetic and Natural Protease Inhibitors Are Mediated by Annexin A1.

    PubMed

    Vago, Juliana P; Tavares, Luciana P; Sugimoto, Michelle A; Lima, Graziele Letícia N; Galvão, Izabela; de Caux, Thais R; Lima, Kátia M; Ribeiro, Ana Luíza C; Carneiro, Fernanda S; Nunes, Fernanda Freire C; Pinho, Vanessa; Perretti, Mauro; Teixeira, Mauro M; Sousa, Lirlândia P

    2016-02-15

    Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis. PMID:26800869

  18. 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

  19. A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis suaveolens L.) inhibits proteases from the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae).

    PubMed

    Aguirre, Cesar; Valdés-Rodríguez, Silvia; Mendoza-Hernández, Guillermo; Rojo-Domínguez, Arturo; Blanco-Labra, Alejandro

    2004-05-01

    A novel trypsin inhibitor purified from chan seeds (Hyptis suaveolens, Lamiaceae) was purified and characterized. Its apparent molecular mass was 8700 Da with an isoelectric point of 3.4. Its N-terminal sequence showed a high content of acidic amino acids (seven out of 18 residues). Its inhibitory activity was potent toward all trypsin-like proteases extracted from the gut of the insect Prostephanus truncatus (Coleoptera: Bostrichidae), a very important pest of maize. This activity was highly specific, because among proteases from seven different insects, only those from P. truncatus and Manduca sexta (Lepidoptera: Sphingidae) were inhibited. This inhibitor has potential to enhance the defense mechanism of maize against the attack of P. truncatus. PMID:15142539

  20. Ombitasvir, Paritaprevir, Ritonavir, and Dasabuvir

    MedlinePlus

    Viekira Pak® (as a combination product containing Ombitasvir, Paritaprevir, Ritonavir, Dasabuvir) ... C infection (swelling of the liver caused by a virus). Ombitasvir is a hepatitis C virus (HCV) ...

  1. 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.

  2. 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.

  3. Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity.

    PubMed

    Schock, H B; Garsky, V M; Kuo, L C

    1996-12-13

    Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W. A., Blahy, O. M. , Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). These four substitutions are among the prominent mutations found in primary HIV isolates obtained from patients undergoing therapy with several protease inhibitors. Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110-360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing Ki), and those away from the active site enhance catalysis (by increasing kcat/KM). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively

  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. Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors.

    PubMed

    Duan, Haichuan; Hu, Tao; Foti, Robert S; Pan, Yongmei; Swaan, Peter W; Wang, Joanne

    2015-11-01

    Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1-3 (OCT1-3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1-3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC₅₀ = 1.4 ± 0.2 µM) over OCT1 (IC₅₀ = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1-3. PMID:26285765

  6. 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

  7. A Phase III Comparative Study of the Efficacy and Tolerability of Three Non-Nucleoside Reverse Transcriptase Inhibitor-Sparing Antiretroviral Regimens for Treatment-Naïve HIV-1-Infected Volunteers: A Randomized, Controlled Trial

    PubMed Central

    Lennox, Jeffrey L.; Landovitz, Raphael J.; Ribaudo, Heather J.; Ofotokun, Ighovwerha; Na, Lumine H.; Godfrey, Catherine; Kuritzkes, Daniel R.; Sagar, Manish; Brown, Todd T.; Cohn, Susan E.; McComsey, Grace A.; Aweeka, Francesca; Fichtenbaum, Carl J.; Presti, Rachel M.; Koletar, Susan L.; Haas, David W.; Patterson, Kristine B.; Benson, Constance A.; Baugh, Bryan P.; Leavitt, Randi Y.; Rooney, James F.; Seekins, Daniel; Currier, Judith S.

    2015-01-01

    Background Non-nucleoside reverse transcriptase (NNRTI) inhibitor-based antiretroviral therapy is not suitable for all treatment-naïve HIV-infected persons. Objective Perform a rigorous evaluation of three NNRTI-sparing initial antiretroviral regimens to demonstrate equivalence for virologic efficacy and tolerability. Design Phase-III, 1:1:1 randomized, open label, >96 week study. Setting Fifty-seven sites in United States and Puerto Rico. Patients Treatment naïve, ≥18 years, HIV-1 RNA >1000 copies/mL, no nucleoside reverse transcriptase or protease inhibitor resistance. Intervention Atazanavir 300 mg with ritonavir 100 mg, daily; or raltegravir 400 mg twice daily; or darunavir 800 mg with ritonavir 100 mg, daily; plus emtricitabine 200 mg + tenofovir disoproxil fumarate 300 mg daily. Measurements Virologic failure defined as confirmed HIV-1 RNA >1000 copies/mL between 16 and 24 weeks, or >200 copies/mL at or after 24 weeks; tolerability failure defined as discontinuation of atazanavir, raltegravir or darunavir for toxicity. A secondary endpoint was a combination of virologic efficacy and tolerability. Results Among 1,809 participants all pairwise comparisons of incidence of virologic failure over 96-weeks demonstrated equivalence within ±10%. Raltegravir and ritonavir-boosted darunavir were equivalent for tolerability, whereas ritonavir-boosted atazanavir resulted in a 12.7% and a 9.2% higher incidence of tolerability discontinuation than raltegravir and ritonavir-boosted darunavir respectively, primarily due to hyperbilirubinemia. For combined virologic efficacy and tolerability ritonavir-boosted darunavir was superior to ritonavir-boosted atazanavir, and raltegravir was superior to both protease inhibitors. Antiretroviral resistance at time of virologic failure was rare but more likely with raltegravir. Limitations Open label; ritonavir not provided Conclusions Over 2 years all three regimens attain high and equivalent rates of virologic control. Regimens

  8. Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2-P4 study.

    PubMed

    Ang, Melgious J Y; Lau, Qiu Ying; Ng, Fui Mee; Then, Siew Wen; Poulsen, Anders; Cheong, Yuen Kuen; Ngoh, Zi Xian; Tan, Yong Wah; Peng, Jianhe; Keller, Thomas H; Hill, Jeffrey; Chu, Justin J H; Chia, C S Brian

    2016-01-01

    Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC50 value 3.4 ± 0.4 versus 7.3 ± 0.8 μM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities. PMID:25792507

  9. Impact of Stereochemistry on Ligand Binding: X-ray Crystallographic Analysis of an Epoxide-Based HIV Protease Inhibitor.

    PubMed

    Benedetti, Fabio; Berti, Federico; Campaner, Pietro; Fanfoni, Lidia; Demitri, Nicola; Olajuyigbe, Folasade M; De March, Matteo; Geremia, Silvano

    2014-09-11

    A new pseudopeptide epoxide inhibitor, designed for irreversible binding to HIV protease (HIV-PR), has been synthesized and characterized in solution and in the solid state. However, the crystal structure of the complex obtained by inhibitor-enzyme cocrystallization revealed that a minor isomer, with inverted configuration of the epoxide carbons, has been selected by HIV-PR during crystallization. The structural characterization of the well-ordered pseudopeptide, inserted in the catalytic channel with its epoxide group intact, provides deeper insights into inhibitor binding and HIV-PR stereoselectivity, which aids development of future epoxide-based HIV inhibitors. PMID:25221650

  10. In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure†

    PubMed Central

    La Seta Catamancio, Simona; De Pasquale, Maria Pia; Citterio, Paola; Kurtagic, Semir; Galli, Massimo; Rusconi, Stefano

    2001-01-01

    We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions. PMID:11230439

  11. 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

  12. Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

    PubMed Central

    Trylska, J.; Antosiewicz, J.; Geller, M.; Hodge, C. N.; Klabe, R. M.; Head, M. S.; Gilson, M. K.

    1999-01-01

    The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed. PMID:10210196

  13. 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

  14. [Presence of lectins, tannins and protease inhibitors in venezuelan marine algae].

    PubMed

    Perez-Lorenzo, S; Levy-Benshimol, A; Gomez-Acevedo, S

    1998-01-01

    The presence of lectins, tannins and protease inhibitors was studied in 27 algae species collected at four Venezuelan coral rift sites. Among the species studied, only six had hemagglutinating activity, apparently due to their lectin content. Higher hemagglutinating titers were obtained when the extracts were tested on pronase-treated erythrocytes. Hemagglutination was inhibited by simple sugars and by bovine submaxillary gland mucine. GaINAc was the only inhibitor of the hemagglutination caused by Grateulopia filicina extracts. None of the compounds tested inhibited the hemagglutination caused by Halimeda opuntia. The polyvinylpolypirrolidone treatment abolished the hemagglutinating activity of both brown and red algae. However, in Grateulopia filicina and Hypnea cervicornis (Rhodophyta) hemagglutinating activity persisted after the polyvinylpolypirrolidone treatment, presumably due to the presence of true lectins in those algae. Tannin content (presumably phlorotannins) was higher in the Phaeophyta as compared to the Rhodophyta. The brown alga Padina gymnospora had the higher content of these polyphenols. Trypsin inhibitors were detected, in minute ammounts, only in Padina gymnospora (Phaeophyta) and Acantophora spicifera (Rhodophyta). No subtilisin inhibition was observed whatsoever. PMID:10030041

  15. 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.

  16. Susceptibility of treatment-naive hepatitis C virus (HCV) clinical isolates to HCV protease inhibitors.

    PubMed

    Bae, Andrew; Sun, Siu-Chi; Qi, Xiaoping; Chen, Xiaowu; Ku, Karin; Worth, Angela; Wong, Kelly A; Harris, Jeanette; Miller, Michael D; Mo, Hongmei

    2010-12-01

    In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses. PMID:20855726

  17. Differential regulation of secretory leukocyte protease inhibitor and elafin by progesterone.

    PubMed

    King, Anne E; Morgan, Kevin; Sallenave, Jean-Michel; Kelly, Rodney W

    2003-10-17

    Elafin and secretory leukocyte protease inhibitor (SLPI) are anti-protease and anti-microbial molecules present at mucosal surfaces. Both molecules are expressed in the female reproductive tract where they may be involved in innate immune defence. This study examines the role of progesterone in the regulation of SLPI and elafin. Progesterone treatment increases expression of SLPI mRNA and protein in the T47D breast epithelial cell line and this upregulation is attenuated in the presence of the anti-gestogens, RU486 and ZK98734, confirming the involvement of the nuclear progesterone receptor. A putative progesterone response element has been identified in the SLPI promoter. Progesterone also acts in synergy with the proinflammatory cytokines, IL-1beta and TNFalpha, to increase SLPI. In contrast, progesterone treatment has no direct effect on elafin mRNA expression. In summary, progesterone has a differential effect on SLPI and elafin expression and although both vary within the uterus throughout the menstrual cycle, progesterone is likely to contribute to the direct regulation of SLPI in the female reproductive tract even in the presence of inflammatory agents. PMID:14521952

  18. 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

  19. Pentapeptide Boronic Acid Inhibitors of Mycobacterium tuberculosis MycP1 Protease

    PubMed Central

    Frasinyuk, Mykhaylo S.; Kwiatkowski, Stefan; Wagner, Jonathan M.; Evans, Timothy J.; Reed, Robert W.; Korotkov, Konstantin V.; Watt, David S.

