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1.
Chemistry ; 26(1): 49-88, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31483909

RESUMO

Drugs in the chemical space beyond the rule of 5 (bRo5) can modulate targets with difficult binding sites while retaining cell permeability and oral absorption. Reviewing the syntheses of bRo5 drugs approved since 1990 highlights synthetic chemistry's contribution to drug discovery in this space. Initially, bRo5 drugs were mainly natural products and semi-synthetic derivatives. Later, peptidomimetics and de novo designed compounds, that include up to seven chiral centres and macrocyclic rings became dominant. These drugs are prepared by total synthesis, sometimes by routes of more than 25 steps with stereocentres originating from the chiral pool, or being installed by chiral induction or enzymatic resolution. Interestingly, ring-closing metathesis proved to be the method of choice for macrocyclisation in hepatitis C virus protease inhibitors. We conclude that structural simplification, planning of synthetic routes regarding incorporation of stereocentres and macrocyclisation, as well as incorporation of structural knowledge and consideration of chameleonic properties in design, should facilitate drug discovery in bRo5 space.


Assuntos
Descoberta de Drogas , Preparações Farmacêuticas/síntese química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Hepacivirus/enzimologia , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Peptidomiméticos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
2.
Comput Biol Chem ; 84: 107167, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855781

RESUMO

BACKGROUND: Hepatitis C Virus (HCV) infection is a major public health concern across the globe. At present, direct-acting antivirals are the treatment of choice. However, the long-term effect of this therapy has yet to be ascertained. Previously, fluoroquinolones have been reported to inhibit HCV replication by targeting NS3 protein. Therefore, it is logical to hypothesize that the natural analogs of fluoroquinolones will exhibit NS3 inhibitory activity with substantially lesser side effects. METHOD: In this study, we tested the application of a recently devised integrated in-silico Cheminformatics-Molecular Docking approach to identify physicochemically similar natural analogs of fluoroquinolones from the available databases (Ambinter, Analyticon, Indofines, Specs, and TimTec). Molecular docking and ROC curve analyses were performed, using PatchDock and Graphpad software, respectively, to compare and analyze drug-protein interactions between active natural analogs, Fluoroquinolones, and HCV NS3 protein. RESULT: In our analysis, we were able to shortlist 18 active natural analogs, out of 10,399, that shared physicochemical properties with the template drugs (fluoroquinolones). These analogs showed comparable binding efficacy with fluoroquinolones in targeting 32 amino acids in the HCV NS3 active site that are crucial for NS3 activity. Our approach had around 80 % sensitivity and 70 % specificity in identifying physicochemically similar analogs of fluoroquinolones. CONCLUSION: Our current data suggest that our approach can be efficiently applied to identify putative HCV drug inhibitors that can be taken for in vitro testing. This approach can be applied to discover physicochemically similar analogs of virtually any drug, thus providing a speedy and inexpensive approach to complement drug discovery and design, which can tremendously economize on time and money spent on the screening of putative drugs.


Assuntos
Antivirais/metabolismo , Descoberta de Drogas/métodos , Inibidores Enzimáticos/metabolismo , Fluoroquinolonas/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Domínio Catalítico , Quimioinformática , Inibidores Enzimáticos/química , Fluoroquinolonas/química , Hepacivirus/enzimologia , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
3.
Asian Pac J Cancer Prev ; 20(8): 2311-2317, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450900

