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1.
PLoS One ; 15(5): e0223464, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379830

RESUMO

Resistance associated mutations (RAMs) threaten the long-term success of combination antiretroviral therapy (cART) outcomes for HIV-1 treatment. HIV-1 Integrase (IN) strand transfer inhibitors (INSTIs) have proven to be a viable option for highly specific HIV-1 therapy. The INSTI, Dolutegravir is recommended by the World Health Organization for use as first-line cART. This study aims to understand how RAMs affect the stability of IN, as well as the binding of the drug Dolutegravir to the catalytic pocket of the protein. A homology model of HIV-1 subtype C IN was successfully constructed and validated. The site directed mutator webserver was used to predict destabilizing and/or stabilizing effects of known RAMs while FoldX confirmed any changes in protein energy upon introduction of mutation. Also, interaction analysis was performed between neighbouring residues. Three mutations known to be associated with Raltegravir, Elvitegravir and Dolutegravir resistance were selected; E92Q, G140S and Y143R, for molecular dynamics simulations. The structural quality assessment indicated high reliability of the HIV-1C IN tetrameric structure, with more than 90% confidence in modelled regions. Change in free energy for the three mutants indicated different effects, while simulation analysis showed G140S to have the largest affect on protein stability and flexibility. This was further supported by weaker non-bonded pairwise interaction energy and binding free energy values between the drug DTG and E92Q, Y143R and G140S mutants suggesting reduced binding affinity, as indicated by interaction analysis in comparison to the WT. Our findings suggest the G140S mutant has the strongest effect on the HIV-1C IN protein structure and Dolutegravir binding. To the best of our knowledge, this is the first study that uses the consensus wild type HIV-1C IN sequence to build an accurate 3D model to understand the effect of three known mutations on DTG drug binding in a South Africa context.


Assuntos
Farmacorresistência Viral/genética , Infecções por HIV/metabolismo , Inibidores de Integrase de HIV/metabolismo , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1/enzimologia , Compostos Heterocíclicos com 3 Anéis/metabolismo , Mutação , Sequência de Aminoácidos , Domínio Catalítico/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Integrase de HIV/química , Inibidores de Integrase de HIV/uso terapêutico , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Estabilidade Proteica , Quinolonas/metabolismo , Quinolonas/uso terapêutico , Raltegravir Potássico/metabolismo , Raltegravir Potássico/uso terapêutico , África do Sul , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
2.
Acta Crystallogr D Struct Biol ; 76(Pt 3): 302-310, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133994

RESUMO

Twinning is a crystal-growth anomaly in which protein monomers exist in different orientations but are related in a specific way, causing diffraction reflections to overlap. Twinning imposes additional symmetry on the data, often leading to the assignment of a higher symmetry space group. Specifically, in merohedral twinning, reflections from each monomer overlap and require a twin law to model unique structural data from overlapping reflections. Neglecting twinning in the crystallographic analysis of quasi-rotationally symmetric homo-oligomeric protein structures can mask the degree of structural non-identity between monomers. In particular, any deviations from perfect symmetry will be lost if higher than appropriate symmetry is applied during crystallographic analysis. Such cases warrant choosing between the highest symmetry space group possible or determining whether the monomers have distinguishable structural asymmetries and thus require a lower symmetry space group and a twin law. Using hexagonal cocrystals of HIV-1 protease, a C2-symmetric homodimer whose symmetry is broken by bound ligand, it is shown that both assigning a lower symmetry space group and applying a twin law during refinement are critical to achieving a structural model that more accurately fits the electron density. By re-analyzing three recently published HIV-1 protease structures, improvements in nearly every crystallographic metric are demonstrated. Most importantly, a procedure is demonstrated where the inhibitor can be reliably modeled in a single orientation. This protocol may be applicable to many other homo-oligomers in the PDB.