    2014-01-01

    Mycosin protease-1 (MycP1) cleaves ESX secretion-associated protein B (EspB) that is a virulence factor of Mycobacterium tuberculosis, and accommodates an octapeptide, AVKAASLG, as a short peptide substrate. Because peptidoboronic acids are known inhibitors of serine proteases, the synthesis and binding of a boronic acid analog of the pentapeptide cleavage product, AVKAA, was studied using MycP1 variants from M. thermoresistible (MycP1mth), M. smegmatis (MycP1msm) and M. tuberculosis (MycP1mtu). We synthesized the boropentapeptide, HAlaValLysAlaAlaB(OH)2 (1) and the analogous pinanediol PD-protected HAlaValLysAlaAlaBO2(PD) (2) using an Fmoc/Boc peptide strategy. The pinanediol boropentapeptide 2 displayed IC50 values 121.6±25.3 μM for MycP1mth, 93.2±37.3 μM for MycP1msm and 37.9±5.2 μM for MycP1mtu. Such relatively strong binding creates a chance for crystalizing the complex with 2 and finding the structure of the unknown MycP1 catalytic site that would potentially facilitate the development of new anti-tuberculosis drugs. PMID:24915878

  20. Antiviral properties of palinavir, a potent inhibitor of the human immunodeficiency virus type 1 protease.

    PubMed Central

    Lamarre, D; Croteau, G; Wardrop, E; Bourgon, L; Thibeault, D; Clouette, C; Vaillancourt, M; Cohen, E; Pargellis, C; Yoakim, C; Anderson, P C

    1997-01-01

    Palinavir is a potent inhibitor of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases. Replication of laboratory strains (HIV-1, HIV-2, and simian immunodeficiency virus) and HIV-1 clinical isolates is inhibited by palinavir with 50% effective concentrations ranging from 0.5 to 30 nM. The average cytotoxic concentration of palinavir (35 microM) in the various target cells indicates a favorable therapeutic index. Potent antiviral activity is retained with increased doses of virus and with clinical isolates resistant to zidovudine (AZT), didanosine (ddI), or nevirapine. Combinations of palinavir with either AZT, ddI, or nevirapine demonstrate synergy or additivity in the inhibition of HIV-1 replication. Palinavir retains anti-HIV-1 activity when administered postinfection until times subsequent to the reverse transcription step. In chronically infected CR-10 cells, palinavir blocks Gag precursor polyprotein processing completely, reducing greater than 99% of infectious particle production. The results indicate that the antiviral activity of palinavir is specific to inhibition of the viral protease and occurs at a late stage in the replicative cycle of HIV-1. On the basis of the potent in vitro activity, low-level cytotoxicity, and other data, palinavir was selected for in-depth preclinical evaluation. PMID:9145853

  1. Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies.

    PubMed

    Sabotič, Jerica; Kilaru, Sreedhar; Budič, Maruška; Gašparič, Meti Buh; Gruden, Kristina; Bailey, Andy M; Foster, Gary D; Kos, Janko

    2011-10-01

    Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators. PMID:21672601

  2. 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

  3. Protease inhibitor 15, a candidate gene for abdominal aortic internal elastic lamina ruptures in the rat

    PubMed Central

    Falak, Samreen; Schafer, Sebastian; Baud, Amelie; Hummel, Oliver; Schulz, Herbert; Gauguier, Dominique; Osborne-Pellegrin, Mary

    2014-01-01

    The inbred Brown Norway (BN) rat develops spontaneous ruptures of the internal elastic lamina (RIEL) of the abdominal aorta (AA) and iliac arteries. Prior studies with crosses of the BN/Orl RJ (susceptible) and LOU/M (resistant) showed the presence of a significant QTL on chromosome 5 and the production of congenic rats proved the involvement of this locus. In this study, we further dissected the above-mentioned QTL by creating a new panel of LOU.BN(chr5) congenic and subcongenic lines and reduced the locus to 5.2 Mb. Then we studied 1,002 heterogeneous stock (HS) rats, whose phenotyping revealed a low prevalence and high variability for RIEL. High-resolution mapping in the HS panel detected the major locus on chromosome 5 (log P > 35) and refined it to 1.4 Mb. Subsequently, RNA-seq analysis on AA of BN, congenics, and LOU revealed expression differences for only protease inhibitor 15 (Pi15) gene and a putative long intergenic noncoding RNA (lincRNA) within the linkage region. The high abundance of lincRNA with respect to reduced Pi15 expression, in conjunction with exertion of longitudinal strain, may be related to RIEL, indicating the potential importance of proteases in biological processes related to defective aortic internal elastic lamina structure. Similar mechanisms may be involved in aneurysm initiation in the human AA. PMID:24790086

  4. Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

    PubMed

    Kesteleyn, Bart; Amssoms, Katie; Schepens, Wim; Hache, Geerwin; Verschueren, Wim; Van De Vreken, Wim; Rombauts, Klara; Meurs, Greet; Sterkens, Patrick; Stoops, Bart; Baert, Lieven; Austin, Nigel; Wegner, Jörg; Masungi, Chantal; Dierynck, Inge; Lundgren, Stina; Jönsson, Daniel; Parkes, Kevin; Kalayanov, Genadiy; Wallberg, Hans; Rosenquist, Asa; Samuelsson, Bertil; Van Emelen, Kristof; Thuring, Jan Willem

    2013-01-01

    The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS. PMID:23177258

  5. The potency-insolubility conundrum in pharmaceuticals: Mechanism and solution for hepatitis C protease inhibitors.

    PubMed

    Connelly, Patrick R; Snyder, Phillip W; Zhang, Yuegang; McClain, Brian; Quinn, Brian P; Johnston, Steve; Medek, Ales; Tanoury, Jerry; Griffith, James; Patrick Walters, W; Dokou, Eleni; Knezic, Dragutin; Bransford, Philip

    2015-01-01

    As compounds are optimized for greater potency during pharmaceutical discovery, their aqueous solubility often decreases, making them less viable as orally-administered drugs. To investigate whether potency and insolubility share a common origin, we examined the structural and thermodynamic properties of telaprevir, a sparingly soluble inhibitor of hepatitis C virus protease. Comparison of the hydrogen bond motifs in crystalline telaprevir with those present in the protease-telaprevir complex revealed striking similarities. Additionally, the thermodynamics of telaprevir dissolution closely resembles those of protein-ligand dissociation. Together, these findings point to a common origin of potency and insolubility rooted in particular amide-amide hydrogen bond patterns. The insolubility of telaprevir is shown by computational analysis to be caused by interactions in the crystal, not unfavorable hydrophobic hydration. Accordingly, competing out the particular amide-amide hydrogen bond motifs in crystalline telaprevir with 4-hydroxybenzoic acid yielded a co-crystalline solid with excellent aqueous dissolution and oral absorption. The analysis suggests a generalizable approach for identifying drug candidate compounds that either can or cannot be rendered orally bioavailable by alteration of their crystalline solid phases, in an approach that provides a pragmatic way to attain substantial enhancements in the success rate of drug discovery and development. PMID:25451684

  6. 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

  7. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  8. Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

    PubMed Central

    Mulenga, A.; Kim, T.; Ibelli, A. M. G.

    2013-01-01

    We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross-class inhibitor of both serine- and papain-like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics analyses revealed that Pichia pastoris-expressed recombinant (r) AamS6 reduced initial velocities of substrate hydrolysis (V0) and/or maximum enzyme velocity (Vmax) of trypsin, chymotrypsin, elastase, chymase, and papain in a dose–response manner. We speculate that rAamS6 inhibited plasmin in a temporary fashion in that while rAamS6 reduced V0 of plasmin by up to ~53%, it had no effect on Vmax. Our data also suggest that rAmS6 has minimal or no apparent effect on V0 or Vmax of thrombin, factor Xa, and kallikrein. We speculate that AamS6 is apparently involved in facilitating blood meal feeding in that various amounts of rAamS6 reduced platelet aggregation by up to ~47% and delayed plasma clotting time in the recalcification time assay by up to ~210 s. AamS6 is most likely not involved with the tick’s evasion of the host’s complement defense mechanism, in that rAamS6 did not interfere with the complement activation pathway. Findings in this study are discussed in the context of expanding our understanding of tick proteins that control bloodmeal feeding and hence tick-borne disease transmission by ticks. PMID:23521000

  9. Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

    PubMed

    Meyer-Hoffert, Ulf; Wu, Zhihong; Schröder, Jens-Michael

    2009-01-01

    Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5. PMID:19190773

  10. Quantification of secretory leukocyte protease inhibitor (SLPI) in oral gargle specimens collected using mouthwash

    PubMed Central

    Pierce Campbell, Christine M.; Guan, Wei; Sprung, Robert; Koomen, John M.; O’Keefe, Michael T.; Ingles, Donna J.; Abrahamsen, Martha; Giuliano, Anna R.

    2014-01-01

    Background Secretory leukocyte protease inhibitor (SLPI) is an innate immunity-associated protein known to inhibit HIV transmission, and is thought to inhibit a variety of infectious agents, including human papillomaviruses (HPVs). We aimed to optimize an established ELISA-based SLPI quantification assay for use with oral gargle specimens collected using mouthwash, and to assess preliminary associations with age, smoking status, and alcohol intake. Methods Oral gargle supernatants from 50 individuals were used to optimize the Human SLPI Quantikine ELISA Kit. Sample suitability was assessed and quality control analyses were conducted. Results Salivary SLPI was successfully recovered from oral gargles with low intra-assay and high inter-individual variability. Initial measurements showed that salivary SLPI varied considerably across individuals, and that SLPI was inversely associated with age. Conclusions This optimized assay can be used to examine the role of SLPI in the acquisition of oral HPV and other infections. PMID:24140751

  11. Vioserpin, a serine protease inhibitor from Gloeobacter violaceus possibly regulated by heparin.

    PubMed

    Oliveira, Jocélia P C; Salazar, Natália; Zani, Marcelo B; de Souza, Lucas R; Passos, Silvia G; Sant'Ana, Aquiles M; de Andrade, Regiane A; Marcili, Arlei; Sperança, Marcia A; Puzer, Luciano

    2016-08-01

    Serine peptidase inhibitor (serpin) is the name given to the superfamily of proteins with wide range of biological functions, and that the main feature is the inhibition of serine proteases. Here we describe the inhibitory characterization of a serpin from Gloeobacter violaceus that we named vioserpin. The serpin presented a high specificity to inhibit trypsin-like enzymes with a rapid inhibition rate constant (2.1 × 10(6) M(-1) s(-1)). We also demonstrated that the inhibitory activity of the vioserpin is influenced by the concentration of heparin, and this finding may throw a new light on understanding the molecular evolution of serpins. PMID:27157268

  12. 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

  13. 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

  14. A new multigene superfamily of Kunitz-type protease inhibitors from sea anemone Heteractis crispa.

    PubMed

    Isaeva, Marina P; Chausova, Victoriya E; Zelepuga, Elena A; Guzev, Konstantin V; Tabakmakher, Valentin M; Monastyrnaya, Margarita M; Kozlovskaya, Emma P

    2012-03-01

    Despite a considerable number of publications devoted to isolation and physicochemical properties of protease inhibitors from sea anemones, virtually nothing is known about the structure of the genes, and the nature of their isoforms diversity. Using the PCR-based cloning approach we discovered the Kunitz-type multigene superfamily composed of distinct gene families (GS-, RG-, GG-, and GN-gene families). It has been identified only three full-length GS-transcripts indicating a much greater variety of Kunitz homologs in Heteractis crispa. We have examined an exon-intron structure of GS-genes; an open reading frame is interrupted by a single intron located at the middle of the signal peptide. 33 deduced mature GS-polypeptides have been categorized into three groups according to the nature of a P1 residue. Some of them corresponded to native Kunitz-type protease inhibitors earlier isolated from H. crispa. The deduced GS-polypeptide sequences demonstrated diverse charge distribution ranging from the local point charges forms to the overall positive ones. We have suggested that the GS-gene family has evolved through gene tandem duplication followed by adaptive divergence of the P1 residue in the reactive site selected for divergent functions in paralogs. The expansion of this Kunitz-type multigene superfamily during evolution is lineage-specific, providing the tropical sea anemone H. crispa with the ability to interact an increasing diversity of the preys and predators. Our results show that the Kunitz-type polypeptides are encoded by a multigene superfamily and realized via a combinatory Kunitz-type library in the H. crispa tentacles venom. PMID:22001835

  15. IrSPI, a tick serine protease inhibitor involved in tick feeding and Bartonella henselae infection.

    PubMed

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

    2014-07-01

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

  16. 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.

  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. 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

  19. 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

  20. 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

  1. Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

    PubMed Central

    Rose, R E; Gong, Y F; Greytok, J A; Bechtold, C M; Terry, B J; Robinson, B S; Alam, M; Colonno, R J; Lin, P F

    1996-01-01

    The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise. Images Fig. 1 PMID:8643685

  2. Cysteine protease inhibition by nitrile-based inhibitors: a computational study

    PubMed Central

    Quesne, Matthew G.; Ward, Richard A.; de Visser, Sam P.

