Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 23
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
3.
J Virol ; 93(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30381490

RESUMO

Drug resistance remains a major concern for human immunodeficiency virus (HIV) treatment. To date, very few resistance mutations have emerged in first-line combination therapy that includes the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). In vitro, DTG selects for several primary mutations that induce low-level DTG resistance; secondary mutations, while increasing the level of resistance, however, further impair replication fitness, which raised the idea that DTG monotherapy may be feasible. The simian immunodeficiency virus (SIV) rhesus macaque model of HIV infection can be useful to explore this concept. Nine macaques were infected with virulent SIVmac251 and started on DTG monotherapy during either acute (n = 2) or chronic infection (n = 7). Within 4 weeks of treatment, all animals demonstrated a reduction in viremia of 0.8 to 3.5 log RNA copies/ml plasma. Continued treatment led to overall sustained benefits, but the outcome after 10 to 50 weeks of treatment was highly variable and ranged from viral rebound to near pretreatment levels to sustained suppression, with viremia being 0.5 to 5 logs lower than expected based on pretreatment viremia. A variety of mutations previously described to confer low-level resistance of HIV-1 to DTG or other INSTI were detected, and these were sometimes followed by mutations believed to be compensatory. Some mutations, such as G118R, previously shown to severely impair the replication capacity in vitro, were associated with more sustained virological and immunological benefits of continued DTG therapy, while other mutations, such as E92Q and G140A/Q148K, were associated with more variable outcomes. The observed variability of the outcomes in macaques warrants avoidance of DTG monotherapy in HIV-infected people.IMPORTANCE A growing number of anti-HIV drug combinations are effective in suppressing virus replication in HIV-infected persons. However, to reduce their cost and risk for toxicity, there is considerable interest in simplifying drug regimens. A major concern with single-drug regimens is the emergence of drug-resistant viral mutants. It has been speculated that DTG monotherapy may be a feasible option, because DTG may have a higher genetic barrier for the development of drug resistance than other commonly used antiretrovirals. To explore treatment initiation with DTG monotherapy, we started SIV-infected macaques on DTG during either acute or chronic infection. Although DTG initially reduced virus replication, continued treatment led to the emergence of a variety of viral mutations previously described to confer low-level resistance of HIV-1 to DTG, and this was associated with variable clinical outcomes. This unpredictability of mutational pathways and outcomes warns against using DTG monotherapy as initial treatment for HIV-infected people.


Assuntos
Inibidores de Integrase de HIV/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores de Integrase de HIV/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Macaca mulatta , Mutação , Oxazinas , Piperazinas , Piridonas , RNA Viral/sangue , RNA Viral/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Resultado do Tratamento
4.
AIDS ; 32(13): 1773-1780, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-29894388

RESUMO

OBJECTIVES: Dolutegravir (DTG) has achieved better long-term suppression of HIV-1 replication than other integrase strand transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG). In in-vitro drug washout experiments, we previously showed that removal of DTG from pretreated MT-2 cells infected with wild-type HIV-1 showed slower rebound in viral replication as compared to removal of RAL. Now, we performed DTG, EVG and RAL washout experiments to compare the recovery of viral integration and production of 2-long terminal repeat (LTR) circles using wild-type HIV-1 clones, R263K viruses with low-level resistance to DTG and viruses with G140S/Q148H mutations showing cross-resistance against all currently approved INSTIs. DESIGN AND METHODS: MT-2 cells infected with wild-type, R263K or G140S/Q148H HIV-1 clones were treated with DTG, RAL or EVG for 3 days. Viral rebound following drug washout was assessed, monitoring viral integration and 2-LTR circle production by qPCR. RESULTS: Viral integration did not resume for up to 8 days after DTG washout from the wild-type or R263K infections but increased soon after washout of either RAL or EVG. With the G140S/Q148H virus, levels of integration were not significantly affected by the presence of either RAL or EVG. With DTG, integration was much lower at 3 days after infection than for the no-drug control. At 8 days after DTG washout, viral integration resumed but remained relatively low. CONCLUSION: DTG antiretroviral activity in tissue culture is more durable than that of either RAL or EVG after drug washout and this is true for both wild-type and drug-resistant viruses.