RESUMO

Background: Even with the fantastic successes of direct-acting antivirals (DAA) in the treatment of Hepatitis C Virus (HCV) infection, natural drug resistance remains a challenging obstacle for their impacts. The data regarding protease inhibitors (PIs) resistance in Iran population are limited. The aim of this study was to investigate the variations in NS3 protease of HCV from non-responder patients. Methods: In this cross-sectional study, 14 HCV infected patients with genotype 1(N=5) and 3(N=9) who have not responded to Interferon-related regime were enrolled from Liver Clinic, Shiraz. The NS3 protease region was amplified by Nested-PCR followed by product gel extraction. Besides, some amplified protease regions were cloned into a cloning vector to improve the sensitivity of mutation detection. Both crude and cloned sequences were then introduced into sequencing. The obtained sequences were compared with the NS3 reference sequences and analyzed by Geno2pheno available software to find possible substitutions. In the end, the phylogenetic tree was constructed. Results: Among variations responsible for PIs resistance, only one out of 14 (7%) sample who was infected with genotype 1a, harbored R117C+N174S double mutation, which causes reduced susceptibility to Telaprevir. Any another resistance mutation was not found among the studied population. The most frequent substitutions were determined as I52M(N=9), S102A(N=9), S166A(8) and V170I(8) for genotype 3a, and F147S/A(4) for genotype 1. However, some uncharacterized substitutions on scored position, including I132L(N=1), I170V(N=3) and N174S(N=2) were also determined among sequences. Phylogenetic analysis demonstrated that the protease region has enough power to correctly classify enrolled samples into relevant clusters on the tree. There were 2, 3 and 9 cases of sub-genotypes 1a, 1b, and 3a, respectively. Conclusion: A low frequency of PIs resistance mutations in our HCV infected population is a hopeful point of starting these drugs in HCV infected patients.


Assuntos
Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Mutação , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Adulto , Idoso , Substituição de Aminoácidos , Antivirais/farmacologia , Estudos Transversais , Feminino , Seguimentos , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia
4.
Indian J Pathol Microbiol ; 62(3): 391-398, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31361226

RESUMO

Background: Hepatitis C virus (HCV) represents a serious worldwide healthcare problem. No protective vaccines against HCV have been developed yet due to the fact that HCV is rapidly mutable, allowing the virus to escape from the neutralizing antibodies. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Given its essential roles in viral polyprotein processing and immune evasion, HCV NS3/4A protease is a prime target for antiviral chemotherapy. We aimed to establish in vivo cell-based assay system for monitoring the activity of NS3/4A protease from HCV genotype 4a, the predominant genotype in Egypt, and the Middle East. Furthermore, the developed system was used to evaluate the inhibitory potency of a series of computer-designed chemically-synthesized compounds against NS3/4A protease from HCV genotype 4a. Materials and Methods: Native as well as mutant cleavage sites to NS3/4A protease were cloned in frame into ß-galactosidase gene of TA cloning vector. The target specificity of HCV NS3/4A was evaluated by coexpression of ß-galactosidase containing the protease cleavage site with NS3/4A protease construct in bacterial cells. The activity of ß-galactosidase was colorimetrically estimated in the cell lysate using orthonitro phenyl ß-D-galactopyanoside (ONPG) as a substrate. Results and Conclusions: We successfully developed an efficient cell-based system based on the blue/white selection of bacterial cells that are able to express functional/nonfunctional ß-galactosidase enzyme.


Assuntos
Bioensaio/métodos , Hepacivirus/enzimologia , Proteínas não Estruturais Virais/metabolismo , Clonagem Molecular , Colorimetria , Escherichia coli , Genótipo , Hepacivirus/genética , Humanos , Proteínas não Estruturais Virais/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
5.
Food Funct ; 10(6): 3758-3767, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31179460

RESUMO

Hepatitis C virus (HCV) is the main agent responsible for chronic liver disease. Recent advances in anti-HCV treatment strategies have significantly increased the viral clearance rate (>90%). However, sustained antiviral responses vary in different cohorts, and high costs limit the broad use of direct-acting antivirals (DAAs). The goal of this study is to evaluate the inhibitory ability of well characterized (LC-QTOF-MS/MS) aqueous extracts obtained from edible mushrooms (Agaricus bisporus) to diminish HCV viral replication. Our data have demonstrated an in vitro inhibitory effect of A. bisporus extracts on NS3/4A protease and HCV replication. Fractionation by ultra-filtration and sequential liquid-liquid extraction showed that the compounds responsible for the inhibition are water-soluble with low molecular weights (<3 kDa) and that action could be through the following five compounds: ergothioneine, adenine, guanine, hypoxanthine, and xanthine, which are present in all fractions (UF-3, AqF-3 kDa and organic fractions) showing NS3/4A inhibition. Low molecular weight aqueous extracts (<3 kDa) from A. bisporus have potential applications in the prophylaxis and treatment of HCV, especially for patients who do not have access to the last generation of DAAs. They may be useful as well for other flaviviruses, which also possess a NS3 serine protease.


