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1.
Phys Chem Chem Phys ; 22(8): 4464-4480, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32057044

RESUMO

Infection by human immunodeficiency virus type 1 (HIV-1) not only destroys the immune system bringing about acquired immune deficiency syndrome (AIDS), but also induces serious neurological diseases including behavioral abnormalities, motor dysfunction, toxoplasmosis, and HIV-1 associated dementia. The emergence of HIV-1 multidrug-resistant mutants has become a major problem in the therapy of patients with HIV-1 infection. Focusing on the wild type (WT) and G48T/L89M mutated forms of HIV-1 protease (HIV-1 PR) in complex with amprenavir (APV), indinavir (IDV), ritonavir (RTV), and nelfinavir (NFV), we have investigated the conformational dynamics and the resistance mechanism due to the G48T/L89M mutations by conducting a series of molecular dynamics (MD) simulations and free energy (MM-PBSA and solvated interaction energy (SIE)) analyses. The simulation results indicate that alterations in the side-chains of G48T/L89M mutated residues cause the inner active site to increase in volume and induce more curling of the flap tips, which provide the main contributions to weaker binding of inhibitors to the HIV-1 PR. The results of energy analysis reveal that the decrease in van der Waals interactions of inhibitors with the mutated PR relative to the wild-type (WT) PR mostly drives the drug resistance of mutations toward these four inhibitors. The energy decomposition analysis further indicates that the drug resistance of mutations can be mainly attributed to the change in van der Waals and electrostatic energy of some key residues (around Ala28/Ala28' and Ile50/Ile50'). Our work can give significant guidance to design a new generation of anti-AIDS inhibitors targeting PR in the therapy of patients with HIV-1 infection.


Assuntos
Protease de HIV/metabolismo , Simulação de Dinâmica Molecular , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Carbamatos/química , Carbamatos/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Protease de HIV/genética , Indinavir/química , Indinavir/metabolismo , Conformação Molecular , Mutação , Nelfinavir/química , Nelfinavir/metabolismo , Ligação Proteica , Ritonavir/química , Ritonavir/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo
2.
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
3.
J Chem Theory Comput ; 16(2): 1284-1299, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-31877249

RESUMO

Over the past several decades, atomistic simulations of biomolecules, whether carried out using molecular dynamics or Monte Carlo techniques, have provided detailed insights into their function. Comparing the results of such simulations for a few closely related systems has guided our understanding of the mechanisms by which changes such as ligand binding or mutation can alter the function. The general problem of detecting and interpreting such mechanisms from simulations of many related systems, however, remains a challenge. This problem is addressed here by applying supervised and unsupervised machine learning techniques to a variety of thermodynamic observables extracted from molecular dynamics simulations of different systems. As an important test case, these methods are applied to understand the evasion by human immunodeficiency virus type-1 (HIV-1) protease of darunavir, a potent inhibitor to which resistance can develop via the simultaneous mutation of multiple amino acids. Complex mutational patterns have been observed among resistant strains, presenting a challenge to developing a mechanistic picture of resistance in the protease. In order to dissect these patterns and gain mechanistic insight into the role of specific mutations, molecular dynamics simulations were carried out on a collection of HIV-1 protease variants, chosen to include highly resistant strains and susceptible controls, in complex with darunavir. Using a machine learning approach that takes advantage of the hierarchical nature in the relationships among the sequence, structure, and function, an integrative analysis of these trajectories reveals key details of the resistance mechanism, including changes in the protein structure, hydrogen bonding, and protein-ligand contacts.


Assuntos
Farmacorresistência Viral , Protease de HIV/metabolismo , HIV-1/enzimologia , Ligantes , Aprendizado de Máquina , Protease de HIV/química , Protease de HIV/genética , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Humanos , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Método de Monte Carlo , Mutação , Ligação Proteica , Eletricidade Estática
4.
Molecules ; 24(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31489889

RESUMO

HIV protease inhibitors against the viral protease are often hampered by drug resistance mutations in protease and in the viral substrate Gag. To overcome this drug resistance and inhibit viral maturation, targeting Gag alongside protease rather than targeting protease alone may be more efficient. In order to successfully inhibit Gag, understanding of its drug resistance mutations and the elicited structural changes on protease binding needs to be investigated. While mutations on Gag have already been mapped to protease inhibitor resistance, there remain many mutations, particularly the non-cleavage mutations, that are not characterized. Through structural studies to unravel how Gag mutations contributes to protease drug resistance synergistically, it is thus possible to glean insights to design novel Gag inhibitors. In this review, we discuss the structural role of both novel and previously reported Gag mutations in PI resistance, and how new Gag inhibitors can be designed.


