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1.
Medicine (Baltimore) ; 99(7): e18777, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32049783

RESUMO

This study sought to determine the dominant circulating human immunodeficiency virus type 1 (HIV-1) subtype and associated drug resistance mutations in Ghana.This cross-sectional study was conducted with archived samples collected from patients who received care at 2 hospitals in Ghana from 2014 to 2016. Blood samples were earlier processed into plasma and peripheral blood mononuclear cells and stored at -80 °C. Ribonucleic acid (RNA) was extracted from the archived plasma. Two HIV-1 genes; protease and reverse transcriptase, were amplified, sequenced using gene-specific primers and analyzed for subtype and drug resistance mutations using the Stanford HIV Database.Of 16 patient samples successfully sequenced, we identified the predominance of HIV-1 subtype CRF02_AG (11/16, 68%). Subtypes G (2/16, 13%), dual CRF02_AG/G (2/16, 13%), and CRF01_AE (1/16, 6%) were also observed. Major nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations, M184I/V, D67N, T215F, and K70R/E were found. Non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations, K103N, Y181C, V90I, F227L, and V106A were also prevalent. Additionally, and at a lower level, protease inhibitor (PI)-resistance mutations, M46I, I54 V, V82A, L90 M, and I471 V, were also present in the sequences from antiretroviral therapy (ART)-experienced individuals. Two NRTI-associated drug resistance mutations (DRMs) (D67N and T69N) were present in sequences from 1 ART-naive individual.HIV-1 subtype CRF02_AG was most frequently detected in this study thus confirming earlier reports of dominance of this subtype in the West-African sub-region and Ghana in particular. The detection of these drug resistance mutations in individuals on first-line regimen composed of NRTI and NNRTI is an indication of prolonged drug exposure without viral load monitoring. Routine viral load monitoring is necessary for early detection of virologic failure and drug resistance testing will inform appropriate choice of regimens for such patients.


Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , HIV-1/classificação , Mutação , Adulto , Fármacos Anti-HIV/uso terapêutico , Estudos Transversais , Evolução Molecular , Feminino , Gana , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Transcriptase Reversa/uso terapêutico , Carga Viral
2.
Nat Methods ; 16(12): 1281-1288, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548705

RESUMO

Chemical modifications to messenger RNA are increasingly recognized as a critical regulatory layer in the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile platform for directed evolution that rapidly selects for reverse transcriptases that install mutations at sites of a given type of RNA modification during reverse transcription, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. The optimal evolved reverse transcriptase enabled detection of well-characterized m1A sites and revealed hundreds of m1A sites in human mRNA. This work develops and validates the reverse transcriptase evolution platform, and provides new tools, analysis methods and datasets to study m1A biology.


Assuntos
Adenosina/análogos & derivados , Transcriptase Reversa do HIV/genética , RNA Mensageiro/análise , Adenosina/análise , Sequência de Bases , Fluorescência , Humanos , Mutação , Transcriptoma
3.
Biochemistry ; 58(16): 2176-2187, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30900874

RESUMO

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are considered noncompetitive inhibitors that structurally alter reverse transcriptase (RT) and dramatically decrease catalysis. In this report, biochemical analysis with various divalent cations was used to demonstrate that NNRTIs and divalent cation-dNTP complexes are mutually exclusive, inhibiting each other's binding to RT/primer/template (RT-P/T) complexes. The binding of catalytically competent divalent cation-dNTP complexes to RT-P/T was measured with Mg2+, Mn2+, Zn2+, Co2+, and Ni2+ using Ca2+, a noncatalytic cation, for displacement. Binding strength order was Mn2+ ≈ Zn2+ ≫ Co2+ > Mg2+ ≈ Ni2+. Consistent with but not exclusive to mutually exclusive binding, primer extension assays showed that stronger divalent cation-dNTP complexes were more resistant to NNRTIs (efavirenz (EFV), rilpivirine (RPV), and nevirapine (NVP)). Filtration assays demonstrated that divalent cation-dNTP complexes inhibited the binding of 14C-labeled EFV to RT-P/T with stronger binding complexes formed with Mn2+ inhibiting more potently than those with Mg2+. Conversely, filter binding assays demonstrated that EFV inhibited 3H-labeled dNTP binding to RT-P/T complexes with displacement of Mn2+-dNTP complexes requiring much greater concentrations of EFV than the more weakly bound Mg2+-dNTP complexes. EFV bound relatively weakly to the NNRTI resistant K103N RT; but, binding was modestly enhanced in the presence of P/T, and EFV was easily displaced by divalent cation-dNTP complexes. This suggests that K103N overcomes EFV inhibition mostly by binding more weakly to the drug and is in contrast to other reports that indicate K103N has little to no effect on drug or dNTP binding. Overall, this biochemical analysis supports recent biophysical analyses of NNRTI-RT interactions that indicate mutually exclusive binding.


