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1.
Nature ; 531(7594): 381-5, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26934220

RESUMO

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.


Assuntos
Alanina/análogos & derivados , Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca mulatta/virologia , Ribonucleotídeos/uso terapêutico , Monofosfato de Adenosina/análogos & derivados , Alanina/farmacocinética , Alanina/farmacologia , Alanina/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular Tumoral , Ebolavirus/efeitos dos fármacos , Feminino , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Ribonucleotídeos/farmacocinética , Ribonucleotídeos/farmacologia
2.
J Infect Dis ; 222(9): 1468-1477, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31971597

RESUMO

BACKGROUND: Presatovir is an oral respiratory syncytial virus (RSV) fusion inhibitor targeting RSV F protein. In a double-blind, placebo-controlled study in healthy adults experimentally infected with RSV (Memphis-37b), presatovir significantly reduced viral load and clinical disease severity in a dose-dependent manner. METHODS: Viral RNA from nasal wash samples was amplified and the F gene sequenced to monitor presatovir resistance. Effects of identified amino acid substitutions on in vitro susceptibility to presatovir, viral fitness, and clinical outcome were assessed. RESULTS: Twenty-eight treatment-emergent F substitutions were identified. Of these, 26 were tested in vitro; 2 were not due to lack of recombinant virus recovery. Ten substitutions did not affect presatovir susceptibility, and 16 substitutions reduced RSV susceptibility to presatovir (2.9- to 410-fold). No substitutions altered RSV susceptibility to palivizumab or ribavirin. Frequency of phenotypically resistant substitutions was higher with regimens containing lower presatovir dose and shorter treatment duration. Participants with phenotypic presatovir resistance had significantly higher nasal viral load area under the curve relative to those without, but substitutions did not significantly affect peak viral load or clinical manifestations of RSV disease. CONCLUSIONS: Emergence of presatovir-resistant RSV occurred during therapy but did not significantly affect clinical efficacy in participants with experimental RSV infection.


Assuntos
Indazóis/uso terapêutico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Sulfonamidas/uso terapêutico , Inibidores de Proteínas Virais de Fusão/uso terapêutico , Adolescente , Adulto , Substituição de Aminoácidos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Farmacorresistência Viral/genética , Humanos , Pessoa de Meia-Idade , Vírus Sinciciais Respiratórios/genética , Carga Viral/efeitos dos fármacos , Adulto Jovem
3.
Antimicrob Agents Chemother ; 60(1): 316-22, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26503655

RESUMO

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.


Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Pró-Fármacos/metabolismo , Tenofovir/metabolismo , Adenina/metabolismo , Adenina/farmacologia , Alanina , Fármacos Anti-HIV/farmacologia , Biotransformação , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/virologia , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Catepsina A/antagonistas & inibidores , Catepsina A/genética , Catepsina A/metabolismo , Cobicistat/farmacologia , Interações Medicamentosas , Expressão Gênica , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Oligopeptídeos/farmacologia , Cultura Primária de Células , Pró-Fármacos/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Inibidores de Serina Proteinase/farmacologia , Tenofovir/farmacologia
4.
Antimicrob Agents Chemother ; 60(12): 7086-7097, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27645238

RESUMO

Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Amidas , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Sinergismo Farmacológico , Integrase de HIV/genética , HIV-1/genética , HIV-1/isolamento & purificação , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Mutação , Oxazinas , Piperazinas , Piridonas , Raltegravir Potássico/farmacologia
5.
PLoS Pathog ; 10(4): e1004071, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24722454

RESUMO

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.


Assuntos
Linfócitos T CD4-Positivos/virologia , Depsipeptídeos/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Modelos Biológicos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Depsipeptídeos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Infecções por HIV/virologia , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/metabolismo , Humanos , Memória Imunológica/efeitos dos fármacos , Isoenzimas/metabolismo , Masculino
6.
Antimicrob Agents Chemother ; 60(3): 1264-73, 2015 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-26666922