    2013-01-01

    Cysteine protease enzymes are important for human physiology and catalyze key protein degradation pathways. These enzymes react via a nucleophilic reaction mechanism that involves a cysteine residue and the proton of a proximal histidine. Particularly efficient inhibitors of these enzymes are nitrile-based, however, the details of the catalytic reaction mechanism currently are poorly understood. To gain further insight into the inhibition of these molecules, we have performed a combined density functional theory and quantum mechanics/molecular mechanics study on the reaction of a nitrile-based inhibitor with the enzyme active site amino acids. We show here that small perturbations to the inhibitor structure can have dramatic effects on the catalysis and inhibition processes. Thus, we investigated a range of inhibitor templates and show that specific structural changes reduce the inhibitory efficiency by several orders of magnitude. Moreover, as the reaction takes place on a polar surface, we find strong differences between the DFT and QM/MM calculated energetics. In particular, the DFT model led to dramatic distortions from the starting structure and the convergence to a structure that would not fit the enzyme active site. In the subsequent QM/MM study we investigated the use of mechanical vs. electronic embedding on the kinetics, thermodynamics and geometries along the reaction mechanism. We find minor effects on the kinetics of the reaction but large geometric and thermodynamics differences as a result of inclusion of electronic embedding corrections. The work here highlights the importance of model choice in the investigation of this biochemical reaction mechanism. PMID:24790966

  3. The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73.

    PubMed

    Pavlova, Alona; Björk, Ingemar

    2002-11-01

    The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by approximately 300-fold, >10-fold and approximately 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by approximately 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, approximately 45% of the total binding energy, for inhibition of cathepsin B. PMID:12423365

  4. 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

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

    PubMed Central

    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.

    2013-01-01

    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 since 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. PMID:23590295

  6. Origin of decrease in potency of darunavir and two related antiviral inhibitors against HIV-2 compared to HIV-1 protease.

    PubMed

    Kar, Parimal; Knecht, Volker

    2012-03-01

    Acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV) type 1 and 2 (HIV-1 and HIV-2). HIV-1 is observed worldwide while HIV-2 though prevalent in West Africa is persistently spreading to other parts of the world. An important target for AIDS treatment is the use of HIV protease (PR) inhibitors preventing the replication of the virus. In this work, the popular molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method has been used to investigate the effectiveness of the HIV-1 PR inhibitors darunavir, GRL-06579A, and GRL-98065 against HIV-2 and HIV-1 protease. The affinity of the inhibitors for both HIV-1 and HIV-2 PR decreases in the order GRL-06579A > darunavir > GRL-98065, in accordance with experimental data. On the other hand, our results show that all these inhibitors bind less strongly to HIV-2 than to HIV-1 protease, again in agreement with experimental findings. The decrease in binding affinity for HIV-2 relative to HIV-1 PR is found to arise from an increase in the energetic penalty from the desolvation of polar groups (DRV) or a decrease in the size of the electrostatic interactions between the inhibitor and the PR (GRL-06579A and GRL-98065). For GRL-98065, also a decrease in the magnitude of the van der Waals interactions contributes to the reduction in binding affinity. A detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of efficient inhibitors against HIV-2 protease. PMID:22280246

  7. 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

  8. A lack of secretory leukocyte protease inhibitor (SLPI) causes defects in granulocytic differentiation.

    PubMed

    Klimenkova, Olga; Ellerbeck, Wienke; Klimiankou, Maksim; Ünalan, Murat; Kandabarau, Siarhei; Gigina, Anna; Hussein, Kais; Zeidler, Cornelia; Welte, Karl; Skokowa, Julia

    2014-02-20

    We identified diminished levels of the natural inhibitor of neutrophil elastase (NE), secretory leukocyte protease inhibitor (SLPI), in myeloid cells and plasma of patients with severe congenital neutropenia (CN). We further found that downregulation of SLPI in CD34(+) bone marrow (BM) hematopoietic progenitors from healthy individuals resulted in markedly reduced in vitro myeloid differentiation accompanied by cell-cycle arrest and elevated apoptosis. Reciprocal regulation of SLPI by NE is well documented, and we previously demonstrated diminished NE levels in CN patients. Here, we found that transduction of myeloid cells with wild-type NE or treatment with exogenous NE increased SLPI messenger RNA and protein levels, whereas transduction of mutant forms of NE or inhibition of NE resulted in downregulation of SLPI. An analysis of the mechanisms underlying the diminished myeloid differentiation caused by reduced SLPI levels revealed that downregulation of SLPI with short hairpin RNA (shRNA) upregulated nuclear factor κB levels and reduced phospho-extracellular signal-regulated kinase (ERK1/2)-mediated phosphorylation and activation of the transcription factor lymphoid enhancer-binding factor-1 (LEF-1). Notably, microarray analyses revealed severe defects in signaling cascades regulating the cell cycle, including c-Myc-downstream signaling, in myeloid cells transduced with SLPI shRNA. Taken together, these results indicate that SLPI controls the proliferation, differentiation, and cell cycle of myeloid cells. PMID:24352879

  9. Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin.

    PubMed

    Christoffers, Keith H; Khokhar, Arshia; Chaturvedi, Kirti; Howells, Richard D

    2002-04-15

    We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions. PMID:11853866

  10. The effects of protease inhibitor homologues from mamba snake venoms on autonomic neurotransmission.

    PubMed

    Anderson, A J

    1985-01-01

    Five protease inhibitor homologues isolated from mamba venoms were tested for facilitatory actions on autonomic neurotransmission using isolated smooth muscle preparations. Dendrotoxin from the eastern green mamba (Dendroaspis angusticeps) was the most consistent in augmenting the responses to sympathetic stimulation in vas deferens preparations and to parasympathetic stimulation in chick oesophagus preparations. Toxin I from the black mamba (D. polylepis) venom augmented the neurally evoked responses in vas deferens preparations, and toxin K from the same venom augmented neurally evoked responses in chick oesophagus preparations. Proteins B and E from D. polylepis venom, as well as bovine pancreas trypsin inhibitor, had no significant facilitatory action on either smooth muscle preparation. The mechanism of the augmentation of neurally evoked responses produced by toxin I on vas deferens preparations, and dendrotoxin on chick oesophagus preparations, was investigated using a variety of drugs which interfere with cholinergic and adrenergic transmission. It is concluded that dendrotoxin and toxin I increase evoked transmitter release in the autonomic nervous system by a direct action on nerves. PMID:3006283

  11. 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

  12. Four proteolytic processes of myocardium, one insensitive to thiol reactive agents and thiol protease inhibitor.

    PubMed

    Thorne, D P; Lockwood, T D

    1993-07-01

    Four distinct processes mediating protein degradation were identified in the Langendorff perfused rat heart. Hearts were biosynthetically labeled in vitro with [3H]leucine for 10 min. The subsequent release of [3H]leucine at 1.5-min intervals (2 mM nonradioactive leucine) was determined from 20 min to 8 h after labeling in rhythmically contracting hearts. Rapid turnover proteins were eliminated during the first 3 h; this degradation was not inhibited by insulin (5 nM) or isoproterenol (0.5 microM). However, the nontoxic thiol reactive agent diamide (100 microM) caused a complete inhibition of the [3H]leucine release from rapidly degraded proteins. After the elimination of rapidly degraded proteins at 3 h, the release of [3H]leucine was inhibited 35-40% by insulin (5 nM) or the lysosomal inhibitor chloroquine (30 microM), thereby defining a second vesicular process. The beta-agonist isoproterenol (0.5 microM) or the nonselective alpha-agonist naphazoline (100 microM) caused 30-35% proteolytic inhibitions, defining a third adrenergic-responsive process. The inhibitory effects of simultaneously combined insulin and chloroquine did not exceed the effect of either agent alone. However, the combined effects of insulin and isoproterenol were additive, inhibiting two-thirds of basal degradation. Beginning at 3 h after labeling a 75% proteolytic inhibition resulted from the thiol reactive agents diamide (100 microM) or N-ethylmaleimide (10 microM); the thiol protease active site inhibitor trans-epoxysuccinly-L-leucylamino-(4-quinidino)butane (50 microM) caused 65% inhibition. The 75% inhibition caused by diamide includes both the insulin-responsive and beta-adrenergic-responsive pathways. A novel fourth proteolytic process (25% of proteolysis) was thereby distinguished from the above three by its resistance to inhibition by insulin, adrenergic agonists, thiol reactive agents, or thiol protease inhibitor. Only the adrenergic-responsive process was correlated with changes in

  13. 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.

  14. Same fold with different mobility: backbone dynamics of small protease inhibitors from the desert locust, Schistocerca gregaria.

    PubMed

    Szenthe, Borbála; Gáspári, Zoltán; Nagy, Attila; Perczel, András; Gráf, László

    2004-03-30

    SGCI (Schistocerca gregaria chymotrypsin inhibitor) and SGTI (Sch. gregaria trypsin inhibitor) are small, 35-residue serine protease inhibitors with intriguing taxon specificity: SGTI is specific for arthropod proteases while SGCI is an excellent inhibitor on both mammalian and arthropodal enzymes. Here we report the cloning, expression, and (15)N backbone dynamics investigations of these peptides. Successful expression could be achieved by a "dimeric" construct similar to the natural precursor of the inhibitors. An engineered methionine residue between the two modules served as a unique cyanogen bromide cleavage site to cleave the precursor and physically separate SGCI and SGTI. The overall correlation time of the precursor (5.29 ns) as well as the resulted SGCI (3.14 ns) and SGTI (2.96 ns) are as expected for proteins of this size. General order parameters (S(2)) for the inhibitors are lower than those characteristic of well-folded proteins. Values in the binding loop region are even lower. Interestingly, the distribution of residues for which a chemical exchange (R(ex)) term should be considered is strikingly different in SGCI and SGTI. Together with H-D exchange studies, this indicates that the internal dynamics of the two closely related molecules differ. We suggest that the dynamic properties of these inhibitors is one of the factors that determine their specificity. PMID:15035609

  15. Full quantum mechanical study of binding of HIV-1 protease drugs

    NASA Astrophysics Data System (ADS)

    Zhang, Da W.; Zhang, John Z. H.

    Fully quantum mechanical studies of detailed binding interactions between HIV-1 protease and six FDA (Food and Drug Administration)-approved drugs (saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir) are carried out using a recently developed MFCC (molecular fractionation with conjugate caps) method. The MFCC calculation produces a quantum mechanical interaction spectrum for any protease drug binding complex. Detailed quantitative analysis on binding of lopinavir to specific residues of the protease is given from the current study. The present calculation shows that the dominant binding of lopinavir to the protease is through the formation of a strong hydrogen bond between the central hydroxyl group of the drug to the aspartate oxygen of Asp25 in one of the two chains of the protease (A chain). This is closely followed by hydrogen binding of the drug to Asp29 in the B chain and somewhat weak hydrogen bonding to Asp30, Gly27, Gly48, and Ile50 in both chains. By partitioning all six drugs into four building blocks besides the central component containing the hydroxyl group, MFCC calculation finds that block III has essentially no binding interaction with the protease and the major binding interactions of these drugs are from blocks II and IV, in addition to the dominant central hydroxyl group. This detailed quantitative information on drug binding to the protease is very useful in rational design of new and improved inhibitors of HIV-1 protease and its mutants.

  16. Naturally occurring NS3-protease-inhibitor resistant mutant A156T in the liver of an untreated chronic hepatitis C patient.

    PubMed

    Cubero, Maria; Esteban, Juan Ignacio; Otero, Teresa; Sauleda, Silvia; Bes, Marta; Esteban, Rafael; Guardia, Jaume; Quer, Josep

    2008-01-20

    An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3-protease inhibitors. PMID:18006035

  17. 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

  18. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    SciTech Connect

    Xiang, Jin; Wang, Ying; Su, Ke; Liu, Min; Hu, Peng-Chao; Ma, Tian; Li, Jia-Xi; Wei, Lei; Zheng, Zhongliang; Yang, Fang

    2014-10-01

    Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis. - Graphical abstract: RTV directly binds to ERα and Leu536 in the hydrophobic core of ligand binding domain is essential for the interaction. - Highlights: • RTV increases the thickness of rat coronary artery wall and foam cell formation. • RTV downregulates the expression of ERα and ERβ. • RTV inhibits ERα promoter activity. • RTV directly binds to ERα and the key amino acid is Leu536. • RTV inhibits the nuclear translocation of ERα and GPER.