Assuntos
Agaricus/química , Antivirais/isolamento & purificação , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Hepacivirus/enzimologia , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/virologia , Humanos , Extratos Vegetais/química , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Espectrometria de Massas em Tandem , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
6.
Gastroenterology ; 157(3): 692-704.e9, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31078622

RESUMO

BACKGROUND & AIMS: Sofosbuvir is a frequently used pan-genotype inhibitor of hepatitis C virus (HCV) polymerase. This drug eliminates most chronic HCV infections, and resistance-associated substitutions in the polymerase are rare. However, HCV genotype 3 responds slightly less well to sofosbuvir-based therapies than other genotypes. We collected data from England's National Health Service Early Access Program to search for virus factors associated with sofosbuvir treatment failure. METHODS: We collected patient serum samples and used the capture-fusion assay to assess viral sensitivity to sofosbuvir in 14 HCV genotype 3 samples. We identified polymorphisms associated with reduced response and created modified forms of HCV and replicons containing the substitutions of interest and tested their sensitivity to sofosbuvir and ribavirin. We examined the effects of these polymorphisms by performing logistic regression multivariate analysis on their association with sustained virologic response in a separate cohort of 411 patients with chronic HCV genotype 3 infection who had been treated with sofosbuvir and ribavirin, with or without pegylated interferon. RESULTS: We identified a substitution in the HCV genotype 3a NS5b polymerase at amino acid 150 (alanine [A] to valine [V]), V at position 150 was observed in 42% of patients) with a reduced response to sofosbuvir in virus replication assays. In patients treated with sofosbuvir-containing regimens, the A150V variant was associated with a reduced response to treatment with sofosbuvir and ribavirin, with or without pegylated interferon. In 326 patients with V at position 150, 71% achieved an sustained virologic response compared to 88% with A at position 150. In cells, V at position 150 reduced the response to sofosbuvir 7-fold. We found that another rare substitution, glutamic acid (E) at position 206, significantly reduced the response to sofosbuvir (8.34-fold reduction); the combinations of V at position 150 and E at position 206 reduced the virus response to sofosbuvir 35.77-fold. Additionally, in a single patient, we identified 5 rare polymorphisms that reduced sensitivity to sofosbuvir our cell system. CONCLUSIONS: A common polymorphism, V at position 150 in the HCV genotype 3a NS5b polymerase, combined with other variants, reduces the virus response to sofosbuvir. Clinically, infection with HCV genotype 3 containing this variant reduces odds of sustained virologic response. In addition, we identified rare combinations of variants in HCV genotype 3 that reduce response to sofosbuvir.


Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica/tratamento farmacológico , Mutação , Polimorfismo Genético , Sofosbuvir/uso terapêutico , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Antivirais/efeitos adversos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Farmacorresistência Viral/genética , Quimioterapia Combinada , Genótipo , Hepacivirus/enzimologia , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/virologia , Humanos , Fenótipo , Sofosbuvir/efeitos adversos , Resposta Viral Sustentada , Fatores de Tempo , Resultado do Tratamento , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
7.
Eur J Med Chem ; 171: 401-419, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928711

RESUMO

GSK3082 - a hepatitis C virus RNA polymerase inhibitor - and a series of analogues with structural diversity at the 5-position were prepared from a 2,2,4,5-tetrasubstituted pyrrolidine obtained with a well-defined stereochemistry from the 1,3-dipolar cycloaddition of the chiral imino ester derived from leucine tert-butyl ester and (R)-2,3-O-isopropylideneglyceraldehyde with methyl acrylate. The chiral 2,2-dimethyl-1,3-dioxolane moiety provided by the glyceraldehyde served as a synthetic equivalent for different substituents and functional groups and these transformations usually required mild reaction conditions and simple work-up procedures. The inhibitory activity of the resulting GSK3082 analogues was studied in vitro in a cell-based assay of the subgenomic HCV RNA replication system. Some of the analogues showed good inhibitory activity with IC50 values in the nanomolar concentration range.


Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Hepacivirus/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
8.
J Biol Chem ; 294(19): 7573-7587, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30867194

RESUMO

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.


Assuntos
Guanosina Trifosfato/química , Hepacivirus/enzimologia , Simulação de Dinâmica Molecular , RNA Replicase/química , Uridina Trifosfato/química , Proteínas não Estruturais Virais/química , Motivos de Aminoácidos , Domínio Catalítico , Guanosina Trifosfato/metabolismo , RNA Replicase/metabolismo , Uridina Trifosfato/metabolismo , Proteínas não Estruturais Virais/metabolismo
9.
Virology ; 529: 226-233, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30738360

RESUMO

Hepatitis C virus (HCV) was shown to activate protein kinase R (PKR), which inhibits expression of interferon (IFN) and IFN-stimulated genes by controlling the translation of newly transcribed mRNAs. However, it is unknown exactly how HCV activates PKR. To address the molecular mechanism(s) of PKR activation mediated by HCV infection, we examined the effects of viral proteins on PKR activation. Here, we show that expression of HCV NS5B strongly induced PKR and eIF2α phosphorylation, and attenuated MHC class I expression. In contrast, expression of Japanese encephalitis virus RNA-dependent RNA polymerase did not induce phosphorylation of PKR. Co-immunoprecipitation analyses showed that HCV NS5B interacted with PKR. Furthermore, expression of NS5B with polymerase activity-deficient mutation failed to phosphorylate PKR, suggesting that RNA polymerase activity is required for PKR activation. These results suggest that HCV activates PKR by association with NS5B, resulting in translational suppression of MHC class I to establish chronic infection.


Assuntos
Hepacivirus/enzimologia , RNA Replicase/metabolismo , eIF-2 Quinase/metabolismo , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/virologia , Linhagem Celular Tumoral , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Neoplasias Hepáticas , Plasmídeos , RNA Replicase/genética , RNA Viral/genética , eIF-2 Quinase/genética
10.
J Virol Methods ; 264: 11-17, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30381240

RESUMO

Hepatitis C Virus c33, a recombinant protein comprising residues 1192-1457 of NS3 helicase, has been a mainstay of HCV serology for decades. With seven unpaired cysteines, seroreactivity of E. coli expressed c33 is dependant on reductants. While engineering a c33 replacement for new anti-HCV serological tests, we sought to reduce oxidation sensitivity, a liability for immunodiagnostic reagent stability. A series of cysteine-to-serine substituted variants of a c33-like antigen was constructed and evaluated for reactivity against a panel of HCV-positive sera. Several variants were essentially nonreactive while others exhibited reactivity similar to or better than the wild-type construct. One demonstrated equivalent potency to wild-type but also diminished DTT dependence. To explore enhanced anti-NS3 reactivity, we constructed and examined an expanded series of antigens comprising individual helicase domains, the full-length helicase, additional cysteine-to-serine variants, and variants at positions critical to catalytic activity. Immunoassays using these latter NS3 helicase recombinants demonstrated that domain 1 possessed significantly more seroreactivity than previously believed, that the use of soluble full-length helicase protein enhanced sensitivity by several-fold over c33, and that anti-NS3 helicase seroreactivity was further enhanced by the introduction of point mutations which altered the catalytic activity or oxidation sensitivity of the antigen.