Assuntos
HIV-1/enzimologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Cristalografia por Raios X , Inibidores da Protease de HIV/química , Modelos Moleculares
3.
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
4.
Lancet HIV ; 7(3): e164-e172, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31911147

RESUMO

BACKGROUND: Islatravir (also known as ISL and MK-8591) is a unique nucleoside reverse transcriptase translocation inhibitor in clinical development for treatment of people with HIV-1 infection. In preclinical studies, intracellular islatravir-triphosphate exhibits a long half-life and prolonged virological effects. In this study, we aimed to assess islatravir safety, pharmacokinetics, and antiretroviral activity in treatment-naive adults with HIV-1 infection. METHODS: This open-label, consecutive-panel, phase 1b trial was done at Charité Research Organisation (Berlin, Germany) and included men and women (aged 18-60 years, inclusive) with HIV-1 infection who were ART naive. Participants were required to have plasma HIV-1 RNA counts of at least 10 000 copies per mL within 30 days before the trial treatment phase, without evidence of resistance to nucleoside reverse transcriptase inhibitors. Participants were enrolled in one of five consecutive dosing panels, receiving a single oral dose of islatravir (0·5-30 mg). The primary outcomes were safety and tolerability of islatravir and change from baseline in HIV-1 plasma RNA; secondary outcomes were islatravir plasma and islatravir-triphosphate intracellular pharmacokinetics. We obtained descriptive safety and pharmacokinetics statistics, and estimated efficacy results from a longitudinal data analysis model. This study is registered with ClinicalTrials.gov, NCT02217904, and EudraCT, 2014-002192-28. FINDINGS: Between Sept 17, 2015, and May 11, 2017, we enrolled 30 participants (six per panel). Islatravir was generally well tolerated. 27 (90%) participants had 60 adverse events after receipt of drug, of which 21 (35%) were deemed to be drug related. The most common (n>1) drug-related adverse events were headache (in nine [30%] participants) and diarrhoea (in two [7%]). No serious adverse events were reported, and no participants discontinued due to an adverse event. Plasma islatravir pharmacokinetics and intracellular islatravir-triphosphate pharmacokinetics were approximately dose proportional. The islatravir-triphosphate intracellular half-life was 78·5-128·0 h. Least-squares mean HIV-1 RNA at 7 days after dose decreased from 1·67 log10 copies per mL (95% CI 1·42-1·92) at 10 mg dose to 1·20 log10 copies per mL (0·95-1·46) at 0·5 mg dose. No genetic changes consistent with development of viral resistance were detected. INTERPRETATION: Single doses of islatravir as low as 0·5 mg significantly suppressed HIV-1 RNA by more than 1·0 log at day 7 in treatment-naive adults with HIV-1 infection and were generally well tolerated, supporting the further development of islatravir as a flexible-dose treatment for individuals with HIV-1 infection. FUNDING: Merck Sharp & Dohme Corp, a subsidiary of Merck & Co Inc, Kenilworth, NJ, USA.


Assuntos
Fármacos Anti-HIV/farmacocinética , Desoxiadenosinas/farmacocinética , Infecções por HIV/tratamento farmacológico , Inibidores da Transcriptase Reversa/farmacocinética , Adulto , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/efeitos adversos , Desoxiadenosinas/administração & dosagem , Desoxiadenosinas/efeitos adversos , Feminino , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/efeitos adversos , Adulto Jovem
5.
Phys Chem Chem Phys ; 22(4): 2530-2539, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31942584

RESUMO

It is well known that understanding the catalytic mechanism of HIV-1 PR is the rationale on which its inhibitors were developed; therefore, a better understanding of the mechanism of natural substrate hydrolysis is important. Herein, the reaction mechanism of HIV-1 natural substrates with subtypes B and common mutant in South Africa (subtype C-SA) protease were studied through transition state modelling, using a general acid-general base (GA-GB) one-step concerted process. The activation free energies of enzyme-substrate complexes were compared based on their rate of hydrolysis using a two-layered ONIOM (B3LYP/6-31++G(d,p):AMBER) method. We expanded our computational model to obtain a better understanding of the mechanism of hydrolysis as well as how the enzyme recognises or chooses the cleavage site of the scissile bonds. Using this model, a potential substrate-based inhibitor could be developed with better potency. The calculated activation energies of natural substrates in our previous study correlated well with experimental data. A similar trend was observed for the Gag and Gag-Pol natural substrates in the present work for both enzyme complexes except for the PR-RT substrate. Natural bond orbital (NBO) analysis was also applied to determine the extent of charge transfer within the QM part of both enzymes considered and the PR-RT natural substrate. The result of this study shows that the method can be utilized as a dependable computational technique to rationalize lead compounds against specific targets.