Assuntos
Benzoxazinas/metabolismo , Cátions Bivalentes/metabolismo , Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Rilpivirina/metabolismo , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Terapia Antirretroviral de Alta Atividade , Sequência de Bases , Benzoxazinas/farmacologia , Ligação Competitiva , Cátions Bivalentes/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Inibidores da Transcriptase Reversa/farmacologia , Rilpivirina/farmacologia
4.
Int J Antimicrob Agents ; 53(4): 515-519, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30769200

RESUMO

This study investigated the prevalence of doravirine (DOR) resistance mutations in non-nucleoside reverse transcriptase inhibitor (NNRTI)-experienced patients. DOR resistance was assessed in samples from NNRTI-experienced patients who underwent genotypic testing for virological failure from the Antiretroviral Response Cohort Analysis (ARCA) database. Intermediate DOR resistance was defined as detection of any of V106A/M, Y188C/H, V108I, and K103N+P225H. High-level DOR resistance was defined as detection of any of Y188L, M230L, G190E, V106A/M+F227L, and V106A/M+L234I. Overall, 6893 patients were included in the study: 64.2% had experienced efavirenz (EFV), 54.4% nevirapine (NVP), 6.8% etravirine (ETR), 7.7% rilpivirine (RPV) and 0.7% delavirdine. Among NNRTI-experienced patients, 12.7% and 6.1% of subjects had intermediate and high-level DOR resistance, respectively. The most common DOR resistance mutation was Y188L. In multivariable analysis, previous EFV use (OR = 1.52, 95% CI 1.15-2.02) and ETR use (OR = 1.91, 95% CI 1.34-2.73) were associated with detection of high-level DOR resistance, whilst RPV use was associated with a lower probability of high-level DOR resistance (OR = 0.39, 95% CI 0.22-0.71). Moreover, EFV use (OR = 1.76, 95% CI 1.19-2.58) and ETR use (OR = 1.72, 95% CI 1.10-2.68) were associated with detection of the Y188L mutation, whereas RPV use was not (OR = 0.16, 95% CI 0.05-0.50). In Italy, DOR resistance is uncommon among NNRTI-experienced patients, confirming a distinguishing resistance pattern within NNRTIs. However, previous EFV and ETR experience poses a higher risk of DOR resistance. These results support the use of DOR in NNRTI-experienced patients.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Piridonas/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Triazóis/uso terapêutico , Adulto , Benzoxazinas/uso terapêutico , Estudos Transversais , Delavirdina/uso terapêutico , Feminino , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Masculino , Nevirapina/uso terapêutico , Piridazinas/uso terapêutico , Rilpivirina/uso terapêutico , Resultado do Tratamento
5.
Biochem Biophys Res Commun ; 509(4): 943-948, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30648556

RESUMO

Nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs) are major antiviral agents against hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1). However, the notorious insoluble property of HBV RT has prevented atomic-resolution structural studies and rational anti-HBV drug design. Here, we created HIV-1 RT mutants containing HBV-mimicking sextuple or septuple amino acid substitutions at the nucleoside-binding site (N-site) and verified that these mutants retained the RT activity. The most active RT mutant, HIV-1 RT7MC, carrying Q151M/G112S/D113A/Y115F/F116Y/F160L/I159L was successfully crystallized, and its three-dimensional structure was determined in complex with DNA:dGTP/entecavir-triphosphate (ETV-TP), a potent anti-HBV guanosine analogue RT inhibitor, at a resolution of 2.43 Šand 2.60 Å, respectively. The structures reveal significant positional rearrangements of the amino acid side-chains at the N-site, elucidating the mechanism underlying the differential susceptibility of HIV-1 and HBV against recently reported 4'-modified NRTIs.