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. In addition, RSV causes significant morbidity and mortality in hospitalized elderly and immunocompromised patients. Currently, only palivizumab, a monoclonal antibody against the RSV fusion (F) protein, and inhaled ribavirin are approved for the prophylactic and therapeutic treatment of RSV, respectively. Therefore, there is a clinical need for safe and effective therapeutic agents for RSV infections. GS-5806, discovered via chemical optimization of a hit from a high-throughput antiviral-screening campaign, selectively inhibits a diverse set of 75 RSV subtype A and B clinical isolates (mean 50% effective concentration [EC50] = 0.43 nM). The compound maintained potency in primary human airway epithelial cells and exhibited low cytotoxicity in human cell lines and primary cell cultures (selectivity > 23,000-fold). Time-of-addition and temperature shift studies demonstrated that GS-5806 does not block RSV attachment to cells but interferes with virus entry. Follow-up experiments showed potent inhibition of RSV F-mediated cell-to-cell fusion. RSV A and B variants resistant to GS-5806, due to mutations in F protein (RSV A, L138F or F140L/N517I, and RSV B, F488L or F488S), were isolated and showed cross-resistance to other RSV fusion inhibitors, such as VP-14637, but remained fully sensitive to palivizumab and ribavirin. In summary, GS-5806 is a potent and selective RSV fusion inhibitor with antiviral activity against a diverse set of RSV clinical isolates. The compound is currently under clinical investigation for the treatment of RSV infection in pediatric, immunocompromised, and elderly patients.


Assuntos
Antivirais/farmacologia , Pirazóis/farmacologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Sulfonamidas/farmacologia , Brônquios/citologia , Brônquios/virologia , Fusão Celular , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Humanos , Indazóis , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Vírus Sincicial Respiratório Humano/patogenicidade , Internalização do Vírus/efeitos dos fármacos
7.
J Virol ; 88(6): 3586-90, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24371077

RESUMO

Insertions in the protease (PR) region of human immunodeficiency virus (HIV) represent an interesting mechanism of antiviral resistance against HIV PR inhibitors (PIs). Here, we demonstrate the improved ability of a phosphonate-containing experimental HIV PI, GS-8374, relative to that of other PIs, to effectively inhibit patient-derived recombinant HIV strains bearing PR insertions and numerous other mutations. We correlate enzyme inhibition with the catalytic activities of corresponding recombinant PRs in vitro and provide a biochemical and structural analysis of the PR-inhibitor complex.


Assuntos
Infecções por HIV/virologia , Inibidores da Protease de HIV/química , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Mutagênese Insercional , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/química , HIV-1/genética , Humanos , Modelos Moleculares , Organofosfonatos/análise
8.
J Virol ; 87(1): 454-63, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23097440

RESUMO

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.


Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Mutação de Sentido Incorreto , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Substituição de Aminoácidos , Linhagem Celular , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/isolamento & purificação , Humanos , Seleção Genética , Inoculações Seriadas
9.
Bioorg Med Chem Lett ; 24(3): 989-94, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24411125

RESUMO

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.


Assuntos
Inibidores do Citocromo P-450 CYP3A , Diaminas/síntese química , Diaminas/farmacologia , HIV/efeitos dos fármacos , Diaminas/química , Ativação Enzimática/efeitos dos fármacos , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Humanos , Relação Estrutura-Atividade , Resultado do Tratamento
10.
Bioorg Med Chem Lett ; 24(3): 995-9, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24412072

RESUMO

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.


Assuntos
Carbamatos/química , Inibidores do Citocromo P-450 CYP3A , Diaminas/química , Diaminas/farmacologia , Tiazóis/química , Carbamatos/farmacologia , Cobicistat , Ativação Enzimática/efeitos dos fármacos , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/farmacologia
11.
J Med Chem ; 67(15): 12945-12968, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39018526

RESUMO

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.


Assuntos
Antivirais , Picornaviridae , Pró-Fármacos , Infecções por Vírus Respiratório Sincicial , Animais , Antivirais/farmacologia , Antivirais/química , Pró-Fármacos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/síntese química , Chlorocebus aethiops , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Humanos , Picornaviridae/efeitos dos fármacos , Relação Estrutura-Atividade , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Descoberta de Drogas , Nucleosídeos/química , Nucleosídeos/farmacologia , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/virologia
12.
Antimicrob Agents Chemother ; 57(10): 4982-9, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23896476