Assuntos
Farmacorresistência Viral , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mutação de Sentido Incorreto , Linhagem Celular , HIV-1/enzimologia , Humanos , Oxazinas , Piperazinas , Piridonas , Quinolonas/farmacologia , Raltegravir Potássico/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Carga Viral
5.
J Infect Dis ; 218(5): 698-706, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29617824

RESUMO

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.


Assuntos
Substituição de Aminoácidos , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , HIV/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Carga Viral , HIV/genética , HIV/crescimento & desenvolvimento , HIV/isolamento & purificação , Integrase de HIV/genética , Integrase de HIV/metabolismo , Humanos , Quimioterapia de Manutenção/métodos , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto , Oxazinas , Piperazinas , Piridonas , Falha de Tratamento , Replicação Viral
6.
Expert Rev Anti Infect Ther ; 16(2): 143-152, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29347858

RESUMO

INTRODUCTION: Drug adherence has been a recurring issue in the field of HIV treatment, and low treatment adherence is typically associated with emergence of drug resistance, treatment failure and increased risks of transmission. Injectable antiretroviral drugs offer a unique opportunity to counter this issue for the treatment of HIV-positive individuals. In addition, injectables offer a remarkable opportunity to reduce new HIV infections, if applied in the context of both treatment-as-prevention and pre-exposure prophylaxis. Areas covered: Researchers and drug companies are developing long-acting agents that possess long biological half-life and excellent pharmacokinetic profiles that can be administered intramuscularly, intravenously, or subcutaneously. These long-acting injectables are categorized as drugs that target different steps of HIV replication cycle or monoclonal antibodies that target HIV entry. Expert commentary: Injectables against HIV have the potential to revolutionize the fight against HIV by facilitating both treatment and prevention in a wide variety of clinical settings. Several challenges remain including the identification of potent two-drug combinations of drugs that can be formulated as injectables, and thorough drug-drug interaction studies with a broad variety of medications. Finally we believe that the healthcare benefits of injectables will require regulatory changes to allow self-injection before they reach their full potential.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , Adesão à Medicação , Fármacos Anti-HIV/farmacocinética , Preparações de Ação Retardada , Interações Medicamentosas , Farmacorresistência Viral , Infecções por HIV/virologia , Humanos , Injeções , Profilaxia Pré-Exposição/métodos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
7.
Artigo em Inglês | MEDLINE | ID: mdl-28923862

RESUMO

Animal models are essential to study novel antiretroviral drugs, resistance-associated mutations (RAMs), and treatment strategies. Bictegravir (BIC) is a novel potent integrase strand transfer inhibitor (INSTI) that has shown promising results against HIV-1 infection in vitro and in vivo and against clinical isolates with resistance against INSTIs. BIC has a higher genetic barrier to the development of resistance than two clinically approved INSTIs, termed raltegravir and elvitegravir. Another clinically approved INSTI, dolutegravir (DTG) also possesses a high genetic barrier to resistance, while a fourth compound, termed cabotegravir (CAB), is currently in late phases of clinical development. Here we report the susceptibilities of simian immunodeficiency virus (SIV) and HIV-1 integrase (IN) mutants containing various RAMs to BIC, CAB, and DTG. BIC potently inhibited SIV and HIV-1 in single cycle infection with 50% effective concentrations (EC50s) in the low nM range. In single cycle SIV infections, none of the E92Q, T97A, Y143R, or N155H substitutions had a significant effect on susceptibility to BIC (≤4-fold increase in EC50), whereas G118R and R263K conferred ∼14-fold and ∼6-fold increases in EC50, respectively. In both single and multiple rounds of HIV-1 infections, BIC remained active against the Y143R, N155H, R263K, R263K/M50I, and R263K/E138K mutants (≤4-fold increase in EC50). In multiple rounds of infection, the G140S/Q148H combination of substitutions decreased HIV-1 susceptibility to BIC 4.8-fold compared to 16.8- and 7.4-fold for CAB and DTG, respectively. BIC possesses an excellent resistance profile in regard to HIV and SIV and could be useful in nonhuman primate models of HIV infection.