Assuntos
Transcriptase Reversa do HIV/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Substituição de Aminoácidos , Antivirais/farmacologia , Sítios de Ligação/genética , Domínio Catalítico , Cristalografia por Raios X , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Vírus da Hepatite B/química , Vírus da Hepatite B/genética , Humanos , Proteínas Mutantes/química , Conformação Proteica , Inibidores da Transcriptase Reversa/química
6.
Chem Biol Drug Des ; 93(1): 50-59, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30103267

RESUMO

Mutations at HIV-1 reverse transcriptase (RT) codon 184 such as M184V confer resistance to two nucleos(t)ide RT inhibitors (NRTI), lamivudine (3TC) and emtricitabine (FTC). The prevalence of mutations at HIV-1 RT codon 184 was evaluated using three independent RT sequence databases from treatment-experienced (TE) and treatment-naïve (TN) individuals. Data were collected retrospectively from three centers: one in Italy and two in France between 1997 and 2016. In order to highlight the role of these mutations in conferring drug resistance, structural and thermodynamic analyses were conducted by means of computational approaches. Among 32,440 RT sequences isolated from TE and 12,365 isolated from TN patients, the prevalence of HIV-1 RT codon 184 substitutions in each group was 31.21% and 0.72%, respectively. The mutations M184L and M184T have been observed only in TE patients. In all cases but four, M184L and M184T mutations were present during NRTI treatment. Molecular recognition studies on M184L and M184T structures showed both FTC and 3TC thermodynamic profiles unfavorable in comparison with the wild-type sequence, corroborated by molecular dynamic simulations (MDS). In this study, we highlighted two new resistance mutations in vivo for NRTI resistance. The low frequency of this pathway can be related to high impairment of replicative capacity mediated by these mutations.


Assuntos
Farmacorresistência Viral/efeitos dos fármacos , Transcriptase Reversa do HIV/genética , Inibidores da Transcriptase Reversa/farmacologia , Adulto , Idoso , Sítios de Ligação , Emtricitabina/química , Emtricitabina/farmacologia , Feminino , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , HIV-1/metabolismo , Humanos , Lamivudina/química , Lamivudina/farmacologia , Masculino , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Mutação , Estrutura Terciária de Proteína , Estudos Retrospectivos , Inibidores da Transcriptase Reversa/química
7.
AIDS ; 33(2): 315-326, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30325769

RESUMO

OBJECTIVE: To evaluate the effect of primary resistance and selected polymorphic amino-acid substitutions in HIV reverse transcriptase and protease on the CD4 cell count and viral load set point before the start of antiretroviral treatment. DESIGN: Prospective cohort study. METHODS: A total of 6180 individuals with a resistance test prior to starting antiretroviral treatment accessing care in HIV clinics across Europe who had at least one viral load and one CD4+ test available were included in the analysis. The impact of amino-acid substitutions variants on viral load and CD4+ trends was investigated using linear mixed models. Clusters of mutations were studied using principal component analysis. RESULTS: Overall, the detection of any primary resistance was not associated with either the speed of CD4+ cell decline or the viral load set point. However, transmitted nucleoside reverse transcriptase inhibitor and protease inhibitor resistance appeared to be weakly associated with lower viral load set points, as were the polymorphic G16E or Q92K protease mutations. There was some evidence suggesting that these effects varied according to HIV subtype, with the effects of transmitted nucleoside reverse transcriptase inhibitor and protease resistance being particularly marked among individuals with a subtype B virus. A cluster of five polymorphic protease substitutions at position 20, 13, 36, 69 and 89 was associated with less steep CD4+ cell declines and lower viral load set points. CONCLUSION: Although we found little evidence for an association between primary resistance and CD4+ speed of decline and viral load set point, the potential role of polymorphic protease (alone or in clusters) and their interplay with HIV subtype needs to be further evaluated.


Assuntos
Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Farmacorresistência Viral , Infecções por HIV/virologia , HIV/efeitos dos fármacos , Mutação de Sentido Incorreto , Carga Viral , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Antirretrovirais/uso terapêutico , Contagem de Linfócito CD4 , Europa (Continente) , Feminino , HIV/genética , HIV/isolamento & purificação , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Adulto Jovem
8.
AIDS ; 33(2): 339-344, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30325775