Assuntos
Farmacorresistência Viral , HIV-1/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Desenho de Drogas , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Relação Estrutura-Atividade
5.
J Enzyme Inhib Med Chem ; 34(1): 1451-1456, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409143

RESUMO

Herein, we report the effect of nine FDA approved protease inhibitor drugs against a new HIV-1 subtype C mutant protease, E35D↑G↑S. The mutant has five mutations, E35D, two insertions, position 36 (G and S), and D60E. Kinetics, inhibition constants, vitality, Gibbs free binding energies are reported. The variant showed a decreased affinity for substrate and low catalytic efficiency compared to the wild type. There was a significant decrease in the binding of seven FDA approved protease inhibitors against the mutant (p < .0001). Amprenavir and ritonavir showed the least decrease, but still significant reduced activity in comparison to the wildtype (4 and 5 folds, respectively, p = .0021 and .003, respectively). Nelfinavir and atazanavir were the worst inhibitors against the variant as seen from the IC50, with values of 1401 ± 3.0 and 685 ± 3.0 nM, respectively. Thermodynamics data showed less favourable Gibbs free binding energies for the protease inhibitors to the mutant.


Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/efeitos dos fármacos , HIV-1/enzimologia , Termodinâmica , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Concentração Inibidora 50 , Cinética , Simulação de Acoplamento Molecular , Mutação
6.
Intervirology ; 62(2): 72-79, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31311021

RESUMO

BACKGROUND: The therapeutic effect of antiretroviral therapy (ART) is adversely influenced by antiretroviral drug resistance, mainly due to mutations (DRMs) in the human immunodeficiency virus (HIV) genome. These mutations are commonly associated with HIV protease and reverse-transcriptase genes. We sought to determine the frequency of DRMs in a population of ART-experienced patients in the South of Iran. METHOD: A total of 44 HIV-1-positive participants under ART were selected from April 2016 to March 2017. Their DRMs, antiretroviral resistance status, and viral subtypes were determined. RESULTS: At least one DRM was detected in 61.4% of the participants. The highest frequency was related to nucleotide reverse-transcriptase inhibitor (NRTI) mutations (45.45%). In contrast, major protease inhibitor (PI) mutations had the lowest frequency (6.81%). M184V (40.9%) and K103N (25%), respectively related to NRTI and nonnucleoside reverse-transcriptase inhibitor (NNRTI), were the mutations with the highest frequencies. Susceptibility to PI drugs was higher compared to NRTIs and NNRTIs, which was consistent with the results of genotypic DRMs. CONCLUSION: The highest frequency of antiretroviral DRMs was related to NRTIs and NNRTIs. In contrast, PI resistance mutations had the lowest frequency. Laboratory-guided ART to avoid the expansion of mutants as well as investigating DRMs in other viral regions, such as integrase, are recommended.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral Múltipla/genética , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Mutação , Adulto , Estudos Transversais , Feminino , Genótipo , Protease de HIV/genética , Humanos , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Inibidores da Transcriptase Reversa/uso terapêutico , Adulto Jovem
7.
J Biomol NMR ; 73(6-7): 365-374, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31243634

RESUMO

Over the last two decades, both the sensitivity of NMR and the time scale of molecular dynamics (MD) simulation have increased tremendously and have advanced the field of protein dynamics. HIV-1 protease has been extensively studied using these two methods, and has presented a framework for cross-evaluation of structural ensembles and internal dynamics by integrating the two methods. Here, we review studies from our laboratories over the last several years, to understand the mechanistic basis of protease drug-resistance mutations and inhibitor responses, using NMR and crystal structure-based parallel MD simulations. Our studies demonstrate that NMR relaxation experiments, together with crystal structures and MD simulations, significantly contributed to the current understanding of structural/dynamic changes due to HIV-1 protease drug resistance mutations.


