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1.
J Med Chem ; 63(9): 4837-4848, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32293182

RESUMO

The development of efficacious NNRTIs for AIDS therapy commonly encountered the rapid generation of drug-resistant mutations, which becomes a major impediment to effective anti-HIV treatment. Using a structure-based bioisosterism strategy, a series of piperidine-substituted thiophene[2,3-d]pyrimidine derivatives were designed and synthesized. Compound 9a yielded the greatest potency, exhibiting significantly better anti-HIV-1 activity than ETR against all of the tested NNRTI-resistant HIV-1 strains. In addition, the phenotypic (cross)resistance of 9a and other NRTIs to the different selected HIV-1 strains was evaluated. As expected, no phenotypic cross-resistance against the NRTIs (AZT and PMPA) was observed with the mutant 9ares strain. Furthermore, 9a was identified with improved solubility, lower CYP liability, and hERG inhibition. Remarkably, 9a exhibited optimal pharmacokinetic properties in rats (F = 37.06%) and safety in mice (LD50 > 2000 mg/kg), which highlights 9a as a promising anti-HIV-1 drug candidate.


Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Tiofenos/farmacologia , Animais , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacocinética , Linhagem Celular Tumoral , Ensaios Enzimáticos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/farmacocinética , Tiofenos/metabolismo , Tiofenos/farmacocinética
2.
PLoS One ; 15(3): e0229275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119691

RESUMO

BACKGROUND: The presence of drug resistance mutations (DRMs) against antiretroviral agents is one of the main concerns in the clinical management of individuals with human immunodeficiency virus-1 (HIV-1) infection, especially in regions of the world where treatment options are limited. The current study aimed at assessing the prevalence of HIV-1 DRMs among naïve and treatment-experienced HIV-1-infected patients in Iran. METHODS: From April 2013 to September 2018, the HIV-1 protease and reverse transcriptase genes were amplified and sequenced in plasma specimens of 60 newly diagnosed antiretroviral-naive individuals and 46 participants receiving antiretroviral therapies (ARTs) for at least six months with an HIV viral load of more than 1000 IU/mL to determine the HIV-1 DRMs and subtypes. RESULTS: Among the 60 treatment-naïve HIV-1-infected participants, 8.3% were infected with HIV-1 variants with surveillance DRMs (SDRMs). The SDRMs, D67N and D67E, belonged to the NRTIs class in two patients and K103N and V106A belonged to the NNRTIs class in three patients. The phylogenetic analysis showed that 91.7% of the subjects were infected with subtype CRF35_AD, followed by subtype B (5.0%) and CRF01_AE (3.3%). Among the 46 ART-experienced participants, 33 (71.7%) carried HIV-1 variants with SDRMs (9.1% against PIs, 78.8% against NRTIs, and 100% against NNRTIs). M46I and I47V were the most common mutations for PIs, M184V was the most common mutation for the NRTIs, and K103N/S was the most common mutation for NNRTIs. Phylogenetic analysis of the polymerase region showed that all of the 46 HIV-1-infected patients who failed on ART carried CRF35_AD. CONCLUSIONS: The moderate prevalence of SDRMs (8.3%) in treatment-naïve and ART-failed (77.1%) Iranian patients with HIV-1-infection emphasizes the need for systematic viral load monitoring, expanding drug resistance testing, carefully surveilling individuals on ART regimens, and facilitating access to new antiretrovirals by health authorities.


Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Mutação , Adolescente , Adulto , Fármacos Anti-HIV/uso terapêutico , Criança , Pré-Escolar , Estudos Transversais , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/classificação , HIV-1/fisiologia , Humanos , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , Inibidores da Transcriptase Reversa/uso terapêutico , Análise de Sequência de RNA , Carga Viral , Adulto Jovem
3.
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
4.
BMC Infect Dis ; 20(1): 123, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32046664

RESUMO

BACKGROUND: The rate of S68G mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase has increased and is closely related to the K65R mutation among CRF01_AE-infected patients who failed treatment. We aimed to explore the temporal association of S68G and K65R mutations and disclose the role of the former on susceptibility to nucleotide/nucleoside reverse transcriptase inhibitor (NRTI) and viral replication with the K65R double mutations among CRF01_AE-infected patients who failed treatment. METHODS: The occurrence of S68G and K65R mutations was evaluated among HIV-1 of various subtypes in the global HIV Drug Resistance Database. The temporal association of S68G and K65R mutations was analyzed through next-generation sequencing in four CRF01_AE-infected patients who failed treatment with tenofovir/lamivudine/efavirenz. The impact of the S68G mutation on susceptibility to NRTI and replication fitness was analyzed using pseudovirus phenotypic resistance assays and growth competition assays, respectively. RESULTS: The frequency of the S68G mutation increased by 1.4-9.7% in almost all HIV subtypes and circulating recombinant forms in treatment-experienced patients, except subtype F. The S68G mutation often occurred in conjunction with the K65R mutation among RTI-treated patients, with frequencies ranging 21.1-61.7% in various subtypes. Next-generation sequencing revealed that the S68G mutation occurred following the K65R mutation in three of the four CRF01_AE-infected patients. In these three patients, there was no significant change detected in the half maximal inhibitory concentration for zidovudine, tenofovir, or lamivudine between the K65R and K65R/S68G mutations, as demonstrated by the phenotypic resistance assays. Virus stocks of the K65R and K65R/S68G mutations were mixed with 4:6, 1:1, and 9:1 and cultured for 13 days, the K65R/S68G mutants outgrew those of the K65R mutants irrespective of the input ratio. CONCLUSIONS: S68G may be a natural polymorphism and compensatory mutation of K65R selected by NRTIs in the CRF01_AE strain of HIV-1. This mutation does not affect susceptibility to NRTI; however, it improves the replication fitness of K65R mutants. This study deciphers the role of the S68G mutation in the HIV reverse transcriptase of the CRF01_AE strain and provides new evidence for the interpretation of drug-resistant mutations in non-B subtypes of HIV-1.


Assuntos
Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , Mutação , Fármacos Anti-HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Humanos , Lamivudina/uso terapêutico , Taxa de Mutação , Polimorfismo Genético , Inibidores da Transcriptase Reversa/uso terapêutico , Tenofovir/uso terapêutico , Falha de Tratamento , Replicação Viral/efeitos dos fármacos , Zidovudina/uso terapêutico
5.
Int J Mol Sci ; 21(3)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979356

RESUMO

Human Immunodeficiency Virus Type 1 (HIV-1) infection is associated with high mortality if no therapy is provided. Currently, the treatment of an HIV-1 positive patient requires that several drugs should be taken simultaneously. The resistance of the virus to an antiretroviral drug may lead to treatment failure. Our approach focuses on predicting the exposure of a particular viral variant to an antiretroviral drug or drug combination. It also aims at the prediction of drug treatment success or failure. We utilized nucleotide sequences of HIV-1 encoding protease and reverse transcriptase to perform such types of prediction. The PASS (Prediction of Activity Spectra for Substances) algorithm based on the naive Bayesian classifier was used to make a prediction. We calculated the probability of whether a sequence belonged (P1) or did not belong (P0) to the class associated with exposure of the viral sequence to the set of drugs that can be associated with resistance to the set of drugs. The accuracy calculated as the average Area Under the ROC (Receiver Operating Characteristic) Curve (AUC/ROC) for classifying exposure of the sequence to the HIV-1 protease inhibitors was 0.81 (±0.07), and for HIV-1 reverse transcriptase, it was 0.83 (±0.07). To predict cases of treatment effectiveness or failure, we used P1 and P0 values, obtained in PASS, along with the binary vector constructed based on short nucleotide descriptors and the applied random forest classifier. Average AUC/ROC prediction accuracy for the prediction of treatment effectiveness or failure for the combinations of HIV-1 protease inhibitors was 0.82 (±0.06) and of HIV-1 reverse transcriptase was 0.76 (±0.09).