RESUMO

OBJECTIVES: MSM comprise ∼30% of new HIV infections in Israel, a country with mixed Jewish and Arab populations. We molecularly characterized HIV-1 in the Arab and Jewish MSM (AMSM, JMSM) populations to reveal possible interethnical connections. DESIGN: Cross-sectional study. METHODS: All Israeli-born, HIV-1-infected MSM diagnosed between 2005 and 2016 (n = 1143) were cross-matched with the National Civil Registry to identify religion (Jews/Muslims/Christians). Transmitted drug-resistance mutations (TDRM) and HIV-1 subtypes were determined on the first partial protease and reverse transcriptase sequences from treatment-naive patients and phylogenetic trees were constructed. RESULTS: Among MSM, 6.4% (73/1143) were Arabs and 93.6% (1070/1143) were Jews. Interestingly, a higher proportion of Arabs was identified among non-MSM (19%, 46/247 versus 6.4%, 73/1143, P < 0.01). Subtype analysis of 62 HIV-1 AMSM and 440 randomly selected HIV-1 JMSM sequences revealed 80.6, 8.1, 4.8 and 6.5% of AMSM and 82.3, 9.5, 4.1 and 4.1% of JMSM had B, A, C and non-A/B/C, respectively. Overall, 13.1% (66/502) had TDRM; reverse transcriptase-K103N/S, M184 V, T215S and protease-L90M were the most common. TDRM prevalence was not significantly higher in JMSM compared to AMSM (P = 0.1) and no temporal changes were observed in their frequency. Phylogenetic analysis demonstrated AMSM and JMSM clusters including L90M, K103N/S or T215S TDRM. CONCLUSION: Intermingling of AMSM and JMSM HIV-1 in clusters of HIV-1 sequences suggest interethnical sexual contacts among these MSM. Interventions aiming to prevent HIV-transmission in MSM should similarly address both populations groups. The high TDRM frequency requires continuation of resistance testing.


Assuntos
Transmissão de Doença Infecciosa , Genótipo , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , HIV-1/classificação , HIV-1/genética , Homossexualidade Masculina , Adulto , Árabes , Estudos Transversais , Farmacorresistência Viral , Infecções por HIV/virologia , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/isolamento & purificação , Humanos , Israel , Judeus , Masculino , Epidemiologia Molecular , Mutação de Sentido Incorreto , Prevalência
9.
Chem Biol Drug Des ; 93(4): 430-437, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30381875

RESUMO

Two novel series of human immunodeficiency virus-1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) bearing a thiophene[3,2-d]pyrimidine scaffold and sulfonamide linker in the right wing have been identified, which demonstrated activity against the wild-type (WT) HIV-1 strain in MT-4 cells with inhibitory concentrations ranging from micromolar to submicromolar. Especially, against the mutant strains K103N and E138K, most compounds exhibited more potent activity than against WT HIV-1. Compound 7 (EC50  = 0.014, 0.031 µM) achieved the most potent activity against the two mutants, being more effective than that of nevirapine (NVP, EC50  = 7.572, 0.190 µM) and comparable to that of etravirine (ETV, EC50  = 0.004, 0.014 µM). Molecular docking experiments on the novel analogs have also suggested that the extensive network of main chain hydrogen bonds are important in the binding mode, which may provide valuable insights for further optimization.


Assuntos
Desenho de Drogas , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/química , Sítios de Ligação , Domínio Catalítico , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Nevirapina/química , Nevirapina/metabolismo , Piridazinas/química , Piridazinas/metabolismo , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/metabolismo , Solventes/química , Relação Estrutura-Atividade
10.
AIDS Res Hum Retroviruses ; 35(2): 129-138, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30430843

RESUMO

There is evidence of increasing levels of pretreatment HIV drug resistance (PDR) in Southern Africa. We used data from two large population-based HIV surveillance studies to estimate prevalence of PDR in KwaZulu-Natal, the province with the highest HIV prevalence in South Africa. Sanger sequencing was performed on samples obtained from a longitudinal HIV surveillance program (study A, 2013-2014) and the HIV Incidence Provincial Surveillance System (study B, 2014-2015). Sequences were included for adult HIV positive participants (age ≥15 years for study A, age 15-49 years for study B) with no documented prior exposure to antiretroviral therapy (ART). Overall and drug class-specific PDR was estimated using the World Health Organization 2009 surveillance drug resistance mutation (SDRM) list, and phylogenetic analysis was performed to establish evidence of drug resistance transmission linkage. A total of 1,845 sequences were analyzed (611 study A; 1,234 study B). An overall PDR prevalence of 9.2% [95% confidence interval (CI) 7.0-11.7] was observed for study A and 11.0% (95% CI 8.9-13.2) for study B. In study B, the prevalence of non-nucleoside reverse-transcriptase inhibitor (NNRTI) PDR exceeded 10% for sequences collected in 2014 (10.2%, 95% CI 7.5-12.9). The most prevalent SDRMs were K103NS (7.5%), M184VI (2.4%), and V106AM (1.4%). There was no evidence of large transmission chains of drug-resistant virus. High level NNRTI PDR (>10%) suggests a need to modify the standard first-line ART regimen and to focus attention on improving the quality of HIV prevention, treatment, and care.