Assuntos
Inibidores da Protease de HIV/química , Protease de HIV/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Sítios de Ligação , Farmacorresistência Viral , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , Humanos , Mutação , Ligação Proteica , Relação Estrutura-Atividade , Água/química
8.
Bioorg Med Chem ; 27(9): 1863-1870, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30922618

RESUMO

New heteroaryl HIV-protease inhibitors bearing a carbamoyl spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core that can have either H or benzyl group. The in vitro inhibition activity against recombinant protease showed a general beneficial effect of both carbamoyl moiety and the benzyl group, ranging the IC50 values between 11 and 0.6 nM. In particular, benzofuryl and indolyl derivatives showed IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such derivatives in terms of number of interactions in the active site, supporting the experimental results. The inhibition activity was also confirmed in HEK293 mammalian cells and was maintained against protease mutants. Furthermore, the metabolic stability was comparable with that of the commercially available inhibitors.


Assuntos
Carbamatos/química , Inibidores da Protease de HIV/síntese química , Protease de HIV/genética , Sítios de Ligação , Carbamatos/farmacologia , Domínio Catalítico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HEK293 , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Mutação
9.
Mol Biol Evol ; 36(4): 798-810, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30721995

RESUMO

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available data set of 32,163 sequenced isolates from drug-naïve individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple-base mutations in HIV-1 protease is strongly influenced by mutational sampling.


Assuntos
Evolução Molecular , Protease de HIV/genética , HIV-1/genética , Mutação Puntual , Código Genético , Seleção Genética
10.
Protein J ; 38(1): 30-36, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30666487

RESUMO

HIV-1 is an infectious virus that causes acquired immunodeficiency syndrome (AIDS) and it is one of the major causes of deaths worldwide. The production of HIV-1 protease (PR) on a large scale has been a problem for scientists due to its cytotoxicity, low yield, insolubility, and low activity. HIV-1 C-SA protease has been cloned, expressed, and purified previously, however, with low recovery (0.25 mg/L). Herein we report an optimal expression and solubilisation procedure to recover active HIV-1 C-SA protease enzyme from inclusion bodies. The HIV protease was expressed in seven different vectors (pET11b, pET15b, pET28a pET32a, pET39b, pET41b and pGEX 6P-1). The highest expression was achieved when the vector pET32a (Trx tag) was employed. A total of 19.5 mg of fusion protein was refolded of which 5.5 mg of active protease was obtained after cleavage. The free protease had a high specific activity of 2.81 µmoles/min/mg. Interestingly the Trx-fusion protein also showed activity closer (1.24 µmoles/min/mg) to that of the free protease suggesting that the pET32a vector (Trx tag) expressed in BL21(DE3) pLysS provides a more efficient way to obtain HIV-1 protease.


Assuntos
Protease de HIV/química , Protease de HIV/isolamento & purificação , HIV-1/enzimologia , Corpos de Inclusão/enzimologia , Escherichia coli/química , Escherichia coli/genética , Protease de HIV/genética , HIV-1/genética , Corpos de Inclusão/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
11.
Arch Virol ; 164(4): 949-960, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30680529

RESUMO

HIV is one of the most lethal viral diseases in the human population. Patients often suffer from drug resistance, which hampers HIV therapy. Eleven different structural analogues of saquinavir (SQV), designed using ChemSketch™ and named S1 through S11, were compared with SQV with respect to their pharmacodynamic and pharmacokinetic properties. Pharmacokinetic predictions were carried out using AutoDock, and molecular docking between macromolecule HIV protease (PDB ID: 3IXO) and analogues S1 - S11 as ligands was performed. Analogues S1, S3, S4, S9 and S11 had lower binding scores when compared with saquinavir, whereas that of analogue S5 was similar. Pharmacokinetic predictions made using ACDilab2, including the Lipinski profile, general physical features, absorption, distribution, metabolism and excretion parameters, and toxicity values, for the eleven analogues and SQV suggested that S1 and S5 are pharmacodynamically and pharmacokinetically robust molecules that could be developed and established as lead molecules after in vitro and in vivo studies.


Assuntos
Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacocinética , HIV-1/enzimologia , Saquinavir/análogos & derivados , Saquinavir/farmacocinética , Animais , Infecções por HIV/virologia , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Inibidores da Protease de HIV/administração & dosagem , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Camundongos , Simulação de Acoplamento Molecular , Saquinavir/administração & dosagem
12.
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
13.
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
14.
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
15.
ACS Infect Dis ; 5(2): 316-325, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30543749

RESUMO

HIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82, and I84 that line the S1/S1' pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogues to primary S1' mutations. The analogues had modifications at the hydrophobic P1' moiety compared to DRV to better occupy the unexploited space in the S1' pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analogue with the largest P1' moiety, indicating interdependency between the S1' subsite and the flap region. Collective analysis of protease-inhibitor interactions in the crystal structures using principle component analysis was able to distinguish inhibitor identity and relative potency solely based on van der Waals contacts. Our results reveal the complexity of the interplay between inhibitor P1' moiety and S1' mutations and validate principle component analyses as a useful tool for distinguishing resistance and inhibitor potency.