Assuntos
DNA Helicases/genética , DNA Helicases/imunologia , Hepacivirus/enzimologia , Hepacivirus/genética , Testes Sorológicos , Proteínas não Estruturais Virais/genética , Anticorpos Antivirais/sangue , Cisteína/genética , Cisteína/imunologia , DNA Helicases/metabolismo , Escherichia coli/genética , Engenharia Genética , Hepacivirus/imunologia , Humanos , Testes Imunológicos , Mutação Puntual , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Soroconversão , Proteínas não Estruturais Virais/imunologia
12.
Med Chem ; 15(2): 130-137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30324891

RESUMO

BACKGROUND: IDX-184 is a guanosine derivative having a potent inhibitory performance against HCV NS5b polymerase. OBJECTIVE: To test three different groups of 2'C - modified analogues of guanosine nucleotide against HCV polymerase. METHOD: Using combined Quantitative Structure-Activity Relationships (QSAR) and molecular docking, the suggested compounds are studied. RESULTS: Examining the docked structures of the compounds with experimentally solved NS5b structure (PDB ID: 2XI3) revealed that most of the compounds have the same mode of interaction as that of guanosine nucleotide and hence, NS5b inhibition is possible. CONCLUSION: It is revealed that sixteen modifications have a better binding affinity to NS5b compared to guanosine. In addition, seven more compounds are better in NS5b binding compared to the approved drug, sofosbuvir, and the compound under clinical trials, IDX-184. Hence, these compounds could be potent HCV NS5b inhibitors. Summary Points: Novel guanosine modifications were introduced in silico and optimized using QM. QSAR and docking calculations are performed to test the binding affinity of the compounds to HCV NS5b active site. Comparison between the binding affinities and the mode of interactions of the compounds and both GTP and IDX-184 is performed. Structural mining to quantify the mode of binding of the compounds to NS5b active site pocket.


Assuntos
Antivirais/química , Antivirais/farmacologia , Guanosina/química , Guanosina/farmacologia , Hepacivirus/enzimologia , Simulação de Acoplamento Molecular , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/metabolismo , Guanosina/metabolismo , Hepacivirus/efeitos dos fármacos , Conformação Proteica , Relação Quantitativa Estrutura-Atividade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
13.
Med Microbiol Immunol ; 208(1): 3-24, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30298360

RESUMO

Chronic hepatitis C virus (HCV) infections affect 71 million people worldwide, often resulting in severe liver damage. Since 2014 highly efficient therapies based on directly acting antivirals (DAAs) are available, offering cure rates of almost 100%, if the infection is diagnosed in time. It took more than a decade to discover HCV in 1989 and another decade to establish a cell culture model. This review provides a personal view on the importance of HCV cell culture models, particularly the replicon system, in the process of therapy development, from drug screening to understanding of mode of action and resistance, with a special emphasis on the contributions of Ralf Bartenschlager's group. It summarizes the tremendous efforts of scientists in academia and industry required to achieve efficient DAAs, focusing on the main targets, protease, polymerase and NS5A. It furthermore underpins the importance of strong basic research laying the ground for translational medicine.


Assuntos
Descoberta de Drogas/métodos , Hepacivirus/enzimologia , Hepacivirus/crescimento & desenvolvimento , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Modelos Biológicos , Antivirais/isolamento & purificação , Antivirais/farmacologia , Antivirais/uso terapêutico , Pesquisa Biomédica/história , Hepacivirus/genética , História do Século XXI , Humanos , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico
14.
J Gastroenterol Hepatol ; 34(1): 12-21, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30311701