Assuntos
Farmacorresistência Viral/genética , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Mutação , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Amidas , Substituição de Aminoácidos , Células HEK293 , Integrase de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Mutagênese Sítio-Dirigida , Oxazinas , Piperazinas , Piridonas/farmacologia , Raltegravir Potássico/farmacologia , Genética Reversa , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Sci Rep ; 7(1): 6345, 2017 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-28740124

RESUMO

We evaluated Sofosbuvir (SOF), the anti-hepatitis C virus prodrug of ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine-5'-monophosphate, for potential inhibitory activity against DENV replication. Both cell-based and biochemical assays, based on use of purified DENV full-length NS5 enzyme, were studied. Cytopathic effect protection and virus yield reduction assays confirmed that SOF possessed anti-DENV activity in cell culture with a 50% effective concentration (EC50) of 4.9 µM and 1.4 µM respectively. Real-time RT-PCR verified that SOF inhibits generation of viral RNA with an EC50 of 9.9 µM. Purified DENV NS5 incorporated the active triphosphate form (SOF-TP) into nascent RNA, causing chain-termination. Relative to the natural UTP, the incorporation efficiency of SOF-TP was low (discrimination value = 327.5). In a primer extension assay, SOF-TP was active against DENV NS5 wild-type polymerase activity with an IC50 of 14.7 ± 2.5 µM. The S600T substitution in the B Motif of DENV polymerase conferred 4.3-fold resistance to SOF-TP; this was due to decreased incorporation efficiency rather than enhanced excision of the incorporated SOF nucleotide. SOF has antiviral activity against DENV replication. The high discrimination value in favor of UTP in enzyme assays may not necessarily preclude antiviral activity in cells. SOF may be worthy of evaluation against severe DENV infections in humans.


Assuntos
Antivirais/farmacologia , Vírus da Dengue/fisiologia , Sofosbuvir/farmacologia , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Dengue/tratamento farmacológico , Dengue/virologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Avaliação de Medicamentos , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Uridina Trifosfato/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
9.
J Antimicrob Chemother ; 72(9): 2570-2573, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28637235

RESUMO

Objectives: To compare the effectiveness of HIV integrase inhibitor monotherapy between raltegravir and dolutegravir as an approach to simplify therapy. Methods: We evaluated and compared the efficacy of 20 week monotherapy with dolutegravir or raltegravir in humanized mice (HSC-NSG) infected with HIVBaL. Plasma HIV RNA was measured by quantitative RT-PCR (limit of detection of 150 copies/45 µL of plasma) and drug levels by LC-MS/MS. Escape viruses were genotyped and analysed for replication capacity and drug susceptibility in tissue culture. Results: Drug-untreated control mice maintained constant viraemia throughout the study. Virus isolates from these mice were susceptible to both raltegravir (EC50 of <8 nM) and dolutegravir (EC50 of <1 nM). Mice treated with raltegravir or dolutegravir had plasma drug levels comparable to those in humans. Monotherapy with raltegravir initially suppressed HIV viraemia, but failed to maintain suppression in 4/4 mice. Viruses from raltegravir failing mice developed mutations G140G/S and Q148H/K, and were resistant to both raltegravir (EC50 values of >100 nM) and dolutegravir (EC50 values ranging from 8.8 to 13.3 nM). Monotherapy with dolutegravir suppressed viraemia in 5/5 of mice, but viraemia rebounded in one animal. The virus from this mouse had mutations E138K, G140S, Q148H, N155H and S230R, was highly resistant to both raltegravir (EC50 of >1000 nM) and dolutegravir (EC50 of 550 nM), and replicated to levels similar to those of control viruses in PBMCs. Conclusions: Monotherapy with either raltegravir or dolutegravir does not consistently maintain HIV suppression, suggesting that dual therapy may be required in simplification strategies.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Raltegravir Potássico/uso terapêutico , Viremia/tratamento farmacológico , Animais , Genótipo , Infecções por HIV/virologia , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/genética , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Oxazinas , Piperazinas , Piridonas , RNA Viral/sangue , Viremia/virologia , Replicação Viral/efeitos dos fármacos
10.
J Antimicrob Chemother ; 72(3): 727-734, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28069884