Assuntos
Farmacorresistência Viral , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/genética , Mutação , Adolescente , Adulto , Monitoramento Epidemiológico , Feminino , Genótipo , Infecções por HIV/transmissão , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prevalência , RNA Viral/genética , África do Sul/epidemiologia , Adulto Jovem
11.
J Med Chem ; 62(2): 604-621, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30525601

RESUMO

Conformational restriction applied to dihydrobenzylpyrimidin-4-(3 H)-ones (DABOs) by the intoduction of a methyl group at the α-benzylic position is known to massively improve the anti-HIV-1 activity of these compounds. Here, we report the effects of methoxy substitution at the α-benzylic position in S-, NH-, and N, N-DABOs carrying 2,6-difluoro, 2-chloro-6-fluoro, or 2,6-dichloro substituted benzyl moieties. The various α-methoxy DABO series (12-14) present different SAR at the dihalo benzyl substitution, with the most potent compounds (12d,e and 13c) showing similar (picomolar/nanomolar) anti-HIV-1 potency as the corresponding α-methyl analogues against wt HIV-1, and 10-100-fold increased potency (up to low nanomolar) against clinically relevant K103N, Y181C, Y188L, IRLL98, and K103N+Y181C HIV-1 mutant strains, highlighting the importance of the α-methoxy substitution to provide highly efficient DABOs as "second generation" NNRTIs. HPLC enantioseparation of three of the most potent derivatives (12d, 13c, and 14c) provided single enantiomers with significant enantioselectivity in HIV-1 inhibition. Computational studies allowed to correlate the best antiviral activity with the ( R) absolute configuration at the α-methoxy stereogenic center.


Assuntos
Fármacos Anti-HIV/química , Pirimidinonas/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Simulação de Acoplamento Molecular , Mutação , Estrutura Terciária de Proteína , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade
12.
Bioorg Med Chem Lett ; 28(22): 3491-3495, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30318436

RESUMO

A novel series of substituted piperazine-1-yl-pyrimidine derivatives were designed and synthesized as a new type of HIV-1 non-nucleoside inhibitors. Various N-substituted aromatic groups were incorporated into the piperazine ring through a simple and practical route to investigate the biological activity of these target compounds against wild-type and resistant strains of HIV-1. All of the target compounds were also evaluated as HIV-1 reverse transcriptase inhibitors in MT-4 cell cultures. The biological results showed that six of these compounds displayed inhibitory activities against the wild-type strain, among of which 7q and 7t were found to be the two most active analogues possessing EC50 values of 31.50 µM and 3.36 µM, respectively. Molecular modeling studies of 7q provide valuable information for developing new anti-HIV-1 inhibitors.


Assuntos
Desenho de Drogas , Transcriptase Reversa do HIV/antagonistas & inibidores , Piperazina/química , Pirimidinas/química , Inibidores da Transcriptase Reversa/síntese química , Sítios de Ligação , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Simulação de Acoplamento Molecular , Nitrogênio/química , Estrutura Terciária de Proteína , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade
13.
BMC Infect Dis ; 18(1): 514, 2018 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-30314470

RESUMO

BACKGROUND: Incomplete virologic suppression results in mutations associated with resistance and is a major obstacle to disease control. We analyzed the genotypic profiles of HIV-1 patients at the time of the first virologic failure and the response to a salvage regimen after 48 weeks. METHODS: This work was a cross-sectional, retrospective, analytical study based on data collected from medical records and genotyping tests between 2006 and 2016. The sample consisted of data on individuals living with HIV (PLWH) from three major reference centers. RESULTS: A total of 184 patients were included in the data analysis. Viral subtype B was the most common (81.3%) as well as M184 V/I (85.3%) and K103 codon mutations (65.8%). Forty-eight weeks after switching to a salvage regimen, 67.3% of patients achieved an undetectable viral load. DISCUSSION: The number of mutations associated with nucleos(t)ide reverse transcriptase inhibitors (NRTI(t)s) did not affect virologic suppression (9.3% for zero NRTI(t)-associated mutations vs 48.6% for 1-2 NRTI(t)-associated mutations vs 42.1% for ≥3 NRTI(t)-associated mutations, p = 0.179). An ARV time (the beginning of the first ARV regimen up to genotyping) of > 36 months was a protective factor for detectable viral load (PR = 0.60, 95% CI = 0.39-0.92, p = 0.020) and a risk factor for developing ≥3 NRTI(t)-associated mutations (PR = 2.43, 95% CI 1.38-4.28, p = 0.002). CONCLUSIONS: We found that extensive resistance to NRTI(t)s at the time of the first virologic failure did not impact virologic suppression at 48 weeks after switching to a second-line therapy based on NRTI(t)s plus protease inhibitors.