Assuntos
Darunavir/análogos & derivados , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Darunavir/química , HIV-1/enzimologia , Humanos , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Especificidade por Substrato
16.
Biochem J ; 476(2): 375-384, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30573649

RESUMO

HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38↑N↑L) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naïve infant. Isothermal titration calorimetry showed that 10% less of L38↑N↑L protease was in the active conformation as compared with a reference strain. L38↑N↑L protease displayed a ±50% reduction in K M and k cat The catalytic efficiency (k cat/K M) of L38↑N↑L protease was not significantly different from that of wild type although there was a 42% reduction in specific activity for the variant. An in vitro phenotypic assay showed the L38↑N↑L protease to be susceptible to lopinavir (LPV), atazanavir (ATV) and darunavir in the context of an unrelated Gag. However, in the presence of the related Gag, L38↑N↑L showed reduced susceptibility to darunavir while remaining susceptible to LPV and ATV. Furthermore, a reduction in viral replication capacity (RC) was observed in combination with the related Gag. The reduced susceptibility to darunavir and decrease in RC may be due to PTAPP duplication in the related Gag. The present study shows the importance of considering the Gag region when looking at drug susceptibility of HIV-1 protease variants.


Assuntos
Darunavir/química , Inibidores da Protease de HIV/química , Protease de HIV/química , Protease de HIV/genética , HIV-1 , Lopinavir/química , Mutagênese Insercional , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Darunavir/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Infecções por HIV/genética , Protease de HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , Humanos , Lopinavir/farmacologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
17.
Sci Rep ; 8(1): 17938, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560871

RESUMO

The use of antiretrovirals (ARVs) has drastically improved the life quality and expectancy of HIV patients since their introduction in health care. Several millions are still afflicted worldwide by HIV and ARV resistance is a constant concern for both healthcare practitioners and patients, as while treatment options are finite, the virus constantly adapts via complex mutation patterns to select for resistant strains under the pressure of drug treatment. The HIV protease is a crucial enzyme for viral maturation and has been a game changing drug target since the first application. Due to similarities in protease inhibitor designs, drug cross-resistance is not uncommon across ARVs of the same class. It is known that resistance against protease inhibitors is associated with a wider active site, but results from our large scale molecular dynamics simulations combined with statistical tests and network analysis further show, for the first time, that there are regions of local expansions and compactions associated with high levels of resistance conserved across eight different protease inhibitors visible in their complexed form within closed receptor conformations. The observed conserved expansion sites may provide an alternative drug-targeting site. Further, the method developed here is novel, supplementary to methods of variation analysis at sequence level, and should be applicable in analysing the structural consequences of mutations in other contexts using molecular ensembles.


Assuntos
Farmacorresistência Viral , Inibidores da Protease de HIV/química , Protease de HIV/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Sítios de Ligação , Desenho de Drogas , Protease de HIV/genética , Inibidores da Protease de HIV/farmacologia , Humanos , Conformação Molecular , Mutação , Ligação Proteica , Relação Estrutura-Atividade , Fluxo de Trabalho
18.
PLoS One ; 13(11): e0206234, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30408827