RESUMO

BACKGROUND AND AIM: Although treatment with direct-acting antivirals has dramatically improved morbidity and mortality attributable to chronic hepatitis C virus infection, universal access to these medicines has been slow in the Asia-Pacific region and Russia. This study evaluated efficacy and safety of elbasvir/grazoprevir in participants with hepatitis C virus infection from Asia-Pacific countries and Russia (C-CORAL). METHODS: C-CORAL was a phase 3, randomized, placebo-controlled study (NCT02251990). Treatment-naive, HIV-negative, cirrhotic and non-cirrhotic participants with chronic hepatitis C genotype 1, 4, or 6 infection were randomized to elbasvir 50 mg/grazoprevir 100 mg once daily for 12 weeks (immediate-treatment group) or placebo followed by deferred treatment with elbasvir/grazoprevir (deferred-treatment group). The primary efficacy outcome was sustained virologic response at 12 weeks, and the primary safety outcome was a comparison between the immediate-treatment group and placebo phase of the deferred-treatment group. RESULTS: A total of 489 participants were randomized (immediate-treatment group, n = 366; deferred-treatment group, n = 123). Sustained virologic response at 12 weeks in the combined immediate/deferred-treatment groups was 94.4% (459/486; 95% confidence interval = 92.4-96.5%). Sustained virologic response at 12 weeks was 98.2% in participants with genotype 1b, 91.9% with genotype 1a, and 66.7% with genotype 6 infection. Similar rates of adverse events and drug-related adverse events were seen in the immediate-treatment group versus placebo phase of the deferred-treatment group (51.0% vs 50.4% and 21.4% vs 21.1%). CONCLUSIONS: Elbasvir/grazoprevir for 12 weeks represents an effective and well-tolerated treatment option for treatment-naive people with genotype 1 infection from Asia-Pacific countries and Russia.


Assuntos
Antivirais/uso terapêutico , Benzofuranos/uso terapêutico , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Imidazóis/uso terapêutico , Quinoxalinas/uso terapêutico , Adulto , Alanina Transaminase/sangue , Antivirais/efeitos adversos , Aspartato Aminotransferases/sangue , Austrália , Benzofuranos/efeitos adversos , Método Duplo-Cego , Combinação de Medicamentos , Farmacorresistência Viral/genética , Extremo Oriente , Feminino , Genótipo , Hepacivirus/enzimologia , Humanos , Imidazóis/efeitos adversos , Masculino , Pessoa de Meia-Idade , Quinoxalinas/efeitos adversos , Federação Russa , Resposta Viral Sustentada , Tailândia , Vietnã , Proteínas não Estruturais Virais/metabolismo , Adulto Jovem
15.
Xenobiotica ; 49(8): 935-944, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30227770

RESUMO

Paritaprevir (PTV) is a non-structural protein 3/4A protease inhibitor developed for the treatment of hepatitis C disease as a fixed dose combination of ombitasvir (OBV) and ritonavir (RTV) with or without dasabuvir. The aim of this study was to evaluate the effects of cytochrome P450 (CYP) 3A5 on in vitro PTV metabolism using human recombinant CYP3A4, CYP3A5 (rCYP3A4, rCYP3A5) and human liver microsomes (HLMs) genotyped as either CYP3A5*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3. The intrinsic clearance (CLint, Vmax/Km) for the production of a metabolite from PTV in rCYP3A4 was 1.5 times higher than that in rCYP3A5. The PTV metabolism in CYP3A5*1/*1 and CYP3A5*1/*3 HLMs expressing CYP3A5 was comparable to that in CYP3A5*3/*3 HLMs, which lack CYP3A5. CYP3A4 expression level was significantly correlated with PTV disappearance rate and metabolite formation. In contrast, there was no such correlation found for CYP3A5 expression level. This study represents that the major CYP isoform involved in PTV metabolism is CYP3A4, with CYP3A5 having a minor role in PTV metabolism. The findings of the present study may provide foundational information on PTV metabolism, and may further support dosing practices in HCV-infected patients prescribed PTV-based therapy.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Hepacivirus/enzimologia , Compostos Macrocíclicos/metabolismo , Inibidores de Proteases/metabolismo , Anilidas/química , Anilidas/metabolismo , Carbamatos/química , Carbamatos/metabolismo , Humanos , Compostos Macrocíclicos/química , Microssomos Hepáticos/metabolismo , Inibidores de Proteases/química
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 210: 290-297, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30469132