Assuntos
Antirretrovirais/uso terapêutico , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/genética , HIV-1/genética , Adulto , Estudos Transversais , Feminino , Genótipo , HIV-1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estudos Retrospectivos , Fatores de Risco , Terapia de Salvação , Falha de Tratamento , Carga Viral , Adulto Jovem
14.
Molecules ; 23(11)2018 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-30355996

RESUMO

The high variability of the human immunodeficiency virus (HIV) is an important cause of HIV resistance to reverse transcriptase and protease inhibitors. There are many variants of HIV type 1 (HIV-1) that can be used to model sequence-resistance relationships. Machine learning methods are widely and successfully used in new drug discovery. An emerging body of data regarding the interactions of small drug-like molecules with their protein targets provides the possibility of building models on "structure-property" relationships and analyzing the performance of various machine-learning techniques. In our research, we analyze several different types of descriptors in order to predict the resistance of HIV reverse transcriptase and protease to the marketed antiretroviral drugs using the Random Forest approach. First, we represented amino acid sequences as a set of short peptide fragments, which included several amino acid residues. Second, we represented nucleotide sequences as a set of fragments, which included several nucleotides. We compared these two approaches using open data from the Stanford HIV Drug Resistance Database. We have determined the factors that modulate the performance of prediction: in particular, we observed that the prediction performance was more sensitive to certain drugs than a type of the descriptor used.


Assuntos
Aminoácidos/química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Biologia Computacional/métodos , Farmacorresistência Viral , Protease de HIV/química , Transcriptase Reversa do HIV/química , Aminoácidos/genética , Simulação por Computador , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Mutação
15.
Cell Chem Biol ; 25(10): 1268-1278.e3, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30174310

RESUMO

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA/MK-8591), a nucleoside reverse transcriptase inhibitor (NRTI) under clinical trials, is a potent and promising long-acting anti-HIV type 1 (HIV-1) agent. EFdA and its derivatives possess a modified 4'-moiety and potently inhibit the replication of a wide spectrum of HIV-1 strains resistant to existing NRTIs. Here, we report that EFdA and NRTIs with a 4'-ethynyl- or 4'-cyano-moiety exerted activity against HIV-1 with an M184V mutation and multiple NRTI-resistant HIV-1s, whereas NRTIs with other moieties (e.g., 4'-methyl) did not show this activity. Structural analysis indicated that EFdA and 4'-ethynyl-NRTIs (but not other 4'-modified NRTIs), formed strong van der Waals interactions with critical amino acid residues of reverse transcriptase. Such interactions were maintained even in the presence of a broad resistance-endowing M184V substitution, thus potently inhibiting drug-resistant HIV-1 strains. These findings also explain the mechanism for the potency of EFdA and provide insights for further design of anti-HIV-1 therapeutics.


Assuntos
Domínio Catalítico/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Mutação Puntual , Inibidores da Transcriptase Reversa/farmacologia , Linhagem Celular , Desoxiadenosinas/química , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , Humanos , Simulação de Acoplamento Molecular , Inibidores da Transcriptase Reversa/química
16.
Chem Biol Drug Des ; 92(6): 2009-2021, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30079476

RESUMO

In the previous studies of our laboratory, the thiophene[3,2-d]pyrimidine was identified as a promising scaffold for seeking highly potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). In this study, we designed, synthesized, and biologically evaluated a series of thiophene[3,2-d]pyrimidine derivatives with changed linker between the thiophenepyrimidine core and the right wing. Some of the synthesized compounds exhibited excellent HIV-1 inhibitory potency with low (double-digit) nanomolar 50% effective concentration (EC50 ) values. Among them, compound 13a exhibited the most potent anti-HIV-1 activity (EC50  = 21.2 nM), which was 10-fold greater than that of NVP (EC50  = 281 nM). Moreover, 13a showed much lower cytotoxicity (CC50  = 183 µM) and higher selection index (SI = 8,632) than NVP, ETV, and AZT. Besides, some physicochemical properties and water solubility were calculated or measured. The preliminary structure-activity relationships and molecular simulation studies of these compounds were also discussed comprehensively to provide valuable direction for further design and optimization.