  19. Molecular characterization of serine protease inhibitor isoform 3, SmSPI, from Schistosoma mansoni.

    PubMed

    Pakchotanon, Pattarakul; Molee, Patamaporn; Nuamtanong, Supaporn; Limpanont, Yanin; Chusongsang, Phiraphol; Limsomboon, Jareemate; Chusongsang, Yupa; Maneewatchararangsri, Santi; Chaisri, Urai; Adisakwattana, Poom

    2016-08-01

    Serine protease inhibitors, known as serpins, are pleiotropic regulators of endogenous and exogenous proteases, and molecule transporters. They have been documented in animals, plants, fungi, bacteria, and viruses; here, we characterize a serpin from the trematode platyhelminth Schistosoma mansoni. At least eight serpins have been found in the genome of S. mansoni, but only two have characterized molecular properties and functions. Here, the function of S. mansoni serpin isoform 3 (SmSPI) was analyzed, using both computational and molecular biological approaches. Phylogenetic analysis showed that SmSPI was closely related to Schistosoma haematobium serpin and Schistosoma japonicum serpin B10. Structure determined in silico confirmed that SmSPI belonged to the serpin superfamily, containing nine α-helices, three β-sheets, and a reactive central loop. SmSPI was highly expressed in schistosomules, predominantly in the head gland, and in adult male and female with intensive accumulation on the spines, which suggests that it may have a role in facilitating intradermal and intravenous survival. Recombinant SmSPI was overexpressed in Escherichia coli; the recombinant protein was of the same size (46 kDa) as the native protein. Immunological analysis suggested that mice infected with S. mansoni responded to rSmSPI at 8 weeks postinfection (wpi) but not earlier. The inhibitory activity of rSmSPI was specific to chymotrypsin but not trypsin, neutrophil elastase, and porcine pancreatic elastase. Elucidating the biological and physiological functions of SmSPI as well as other serpins will lead to further understanding of host-parasite interaction machinery that may provide novel strategies to prevent and control schistosomiasis in the future. PMID:27083187

  20. 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

  1. Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

    PubMed Central

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease. PMID:24015196

  2. Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins)

    PubMed Central

    Porter, Lindsay; Radulovic, Zeljko; Kim, Tae; Braz, Gloria R. C.; Da Silva Vaz, Itabajara; Mulenga, Albert

    2014-01-01

    Serine protease inhibitors (serpins) are a diverse family of proteins that is conserved across taxa. The diversity of Amblyomma americanum serpins (AAS) is far more complex than previously thought as revealed by discovery of 57 and 33 AAS transcripts that are respectively expressed in male and female A. americanum ticks, with 30 found in both. While distinct reproductively, both male and female metastriate ticks, such as A. americanum, require a blood meal. Thus, 30 AAS sequences found in both male and female ticks could play important role(s) in regulating tick feeding and thus represent attractive candidates for anti-tick vaccine development. Of significant interest, 19 AAS sequences expressed in male and female ticks are also part of the 48 AAS sequences expressed in fed female tick salivary glands or midguts; two organs through which the tick interacts with host blood and immune response factors. Considered the most important domain for serpin function, the reactive center loop (RCL) is further characterized by a single ‘P1’ site amino acid residue, which is central to determining the protease regulated by the serpin. In this study, a diversity of 17 different P1 site amino acid residues were predicted, suggesting that A. americanum serpins potentially regulate a large number of proteolytic pathways. Our data also indicate that some serpins in this study could regulate target protease common to all tick species, in that more than 40% of AAS show 58–97% inter-species amino acid conservation. Of significance, 24% of AAS showed 62–100% inter-species conservation within the functional RCL domain, with 10 RCLs showing ≥90–100% conservation. In vertebrates, serpins with basic residues at the P1 site regulate key host defense pathways, which the tick must evade to feed successfully. Interestingly, we found that AAS sequences with basic or polar uncharged residues at the putative P1 site are more likely to be conserved across tick species. Another notable

  3. 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

  4. 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

  5. Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

    PubMed

    Boudida, Yasmine; Gagaoua, Mohammed; Becila, Samira; Picard, Brigitte; Boudjellal, Abdelghani; Herrera-Mendez, Carlos H; Sentandreu, Miguel; Ouali, Ahmed

    2016-04-25

    Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50-70 kDa), F2 (Mr:40-60 kDa) and F3 (Mr:10-15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear. PMID:25085261

  6. 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.

  7. [Efficacy of initial antiretroviral therapy based on lopinavir/ritonavir plus 2 nucleoside/nucleotide analogs in patients with human immunodeficiency virus type 1 infection].

    PubMed

    Zamora, Laura; Gatell, José M

    2014-11-01

    Triple combination regimens consisting of lopinavir/ritonavir (LPV/r) plus 2 nucleoside/nucleotide analogs continue to be a valid option in initial antiretroviral therapy. Other protease inhibitors boosted with ritonavir (and in future with cobicistat) have been introduced, as well as other non-nucleoside analogs (rilpivirin) and 3 integrase inhibitors. None of the new regimens have shown superiority over LPV/r or comparisons are lacking. Therefore, regimens including LPV/r continue to be recommended as initial first-line or alternative strategies in most treatment guidelines. Dual combinations with LPV/r (plus raltegravir or lamivudine) are described in another article and can provide a similar response rate to triple combinations, better tolerance, and an improved cost-efficacy ratio, both for initial therapy and in simplification strategies. In contrast, LPV/r or darunavir/r monotherapy does not seem an acceptable option in treatment-naïve patients and is becoming increasingly less acceptable in simplification strategies. PMID:25542868

  8. 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

  9. Cerebrospinal fluid analysis for HIV replication and biomarkers of immune activation and neurodegeneration in long-term atazanavir/ritonavir monotherapy treated patients

    PubMed Central

    Ferretti, Francesca; Bigoloni, Alba; Passeri, Laura; Galli, Laura; Longo, Valeria; Gerevini, Simonetta; Spagnuolo, Vincenzo; Gisslen, Magnus; Zetterberg, Henrik; Fuchs, Dietmar; Cattaneo, Dario; Caramatti, Giada; Lazzarin, Adriano; Cinque, Paola; Castagna, Antonella

    2016-01-01

    Abstract Background: Cerebrospinal fluid (CSF) viral escape is a concern in ritonavir-boosted protease inhibitors monotherapy. The aim was to assess HIV-RNA, biomarkers of immune activation and neurodegeneration, and atazanavir concentrations in CSF of patients on successful long-term atazanavir/ritonavir (ATV/r) monotherapy. Methods: This is a substudy of the multicentric, randomized, open-label, noninferiority trial monotherapy once a day with atazanavir/ritonavir (NCT01511809), comparing the ongoing ATV/r along with 2 nucleoside retrotranscriptase inhibitors (NRTIs) regimen to a simplified ATV/r monotherapy. Patients with plasma HIV-RNA < 50 copies/mL after at least 96 study weeks were eligible. We assessed HIV-RNA, soluble (s)CD14, sCD163, CCL2, CXCL10, interleukin-6, and YKL40 by enzyme-linked immunosorbent assay; neopterin, tryptophan, kynurenine, and neurofilament by immunoassays; and ATV concentrations by liquid chromatography–mass spectrometry in paired plasma and CSF samples. Variables were compared with Wilcoxon rank-sum or Fisher exact test, as appropriate. Results: HIV-RNA was detected in the CSF of 1/11 patients on ATV/r monotherapy (114 copies/mL), without neurological symptoms, who was successfully reintensified with his previous 2NRTIs, and in none of the 12 patients on ATV/r + 2NRTIs. CSF biomarkers and ATV concentrations did not differ between the 2 arms. Conclusions: CSF escape was uncommon in patients on long-term ATV/r monotherapy and was controlled with reintensification. PMID:27428202

  10. Mutation in promoter region of a serine protease inhibitor confers Perkinsus marinus resistance in the eastern oyster (Crassostrea virginica).

    PubMed

    He, Yan; Yu, Haiyang; Bao, Zhenmin; Zhang, Quanqi; Guo, Ximing

    2012-08-01

    Protease inhibitors from the host may inhibit proteases from invading pathogens and confer resistance. We have previously shown that a single-nucleotide polymorphism (SNP198C) in a serine protease inhibitor gene (cvSI-1) is associated with Perkinsus marinus resistance in the eastern oyster. As SNP198 is synonymous, we studied whether its linkage to polymorphism at the promoter region could explain the resistance. A 631 bp fragment of the promoter region was cloned by genome-walking and resequenced, revealing 22 SNPs and 3 insertion/deletions (indels). A 25 bp indel at position -404 was genotyped along with SNP198 for association analysis using before- and after-mortality samples. After mortalities that were primarily caused by P. marinus, the frequency of deletion allele at -404indel increased by 15.6% (p = 0.0437), while that of SNP198C increased by only 3.4% (p = 0.5756). The resistance alleles at the two loci were coupled in 79.6% of the oysters. Oysters with the deletion allele at -404indel showed significant (p = 0.0189) up-regulation of cvSI-1 expression under P. marinus challenge. Our results suggest that mutation at the promoter region causes increased transcription of cvSI-1, which in turn confers P. marinus resistance in the eastern oyster likely through inhibiting pathogenic proteases from the parasite. PMID:22683517

  11. Multistage virtual screening and identification of novel HIV-1 protease inhibitors by integrating SVM, shape, pharmacophore and docking methods.

    PubMed

    Wei, Yu; Li, Jinlong; Chen, Zeming; Wang, Fengwei; Huang, Weiqiang; Hong, Zhangyong; Lin, Jianping

    2015-08-28

    The HIV-1 protease has proven to be a crucial component of the HIV replication machinery and a reliable target for anti-HIV drug discovery. In this study, we applied an optimized hierarchical multistage virtual screening method targeting HIV-1 protease. The method sequentially applied SVM (Support Vector Machine), shape similarity, pharmacophore modeling and molecular docking. Using a validation set (270 positives, 155,996 negatives), the multistage virtual screening method showed a high hit rate and high enrichment factor of 80.47% and 465.75, respectively. Furthermore, this approach was applied to screen the National Cancer Institute database (NCI), which contains 260,000 molecules. From the final hit list, 6 molecules were selected for further testing in an in vitro HIV-1 protease inhibitory assay, and 2 molecules (NSC111887 and NSC121217) showed inhibitory potency against HIV-1 protease, with IC50 values of 62 μM and 162 μM, respectively. With further chemical development, these 2 molecules could potentially serve as HIV-1 protease inhibitors. PMID:26185005

  12. Protease inhibitors and haemagglutinins associated with resistance to the protozoan parasite, Perkinsus marinus, in the Pacific oyster, Crassostrea gigas.