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Algoritmos , Fármacos Anti-HIV/farmacologia , Área Sob a Curva , Teorema de Bayes , Farmacorresistência Viral , Quimioterapia Combinada , Protease de HIV/química , Protease de HIV/genética , Inibidores da Protease de HIV/farmacologia , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Inibidores da Transcriptase Reversa/farmacologia , Falha de Tratamento , Resultado do Tratamento
7.
Commun Biol ; 2: 469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31872074

RESUMO

Emtricitabine (FTC) and lamivudine (3TC), containing an oxathiolane ring with unnatural (-)-stereochemistry, are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in anti-HIV therapy. Treatment with FTC or 3TC primarily selects for the HIV-1 RT M184V/I resistance mutations. Here we provide a comprehensive kinetic and structural basis for inhibiting HIV-1 RT by (-)-FTC-TP and (-)-3TC-TP and drug resistance by M184V. (-)-FTC-TP and (-)-3TC-TP have higher binding affinities (1/K d) for wild-type RT but slower incorporation rates than dCTP. HIV-1 RT ternary crystal structures with (-)-FTC-TP and (-)-3TC-TP corroborate kinetic results demonstrating that their oxathiolane sulfur orients toward the DNA primer 3'-terminus and their triphosphate exists in two different binding conformations. M184V RT displays greater (>200-fold) K d for the L-nucleotides and moderately higher (>9-fold) K d for the D-isomers compared to dCTP. The M184V RT structure illustrates how the mutation repositions the oxathiolane of (-)-FTC-TP and shifts its triphosphate into a non-productive conformation.


Assuntos
Farmacorresistência Viral , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Nucleotídeos/química , Inibidores da Transcriptase Reversa/química , Alelos , Substituição de Aminoácidos , Bases de Dados Genéticas , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Mutação , Nucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia
8.
Oxid Med Cell Longev ; 2019: 6016278, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885806

RESUMO

HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.


Assuntos
Adenocarcinoma/virologia , Neoplasias da Mama/virologia , Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/metabolismo , Neoplasias Mamárias Experimentais/virologia , Proteína 1 Relacionada a Twist/metabolismo , Animais , Carcinogênese , Processos de Crescimento Celular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Infecções por HIV/patologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Metástase Neoplásica , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 Relacionada a Twist/genética , Regulação para Cima
9.
Acta Med Indones ; 51(3): 197-204, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31699942

RESUMO

BACKGROUND: the global scale-up of antiretroviral therapy (ART) is the primary factor contributing to the decline in deaths from acquired immune deficiency syndrome (AIDS)-related illnesses. However, the emergence of transmitted drug resistance (TDR) compromises the effects of ART in treatment-naïve individuals, which may hinder treatment success. The present study aimed to identify the presence of TDR among treatment-naive individuals in Buleleng, Bali, which is currently ranked sixth among Indonesian provinces with the highest cumulative human immunodeficiency virus type 1 (HIV-1) infection cases. METHODS: thirty-nine ART-naive individuals in Buleleng Regency General Hospital were enrolled in the present study. Blood samples from participants were subjected to a genotypic analysis. RESULTS: 28 protease (PR) and 30 reverse transcriptase (RT) genes were successfully amplified and sequenced from 37 samples. HIV-1 subtyping revealed CRF01_AE as the dominant circulating recombinant form in the region. No TDR for PR inhibitors was detected; however, TDR for RT inhibitors was identified in five out of 30 samples (16.7%). CONCLUSION: these results indicate the emergence of TDR among ART-naive individuals in Buleleng, Bali. This issue warrants serious consideration because TDR may hamper treatment success and reduce ART efficacy among newly diagnosed individuals. Continuous surveillance with a larger sample size is necessary to monitor TDR among ART-naive individuals.