RESUMO

A once-daily single-tablet antiretroviral regimen containing tenofovir (TFV) disoproxil fumarate, emtricitabine (FTC), elvitegravir (EVG), and cobicistat (COBI) is an approved combination for the treatment of patients infected with HIV. COBI and TFV have been reported to interact with distinct transporters in renal proximal tubules; while TFV is renally eliminated by a combination of glomerular filtration and tubular secretion via anion transporters OAT1, OAT3, and MRP4, COBI inhibits renal cation transporters, particularly MATE1, resulting in a measurable decrease in the tubular secretion of creatinine. To investigate the potential for a renal drug-drug interaction between TFV and COBI in vitro, the uptake of TFV in the presence and absence of COBI was determined in fresh human renal cortex tissue and in cells expressing the relevant renal transporters. At concentrations exceeding clinical protein-unbound plasma levels, COBI did not significantly inhibit the transport of TFV by the anion transporters OAT1, OAT3, and MRP4 (50% inhibitory concentrations [IC50s] of >15, 6.6, and 8.5 µM, respectively). Conversely, TFV had little or no effect on the cation transporters OCT2 and MATE1 (IC50 > 100 µM). Consistent with studies using individual transporters, no increase in the accumulation of TFV in freshly isolated human renal cortex tissue or renal proximal tubule cells (RPTECs) was observed in the presence of COBI. Finally, COBI alone or in combination with FTC and EVG did not affect the sensitivity to TFV of cultured primary RPTECs or cells coexpressing OAT1 and MRP4. These results illustrate that COBI and TFV interact primarily with distinct renal transporters and indicate a low potential for pharmacokinetic renal drug-drug interaction.


Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Carbamatos/farmacologia , Organofosfonatos/farmacologia , Tiazóis/farmacologia , Adenina/farmacologia , Adulto , Linhagem Celular , Cobicistat , Interações Medicamentosas , Humanos , Técnicas In Vitro , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico , Tenofovir
13.
Antimicrob Agents Chemother ; 55(4): 1366-76, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21245449

RESUMO

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 (K(i) = 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 [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 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 EC(50)s 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.


Assuntos
Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , Protease de HIV/metabolismo , Organofosfonatos/química , Linfócitos T CD4-Positivos/efeitos dos fármacos , Calorimetria , Células Cultivadas , Cristalografia por Raios X , Células HEK293 , HIV-1/efeitos dos fármacos , Células Hep G2 , Humanos , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo
14.
J Med Chem ; 64(8): 5001-5017, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33835812

RESUMO

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , Pró-Fármacos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Antivirais/química , Antivirais/farmacocinética , Células CACO-2 , Células Cultivadas , Chlorocebus aethiops , Modelos Animais de Doenças , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais/virologia , Humanos , Macaca fascicularis , Masculino , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Ratos Sprague-Dawley , Infecções por Vírus Respiratório Sincicial/virologia , Relação Estrutura-Atividade , Distribuição Tecidual , Tubercidina/análogos & derivados , Tubercidina/química , Carga Viral
15.
J Cell Biol ; 162(3): 425-34, 2003 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-12900394

RESUMO

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the "late domain") binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Eucarióticas/virologia , Produtos do Gene gag/metabolismo , HIV/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral/fisiologia , Eliminação de Partículas Virais/fisiologia , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Endossomos/ultraestrutura , Endossomos/virologia , Células Eucarióticas/metabolismo , HIV/patogenicidade , HIV/ultraestrutura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica , Mimetismo Molecular/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Vesículas Transportadoras/virologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana
16.
J Mol Biol ; 363(3): 635-47, 2006 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-16979654

RESUMO

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold. The resulting phosphonate analogs maintain high binding affinity to HIV-1 protease, potent antiretroviral activity, and unlike the parent molecules, display no loss of potency against a panel of clinically important PI-resistant HIV-1 strains. As shown by crystallographic analysis, the phosphonate moiety is highly exposed to solvent with no discernable interactions with any of the enzyme active site or surface residues. We term this effect "solvent anchoring" and demonstrate that it is driven by a favorable change in the inhibitor binding entropy upon the interaction with mutant enzymes. This type of thermodynamic behavior, which was not found with the parent scaffold fully buried in the enzyme active site, is a result of the increased degeneracy of inhibitor binding states, allowing effective molecular adaptation to the expanded cavity volume of mutant proteases. This strategy, which is applicable to various PI scaffolds, should facilitate the design of novel PIs and potentially other antiviral therapeutics.


Assuntos
Desenho de Fármacos , Farmacorresistência Viral Múltipla , Inibidores da Protease de HIV/química , Protease de HIV/química , Organofosfonatos/química , Solventes , Sulfato de Atazanavir , Sítios de Ligação , Infecções por HIV/tratamento farmacológico , Protease de HIV/metabolismo , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/uso terapêutico , Humanos , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Piridinas/química , Piridinas/metabolismo , Termodinâmica
17.
Antiviral Res ; 140: 116-120, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28131805