    PubMed

    Romestand, B; Corbier, F; Roch, P

    2002-10-01

    Perkinsus marinus is a protozoan responsible for dramatic mortality in the Eastern oyster, Crassostrea virginica, but not in the Pacific oyster, C. gigas. To understand the host-parasite relationship, we inoculated P. marinus trophozoites into the shell cavity of C. gigas and measured, over 2 months, (i) intensity of infection, (ii) protease inhibitory activities against P. marinus proteases and against bovine z-chymotrypsin, (iii) plasma haemagglutinin titre, (iv) plasma protein concentration, (v) plasma lysozyme activity and (vi) total haemocyte count. We observed that the highest protease inhibitory activities and haemagglutinin titres (3-10 days post-challenge) preceded parasite elimination (initiated 7 days post-challenge). In contrast, plasma protein concentration, lysozyme activity and total haemocyte count showed no significant modification following the challenge. It is hypothesized that the capacity of C. gigas to increase its protease inhibitors represents the key event in resistance to parasite infection by neutralizing the proteases secreted by P. marinus, thus preserving the oyster haemagglutinins from degradation. Such haemagglutinins will be ready to act as opsonins stimulating phagocytosis of parasites. PMID:12403320

  13. 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

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

    PubMed

    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 (Cys(8) to Cys(16)) was identified. PMID:25978745

  15. Cellular HIV-1 DNA quantitation in patients during simplification therapy with protease inhibitor-sparing regimens.

    PubMed

    Sarmati, Loredana; Parisi, Saverio Giuseppe; Nicastri, Emanuele; d'Ettorre, Gabriella; Andreoni, Carolina; Dori, Luca; Gatti, Francesca; Montano, Marco; Buonomini, Anna Rita; Boldrin, Caterina; Palù, Giorgio; Vullo, Vincenzo; Andreoni, Massimo

    2007-07-01

    Simplified regimens containing protease-inhibitors (PI)-sparing combinations were used in patients with virological suppression after prolonged highly active antiretroviral therapy. This study evaluated the total HIV-1 DNA quantitation as a predictor of long-term success for PI-sparing simplified therapy. Sixty-two patients were enrolled in a prospective non-randomized cohort. All patients have been receiving a triple-therapy regimen, two nucleoside reverse transcriptase inhibitors (NRTIs) plus one PI, for at least 9 months and were characterized by undetectable plasma HIV-1 RNA levels (<50 cp/ml) for at least 6 months. Patients were changed to a simplified PI-sparing regimen to overcome PI-associated adverse effects. HIV-DNA levels in peripheral blood mononuclear cells (PBMCs) were evaluated at baseline and at the end of follow-up. Patients with proviral DNA levels below the median value (226 copies/10(6) PBMCs) had a significant higher CD4 cell count at nadir (P = 0.003) and at enrolment (P = 0.001) with respect to patients with HIV-DNA levels above the median value. At month 18, 53 out of 62 (85%) patients on simplified regimen showed virological success, 4 (6.4%) patients experienced virological failure and 5 (8%) patients showed viral blip. At logistic regression analysis, HIV-DNA levels below 226 copies/10(6) PBMCs at baseline were associated independently to a reduced risk of virological failure or viral blip during simplified therapy (OR 0.002, 95% CI 0.001-0.46, P = 0.025). The substitution of PI with NRTI or non-NRTIs may represent an effective treatment option. Indeed, treatment failure or viral blip were experienced by 6% and 8% of the patients on simplified therapy, respectively. In addition, sustained suppression of the plasma viral load was significantly correlated with low levels of proviral DNA before treatment simplification. PMID:17516532

  16. 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

  17. 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

  18. Reversible Cysteine Protease Inhibitors Show Promise for a Chagas Disease Cure

    PubMed Central

    Beaulieu, Christian; Black, W. Cameron; Isabel, Elise; Vasquez-Camargo, Fabio; Nath-Chowdhury, Milli; Massé, Frédéric; Mellon, Christophe; Methot, Nathalie

    2014-01-01

    The cysteine protease cruzipain is essential for the viability, infectivity, and virulence of Trypanosoma cruzi, the causative agent of Chagas disease. Thus, inhibitors of cruzipain are considered promising anti-T. cruzi chemotherapeutic agents. Reversible cruzipain inhibitors containing a nitrile “warhead” were prepared and demonstrated 50% inhibitory concentrations (IC50s) as potent as 1 nM in baculovirus-generated cruzipain enzyme assays. In epimastigote and intracellular amastigote in vitro assays, the most potent compounds demonstrated antiparasitic behavior in the 5 to 10 μM IC50 range; however, trypomastigote production from the amastigote form was ∼90 to 95% inhibited at 2 μM. Two key compounds, Cz007 and Cz008, with IC50s of 1.1 and 1.8 nM, respectively, against the recombinant enzyme were tested in a murine model of acute T. cruzi infection, with oral dosing in chow for 28 days at doses from 3 to 50 mg/kg of body weight. At 3 mg/kg of Cz007 and 3 mg/kg of Cz008, the blood parasitemia areas under the concentration-time curves were 16% and 25% of the untreated group, respectively. At sacrifice, 24 days after immunosuppression with cyclophosphamide, parasite presence in blood, heart, and esophagus was evaluated. Based on negative quantitative PCR results in all three tissues, cure rates in surviving animals were 90% for Cz007 at 3 mg/kg, 78% for Cz008 at 3 mg/kg, and 71% for benznidazole, the control compound, at 50 mg/kg. PMID:24323474

  19. Secretory Leukocyte Protease Inhibitor Is an Inducible Antimicrobial Peptide Expressed in Staphylococcus aureus Endophthalmitis

    PubMed Central

    Reviglio, Victor E.; Sambuelli, Ruben H.; Olmedo, Alejandra; Falco, Micaela; Echenique, Jose; O'Brien, Terrence P.; Kuo, Irene C.

    2007-01-01

    Purpose. To describe the presence of secretory leukocyte protease inhibitor (SLPI), a cationic peptide with antimicrobial and antiprotease activity, in the innate ocular immune reaction in a rat model of Staphylococcus aureus endophthalmitis. Methods. Seventy-five female Lewis rats were divided into three groups: the endophthalmitis group received an intravitreal injection of 65 colony-forming units of viable S. aureus, the vehicle-injected group received balanced sterile saline solution (BSS), and the control group was not injected. Eyes were enucleated at 24 and 48 hours and processed for immunohistochemical staining and Western blot studies for SLPI. Results. In S. aureus endophthalmitis eyes, there was strong immunostaining for SLPI in the retina and vitreous with associated neutrophilic infiltrates. At 48 hours, corneas also stained for SLPI. Western blots confirmed increased SLPI expression in all infected eyes. By immunohistochemical assays, SLPI was absent in the BSS and control eyes. The causative pathogen was identified in all samples from the endophthalmitis group by traditional culture methods. Conclusions. To our knowledge, this report is the first to demonstrate the presence of SLPI in the inflamed cornea, vitreous, and retina tissues of rat eyes with S. aureus endophthalmitis, suggesting that SLPI has an active role in the innate immunity of the eye. Release of SLPI by inflammatory cells in the anterior and posterior segments may contribute to the host defense response against infectious endophthalmitis. PMID:18274645

  20. 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

  1. Prototypical Recombinant Multi-Protease Inhibitor Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type-1.

    PubMed

    Varghese, Vici; Mitsuya, Yumi; Fessel, W Jeffrey; Liu, Tommy F; Melikian, George L; Katzenstein, David A; Schiffer, Celia A; Holmes, Susan P; Shafer, Robert W

    2013-06-24

    The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI-resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI-resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI-resistance amino acid substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activity of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most, if not all clinically relevant PI-resistant viruses. PMID:23796938

  2. A phase I trial of the HIV protease inhibitor nelfinavir in adults with solid tumors

    PubMed Central

    Blumenthal, Gideon M.; Gills, Joell J.; Ballas, Marc S.; Bernstein, Wendy B.; Komiya, Takefumi; Dechowdhury, Roopa; Morrow, Betsy; Root, Hyejeong; Chun, Guinevere; Helsabeck, Cynthia; Steinberg, Seth M.; LoPiccolo, Jaclyn; Kawabata, Shigeru; Gardner, Erin R.; Figg, William D.; Dennis, Phillip A.

    2014-01-01

    Nelfinavir is an HIV protease inhibitor being repurposed as an anti-cancer agent in preclinical models and in small oncology trials, yet the MTD of nelfinavir has not been determined. Therefore, we conducted a Phase Ia study to establish the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of nelfinavir in subjects with advanced solid tumors. Adults with refractory cancers were given oral nelfinavir twice daily with pharmacokinetic and pharmacodynamic analyses. Twenty-eight subjects were enrolled. Nelfinavir was generally well tolerated. Common adverse events included diarrhea, anemia, and lymphopenia, which were mostly mild. The DLT was rapid-onset neutropenia that was reversible. The MTD was established at 3125 mg twice daily. In an expansion cohort at the MTD, one of 11 (9%) evaluable subjects had a confirmed partial response. This, plus two minor responses, occurred in subjects with neuroendocrine tumors of the midgut or pancreatic origin. Thirty-six percent of subjects had stable disease for more than 6 months. In peripheral blood mononuclear cells, Nelfinavir inhibited AKT and induced markers of ER stress. In summary, nelfinavir is well tolerated in cancer patients at doses 2.5 times the FDA-approved dose for HIV management and showed preliminary activity in tumors of neuroendocrine origin. PMID:25327558

  3. Delayed healing and induction of secretory leukocyte protease inhibitor in polycystic ovary syndrome rat skin wounds.

    PubMed

    Jeong, Soon-Jeong; Kim, Sung-Shin; Bae, Chun-Sik; Park, Jin-Ju; Choi, Baik-Dong; Wang, Guanlin; Jung, Myung-Ju; Jang, Hyun-Sun; Kim, Byung-Ock; Lim, Do-Seon; Cho, Young-Sik; Jeong, Moon-Jin

    2012-02-01

    Secretory leukocyte protease inhibitor (SLPI) and estrogen promote wound healing through a decrease in the excessive inflammatory response, accelerating re-epithelialization and increasing the amount of collagen deposition. The excessive administration of estradiol valerate (EV) using hormonal therapy decreases the concentration of estrogen abruptly and induces the polycystic ovary syndrome (PCOS). In this study, the PCOS rat skin wound area was wider than that of the normal groups and the rate of keratinocyte migration in PCOS was lower than the normal group. The numbers of inflammatory cells and macrophages recruited in the PCOS group were larger than that of the normal group. More collagen was deposited in the healing area of the normal group than in the PCOS group. The level of SLPI expression was higher in the PCOS group than the normal group after wounding, with the exception of the epithelium. On the other hand, mRNA and protein expression levels of transforming growth factor-β1 (TGF-β1) were lower in the PCOS group than in the normal group. Matrix metalloproteinase-2 (MMP-2) and MMP-9 levels in the PCOS group were significantly lower than that of the normal group. Therefore, increased SLPI in PCOS skin wounds may help prevent an excessive inflammatory response and aberrant collagen deposition but not are sufficient to accelerate PCOS skin wound healing, suggesting that SLPI may act as a local rather than a systemic modulating molecule in PCOS rat skin wounds. PMID:22020578

  4. Mammary serine protease inhibitor and CD138 immunohistochemical expression in ovarian serous and clear cell carcinomas.

    PubMed

    Hasby, Eiman Adel

    2016-04-01

    This study aims to investigate the immunohistochemical expression of mammary serine protease inhibitor (maspin) and CD138 in primary ovarian high-grade serous carcinomas (HGSC) as compared to low-grade serous carcinomas (LGSC) and clear cell carcinomas and investigate if the studied markers have a correlation to International Federation of Gynaecology and Obstetrics (FIGO) stage, Ki67 proliferation index, and to each other. Maspin cellular location varied significantly between studied groups with only nuclear expression seen in 46.7 % of LGSC group, mixed nuclear and cytoplasmic in 13.3, 28.6, and 20 % of LGSC, HGSC, and clear cell carcinoma, respectively, and was only cytoplasmic in 26.7, 71.4, and 80 % of LGSC, HGSC, and clear cell carcinoma, respectively. Mean maspin and CD138 counts were significantly higher in HGSC and clear cell carcinoma compared to LGSC. Both maspin and CD138 scores varied significantly between studied groups and were positively correlated with adverse prognostic factors in studied carcinomas including FIGO stage and Ki67 proliferation index. Besides, both maspin and CD138 had significant correlation to each other. These findings suggest that epithelial cytoplasmic expression of maspin and CD138 may have a significant role in tumorigenesis in ovarian high-grade serous carcinomas and clear cell carcinomas; these markers may regulate tumor cell proliferation, and their significant correlation to each other may suggest that CD138 probably induces maspin expression to protect tumor growth factors from being lysed by proteolytic enzymes. PMID:26526579

  5. 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.