Assuntos
Protease de HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , Simulação de Dinâmica Molecular , Teoria Quântica , HIV-1/enzimologia , Ligação de Hidrogênio , Hidrólise , Cinética , Ligação Proteica , Especificidade por Substrato , Termodinâmica
6.
Eur J Med Chem ; 186: 111900, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31771827

RESUMO

Since dual inhibitors may yield lower toxicity and reduce the likelihood of drug resistance, as well as inhibitors of HIV-1 PR and RT constitute the core of chemotherapy for AIDS treatment, we herein designed and synthesized new coumarin derivatives characterized by various linkers that exhibited excellent potency against PR and a weak inhibition of RT. Among which, compounds 6f and 7c inhibited PR with IC50 values of 15.5 and 62.1 nM, respectively, and weakly affected also RT with IC50 values of 241.8 and 188.7 µM, respectively, showing the possibility in the future of developing dual HIV-1 PR/RT inhibitors. Creative stimulation for further research of more potent dual HIV-1 inhibitors was got according to the molecular docking studies.


Assuntos
Fármacos Anti-HIV/farmacologia , Cumarínicos/farmacologia , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Linhagem Celular , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade
7.
Arch Virol ; 165(1): 115-125, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31741096

RESUMO

The latest class of antiretrovirals (ARVs), including integrase strand transfer inhibitors (INSTIs), has been demonstrated to be effective for antiretroviral therapy (ART). Despite all the distinguishing characteristics of these drugs, including a high genetic barrier to resistance and lower toxicity than other ARVs, unfortunately, INSTI drug resistance mutations (DRMs) have occasionally been observed. The aim of this study was to investigate the presence of DRMs associated with INSTIs among treatment-experienced HIV-1-infected patients. From June 2012 to December 2018, a total of 655 treatment-experienced HIV-1-infected patients enrolled in this cross-sectional survey. Following amplification and sequencing of the HIV-1 integrase region of the pol gene, DRM and phylogenetic analysis were successfully carried out on the plasma samples of patients who had a viral load over 1,000 IU/ml after at least 6 months of ART. Out of the 655 patients evaluated, 62 (9.5%) had a viral load higher than 1,000 IU/ml after at least 6 months of ART. Phylogenetic analysis showed that all of the 62 HIV-1 patients experiencing treatment failure were infected with CRF35_AD, and one of these patients (1.6%) was infected with HIV-1 variants with DRMs. The DRMs that were identified belonged to the INSTI class, including E138K, G140A, S147G, and Q148R. This survey shows that DRMs belonging to the INSTI class were detected in an Iranian HIV patient who has experienced treatment failure. Therefore, regarding the presence of DRMs to INSTIs in ART-experienced patients, it seems better to perform drug resistance mutation testing in HIV patients experiencing treatment failure before changing the ART regimen and prescribing this class of medication.


Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , Integrase de HIV/genética , HIV-1/classificação , Mutação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Estudos Transversais , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/enzimologia , HIV-1/genética , Humanos , Lactente , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Filogenia , Análise de Sequência de RNA , Adulto Jovem
8.
Eur J Med Chem ; 185: 111866, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734023

RESUMO

Introducing pyrimidine bases, the basic components of nucleic acid, to P2 ligands might enhance the potency of Human Immunodeficiency Virus-1 (HIV-1) protease inhibitors because of the carbonyl and amino groups promoting the formation of extensive hydrogen bonding interactions. In this work, we provide evidence that inhibitor 10e, with N-2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide as the P2 ligand and a 4-methoxylphenylsulfonamide as the P2' ligand, displayed remarkable enzyme inhibitory and antiviral activity, with the IC50 2.53 nM in vitro and a promising inhibition ratio with 68% against wild-type HIV-1 in vivo, with low cytotoxicity. This inhibitor also exhibited appreciable antiviral activity against DRV-resistant HIV-1 variants, which was of great value for further study.


Assuntos
Acetamidas/farmacologia , Aminas/farmacologia , Fármacos Anti-HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Pirimidinas/farmacologia , Acetamidas/síntese química , Acetamidas/química , Aminas/síntese química , Aminas/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , HIV-1/enzimologia , Humanos , Ligantes , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
9.
Eur J Med Chem ; 185: 111874, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31735575

RESUMO

The fragment hopping approach is widely applied in drug development. A series of diarylpyrimidines (DAPYs) were obtained by hopping the thioacetamide scaffold to novel human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitors (NNRTIs) to address the cytotoxicity issue of Etravirine and Rilpivirine. Although the new compounds (11a-l) in the first-round optimization possessed less potent anti-viral activity, they showed much lower cytotoxicity. Further optimization on the sulfur led to the sulfinylacetamide-DAPYs exhibiting improved anti-viral activity and a higher selectivity index especially toward the K103N mutant strain. The most potent compound 12a displayed EC50 values of 0.0249 µM against WT and 0.0104 µM against the K103N mutant strain, low cytotoxicity (CC50 > 221 µM) and a high selectivity index (SI WT > 8873, SI K103N > 21186). In addition, this compound showed a favorable in vitro microsomal stability across species. Computational study predicted the binding models of these potent compounds with HIV-1 reverse transcriptase thus providing further insights for new developments.