RESUMO

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 µM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 µM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Zika virus/enzimologia , Descoberta de Drogas/métodos , Integrase de HIV/metabolismo , Transcriptase Reversa do HIV/antagonistas & inibidores , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes/metabolismo , Sofosbuvir/farmacologia , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Zika virus/fisiologia
11.
Antimicrob Agents Chemother ; 60(1): 600-8, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26574011

RESUMO

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 µM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 µM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.


Assuntos
Antivirais/farmacologia , Quelantes/farmacologia , Vírus da Dengue/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Picolinas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Aedes , Substituição de Aminoácidos , Animais , Antivirais/síntese química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Quelantes/síntese química , Cricetinae , Vírus da Dengue/enzimologia , Vírus da Dengue/genética , Relação Dose-Resposta a Droga , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Expressão Gênica , Histidina/genética , Histidina/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Cinética , Simulação de Acoplamento Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Picolinas/síntese química , Ligação Proteica , Estrutura Secundária de Proteína , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Sulfonas/síntese química , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
12.
Pathog Immun ; 1(1): 41-67, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-30993244

RESUMO

Since the discovery of the first inhibitors of HIV replication, drug resistance has been a major problem in HIV therapy due in part to the high mutation rate of HIV. Therefore, the development of a predictive animal model is important to identify impending resistance mutations and to possibly inform treatment decisions. Significant advances have been made possible through use of nonhuman primates infected by SIV, SHIV, and simian-tropic HIV-1 (stHIV-1), and use of humanized mouse models of HIV-1 infections. In this review, we describe some of the findings from animal models used for the preclinical testing of integrase strand transfer inhibitors. These models have led to important findings about the potential role of integrase strand transfer inhibitors in both the prevention and treatment of HIV-1 infection.

13.
J Virol ; 89(23): 12002-13, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26378179

RESUMO

UNLABELLED: We previously showed that the simian immunodeficiency virus SIVmac239 is susceptible to human immunodeficiency virus (HIV) integrase (IN) strand transfer inhibitors (INSTIs) and that the same IN drug resistance mutations result in similar phenotypes in both viruses. Now we wished to determine whether tissue culture drug selection studies with SIV would yield the same resistance mutations as in HIV. Tissue culture selection experiments were performed using rhesus macaque peripheral blood mononuclear cells (PBMCs) infected with SIVmac239 viruses in the presence of increasing concentrations of dolutegravir (DTG), elvitegravir (EVG), and raltegravir (RAL). We now show that 22 weeks of selection pressure with DTG yielded a mutation at position R263K in SIV, similar to what has been observed in HIV, and that selections with EVG led to emergence of the E92Q substitution, which is a primary INSTI resistance mutation in HIV associated with EVG treatment failure. To study this at a biochemical level, purified recombinant SIVmac239 wild-type (WT) and E92Q, T97A, G118R, Y143R, Q148R, N155H, R263K, E92Q T97A, E92Q Y143R, R263K H51Y, and G140S Q148R recombinant substitution-containing IN enzymes were produced, and each of the characteristics strand transfer, 3'-processing activity, and INSTI inhibitory constants was assessed in cell-free assays. The results show that the G118R and G140S Q148R substitutions decreased Km' and Vmax'/Km' for strand transfer compared to those of the WT. RAL and EVG showed reduced activity against both viruses and against enzymes containing Q148R, E92Q Y143R, and G140S Q148R. Both viruses and enzymes containing Q148R and G140S Q148R showed moderate levels of resistance against DTG. This study further confirms that the same mutations associated with drug resistance in HIV display similar profiles in SIV. IMPORTANCE: Our goal was to definitively establish whether HIV and simian immunodeficiency virus (SIV) share similar resistance pathways under tissue culture drug selection pressure with integrase strand transfer inhibitors and to test the effect of HIV-1 integrase resistance-associated mutations on SIV integrase catalytic activity and resistance to integrase strand transfer inhibitors. Clinically relevant HIV integrase resistance-associated mutations were selected in SIV in our tissue culture experiments. Not only do we report on the characterization of SIV recombinant integrase enzyme catalytic activities, we also provide the first research anywhere on the effect of mutations within recombinant integrase SIV enzymes on drug resistance.