  6. Identification of a serine protease inhibitor homologue in Bird's Nest by an integrated proteomics approach.

    PubMed

    Ou, K; Seow, T K; Liang, R C; Lee, B W; Goh, D L; Chua, K Y; Chung, M C

    2001-10-01

    For centuries, the edible nests of Collocalia spp. ("Bird's Nests") have been used as a Chinese delicacy that had been claimed to be an effective health-giving tonic. However, clinical studies indicated that in Singapore, Bird's Nest is the most common cause of food-induced anaphylaxis in children, which could lead to potentially life-threatening allergenic reactions. The purpose of this study was to characterize the major allergens in Bird's Nest by using the combined technologies of two-dimensional gel electrophoresis (2-DE), immunochemistry, N-terminal protein sequencing, and mass spectrometry. Results from the immunostaining of the Western blots of the Bird's Nest 2-DE separated proteins with the sera from allergic patients indicated the presence of a major allergen of 66 kDa. Initial searches of the matrix assisted laser desorption/ionization--time of flight--mass spectrometry (MALDI-TOF-MS) tryptic peptide masses of the allergen in the SWISS-PROT and NCBI nonredundant databases revealed that this protein was novel. Based on the partial protein sequence information obtained from N-terminal microsequencing and nanoelectrospray-tandem MS, the 66 kDa immunoreactive allergen was found to be homologous to ovoinhibitor, a Kazal-type serine protease inhibitor, which is one of the dominant allergens found in chicken egg white. PMID:11669547

  7. Exploitation of HIV protease inhibitor Indinavir as a memory restorative agent in experimental dementia.

    PubMed

    Sharma, Bhupesh; Singh, Nirmal; Singh, Manjeet; Jaggi, Amteshwar Singh

    2008-06-01

    The present study was undertaken to investigate the beneficial effect of HIV protease inhibitor Indinavir on memory deficits associated with experimental dementia of Alzheimer disease's (AD) type. Dementia was induced in Swiss albino mice by administration of Celecoxib (100 mg kg(-1) orally, daily for 9 days) or Streptozotocin (3 mg kg(-1) administered intracerebroventricularly on 1st and 3rd day) and the cognitive behaviors of Swiss albino mice were assessed using Morris water maze test. Brain acetyl cholinesterase (AChE) activity was measured by Ell Mann's method. Brain thiobarbituric acid reactive species (TBARS) levels and reduced glutathione (GSH) levels were measured by Ohokawa's and Beutler's method respectively to assess total oxidative stress. Donepezil (0.1 mg kg(-1) i.p.) served as positive control in the present investigation. Celecoxib as well as Streptozotocin (STZ) produced a significant loss of learning and memory. Indinavir (100 and 200 mg kg(-1) orally) successfully attenuated Celecoxib as well as STZ induced cognitive deficits. Higher levels of brain AChE activity, TBARS and lower levels of GSH were observed in Celecoxib as well as STZ treated animals, which were significantly attenuated by Donepezil and Indinavir. Study highlights the potential of Indinavir in memory dysfunctions associated with dementia of AD. PMID:18343489

  8. Prevalence and incidence of diabetes in HIV-infected minority patients on protease inhibitors.

    PubMed Central

    Salehian, Behrouz; Bilas, Josephine; Bazargan, Mohsen; Abbasian, Mohammad

    2005-01-01

    In HIV-infected patients, the use of protease inhibitors (PIs) is associated with a constellation of abdominal obesity; buffalo hump; decreased facial and subcutaneous fat; hyperlipidemia and type-2 diabetes mellitus, a so-called HAART-associated dysmetabolic syndrome. The incidence and prevalence of one of its components, the type-2 diabetes mellitus, among minority population is unknown. In August and September 1999, we reviewed 101 charts of HIV-infected patients who visited an inner-city HIV outpatient clinic. The age, gender, ethnicity, BMI, fasting plasma glucose, random serum glucose, triglycerides, CD4 counts, and the type and duration of antiretroviral drugs were recorded. Three years later (2002), the same patient charts were reviewed for evidence of new-onset diabetes. Ten percent of the subjects were identified as diabetic at baseline. The prevalence of diabetes was 12% among those who were taking PIs, compared to 0% among those who were not taking PIs. The incidence of newly diagnosed diabetes during this three-year period was 7.2%. Diabetes occurred only in the group taking PIs. Diabetic subjects were older than their nondiabetic counterparts. All were African Americans. Our study suggests that PIs increase the likelihood of diabetes developing with increasing age in African Americans infected with HIV. PMID:16173323

  9. 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. PMID:25534233

  10. Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

    PubMed

    Xue, Song; Javor, Sacha; Hixon, Mark S; Janda, Kim D

    2014-11-01

    Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo. PMID:25295706

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Lipopolysaccharide-Related Stimuli Induce Expression of the Secretory Leukocyte Protease Inhibitor, a Macrophage-Derived Lipopolysaccharide Inhibitor

    PubMed Central

    Jin, Fenyu; Nathan, Carl F.; Radzioch, Danuta; Ding, Aihao

    1998-01-01

    Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-κB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417–426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins—interleukin-10 (IL-10) and IL-6—also induced SLPI, while TNF and IL-1β did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules—taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls—also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI’s slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria. PMID:9596701

  16. 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

  17. 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. PMID:26916437

  18. A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

    PubMed

    Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

    1996-09-01

    The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues. PMID:8894111

  19. 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

  20. HCV-1b intra-subtype variability: Impact on genetic barrier to protease inhibitors.

    PubMed

    Ferraro, Donatella; Urone, Noemi; Di Marco, Vito; Craxì, Antonio

    2014-04-01

    Due to error-prone RNA polymerase and the lack of proofreading mechanisms, to the spread worldwide and probable long-term presence in human population, HCV showed a high degree of inter- and intra-subtype genetic variability. Protease inhibitors (PIs), a new class of drugs, have been designed specifically on the HCV genotype 1 NS3 protease three-dimensional structure. The viral genetic barrier limits the efficacy of PIs, and fourteen loci in the HCV NS3 gene are involved in resistance to PIs. A sensitive method (15UI/ml) for study the HCV genetic profile of 125 strains from patients naïve to PIs, was developed through the use of new degenerate primers for subtype 1b. We observed the presence of naturally resistance-associated variants in 14% of the HCV strains (V36L, F43S, T54S, I153V, R155Q, D168A/G). T54S was the most common mutation (4%) detected. We investigated, through minimal score (m.s.) calculating, how the HCV intra-subtype 1b variability modifies the genetic barrier to PIs. For >60% of strains a single transition (m.s. of 1) was required for selection of low to medium resistance mutations, while more than one transition/transversion (m.s. ⩾2.5) or one transition plus one transversion (m.s. ⩾3.5) was necessary for most of the high level PI-resistant-associated mutations, except for A156V, for which a single transition was sufficient (m.s. of 1). However, the presence at locus 36 of the amino acid polymorphism S36 in one case and the wild type V36 in 6 isolates, encoded by unusual GTA or GTG codons, might determined a higher probability of V36L/M mutations because of the reduction of the genetic barrier. Instead, the presence of the CGA and CGT codons in the 155(th) position increases the genetic barrier for R155M or R155Q/M. The large intra-subtype variability, suggests that a routine baseline resistance test must be used before PIs-treatment. PMID:24508244

  1. HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants.

    PubMed

    Altman, Michael D; Ali, Akbar; Reddy, G S Kiran Kumar; Nalam, Madhavi N L; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X; Gilson, Michael K; Schiffer, Celia A; Rana, Tariq M; Tidor, Bruce

    2008-05-14

    The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance. PMID:18412349

  2. HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibit Subnanomolar Binding to Drug-Resistant Variants

    PubMed Central

    Altman, Michael D.; Ali, Akbar; Reddy, G. S. Kiran Kumar; Nalam, Madhavi N. L.; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X.; Gilson, Michael K.; Schiffer, Celia A.; Rana, Tariq M.; Tidor, Bruce

    2010-01-01

    The acquisition of drug-resistance mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from Ki of 30–50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6–13 fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors — robust binders (maximum affinity loss of 14–16 fold), moderate binders (35–80 fold), and susceptible binders (greater than 100 fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance. PMID:18412349

  3. 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

  4. 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. PMID:12535099

  5. 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.

  6. The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

    PubMed

    Fong, Clifford W

    2016-08-01

    Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. PMID:27317642

  7. 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

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

    PubMed

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

    2001-05-01

    A recombinant human serine protease inhibitor known as Kunitz protease inhibitor (KPI) wild type has functional similarities to the bovine Kunitz inhibitor, aprotinin, and had shown a potential to reduce bleeding in an ovine model of cardiopulmonary bypass (CPB). The aim of this study was to assess KPI-185, a modification of KPI-wild type that differs from KPI-wild type in two amino acid residues and which enhances anti-kallikrein activity in a further double-blind, randomized study in an ovine model of CPB, and to compare with our previous study of KPI-wild type and aprotinin in the same ovine model. Post-operative drain losses and subjective assessment of wound 'dryness' showed no significant differences between KPI-185 and KPI-wild type, despite the significant enhancement of kallikrein inhibition using KPI-185 seen in serial kallikrein inhibition assays. These preliminary findings support the hypothesis that kallikrein inhibition is not the major mechanism by which Kunitz inhibitors such as aprotinin reduce perioperative bleeding. PMID:11419655

  9. 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.

  10. 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. PMID:27075731

  11. Novel role of the serine protease inhibitor elafin in gluten-related disorders

    PubMed Central

    Galipeau, Heather J.; Wiepjes, Michelle; Motta, Jean-Paul; Schulz, Jessica D.; Jury, Jennifer; Natividad, Jane M.; Pinto-Sanchez, Ines; Sinclair, Daniel; Rousset, Perrine; Martin-Rosique, Rebeca; Bermudez-Humaran, Luis; Leroux, Jean Christophe; Murray, Joseph; Smecuol, Edgardo; Bai, Julio C.; Vergnolle, Nathalie; Langella, Philippe; Verdu, Elena F

    2014-01-01

    Objectives Elafin, an endogenous serine protease inhibitor, modulates colonic inflammation. We investigated the role of elafin in celiac disease (CD) using human small intestinal tissues and in vitro assays of gliadin deamidation. We also investigated potential beneficial effects of elafin in a mouse model of gluten sensitivity. Methods Epithelial elafin expression in the small intestine of patients with active CD, treated CD and controls without CD was determined by immunofluorescence. Interaction of elafin with human tissue transglutaminase-2 (TG-2) was investigated in vitro. The 33-mer peptide, a highly immunogenic gliadin peptide, was incubated with TG-2 and elafin at different concentrations. The degree of deamidation of the 33-mer peptide was analyzed by liquid chromatography-mass spectrometry. Elafin was delivered to the intestine of gluten-sensitive mice using a recombinant Lactococcus lactis vector. Small intestinal barrier function, inflammation, proteolytic activity, and zonula occludens-1 (ZO-1) expression were assessed. Results Elafin expression in the small intestinal epithelium was lower in patients with active CD compared to control patients. In vitro, elafin significantly slowed the kinetics of the deamidation of the 33-mer peptide to its more immunogenic form. Treatment of gluten-sensitive mice with elafin delivered by the L. lactis vector normalized inflammation, improved permeability and maintained ZO-1 expression. Conclusions The decreased elafin expression in small intestine of patients with active CD, the reduction of 33-mer peptide deamidation by elafin, coupled to the barrier enhancing and anti-inflammatory effects observed in gluten sensitive mice, suggest this molecule may have pathophysiological and therapeutic importance in gluten-related disorders. PMID:24710505

  12. Secretory leukocyte protease inhibitor plays an important role in the regulation of allergic asthma in mice.

    PubMed

    Marino, Rafael; Thuraisingam, Thusanth; Camateros, Pierre; Kanagaratham, Cynthia; Xu, Yong Zhong; Henri, Jennifer; Yang, Jingxuan; He, Guoan; Ding, Aihao; Radzioch, Danuta

    2011-04-01

    Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice. PMID:21335488

  13. Antiviral properties of aminodiol inhibitors against human immunodeficiency virus and protease.

    PubMed Central

    Bechtold, C M; Patick, A K; Alam, M; Greytok, J; Tino, J A; Chen, P; Gordon, E; Ahmad, S; Barrish, J C; Zahler, R

    1995-01-01

    A series of aminodiol inhibitors of human immunodeficiency virus type 1 (HIV-1) protease were identified by using an in vitro peptide cleavage assay. BMS 182,193, BMS 186,318, and BMS 187,071 protected cells against HIV-1, HIV-2, and simian immunodeficiency virus infections, with 50% effective doses ranging from 0.05 to 0.33 microM, while having no inhibitory effect on cells infected with unrelated viruses. These compounds were also effective in inhibiting p24 production in peripheral blood mononuclear cells infected with HIV-1 IIIB and against the zidovudine-resistant HIV-1 strain A018C. Time-of-addition studies indicated that BMS 182,193 could be added as late as 27 h after infection and still retain its antiviral activity. To directly show that the activity of these compounds in culture was due to inhibition of proteolytic cleavage, the levels of HIV-1 gag processing in chronically infected cells were monitored by Western blot (immunoblot) analysis. All compounds blocked the processing of p55 in a dose-dependent manner, with 50% effective doses of 0.4 to 2.4 microM. To examine the reversibility of BMS 186,318, chronically infected CEM-SS cells were treated with drug and virions purified from the culture medium. Incubation of HIV-1 particles in drug-free medium indicated that inhibition of p55 proteolysis was slowly reversible. The potent inhibition of HIV-1 during both acute and chronic infections indicates that these aminodiol compounds are effective anti-HIV-1 compounds. PMID:7726501

  14. [The effects of protease inhibitor upon the ischemia-reperfusion injury].