Assuntos
Acetamidas/farmacologia , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Acetamidas/síntese química , Acetamidas/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade
10.
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 , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Método de Monte Carlo , Mutação , Ligação Proteica , Eletricidade Estática
11.
Molecules ; 24(24)2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31835661

RESUMO

In this study, we isolated nine compounds from the acid hydrolysate of the flower buds of Lonicera fulvotomentosa Hsu et S. C. Cheng and characterized their chemical structures using 1H-NMR, 13C-NMR, and electron ionization mass spectroscopy (EI-MS). These compounds were identified as ß-sitosterol (1), 5,5'-dibutoxy-2,2'-bifuran (2), nonacosane-10-ol (3), ethyl (3ß)-3,23-dihydroxyolean-12-en-28-oate (4), oleanolic acid (5), ethyl caffeate (6), caffeic acid (7), isovanillin (8), and hederagenin (9), with 4 as a new triterpene compound. Inhibitory activity against human immunodeficiency virus (HIV) protease was also evaluated for the compounds, and only ethyl caffeate, caffeic acid, and isovanillin (6, 7, and 8) exhibited inhibitory effects, with IC50 values of 1.0 µM, 1.5 µM, and 3.5 µM, respectively. Molecular docking with energy minimization and subsequent molecular dynamic (MD) simulation showed that ethyl caffeate and caffeic acid bound to the active site of HIV protease, while isovanillin drifted out from the active site and dissociated into bulk water during MD simulations, and most of the binding residues of HIV protease have been previously identified for HIV protease inhibitors. These results suggest that caffeic acid derivatives may possess inhibitory activities towards HIV protease other than previously reported inhibitory activities against HIV integrase, and thus ethyl caffeate and caffeic acid could be used as lead compounds in developing potential HIV protease inhibitors, and possibly even dual-function inhibitors against HIV.


Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/enzimologia , Lonicera/química , Compostos Fitoquímicos/farmacologia , Domínio Catalítico , Protease de HIV/química , Inibidores da Protease de HIV/química , Espectrometria de Massas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Compostos Fitoquímicos/química , Extratos Vegetais/análise
12.
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
13.
Retrovirology ; 16(1): 30, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31690330

RESUMO

BACKGROUND: HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. RESULTS: We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. CONCLUSIONS: Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding-a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.


Assuntos
Reparo do DNA por Junção de Extremidades , Integrase de HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , Autoantígeno Ku/metabolismo , Quebras de DNA de Cadeia Dupla , Integrase de HIV/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Autoantígeno Ku/genética , Redes e Vias Metabólicas
14.
Molecules ; 24(21)2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31652782

RESUMO

BACKGROUND: HIV is the causative agent of Acquired Immunodeficiency Syndrome (AIDS), an infectious disease with increasing incidence worldwide. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) play an important role in the treatment of AIDS. Although, many compounds are already being used as anti-HIV drugs, research for the development of new inhibitors continues as the virus develops resistant strains. METHODS: The best features of available NNRTIs were taken into account for the design of novel inhibitors. PASS (Prediction of activity spectra for substances) prediction program and molecular docking studies for the selection of designed compounds were used for the synthesis. Compounds were synthesized using conventional and microwave irradiation methods and HIV RT inhibitory action was evaluated by colorimetric photometric immunoassay. RESULTS: The evaluation of HIV-1 RT inhibitory activity revealed that seven compounds have significantly lower ΙC50 values than nevirapine (0.3 µΜ). It was observed that the activity of compounds depends not only on the nature of substituent and it position in benzothiazole ring but also on the nature and position of substituents in benzene ring. CONCLUSION: Twenty four of the tested compounds exhibited inhibitory action lower than 4 µΜ. Seven of them showed better activity than nevirapine, while three of the compounds exhibited IC50 values lower than 5 nM. Two compounds 9 and 10 exhibited very good inhibitory activity with IC50 1 nM.