Assuntos
Farmacorresistência Viral , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Adolescente , Adulto , Terapia Antirretroviral de Alta Atividade , Feminino , Genótipo , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , Humanos , Indonésia/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação , Adulto Jovem
10.
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
11.
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
12.
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
13.
J Med Chem ; 62(4): 2083-2098, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30721060

RESUMO

Diarylpyrimidine derivatives (DAPYs) exhibit robust anti-HIV-1 potency, although they have been compromised by E138K variant and severe side-effects and been suffering from poor water solubility. In the present work, hydrophilic morpholine or methylsulfonyl and sulfamide-substituted piperazine/piperidines were introduced into the right wing of DAPYs targeting the solvent-exposed tolerant region I. The anti-HIV-1 activities of 11c (EC50(WT) = 0.0035 µM, EC50(E138K) = 0.0075 µM) were the same as and 2-fold better than that of the lead etravirine against the wild-type and E138K mutant HIV-1, respectively, with a relative low cytotoxicity (CC50 ≥ 173 µM). Further test showed a significant improvement in the water solubility of 11c. Besides, 11c displayed no significant inhibition on main cytochrome P450 enzymes and exhibited no acute/subacute toxicities at doses of 2000 mg·kg-1/50 mg·kg-1 in mice. Taken together, we consider that 11c is a promising lead for further structural optimization.


Assuntos
Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Feminino , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Pirimidinas/síntese química , Pirimidinas/toxicidade , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/toxicidade , Solubilidade , Relação Estrutura-Atividade
14.
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
15.
AIDS Res Hum Retroviruses ; 35(5): 505-508, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30681008

RESUMO

HIV-2 is important due to its unique challenges in diagnosis, treatment, and drug resistance. The data on Indian HIV-2 pol gene as well as resistance to antiretroviral drugs are limited. Here we report sequence data of protease (PR) and reverse transcriptase (RT) genes from HIV-2 infected treatment naive individuals (N = 32) from Maharashtra, India. These sequences were found to be closely related to HIV-2 subtype A sequences from Guinea Bissau. We observed two unique residues at positions 14 and 70 in the PR region specific to Indian HIV-2. Mutations associated with resistance to RT and protease inhibitors were observed in 3 of 32 (9.37%) samples. To our knowledge, this is the first study from India to report drug resistance among treatment naive HIV-2 infected individuals. The results emphasize need for larger nationwide surveillance for HIV-2 drug resistance to better understand the primary drug resistance among HIV-2 infected individuals.


Assuntos
Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-2/genética , Mutação , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética , Adolescente , Adulto , Idoso , Feminino , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-2/isolamento & purificação , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
16.
AIDS Res Hum Retroviruses ; 35(4): 407-413, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30229669

RESUMO

Manado, the capital city of North Sulawesi, is a unique region in Indonesia because of its religion. We collected peripheral blood samples from 63 individuals on antiretroviral therapy. The amplification of viral genomic fragments, viral subtyping, detection of HIV drug resistance-associated mutations (DRAMs), and phylogenetic analyses were performed. Viral subtyping revealed that the most prevalent HIV type 1 (HIV-1) subtype/circulating recombinant form (CRF) was CRF01_AE (84.1%), followed by subtype B (6.8%) and recombinants between CRF01_AE and CRF02_AG (4.5%). Although no major DRAMs were present in protease genes, they were detected in reverse transcriptase (RT) genes. Nine of 38 samples (23.7%) had major DRAMs against nucleoside RT inhibitors (NRTIs) and/or non-NRTIs. The results of phylogenetic analyses indicated that CRF01_AE in North Sulawesi is related to that in Bali. Therefore, Bali may play an important role in circulating CRF01_AE in North Sulawesi.


Assuntos
Farmacorresistência Viral , Variação Genética , Infecções por HIV/virologia , HIV-1/genética , Adulto , Feminino , Genótipo , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Humanos , Indonésia , Masculino , Filogenia
17.
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
18.
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 Fármacos , 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
19.
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
20.
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
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