Assuntos
Desenho de Drogas , HIV-1/efeitos dos fármacos , Hidrazonas/química , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Sítios de Ligação , Genótipo , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , Humanos , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Pirimidinas/síntese química , Pirimidinas/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Solubilidade , Relação Estrutura-Atividade , Tiofenos/química
17.
AIDS ; 32(16): 2353-2361, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30096070

RESUMO

BACKGROUND: The IPERGAY ANRS trial showed that on-demand preexposure prophylaxis (PrEP) with tenofovir (TDF) and emtricitabine (FTC) was highly effective in preventing HIV infection among highly exposed MSM. Here, we analyzed drug resistance-associated mutations (RAMs) among all participants who acquired HIV infection during this trial. METHODS: Resistance was analyzed on frozen plasma at the time of HIV diagnosis among participants enrolled in the double-blind and open-label phases of the ANRS IPERGAY trial. Reverse transcriptase sequencing was performed, using population-based and ultradeep sequencing (454 GS Flex). Adherence was measured by pill counting and by plasma tenofovir and FTC assay. RESULTS: During the trial, 31 participants were diagnosed with HIV-1 infection (subtype B, 64.5%), using antigen/antibody immune assay in 29 cases and plasma HIV RNA assay in two. The median plasma HIV-1 RNA level was 5.52 log10 copies/ml. Drug resistance was tested in 12 participants before starting PrEP, in six assigned to TDF/FTC group and in 13 assigned to placebo group. Primary resistance to nucleoside reverse transcriptase inhibitors (zidovudine) and/or nonnucleoside reverse transcriptase inhibitors was detected in six participants (19%; 95% confidence interval 7-42). No major or minor TDF-resistant or FTC-resistant variants were detected. CONCLUSION: No TDF or FTC resistance-associated mutations were found among participants who acquired HIV in the ANRS IPERGAY trial.


Assuntos
Farmacorresistência Viral , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Mutação , Profilaxia Pré-Exposição/métodos , Ensaios Clínicos como Assunto , Método Duplo-Cego , Genótipo , Transcriptase Reversa do HIV/genética , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Placebos/administração & dosagem , Análise de Sequência de DNA , Falha de Tratamento
18.
Viruses ; 10(7)2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029500

RESUMO

Emergence of human immunodeficiency virus type 1 (HIV-1) drug resistance arises from mutation fixation in the viral genome during antiretroviral therapy. Primary mutations directly confer antiviral drug resistance, while secondary mutations arise that do not confer drug resistance. The A62V amino acid substitution in HIV-1 reverse transcriptase (RT) was observed to be associated with multi-drug resistance, but is not known to be a resistance-conferring mutation. In particular, A62V was observed in various multi-dideoxynucleoside resistant (MDR) mutation complexes, including the Q151M complex (i.e., A62V, V75I, F77L, F116Y, and Q151M), and the T69SSS insertion complex, which has a serine⁻serine insertion between amino acid positions 69 and 70 (i.e., M41L, A62V, T69SSS, K70R, and T215Y). However, what selective advantage is conferred to the virus remains unresolved. In this study, we hypothesized that A62V could influence replication fidelity and viral fitness with viruses harboring the Q151M and T69SSS MDR mutation complexes. A single-cycle replication assay and a dual-competition fitness assay were used to assess viral mutant frequency and viral fitness, respectively. A62V was found to increase the observed lower mutant frequency identified with each of the viruses harboring the MDR mutation complexes in the single-cycle assay. Furthermore, A62V was observed to improve viral fitness of replication-competent MDR viruses. Taken together, these observations indicate an adaptive role of A62V in virus replication fidelity and viral fitness, which would likely enhance virus persistence during drug-selective pressure.