Assuntos
Síndrome de Imunodeficiência Adquirida , Fármacos Anti-HIV , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Simulação de Acoplamento Molecular , Inibidores da Transcriptase Reversa , Tiazóis , Síndrome de Imunodeficiência Adquirida/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida/enzimologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Transcriptase Reversa do HIV/metabolismo , Humanos , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Tiazóis/síntese química , Tiazóis/química , Tiazóis/farmacologia
15.
J Agric Food Chem ; 67(43): 11942-11947, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31622090

RESUMO

Manilkara zapota, usually known as Sapodilla, is a fairly slow-growing evergreen tropical tree which belongs to the genus Manilkara (Sapotaceae), indigenous to Central America, southern Mexico, and the Caribbean. The ripe fruits of M. zapota have been widely consumed as an uniquely flavored tropical fruit and verified to hold a variety of health benefits. In order to investigate the potential health-promoting chemical compositions from the fruits of M. zapota cultivated in Hainan Island of China, a systematic and in-depth phytochemical study on this fruit was accordingly implemented. In our current study, three new prenylated coumarins, manizapotins A-C (1-3), together with seven known prenylated coumarins (4-10), were separated from the fruits of M. zapota. The chemical structures of new prenylated coumarins 1-3 were unambiguously established by means of comprehensive spectroscopic analyses, and the known compounds 4-10 were determined by comparing their experimental spectral data with those described data in the literature. This is the first time to discover prenylated coumarins occurring in M. zapota. The potential anti-inflammatory effects and anti-HIV (human immunodeficiency virus) activities of all these separated prenylated coumarins were assessed. Prenylated coumarins 1-10 dispalyed remarkable inhibitory effects against nitric oxide production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells with the IC50 values equivalent to that of hydrocortisone in vitro. Meanwhile, prenylated coumarins 1-10 exhibited pronounced anti-HIV-1 reverse transcriptase activities with the EC50 values in range of 0.12-8.69 µM. These results suggest that appropriate and reasonable consumption of the fruits of M. zapota might assist people to prevent and reduce the occurrence of inflammatory diseases together with the infection of HIV. Furthermore, the discovery of these prenylated coumarins from the fruits of M. zapota holding pronounced anti-inflammatory effects along with anti-HIV activities could be of great significance to the research and development of new natural anti-inflammatory and anti-HIV agents.


Assuntos
Fármacos Anti-HIV/química , Anti-Inflamatórios/química , Cumarínicos/química , Manilkara/química , Extratos Vegetais/química , Animais , Fármacos Anti-HIV/isolamento & purificação , Fármacos Anti-HIV/farmacologia , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , China , Cumarínicos/isolamento & purificação , Cumarínicos/farmacologia , Frutas/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Estrutura Molecular , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Prenilação , Células RAW 264.7
16.
ACS Chem Biol ; 14(10): 2166-2175, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31560515

RESUMO

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2'-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of ∼50-100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5' DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2'-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.


Assuntos
Aptâmeros de Nucleotídeos/química , Arabinonucleotídeos/química , Integrase de HIV/química , HIV-1/enzimologia , RNA/química , Sequência de Bases , Técnica de Seleção de Aptâmeros , Alinhamento de Sequência
17.
Virol J ; 16(1): 103, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416460

RESUMO

BACKGROUND: The high genetic diversity of HIV-1 has been shown to influence the global distribution, disease progression, treatment success, and the development of an effective vaccine. Despite the low HIV prevalence in Cameroon, all the major HIV subtypes alongside several circulating recombinant forms (CRFs) and unique recombinant forms (URFs) have been reported in Cameroon. To date, HIV-1 diversity in some parts of Cameroon has been largely studied however, information on circulating HIV-1 subtypes in the Northwest region (NWR) of Cameroon is dearth. Therefore the aim of this study was to determine the current circulating HIV-1 subtypes among adults in the NWR of Cameroon. METHODS: The genetic analysis of the reverse transcriptase region of the pol gene was performed on 81 samples. The samples were collected from drug naïve patients aged between 18 and 61 years residing within the rural and urban towns in the NWR during the period between February and April 2016. Viral RNA was extracted from plasma, reverse-transcribed, further amplified by nested-PCR before sequencing using an in-house protocol. Generated sequences were then phylogenetically analyzed together with references using MEGA 7. RESULTS: Phylogenetic analysis revealed a broad viral diversity including CRF02 _AG (74.1%), F2 (7.4%), D (7.4%), G (3.7%), A1 (1.2%), CRF22_01A1 (2.5%), CRF06_cpx (1.2%), CRF09_cpx (1.2%), CRF11_cpx (1.2%). Three close epidemic clusters were found among F2 (1) and CRF02_AG (2) variants. For the first time we are reporting the CRF22_01A1 subtype in this region. CONCLUSION: Our findings update HIV-1 subtypes information in Cameroon and uphold previous studies that CRF02_AG is the most prevalent subtype. This CRF02_AG subtype may have important public health, research, and clinical consequences.