Assuntos
Farmacorresistência Viral/genética , Inibidores de Integrase/farmacologia , Seleção Genética , Vírus da Imunodeficiência Símia/genética , Animais , Clonagem Molecular , Primers do DNA/genética , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Leucócitos Mononucleares/virologia , Macaca mulatta , Mutagênese , Mutação de Sentido Incorreto/genética , Oxazinas , Piperazinas , Piridonas , Quinolonas/farmacologia , Raltegravir Potássico/farmacologia , Especificidade da Espécie
14.
AIDS ; 29(12): 1459-66, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26244385

RESUMO

OBJECTIVES: Dolutegravir is an integrase strand-transfer inhibitor that has shown unprecedented robustness against the emergence of HIV drug-resistant strains in treatment-naive individuals. The R263K substitution in integrase was identified through culture selection as a resistance-associated substitution for dolutegravir and was recently detected in two treatment-experienced participants in the SAILING clinical trial, who experienced dolutegravir-based treatment failure, one of whom was infected by a subtype C virus. The objective of this study was to characterize the R263K substitution in HIV-1 subtype C integrase. DESIGN AND METHODS: We used cell-free strand transfer assays and tissue culture experiments to characterize the R263K substitution in HIV-1 subtype C integrase in comparison with subtype B. RESULTS: Cell-free biochemical assays showed that the R263K substitution diminished subtype C integrase strand-transfer activity by decreasing the affinity of integrase for target DNA. Similarly, both viral infectiousness and replication capacity were reduced by the R263K substitution in tissue culture. Decrease in enzyme activity and viral infectiousness exceeded 35 and 50%, respectively - significantly more than in HIV-1 subtype B. R263K in HIV-1 subtype C also conferred low levels of resistance against dolutegravir and high levels of cross-resistance against elvitegravir, but not raltegravir. CONCLUSIONS: The R263K substitution is more deleterious to integrase strand-transfer activity and viral infectiousness in HIV-1 subtype C than in subtype B. Our results suggest that cross-resistance may prevent treatment-experienced individuals who are experiencing treatment failure with dolutegravir from being subsequently treated with elvitegravir.


Assuntos
Substituição de Aminoácidos , Genótipo , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1/fisiologia , Mutação de Sentido Incorreto , Replicação Viral , Fármacos Anti-HIV/farmacologia , Fenômenos Bioquímicos , Linhagem Celular , Farmacorresistência Viral , HIV-1/classificação , HIV-1/enzimologia , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Oxazinas , Piperazinas , Piridonas , Seleção Genética , Cultura de Vírus
15.
PLoS One ; 10(6): e0128310, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26046987

RESUMO

Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance.


Assuntos
Inibidores de Integrase de HIV/química , Integrase de HIV/química , HIV-1/enzimologia , RNA Viral/química , Sítios de Ligação , Linhagem Celular , Bases de Dados de Compostos Químicos , Dimerização , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/farmacologia , HIV-1/fisiologia , Humanos , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Replicação Viral/efeitos dos fármacos
16.
J Virol ; 89(6): 3163-75, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25552724

RESUMO

UNLABELLED: Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE: Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3' processing indicates the importance of the 3' processing step and highlights a therapeutic angle that needs to be better exploited.