RESUMO

BACKGROUND: Molecular surveillance of newly diagnosed HIV-infections is important for tracking trends in circulating HIV-variants, including those with transmitted drug resistances (TDR) to sustain ART efficacy. METHODS: Dried serum spots (DSS) are received together with the statutory notification of a new diagnosis. 'Recent infections' (<155 days) classified by a 'recent infection test algorithm' (BED-CEIA and clinical data) are genotyped in HIV-protease (PR), reverse transcriptase (RT) and integrase (INT) to determine the HIV-1 subtype, to calculate prevalence and trends of TDR, to predict baseline susceptibility and to identify potential transmission clusters for resistant variants. RESULTS: Between January 2013 and December 2016, 1,885 recent infections were analysed regarding the PR/RT genomic region, with 43.5% of these also being subjected to the analysis of INT. The proportion of HIV-1 non-B viruses (31.3%; 591/1,885) increased from 21.6% to 36.0%, particularly the subtypes A (5.0% to 8.3%) and C (3.2% to 7.7%; all ptrends < 0.01). The subtype A increment is mainly due to transmissions within men who have sex with men (MSM) while subtype C transmissions are associated with heterosexuals and people who inject drugs. The prevalence of TDR was stable at 11.0% (208/1,885) over the study period. Resistances to nucleotide RT inhibitors (NRTI) and PR inhibitors (PI) were 4.5% and 3.2%, respectively, without identifiable trends. In contrast, resistances to non-NRTIs (NNRTI, 4.7%) doubled between 2014 and 2016 from 3.2% to 6.4% (ptrend = 0.02) mainly due to the K103N mutation (from 1.7% to 4.1%; ptrend = 0.03) predominantly detected in recently infected German MSM not linked to transmission clusters. Transmitted INSTI mutations were present in only one case (T66I) and resistance to dolutegravir was not identified at all. Reduced susceptibility to recommended first-line therapies was low with 1.0% for PIs, 1.3% for NRTIs and 0.7% for INSTIs, but high for the NNRTIs efavirence (4.9%) and rilpivirine (6.0%) due to the K103N mutation and the polymorphic mutation E138A. These trends in therapy-naïve individuals impact current first-line regimens and require awareness and vigilant surveillance.


Assuntos
Infecções por HIV/epidemiologia , HIV-1/patogenicidade , Inibidores da Transcriptase Reversa/uso terapêutico , Adulto , Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral/genética , Feminino , Genótipo , Alemanha/epidemiologia , Infecções por HIV/diagnóstico , Infecções por HIV/genética , Infecções por HIV/virologia , Protease de HIV/genética , Homossexualidade Masculina/genética , Humanos , Masculino , Mutação , Minorias Sexuais e de Gênero
19.
BMC Bioinformatics ; 19(Suppl 11): 362, 2018 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-30343664

RESUMO

BACKGROUND: Drug resistance in HIV is the major problem limiting effective antiviral therapy. Computational techniques for predicting drug resistance profiles from genomic data can accelerate the appropriate choice of therapy. These techniques can also be used to select protease mutants for experimental studies of resistance and thereby assist in the development of next-generation therapies. RESULTS: The machine learning produced highly accurate and robust classification of HIV protease resistance. Genotype data were mapped to the enzyme structure and encoded using Delaunay triangulation. Generative machine learning models trained on one inhibitor could classify resistance from other inhibitors with varying levels of accuracy. Generally, the accuracy was best when the inhibitors were chemically similar. CONCLUSIONS: Restricted Boltzmann Machines are an effective machine learning tool for classification of genomic and structural data. They can also be used to compare resistance profiles of different protease inhibitors.


Assuntos
Farmacorresistência Viral/genética , Protease de HIV/genética , Algoritmos , Bases de Dados como Assunto , Farmacorresistência Viral/efeitos dos fármacos , Genótipo , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Humanos , Aprendizado de Máquina , Análise de Componente Principal
20.
SAR QSAR Environ Res ; 29(11): 911-933, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30332922

RESUMO

Multiple Quantitative Structure-Activity Relationship (QSAR) analysis is widely used in drug discovery for lead identification. Human Immunodeficiency Virus (HIV) protease is one of the key targets for the treatment of Acquired Immunodeficiency Syndrome (AIDS). One of the major challenges for the design of HIV-1 protease inhibitors (HIV PRIs) is to increase the inhibitory activities against the enzyme to a level where the problem associated to drug resistance may be considerably delayed. Herein, chemometric analyses were performed with 346 structurally diverse HIV PRIs with experimental bioactivities against a sub-type B mutant to develop highly predictable QSAR models and also to identify the effective structural determinants for higher affinity against HIV PR. The QSAR models were developed using OCHEM-based machine learning tools (ASNN, FSMLR, KNN, RF, MANN and XGBoost), with descriptors calculated by eight different software packages. Simultaneously, a Monte Carlo optimization-based QSAR modelling was performed using SMILES and graph-based descriptors to understand fragment and topochemical contributions. To validate the actual predictability of all these models, an additional set of 104 compounds (also with known experimental activities) with slightly different chemical space were employed. This ligand-based study serves as a crucial benchmark for further development of the HIV protease inhibitors with improved activities.


Assuntos
Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , Relação Quantitativa Estrutura-Atividade , Protease de HIV/genética , Protease de HIV/metabolismo , Método de Monte Carlo
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