Assuntos
Variação Genética , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/classificação , Filogenia , Adolescente , Adulto , Camarões/epidemiologia , Estudos Transversais , Evolução Molecular , Feminino , Genes pol , Genótipo , Geografia , HIV-1/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , DNA Polimerase Dirigida por RNA/genética , Recombinação Genética , Análise de Sequência de DNA , Adulto Jovem
18.
Eur J Med Chem ; 182: 111603, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31421633

RESUMO

Conformational restriction is a promising strategy in the development of DAPY-type non-nucleoside reverse transcriptase inhibitors (NNRTIs). Herein, eighteen thiophene-biphenyl-DAPY derivatives were designed and synthesized as potent HIV-1 NNRTIs in which halogen and methyl groups were introduced to explore the conformationally constrained effects. Molecular docking and dynamic simulation analysis indicated that substituents on different positions of the biphenyl ring induced different dihedral angles and binding conformations, further explaining their anti-viral activities. The 2'-fluoro and 3'-chloro substitutions could form electrostatic or halogen-bonding interactions with adjacent residues of the RT enzyme. The 2'-methyl group contributed to enlarge the dihedral angle of biphenyl ring and was positioned to a space-filling hydrophobic pocket. Notably, compounds 22 and 23 with two methyl groups exhibited potent biological activity against WT HIV-1-infected MT-4 cells (EC50 = 14 and 17 nM, respectively) and RT enzyme (EC50 = 27 and 42 nM, respectively). In particular, 23 exhibited much lower cytotoxicity (CC50 = 264.19 µM) and higher selectivity index (SI = 18,564) than etravirine. Taken together, a rational conformational model for further design of DAPYs is proposed, providing a new guidance for the development of NNRTIs.


Assuntos
Fármacos Anti-HIV/farmacologia , Compostos de Bifenilo/farmacologia , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Pirimidinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Tiofenos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Compostos de Bifenilo/química , Linhagem Celular , Relação Dose-Resposta a Droga , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Conformação Molecular , Pirimidinas/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Tiofenos/química
19.
Eur Biophys J ; 48(7): 685-689, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463540

RESUMO

Cells are crowded with various macromolecules and metabolites, which affect biochemical reactions in many ways, from the diffusion of substrates to catalytic activities of enzymes. We herein investigated the proteolytic activity of the human immunodeficiency virus type 1 protease (HIV-1 PR) under non-crowded and crowded conditions. The latter environment was mimicked with various (poly)ethylene glycol molecules as crowding agents. We found that these crowding agents affect the kinetic parameters of the HIV-1 PR catalyzed reaction by increasing the Michaelis-Menten constant and decreasing the maximum velocity. The influence of crowding was concentration dependent. We explain this effect by the dynamics of the HIV-1 PR flexible flaps that cover the peptide substrate binding site and are crucial for enzyme activity, and by a possibly slower substrate-enzyme association time in the crowded conditions.


Assuntos
Protease de HIV/metabolismo , HIV-1/enzimologia , Biocatálise , Protease de HIV/química , Hidrólise , Cinética , Modelos Moleculares , Estrutura Terciária de Proteína , Soluções
20.
Eur J Med Chem ; 182: 111617, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31442684

RESUMO

A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1 (HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-hydroxy-2H, 5H-pyrano (3, 2-c) chromene-2, 5-dione derivatives were synthesized. The modeling studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds.


Assuntos
Fármacos Anti-HIV/farmacologia , Cumarínicos/farmacologia , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Cumarínicos/síntese química , Cumarínicos/química , Relação Dose-Resposta a Droga , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/química , HIV-1/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Relação Estrutura-Atividade
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