    PubMed

    Shibata, T; Yamamoto, F; Ohashi, T; Shimada, Y; Nakajima, N; Kinoshita, H; Kawashima, Y

    1992-10-01

    The purpose of the study is to investigate the effects of protease inhibitor (Nafamostat mesilate: NM) upon myocardial protection. Hearts were subjected to 20 min working control perfusion followed by 3 min cardioplegic infusion with the St. Thomas Cardioplegic Solution (ST) contained various concentrations of NM, and global ischemia for 33 min at 37 degrees C (Exp. 1) or 150 min at 20 degrees C (Exp. 2). Hearts were then converted to Langendorff reperfusion (the leakage of Creatine Kinase (CK) and Cathepsin B (Cat-B) ware measured) and 20 min working reperfusion. Various concentrations of NM added during Langendorff reperfusion (Exp. 3). During working perfusion cardiac functions (aortic flow (AoF), coronary flow (CoF), heart rate (HR), aortic pressure (AoP)) were measured, and expressed as the percent recovery of pre-ischemic control value. Post-ischemic recovery of AoF (%AoF) showed the bell-shaped dose-response curve, and the optimal dose was 3 microM (Exp. 1) and 10 microM (Exp. 2) respectively. There was a significant (p < 0.05) increase of %AoF in optimal dose compared with that in controls (64.2 +/- 1.2% vs 52.3 +/- 2.5% in Exp. 1, 68.9 +/- 3.1% vs 54.1 +/- 1.4% in Exp. 2). These increase of functional recovery reflected in the values for CK and Cat-B leakage. The addition of NM in ST reduced CK and Cat-B leakage significantly in the concentration of 5 microM (in Exp. 1) and 10 microM (in Exp. 2) respectively. But the addition of NM in reperfusate did not reduced CK leakage significantly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1431402

  15. Human papillomavirus infection in head and neck cancer: The role of the secretory leukocyte protease inhibitor

    PubMed Central

    HOFFMANN, MARKUS; QUABIUS, ELGAR S.; TRIBIUS, SILKE; HEBEBRAND, LENA; GÖRÖGH, TIBOR; HALEC, GORDANA; KAHN, TOMAS; HEDDERICH, JÜRGEN; RÖCKEN, CHRISTOPH; HAAG, JOCHEN; WATERBOER, TIM; SCHMITT, MARKUS; GIULIANO, ANNA R.; KAST, W. MARTIN

    2013-01-01

    We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non-malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases. PMID:23467841

  16. 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

  17. Serpin-serine protease binding kinetics: alpha 2-antiplasmin as a model inhibitor.

    PubMed

    Longstaff, C; Gaffney, P J

    1991-01-29

    We have examined in detail the kinetics of binding of the serpin alpha 2-antiplasmin to the serine proteases alpha-chymotrypsin and plasmin. These represent model systems for serpin binding. We find, in contrast to earlier published results with alpha 2-antiplasmin and plasmin, that binding is reversible, and slow binding kinetics can be observed, under appropriate conditions. Binding follows a two-step process with both enzymes, with the formation of an initial loose complex which then proceeds to a tightly bound complex. In the absence of lysine and analogues, equilibrium between alpha 2-antiplasmin and plasmin is achieved rapidly, with an overall inhibition constant (Ki') of 0.3 pM. In the presence of tranexamic acid or 6-aminohexanoic acid, lysine analogues that mimic the effects of fibrin, plasmin binding kinetics are changed such that equilibrium is reached slowly following a lag phase after mixing of enzyme and inhibitor. The Ki' is also affected, rising to 2 pM in the presence of 6-aminohexanoic acid concentrations above 15 mM. Thus extrapolation to the in vivo situation indicates that complex formation in the presence of fibrin will be delayed, allowing a burst of enzyme activity following plasmin generation, but a tight, pseudoirreversible complex will result eventually. Chymotrypsin is more weakly inhibited by alpha 2-antiplasmin, exhibiting an overall Ki' of 0.1 nM, after two-stage complex formation. The inhibition constant for the initial loose complex (Ki) is very similar for both enzymes. The difference in binding strength between the two enzymes is accounted for by the dissociation rate constant of the second step of complex formation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1703440

  18. HIV Lipodystrophy in Participants Randomised to Lopinavir/Ritonavir (LPV/r) +2–3 Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (N(t)RTI) or LPV/r + Raltegravir as Second-Line Antiretroviral Therapy

    PubMed Central

    Martin, Allison; Moore, Cecilia L.; Mallon, Patrick W. G.; Hoy, Jennifer F.; Emery, Sean; Belloso, Waldo H.; Phanuphak, Praphan; Ferret, Samuel; Cooper, David A.; Boyd, Mark A.

    2013-01-01

    Objective To compare changes over 48 weeks in body fat, lipids, Metabolic Syndrome and cardiovascular disease risk between patients randomised 1∶1 to lopinavir/ritonavir (r/LPV) plus raltegravir (RAL) compared to r/LPV plus 2–3 nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs) as second-line therapy. Methods Participants were HIV-1 positive (>16 years) failing first-line treatment (2 consecutive HIV RNA >500 copies/mL) of NNRTI +2N(t)RTI. Whole body dual energy x-ray absorptiometry was performed at baseline and week 48. Data were obtained to calculate the Metabolic Syndrome and Framingham cardiovascular disease (CVD) risk score. Linear regression was used to compare mean differences between arms. Logistic regression compared incidence of metabolic syndrome. Associations between percent limb fat changes at 48 weeks with baseline variables were assessed by backward stepwise multivariate linear regression. Analyses were adjusted for gender, body mass index and smoking status. Results 210 participants were randomised. The mean (95% CI) increase in limb fat over 48 weeks was 15.7% (5.3, 25.9) or 0.9 kg (0.2, 1.5) in the r/LPV+N(t)RTI arm and 21.1% (11.1, 31,1) or 1.3 kg (0.7, 1.9) in the r/LPV+RAL arm, with no significant difference between treatment arms (−5.4% [−0.4 kg], p>0.1). Increases in total body fat mass (kg) and trunk fat mass (kg) were also similar between groups. Total:HDL cholesterol ratio was significantly higher in the RAL arm (mean difference −0.4 (1.4); p = 0.03), there were no other differences in lipid parameters between treatment arms. There were no statistically significant differences in CVD risk or incidence of Metabolic Syndrome between the two treatment arms. The baseline predictors of increased limb fat were high viral load, high insulin and participant's not taking lipid lowering treatment. Conclusion In patients switching to second line therapy, r/LPV combined with RAL demonstrated similar improvements in limb

  19. Crystallization and preliminary crystallographic study of Feline infectious peritonitis virus main protease in complex with an inhibitor.

    PubMed

    Wang, Jinshan; Wang, Fenghua; Tan, Yusheng; Chen, Xia; Zhao, Qi; Fu, Sheng; Li, Shuang; Chen, Cheng; Yang, Haitao

    2014-12-01

    Feline infectious peritonitis virus (FIPV) causes a lethal systemic granulomatous disease in wild and domestic cats around the world. Currently, no effective vaccines or drugs have been developed against it. As a member of the genus Alphacoronavirus, FIPV encodes two polyprotein precursors required for genome replication and transcription. Each polyprotein undergoes extensive proteolytic processing, resulting in functional subunits. This process is mainly mediated by its genome-encoded main protease, which is an attractive target for antiviral drug design. In this study, the main protease of FIPV in complex with a Michael acceptor-type inhibitor was crystallized. The complex crystals diffracted to 2.5 Å resolution and belonged to space group I422, with unit-cell parameters a = 112.3, b = 112.3, c = 102.1 Å. There is one molecule per asymmetric unit. PMID:25484209

  20. Serine protease inhibitor Kazal type 1 (SPINK1) drives proliferation and anoikis resistance in a subset of ovarian cancers

    PubMed Central

    Mehner, Christine; Oberg, Ann L.; Kalli, Kimberly R.; Nassar, Aziza; Hockla, Alexandra; Pendlebury, Devon; Cichon, Magdalena A.; Goergen, Krista M.; Maurer, Matthew J.; Goode, Ellen L.; Keeney, Gary L.; Jatoi, Aminah; Sahin-Tóth, Miklós; Copland, John A.; Radisky, Derek C.; Radisky, Evette S.

    2015-01-01

    Ovarian cancer represents the most lethal tumor type among malignancies of the female reproductive system. Overall survival rates remain low. In this study, we identify the serine protease inhibitor Kazal type 1 (SPINK1) as a potential therapeutic target for a subset of ovarian cancers. We show that SPINK1 drives ovarian cancer cell proliferation through activation of epidermal growth factor receptor (EGFR) signaling, and that SPINK1 promotes resistance to anoikis through a distinct mechanism involving protease inhibition. In analyses of ovarian tumor specimens from a Mayo Clinic cohort of 490 patients, we further find that SPINK1 immunostaining represents an independent prognostic factor for poor survival, with the strongest association in patients with nonserous histological tumor subtypes (endometrioid, clear cell, and mucinous). This study provides novel insight into the fundamental processes underlying ovarian cancer progression, and also suggests new avenues for development of molecularly targeted therapies. PMID:26437224

  1. 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

  2. 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, ...

  3. 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

  4. 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

  5. Effect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiation

    PubMed Central

    Caso, Giuseppe; Mileva, Izolda; Mcnurlan, Margaret A; Mynarcik, Dennis C; Darras, Frank; Gelato, Marie C

    2010-01-01

    Protease inhibitors (PIs) have been implicated in the development of HIV-associated lipodystrophy through a reduction in the differentiation of preadipocytes. While atazanavir (ATV) is associated with fewer clinical metabolic abnormalities in the short-term, the effects of long-term exposure are not known. ATV effects on preadipocyte replication or differentiation would indicate the potential for long-term problems. This study compared ritonavir (RTV) and ATV effects on preadipocyte replication and differentiation in human primary cultures. Preadipocytes from subcutaneous fat were studied in the presence of therapeutic concentrations of RTV and ATV for replication, differentiation, and adipokine secretion. The effects of the drugs on the expression of PPARγ and related genes during differentiation were also assessed by real time quantitative PCR. RTV induced a significant inhibition of preadipocyte proliferation, differentiation and adiponectin secretion. ATV at concentrations within the range of therapeutic levels did not affect differentiation or adiponectin secretion, but did have inhibitory effects on preadipocyte proliferation. Inhibition of differentiation by PIs was associated with a decrease expression of PPARγ, C/EBPα, and aP2 genes. In summary, although ATV at therapeutic levels has a smaller impact on adipogenesis, alterations in preadipocyte proliferation suggest the potential for adverse effects with long-term use. PMID:20153378

  6. Effect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiation.

    PubMed

    Caso, Giuseppe; Mileva, Izolda; McNurlan, Margaret A; Mynarcik, Dennis C; Darras, Frank; Gelato, Marie C

    2010-05-01

    Protease inhibitors (PIs) have been implicated in the development of HIV-associated lipodystrophy through a reduction in the differentiation of preadipocytes. While atazanavir (ATV) is associated with fewer clinical metabolic abnormalities in the short-term, the effects of long-term exposure are not known. ATV effects on preadipocyte replication or differentiation would indicate the potential for long-term problems. This study compared ritonavir (RTV) and ATV effects on preadipocyte replication and differentiation in human primary cultures. Preadipocytes from subcutaneous fat were studied in the presence of therapeutic concentrations of RTV and ATV for replication, differentiation, and adipokine secretion. The effects of the drugs on the expression of PPARgamma and related genes during differentiation were also assessed by real-time quantitative PCR. RTV induced a significant inhibition of preadipocyte proliferation, differentiation and adiponectin secretion. ATV at concentrations within the range of therapeutic levels did not affect differentiation or adiponectin secretion, but did have inhibitory effects on preadipocyte proliferation. Inhibition of differentiation by PIs was associated with decreased expression of PPARgamma, C/EBPalpha, and aP2 genes. In summary, although ATV at therapeutic levels has a smaller impact on adipogenesis, alterations in preadipocyte proliferation suggest the potential for adverse effects with long-term use. PMID:20153378

  7. Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034.