Assuntos
Farmacorresistência Viral Múltipla/genética , Aptidão Genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , Mutação , Replicação Viral/genética , Fármacos Anti-HIV/farmacologia , Replicação do DNA , Genoma Viral , Células HEK293 , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Reação em Cadeia da Polimerase , Inibidores da Transcriptase Reversa/farmacologia
19.
J Virol ; 92(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29997209

RESUMO

CD8+ T cell-mediated escape mutations in Gag can reduce HIV-1 replication capacity (RC) and alter disease progression, but less is known about immune-mediated attenuation in other HIV-1 proteins. We generated 487 recombinant viruses encoding RT-integrase from individuals with chronic (n = 406) and recent (n = 81) HIV-1 subtype C infection and measured their in vitro RC using a green fluorescent protein (GFP) reporter T cell assay. In recently infected individuals, reverse transcriptase (RT)-integrase-driven RC correlated significantly with viral load set point (r = 0.25; P = 0.03) and CD4+ T cell decline (P = 0.013). Moreover, significant associations between RT integrase-driven RC and viral load (r = 0.28; P < 0.0001) and CD4+ T cell count (r = -0.29; P < 0.0001) remained in chronic infection. In early HIV infection, host expression of the protective HLA-B*81 allele was associated with lower RC (P = 0.05), as was expression of HLA-B*07 (P = 0.02), suggesting early immune-driven attenuation of RT-integrase by these alleles. In chronic infection, HLA-A*30:09 (in linkage disequilibrium with HLA-B*81) was significantly associated with lower RC (P = 0.05), and all 6 HLA-B alleles with the lowest RC measurements represented protective alleles, consistent with long-term effects of host immune pressures on lowering RT-integrase RC. The polymorphisms V241I, I257V, P272K, and E297K in reverse transcriptase and I201V in integrase, all relatively uncommon polymorphisms occurring in or adjacent to optimally described HLA-restricted cytotoxic T-lymphocyte epitopes, were associated with reduced RC. Together, our data suggest that RT-integrase-driven RC is clinically relevant and provide evidence that immune-driven selection of mutations in RT-integrase can compromise RC.IMPORTANCE Identification of viral mutations that compromise HIV's ability to replicate may aid rational vaccine design. However, while certain escape mutations in Gag have been shown to reduce HIV replication and influence clinical progression, less is known about the consequences of mutations that naturally arise in other HIV proteins. Pol is a highly conserved protein, but the impact of Pol function on HIV disease progression is not well defined. Here, we generated recombinant viruses using the RT-integrase region of Pol derived from HIV-1C-infected individuals with recent and chronic infection and measured their ability to replicate in vitro We demonstrate that RT-integrase-driven replication ability significantly impacts HIV disease progression. We further show evidence of immune-mediated attenuation in RT-integrase and identify specific polymorphisms in RT-integrase that significantly decrease HIV-1 replication ability, suggesting which Pol epitopes could be explored in vaccine development.


Assuntos
Infecções por HIV/genética , Integrase de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Alelos , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Estudos de Coortes , Progressão da Doença , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica , Genes Reporter , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Integrase de HIV/imunologia , Transcriptase Reversa do HIV/imunologia , HIV-1/classificação , HIV-1/imunologia , HIV-1/patogenicidade , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Transdução de Sinais , Carga Viral , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
20.
J Biol Chem ; 293(35): 13351-13363, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29991591

RESUMO

During reverse transcription of the HIV-1 genome, two strand-transfer events occur. Both events rely on the RNase H cleavage activity of reverse transcriptases (RTs) and template homology. Using a panel of mutants of HIV-1BH10 (group M/subtype B) and HIV-1ESP49 (group O) RTs and in vitro assays, we demonstrate that there is a strong correlation between RT minus-strand transfer efficiency and template-primer binding affinity. The highest strand transfer efficiencies were obtained with HIV-1ESP49 RT mutants containing the substitutions K358R/A359G/S360A, alone or in combination with V148I or T355A/Q357M. These HIV-1ESP49 RT mutants had been previously engineered to increase their DNA polymerase activity at high temperatures. Now, we found that RTs containing RNase H-inactivating mutations (D443N or E478Q) were devoid of strand transfer activity, whereas enzymes containing F61A or L92P had very low strand transfer activity. The strand transfer defect produced by L92P was attributed to a loss of template-primer binding affinity and, more specifically, to the higher dissociation rate constants (koff) shown by RTs bearing this substitution. Although L92P also deleteriously affected the RT's nontemplated nucleotide addition activity, neither nontemplated nucleotide addition activity nor the RT's clamp activities contributed to increased template switching when all tested mutant and WT RTs were considered. Interestingly, our results also revealed an association between efficient strand transfer and the generation of secondary cleavages in the donor RNA, consistent with the creation of invasion sites. Exposure of the elongated DNA at these sites facilitate acceptor (RNA or DNA) binding and promote template switching.


Assuntos
DNA Viral/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/metabolismo , Ribonuclease H/metabolismo , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Mutação Puntual , Ligação Proteica , RNA Viral/metabolismo , Moldes Genéticos
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