RESUMO

Simeprevir sodium (SMV); a novel hepatitis C inhibitor, quells hepatitis C viral replication by binding to and repressing the protease, hepatitis C infection (HCV) NS3/4A. In this way, it is known as a prompt acting antiviral agent. Calibration curves of SMV were built in various solvents; ethanol, methanol, acetonitrile, chloroform and dichloromethane. It is obeyed up to 60.0 µg/mL; in all solvents at two maximum wavelengths (280 and 327 nm). Several investigations show that, SMV might be present in a mixture of Sofosbuvir (SOF) and/or Ledipasvir (LDP). So far as that is concerned, H-point standard addition strategy (HPSAS) is made to identify it in binary or ternary mixtures. Recovery studies are in the prevalent range (93.0-107.0%) with relative standard deviation <1.5%. A correlation between the developed techniques is carried out and it demonstrates that these strategies are effectively applied for the simultaneous analysis of SMV, SOF and LDP in several synthetic samples and pharmaceutics. Statistical treatment of the acquired data is carried out against a newly published HPLC technique using F- and t-treatments.


Assuntos
Antivirais/análise , Inibidores de Proteases/análise , Simeprevir/análise , Espectrofotometria Ultravioleta/métodos , Artefatos , Benzimidazóis/análise , Calibragem , Misturas Complexas/análise , Fluorenos/análise , Hepacivirus/enzimologia , Reprodutibilidade dos Testes , Sofosbuvir/análise , Espectrofotometria Ultravioleta/estatística & dados numéricos
17.
Nat Prod Res ; 33(23): 3364-3371, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29897265

RESUMO

A new flavonol triglycoside, rhamnazin 3-O-2G-rhamnorutinoside or rhamnazin 3-O-(2″,6″-O-α-di-rhamnosyl)-ß-glucoside (1) was isolated along with known flavonols, rhamnazin 3-O-rutinoside (2), rhamnazin 3-O-(6″-O-α-rhamnosyl)-ß-galactoside (3), isorhamnetin 3-O-(6″-O-α-rhamnosyl)-ß-galactoside (4), isorhamnetin 3-O-(2″,6″-O-α-di-rhamnosyl)-ß-galactoside (5), and isorhamnetin (6), and allantoin (7) from the aqueous methanol extract of Sarcocornia fruticosa leaves. Spectral analyses (UV, MS, and NMR) and acid hydrolysis were used to determine the structures. These compounds in this study except 6 were reported for the first time from the genus Sarcocornia. The extract and flavonol glycosides (1-5) were evaluated for antioxidant and inhibition of HCV protease enzyme. Rhamnazin triglycoside (1) was shown to have a potent HCV protease inhibitor with IC50 value 8.9 µM, while isorhamnetin di- and triglycosides (4 and 5) were effectively scavenged DPPH radicals with IC50 values 3.8 and 4.3 µM, respectively.


Assuntos
Antioxidantes/farmacologia , Chenopodiaceae/química , Flavonoides/farmacologia , Hepacivirus/enzimologia , Inibidores de Proteases/farmacologia , Antioxidantes/química , Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Flavonoides/química , Glicosídeos/química , Glicosídeos/farmacologia , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Extratos Vegetais/química , Folhas de Planta/química , Inibidores de Proteases/química , Arábia Saudita
18.
Methods Mol Biol ; 1911: 3-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593615

RESUMO

The advent of direct-acting antivirals (DAAs) has brought about a sudden renaissance in the treatment of chronic hepatitis C virus (HCV) infection with SVR rates now routinely >90%. However, due to the error-prone nature of the HCV RNA polymerase, resistance-associated substitutions (RASs) to DAAs may be present at baseline and can result in a significant effect on treatment outcomes and hamper the achievement of sustained virologic response. By further understanding the patterns and nature of these RASs, it is anticipated that the incidence of treatment failure will continue to decrease in frequency with the development of drug regimens with increasing potency, barrier to resistance, and genotypic efficacy. This review summarizes our current knowledge of RASs associated with HCV infection as well as the clinical effect of RASs on treatment with currently available DAA regimens.


Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Substituição de Aminoácidos , Animais , Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Genótipo , Hepacivirus/enzimologia , Hepatite C/virologia , Humanos , Modelos Moleculares , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética
19.
Proc Natl Acad Sci U S A ; 116(1): 168-176, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30587591

RESUMO

Biophysical interactions between proteins and peptides are key determinants of molecular recognition specificity landscapes. However, an understanding of how molecular structure and residue-level energetics at protein-peptide interfaces shape these landscapes remains elusive. We combine information from yeast-based library screening, next-generation sequencing, and structure-based modeling in a supervised machine learning approach to report the comprehensive sequence-energetics-function mapping of the specificity landscape of the hepatitis C virus (HCV) NS3/4A protease, whose function-site-specific cleavages of the viral polyprotein-is a key determinant of viral fitness. We screened a library of substrates in which five residue positions were randomized and measured cleavability of ∼30,000 substrates (∼1% of the library) using yeast display and fluorescence-activated cell sorting followed by deep sequencing. Structure-based models of a subset of experimentally derived sequences were used in a supervised learning procedure to train a support vector machine to predict the cleavability of 3.2 million substrate variants by the HCV protease. The resulting landscape allows identification of previously unidentified HCV protease substrates, and graph-theoretic analyses reveal extensive clustering of cleavable and uncleavable motifs in sequence space. Specificity landscapes of known drug-resistant variants are similarly clustered. The described approach should enable the elucidation and redesign of specificity landscapes of a wide variety of proteases, including human-origin enzymes. Our results also suggest a possible role for residue-level energetics in shaping plateau-like functional landscapes predicted from viral quasispecies theory.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Peptídeo Hidrolases/genética , Serina Proteases/genética , Aprendizado de Máquina Supervisionado , Proteínas não Estruturais Virais/genética , Antivirais/farmacologia , Farmacorresistência Viral/genética , Metabolismo Energético , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Hepacivirus/genética , Hepacivirus/metabolismo , Metabolômica/métodos , Peptídeo Hidrolases/metabolismo , Serina Proteases/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas não Estruturais Virais/metabolismo
20.
Curr Pharm Des ; 24(37): 4484-4491, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30501598

RESUMO

BACKGROUND: Hepatitis C virus (HCV) infection poses a considerable threat to the public health. The current standard of care treatment with pegylated interferon-alpha in combination with ribavirin (PEG-IFN- α+RBV) is associated with significant side effects, poorly tolerated, and provides limited efficacy. The development of direct-acting antiviral agents (DAAs) targeting key viral enzymes essential for viral replication represents a significant milestone in the treatment of chronic HCV infection. Given its critical role in the viral polyprotein processing and the evasion of the host innate immunity, the NS3/4A protease has emerged as a promising drug target for the development of anti-HCV therapies. Although several potent NS3/4A protease inhibitors (PIs) have been approved or are in clinical development, the majority of currently available PIs have significant limitations related to untoward adverse events and a lack of pan-genotypic activity, indicating a continuing unmet medical need for the development and optimization of novel PIs with improved efficacy and tolerability, convenient dosing schedules, and shorter treatment durations. METHODS: The inhibitory efficacy of four computer-designed chemically-synthesized compounds was evaluated against in vitro-expressed NS3/4A protease from HCV genotype 4a, the most prevalent genotype in Egypt, using a fluorescence-based enzymatic assay. RESULTS: We successfully identified two non-macrocyclic small molecules, BE113 (7a) and BE114 (7b), which exhibited inhibitory activity against HCV NS3/4A protease from HCV genotype 4a. CONCLUSION: The two compounds presented in this study may be promising inhibitors against NS3/4A protease of HCV genotype 4a and could be novel lead compounds for developing new therapeutics for the treatment of chronic HCV infection.


Assuntos
Antivirais/farmacologia , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Relação Dose-Resposta a Droga , Genótipo , Hepacivirus/genética , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismo
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