RESUMO

HIV-infected patients treated with certain nucleoside reverse transcriptase inhibitors (NRTIs) have experienced adverse effects due to drug-related mitochondrial toxicity. Tenofovir alafenamide (TAF) is a novel prodrug of the NRTI tenofovir (TFV) with an improved safety profile compared to tenofovir disoproxil fumarate (TDF). Prior in vitro studies have demonstrated that the parent nucleotide TFV has no significant effects on mtDNA synthesis. This study investigated whether clinically relevant TAF and TDF exposures affect mtDNA content in human lymphocytes. First, activated or resting peripheral blood mononuclear cells (PBMCs), as well as MT-2 and Jurkat T-cell lines, were continuously treated with ddC for 10 days to establish their susceptibility to mtDNA depletion. PBMCs had low sensitivity to NRTI-mediated mtDNA depletion in vitro. In contrast, ddC treatment of rapidly dividing MT-2 and Jurkat cells resulted in a dose-dependent decrease in mtDNA. Therefore, these two T-cell lines were selected for evaluating TAF and TDF treatment effects. MT-2 and Jurkat cells were pulse-treated with TAF or TDF every 24 h for 10 days to mimic pharmacologically relevant drug exposures. Pulse treatment of cells with 3.3 µM TAF or 1.1 µM TDF for 10 days resulted in 2- to 7-fold greater steady-state intracellular TFV-diphosphate (TFV-DP) levels than those observed clinically in TAF- or TDF-treated patients. At these concentrations, no significant TAF- (106.7% and 84.1% of control; p = 0.77 and 0.12 for MT-2 and Jurkat, respectively) or TDF- (100.6% and 91.0% of control; p = 0.91 and 0.37, respectively) associated reduction in mtDNA content was observed compared with untreated control cells. This study demonstrates that, despite delivering higher intracellular levels of TFV-DP than TDF, TAF does not inhibit mtDNA synthesis in vitro at concentrations exceeding the clinically relevant intracellular drug exposures. Thus, TAF has a low potential for mitochondrial toxicity in T-cells of HIV-infected patients.


Assuntos
Fármacos Anti-HIV/farmacologia , DNA Mitocondrial/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Tenofovir/farmacologia , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/análise , Fármacos Anti-HIV/farmacocinética , Citoplasma/química , DNA Mitocondrial/biossíntese , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Células Jurkat , Leucócitos Mononucleares/química , Leucócitos Mononucleares/citologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/análise , Pró-Fármacos/farmacologia , Linfócitos T/química , Linfócitos T/citologia , Tenofovir/efeitos adversos , Tenofovir/análise , Tenofovir/farmacocinética
18.
J Med Chem ; 60(5): 1648-1661, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28124907

RESUMO

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.


Assuntos
Alanina/análogos & derivados , Amidas/química , Doença pelo Vírus Ebola/tratamento farmacológico , Ácidos Fosfóricos/química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Ribonucleotídeos/química , Viroses/tratamento farmacológico , Monofosfato de Adenosina/análogos & derivados , Alanina/química , Linhagem Celular , Descoberta de Drogas , Humanos , Testes de Sensibilidade Microbiana , Pró-Fármacos/síntese química , Relação Estrutura-Atividade
19.
J Med Chem ; 58(4): 1630-43, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25574686

RESUMO

GS-5806 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a screening hit. The oral absorption properties were optimized by converting to the pyrazolo[1,5-a]-pyrimidine heterocycle, while potency, metabolic, and physicochemical properties were optimized by introducing the para-chloro and aminopyrrolidine groups. A mean EC50 = 0.43 nM was found toward a panel of 75 RSV A and B clinical isolates and dose-dependent antiviral efficacy in the cotton rat model of RSV infection. Oral bioavailability in preclinical species ranged from 46 to 100%, with evidence of efficient penetration into lung tissue. In healthy human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a mean 4.2 log10 reduction in peak viral load and a significant reduction in disease severity compared to placebo. In conclusion, a potent, once daily, oral RSV fusion inhibitor with the potential to treat RSV infection in infants and adults is reported.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Pirazóis/farmacologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Sulfonamidas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Administração Oral , Animais , Antivirais/administração & dosagem , Antivirais/química , Cães , Relação Dose-Resposta a Droga , Humanos , Indazóis , Macaca fascicularis , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirazóis/administração & dosagem , Pirazóis/química , Ratos , Vírus Sinciciais Respiratórios/fisiologia , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/química
20.
PLoS One ; 8(9): e74163, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040198

RESUMO

HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.


Assuntos
DNA Viral/antagonistas & inibidores , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , DNA Viral/genética , Farmacorresistência Viral , Expressão Gênica , Genes Reporter , Vetores Genéticos , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Linfócitos T/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vírion/efeitos dos fármacos , Vírion/genética , Montagem de Vírus/efeitos dos fármacos , Integração Viral/efeitos dos fármacos
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