Assuntos
Substituição de Aminoácidos , Farmacorresistência Viral , Infecções por HIV/virologia , Integrase de HIV/genética , HIV-1/enzimologia , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Integrase de HIV/química , Integrase de HIV/metabolismo , HIV-1/classificação , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Alinhamento de Sequência
17.
Antimicrob Agents Chemother ; 59(4): 1942-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25583721

RESUMO

Drug resistance represents a key aspect of human immunodeficiency virus (HIV) treatment failure. It is important to develop nonhuman primate models for studying issues of drug resistance and the persistence and transmission of drug-resistant viruses. However, relatively little work has been conducted using either simian immunodeficiency virus (SIV) or SIV/HIV recombinant viruses for studying resistance against integrase strand transfer inhibitors (INSTIs). Here, we used a T-cell-tropic SIV/HIV recombinant virus in which the capsid and vif regions of HIV-1 were replaced with their SIV counterparts (simian-tropic HIV-1 [stHIV-1](SCA,SVIF)) to study the impact of a number of drug resistance substitutions in the integrase coding region at positions E92Q, G118R, E138K, Y143R, S153Y, N155H, and R263K on drug resistance, viral infectivity, and viral replication capacity. Our results show that each of these substitutions exerted effects that were similar to their effects in HIV-1. Substitutions associated with primary resistance against dolutegravir were more detrimental to stHIV-1(SCA,SVIF) infectiousness than were resistance substitutions associated with raltegravir and elvitegravir, consistent with data that have been reported for HIV-1. These findings support the role of stHIV-1(SCA,SVIF) as a useful model with which to evaluate the role of INSTI resistance substitutions on viral persistence, transmissibility, and pathogenesis in a nonhuman primate model.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Inibidores de Integrase de HIV/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Animais , Linhagem Celular , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Mutagênese Sítio-Dirigida , Oxazinas , Piperazinas , Piridonas , Quinolonas/farmacologia , Raltegravir Potássico/farmacologia , Replicação Viral/efeitos dos fármacos
18.
J Antimicrob Chemother ; 70(3): 750-6, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25433008

RESUMO

OBJECTIVES: Cenicriviroc is a potent antagonist of the chemokine coreceptors 5 and 2 (CCR5/CCR2) and blocks HIV-1 entry. The CCR5 inhibitor maraviroc has been shown in tissue culture to be able to repel cell-free virions from the cell surface into extracellular space. We hypothesized that cenicriviroc might exhibit a similar effect, and tested this using clinical samples from the Phase IIb study 652-2-202, by measuring rates of intracellular DNA decline. We also monitored viral RNA levels in culture fluids. METHODS: We infected PM-1 cells with CCR5-tropic HIV-1 BaL in the presence or absence of inhibitory concentrations of cenicriviroc (20 nM) or maraviroc (50 nM) or controls. Viral load levels and p24 were measured by ELISA, quantitative PCR and quantitative real-time reverse transcription PCR at 4 h post-infection. Frozen PBMC DNA samples from 30 patients with virological success in the Phase IIb study were studied, as were early and late reverse transcript levels. Docking studies compared binding between cenicriviroc/CCR5 and maraviroc/CCR5. RESULTS: Unlike maraviroc, cenicriviroc did not cause an increase in the amount of virus present in culture fluids at 4 h compared with baseline. The use of cenicriviroc did, however, result in lower levels of intracellular viral DNA after 4 h. Structural modelling indicates that cenicriviroc binds more deeply than maraviroc to the hydrophobic pocket of CCR5, providing an explanation for the absence of viral rebound with cenicriviroc. CONCLUSIONS: In contrast to maraviroc, cenicriviroc does not repel virus back into extracellular space. Differences in results may be due to superior binding of cenicriviroc to CCR5 compared with maraviroc.


Assuntos
Fármacos Anti-HIV/farmacologia , DNA Viral/análise , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Imidazóis/farmacologia , Carga Viral , Fármacos Anti-HIV/uso terapêutico , Linhagem Celular , Ensaios Clínicos Fase II como Assunto , Meios de Cultura , Ensaio de Imunoadsorção Enzimática , Espaço Extracelular/virologia , Infecções por HIV/tratamento farmacológico , Humanos , Imidazóis/uso terapêutico , Leucócitos Mononucleares/virologia , Reação em Cadeia da Polimerase em Tempo Real , Sulfóxidos , Cultura de Vírus
19.
Antimicrob Agents Chemother ; 59(1): 310-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25348535