    PubMed

    Madison, Vincent; Prongay, Andrew J; Guo, Zhuyan; Yao, Nanhua; Pichardo, John; Fischmann, Thierry; Strickland, Corey; Myers, Joseph; Weber, Patricia C; Beyer, Brian M; Ingram, Richard; Hong, Zhi; Prosise, Winifred W; Ramanathan, Lata; Taremi, S Shane; Yarosh-Tomaine, Taisa; Zhang, Rumin; Senior, Mary; Yang, Rong Sheng; Malcolm, Bruce; Arasappan, Ashok; Bennett, Frank; Bogen, Stephane L; Chen, Kevin; Jao, Edwin; Liu, Yi Tsung; Lovey, Raymond G; Saksena, Anil K; Venkatraman, Srikanth; Girijavallabhan, Viyyoor; Njoroge, F George

    2008-05-01

    The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials. PMID:18421139

  8. Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034

    PubMed Central

    Madison, Vincent; Prongay, Andrew J.; Guo, Zhuyan; Yao, Nanhua; Pichardo, John; Fischmann, Thierry; Strickland, Corey; Myers Jr, Joseph; Weber, Patricia C.; Beyer, Brian M.; Ingram, Richard; Hong, Zhi; Prosise, Winifred W.; Ramanathan, Lata; Taremi, S. Shane; Yarosh-Tomaine, Taisa; Zhang, Rumin; Senior, Mary; Yang, Rong-Sheng; Malcolm, Bruce; Arasappan, Ashok; Bennett, Frank; Bogen, Stephane L.; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond G.; Saksena, Anil K.; Venkatraman, Srikanth; Girijavallabhan, Viyyoor; Njoroge, F. George

    2008-01-01

    The structures of both native and S139A holo-HCV NS3/4A protease domain were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contributions to the binding energy arise from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease, which is currently in clinical trials. PMID:18421139

  9. 3-Aminopiperidine-Based Peptide Analogues as the First Selective Noncovalent Inhibitors of the Bacterial Cysteine Protease IdeS

    PubMed Central

    2012-01-01

    A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies. PMID:22369147

  10. Solvent accessibility as a predictive tool for the free energy of inhibitor binding to the HIV-1 protease.

    PubMed Central

    Nauchitel, V.; Villaverde, M. C.; Sussman, F.

    1995-01-01

    We have developed a simple approach for the evaluation of the free energies of inhibitor binding to the protease of the human immunodeficiency virus (HIV-1 PR). Our algorithm is based on the observation that most groups that line the binding pockets of this enzyme are hydrophobic in nature. Based on this fact, we have likened the binding of an inhibitor to this enzyme to its transfer from water to a medium of lower polarity. The resulting expression produced values for the free energy of binding of inhibitors to the HIV-1 PR that are in good agreement with experimental values. The additive nature of this approach has enabled us to partition the free energy of binding into the contributions of single fragments. The resulting analysis clearly indicates the existence of a ranking in the participation of the enzyme's subsites in binding. Although all the enzyme's pockets contribute to binding, the ones that bind the P2-P'2 span of the inhibitor are in general the most critical for high inhibitor potency. Moreover, our method has allowed us to determine the nature of the functional groups that fit into given enzyme binding pockets. Perusal of the energy contributions of single side chains has shown that a large number of hydrophobic and aromatic groups located in the central portion of the HIV-1 PR inhibitors present optimal binding. All of these observations are in agreement with experimental evidence, providing a validation for the physical relevancy of our model. PMID:7670378

  11. 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

  12. Nitrogen-15 NMR spectroscopy of the catalytic-triad histidine of a serine protease in peptide boronic acid inhibitor complexes

    SciTech Connect

    Bachovchin, W.W.; Wong, W.Y.L.; Farr-Jones, S. ); Shenvi, A.B.; Kettner, C.A. )

    1988-10-04

    {sup 15}N NMR spectroscopy was used to examine the active-site histidyl residue of {alpha}-lytic protease in peptide boronic acid inhibitor complexes. Two distinct types of complexes were observed: (1) Boronic acids that are analogues of substrates form complexes in which the active-site imidazole ring is protonated and both imidazole N-H protons are strongly hydrogen bonded. (2) Boronic acids that are not substrate analogues form complexes in which N{sup {epsilon}2} of the active-site histidine is covalently bonded to the boron atom of the inhibitor. The proton bound to N{sup {delta}1} of the histidine in these histidine-boronate adducts remains strongly hydrogen bonded, presumably to the active-site aspartate. In both types of complexes the N-H protons of His-57 exchange unusually slowly as evidenced by the room temperature visibility of the low-field {sup 1}H resonances and the {sup 15}N-H spin couplings. These results indicate that occupancy of the specificity subsites may be required to fully form the transition-state binding site. The significance of these findings for understanding inhibitor binding and the catalytic mechanism of serine proteases is discussed.

  13. Naturally occurring dominant resistance mutations to HCV protease and polymerase inhibitors in treatment-naïve patients

    PubMed Central

    Kuntzen, Thomas; Timm, Joerg; Berical, Andrew; Lennon, Niall; Berlin, Aaron M.; Young, Sarah K.; Lee, Bongshin; Heckerman, David; Carlson, Jonathan; Reyor, Laura L.; Kleyman, Marianna; McMahon, Cory M.; Birch, Christopher; Wiesch, Julian Schulze zur; Ledlie, Timothy; Koehrsen, Michael; Kodira, Chinnappa; Roberts, Andrew D.; Lauer, Georg M.; Rosen, Hugo R.; Bihl, Florian; Cerny, Andreas; Spengler, Ulrich; Liu, Zhimin; Kim, Arthur Y.; Xing, Yanming; Schneidewind, Arne; Madey, Margaret A.; Fleckenstein, Jaquelyn F.; Park, Vicki M.; Galagan, James E.; Nusbaum, Chad; Walker, Bruce D.; Lake-Bakaar, Gerond V.; Daar, Eric S.; Jacobson, Ira M.; Gomperts, Edward D.; Edlin, Brian R.; Donfield, Sharyne M.; Chung, Raymond T.; Talal, Andrew H.; Marion, Tony; Birren, Bruce W.; Henn, Matthew R.; Allen, Todd M.

    2008-01-01

    Resistance mutations to HCV NS3 protease inhibitors in <1% of the viral quasispecies may still allow >1000-fold viral load reductions upon treatment, consistent with their reported reduced replicative fitness in vitro. Recently, however, an R155K protease mutation was reported as the dominant quasispecies in a treatment-naïve individual, raising concerns about possible full drug resistance. To investigate the prevalence of dominant STAT-C resistance mutations in the population we analyzed HCV genome sequences from 507 treatment-naïve HCV genotype 1 infected patients from the US, Germany and Switzerland. Phylogenetic sequence analysis and viral load data were used to identify the possible spread of replication competent, drug resistant viral strains in the population and to infer the consequences of these mutations upon viral replication in vivo. Mutations described to confer resistance to the protease inhibitors Telaprevir, BILN2061, ITMN-191, SCH6 and Boceprevir, the NS5B polymerase inhibitor AG-021541, and to the NS4A antagonist ACH-806 were observed mostly as sporadic, unrelated cases, at frequencies between 0.3% and 2.8% in the population, including two patients with possible multi-drug resistance. Collectively, however, 8.6% of the genotype 1a and 1.4% of the genotype 1b infected patients carried at least one dominant resistance mutation. Viral loads were high in the majority of these patients, suggesting that drug resistant viral strains might achieve replication levels comparable to non-resistant viruses in vivo. Conclusion: Naturally occurring dominant STAT-C resistance mutations are common in HCV genotype 1 infected treatment-naïve patients. Their influence on treatment outcome should further be characterized to evaluate possible benefits of drug resistance testing for individual tailoring of drug combinations when treatment options are limited due to previous non-response to peginterferon and ribavirin. PMID:19026009

  14. A Reduction Grade of Lipodystrophy and Limited Side Effects after HAART Regimen with Raltegravir, Lamivudine, Darunavir and Ritonavir in an HIV-1 Infected Patient after Six Years of Antiretroviral Therapy

    PubMed Central

    Antoni, A Degli; Weimer, LE; Fragola, V; Giacometti, A; Sozio, F

    2015-01-01

    ABSTRACT HIV-associated lipodystrophy commonly presents with fat loss in the face, buttocks, arms and legs, hypocomplementaemia, glomerulonephritis and autoimmune disorders. The exact mechanism of HIV-associated lipodystrophy is not fully elucidated. There is evidence indicating that it can be caused by both antiretroviral medications and HIV infection in the absence of antiretroviral medication. Lipodystrophy seems to be mainly due to HIV-1 protease inhibitors. Interference with lipid metabolism is postulated as pathophysiology. Also, the development of lipodystrophy is associated with specific nucleoside reverse transcriptase inhibitors (NRTI). Mitochondrial toxicity is postulated to be involved in the pathogenesis associated with NRTI. Here, we analyse the side effects and examine the impact of the highly active antiretroviral therapy (HAART) regimen including raltegravir, lamivudine, darunavir and ritonavir in an HIV-1 infected patient with severe lipodystrophy after six years of antiretroviral therapy. PMID:26426188

  15. Alcoholic Extract of Lotus Leaves Improves Lipid Profile in Rats with HIV Protease Inhibitor-induced Dyslipidaemia

    PubMed Central

    Su, QJ; Lu, ZZ; Deng, QY; Wei, BM

    2015-01-01

    ABSTRACT Objective: To examine the effect of the alcoholic extract of lotus leaves (AELL) on antiretroviral treatment-induced dyslipidaemia in a rat model. Methods: Lotus leaves were extracted by 95% ethanol. Seventy male Sprague-Dawley rats were given lopinavir/ritonavir for six weeks. At weeks 0 and 6, sera were collected for measurement of total cholesterol (TC) and triglyceride (TG). Rats meeting the criteria for dyslipidaemia were assigned to four groups and received once daily for another four weeks lopinavir/ritonavir (group A), lopinavir/ritonavir plus 0.52 g/kg AELL (group B), lopinavir/ritonavir plus 0.26 g/kg AELL (group C), or lopinavir/ritonavir plus 0.13 g/kg AELL (group D), respectively. At weeks 8 and 10, blood samples were collected again for measurement of TC and TG. Results: Both TC and TG increased over time in group A during the observation period (weeks 6 to 10), however, TC and TG decreased in group B, and TG declined in group C. Neither TC nor TG could be reduced to a level near baseline. Conclusion: Alcoholic extract of lotus leaves may have the potential to treat dyslipidaemia related to highly active antiretroviral treatment, but may not be potent enough to reduce TC or TG concentrations to goal levels when used alone. PMID:26426169

  16. Probing Lipophilic Adamantyl Group as the P1-Ligand for HIV-1 Protease Inhibitors: Design, Synthesis, Protein X-ray Structural Studies, and Biological Evaluation.

    PubMed

    Ghosh, Arun K; Osswald, Heather L; Glauninger, Kristof; Agniswamy, Johnson; Wang, Yuan-Fang; Hayashi, Hironori; Aoki, Manabu; Weber, Irene T; Mitsuya, Hiroaki

    2016-07-28

    A series of potent HIV-1 protease inhibitors with a lipophilic adamantyl P1 ligand have been designed, synthesized, and evaluated. We have developed an enantioselective synthesis of adamantane-derived hydroxyethylamine isosteres utilizing Sharpless asymmetric epoxidation as the key step. Various inhibitors incorporating P1-adamantylmethyl in combination with P2 ligands such as 3-(R)-THF, 3-(S)-THF, bis-THF, and THF-THP were examined. The S1' pocket was also probed with phenyl and phenylmethyl ligands. Inhibitor 15d, with an isobutyl P1' ligand and a bis-THF P2 ligand, proved to be the most potent of the series. The cLogP value of inhibitor 15d is improved compared to inhibitor 2 with a phenylmethyl P1-ligand. X-ray structural studies of 15d, 15h, and 15i with HIV-1 protease complexes revealed molecular insight into the inhibitor-protein interaction. PMID:27389367

  17. 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 inhib