RESUMO

Clinical studies have shown that integrase strand transfer inhibitors (INSTIs) can be used effectively against HIV-1 infection. To date, no resistance substitution has been found in INSTI-naive patients treated with the new integrase inhibitor dolutegravir (DTG). In a recent selection study with DTG, using a virus bearing the H51Y substitution in integrase, the emergence of an R to K substitution at position 262 (R262K) was observed. We characterized this double mutant with respect to integrase strand transfer activity and susceptibility to DTG both biochemically and in tissue culture. We showed that the addition of R262K to H51Y decreased recombinant integrase strand transfer activity but improved integrase DNA-binding affinity, compared to wild-type or H51Y-containing enzymes. The defect in strand transfer activity did not translate into a decrease in HIV-1 infectivity. The combination of H51Y and R262K substitutions slightly decreased susceptibility to DTG (fold change = 1.87) in cell-based resistance assays. Although viral replication was not affected and enzyme efficiency was impaired by the addition of R262K to H51Y, there was an overall increase in the level of biochemical drug resistance against DTG. Our findings suggest that the R at position 262 plays an important role in DNA binding.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Substituição de Aminoácidos , Sítios de Ligação , Simulação por Computador , DNA Viral/metabolismo , Células HEK293/efeitos dos fármacos , Células HEK293/virologia , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/química , Humanos , Modelos Moleculares , Oxazinas , Piperazinas , Conformação Proteica , Piridonas
20.
J Int AIDS Soc ; 17(4 Suppl 3): 19531, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25394040

RESUMO

INTRODUCTION: Cenicriviroc (CVC), a once-daily, dual CCR5/CCR2 co-receptor antagonist, has completed Phase 2b development. CVC demonstrated favourable safety and similar efficacy compared with efavirenz (EFV) in Study 202 (NCT01338883); an ex vivo sub-analysis evaluated treatment effects on HIV entry, measured by intracellular HIV DNA declines, in subjects with virologic success at Week 24. In addition, in vitro assays determined and compared the extent of any cell-free virion redistribution that CVC or maraviroc (MVC) may cause. METHODS: Ex vivo: intracellular DNA (frozen PBMCs) from 30 subjects with virologic success at Week 24 (10, 13 and 7 subjects on CVC 100 mg, CVC 200 mg and EFV, respectively). Early (strong-stop) and late (full-length) reverse transcript levels were measured by qPCR. In vitro: PM-1 cells were infected with CCR5-tropic HIV-1 BaL in the presence or absence of inhibitory concentrations of CVC (20 nM), MVC (50 nM) or controls. P24 and viral load levels were measured by ELISA and qRT-PCR after 4 hours. RESULTS: Ex vivo analysis showed full-length HIV DNA declines were similar across all groups (CVC 100 mg, CVC 200 mg and EFV) at Week 24. Strong-stop HIV DNA declines (a marker of HIV entry) at Week 24 were pronounced for both CVC arms (CVC 100 mg, 51% decline; CVC 200 mg, 37% decline) compared to no decline for the EFV arm. In vitro experiments revealed that CVC-treated cells had lower levels of supernatant P24 at 4 hours versus baseline (0 hrs: 506 ng/mL; 4 hrs: 192 ng/mL), but P24 levels remained constant for MVC-treated cells after 4 hours (0 hrs: 506 ng/mL; 4 hrs: 520 ng/mL). Viral load levels for CVC-treated cells remained stable after 4 hours (0 hrs: 1.19×10(10) copies/mL; 4 hrs: 1.26×10(10) copies/mL). MVC-treated cells exhibited a slight increase in viral load after 4 hours (0 hrs: 1.19×10(10) copies/mL; 4 hrs: 1.67×10(10) copies/mL). CONCLUSIONS: Ex vivo analysis confirmed that CVC treatment blocks HIV entry (strong-stop HIV DNA declines), while in vitro analysis showed that CVC-treated cells do not repel virus back into the extracellular space, as seen with MVC. Experiments are underway to determine whether or not interactions between CVC and HIV at the binding site may explain these unanticipated findings.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...