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1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199858

RESUMO

The approval of the first HIV-1 protease inhibitors (HIV-1 PRIs) marked a fundamental step in the control of AIDS, and this class of agents still represents the mainstay therapy for this illness. Despite the undisputed benefits, the necessary lifelong treatment led to numerous severe side-effects (metabolic syndrome, hepatotoxicity, diabetes, etc.). The HIV-1 PRIs are capable of interacting with "secondary" targets (off-targets) characterized by different biological activities from that of HIV-1 protease. In this scenario, the in-silico techniques undoubtedly contributed to the design of new small molecules with well-fitting selectivity against the main target, analyzing possible undesirable interactions that are already in the early stages of the research process. The present work is focused on a new mixed-hierarchical, ligand-structure-based protocol, which is centered on an on/off-target approach, to identify the new selective inhibitors of HIV-1 PR. The use of the well-established, ligand-based tools available in the DRUDIT web platform, in combination with a conventional, structure-based molecular docking process, permitted to fast screen a large database of active molecules and to select a set of structure with optimal on/off-target profiles. Therefore, the method exposed herein, could represent a reliable help in the research of new selective targeted small molecules, permitting to design new agents without undesirable interactions.


Assuntos
Desenho de Fármacos , Descoberta de Drogas , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , HIV-1/efeitos dos fármacos , Domínio Catalítico , Simulação por Computador , Infecções por HIV/enzimologia , Infecções por HIV/virologia , HIV-1/enzimologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Conformação Proteica , Relação Estrutura-Atividade
2.
Molecules ; 26(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201561

RESUMO

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated functions.


Assuntos
Fármacos Anti-HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/metabolismo , Ribonuclease H/antagonistas & inibidores , Tiazóis/química , Tiazóis/farmacologia , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Concentração Inibidora 50 , Ligantes , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Tiazóis/síntese química
3.
Molecules ; 26(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202892

RESUMO

Computational analysis of protein-ligand interactions is of crucial importance for drug discovery. Assessment of ligand binding energy allows us to have a glimpse of the potential of a small organic molecule to be a ligand to the binding site of a protein target. Available scoring functions, such as in docking programs, all rely on equations that sum each type of protein-ligand interactions in order to predict the binding affinity. Most of the scoring functions consider electrostatic interactions involving the protein and the ligand. Electrostatic interactions constitute one of the most important part of total interactions between macromolecules. Unlike dispersion forces, they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes and contribute significantly to differences in inhibition strength among related enzyme inhibitors. In this study, complexes of HIV-1 protease with inhibitor molecules (JE-2147 and darunavir) were analyzed by using charge densities from the transferable aspherical-atom University at Buffalo Databank (UBDB). Moreover, we analyzed the electrostatic interaction energy for an ensemble of structures, using molecular dynamic simulations to highlight the main features of electrostatic interactions important for binding affinity.


Assuntos
Bases de Dados de Proteínas , Inibidores da Protease de HIV/química , Protease de HIV/química , HIV-1/enzimologia , Simulação de Dinâmica Molecular
4.
Viruses ; 13(6)2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205716

RESUMO

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR's specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.


Assuntos
Ensaios Enzimáticos/métodos , Protease de HIV/metabolismo , HIV-1/enzimologia , Interferometria/métodos , Técnicas Biossensoriais , Clonagem Molecular , Protease de HIV/genética , Protease de HIV/isolamento & purificação , Humanos , Cinética , Proteólise , Proteínas Recombinantes , Análise de Sequência de DNA , Especificidade por Substrato
5.
J Med Chem ; 64(12): 8579-8598, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34106711

RESUMO

Novel anti-HIV agents are still needed to overcome resistance issues, in particular inhibitors acting against novel viral targets. The ribonuclease H (RNase H) function of the reverse transcriptase (RT) represents a validated and promising target, and no inhibitor has reached the clinical pipeline yet. Here, we present rationally designed non-diketo acid selective RNase H inhibitors (RHIs) based on the quinolinone scaffold starting from former dual integrase (IN)/RNase H quinolinonyl diketo acids. Several derivatives were synthesized and tested against RNase H and viral replication and found active at micromolar concentrations. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, and Mg2+ titration experiments demonstrated that our compounds coordinate the Mg2+ cofactor and interact with amino acids of the RNase H domain that are highly conserved among naïve and treatment-experienced patients. In general, the new inhibitors influenced also the polymerase activity of RT but were selective against RNase H vs the IN enzyme.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/enzimologia , Quinolonas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/metabolismo , Células HeLa , Humanos , Magnésio/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Quinolonas/síntese química , Quinolonas/metabolismo , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Replicação Viral/efeitos dos fármacos
6.
Nat Commun ; 12(1): 2500, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947853

RESUMO

Reverse transcription of the HIV-1 viral RNA genome (vRNA) is an integral step in virus replication. Upon viral entry, HIV-1 reverse transcriptase (RT) initiates from a host tRNALys3 primer bound to the vRNA genome and is the target of key antivirals, such as non-nucleoside reverse transcriptase inhibitors (NNRTIs). Initiation proceeds slowly with discrete pausing events along the vRNA template. Despite prior medium-resolution structural characterization of reverse transcriptase initiation complexes (RTICs), higher-resolution structures of the RTIC are needed to understand the molecular mechanisms that underlie initiation. Here we report cryo-EM structures of the core RTIC, RTIC-nevirapine, and RTIC-efavirenz complexes at 2.8, 3.1, and 2.9 Å, respectively. In combination with biochemical studies, these data suggest a basis for rapid dissociation kinetics of RT from the vRNA-tRNALys3 initiation complex and reveal a specific structural mechanism of nucleic acid conformational stabilization during initiation. Finally, our results show that NNRTIs inhibit the RTIC and exacerbate discrete pausing during early reverse transcription.


Assuntos
Transcriptase Reversa do HIV/química , HIV-1/efeitos dos fármacos , RNA de Transferência de Lisina/química , RNA Viral/química , Inibidores da Transcriptase Reversa/química , Alcinos/química , Alcinos/farmacologia , Benzoxazinas/química , Benzoxazinas/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Ciclopropanos/química , Ciclopropanos/farmacologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/metabolismo , Modelos Moleculares , Nevirapina/química , Nevirapina/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , RNA de Transferência de Lisina/genética , RNA Viral/genética , Inibidores da Transcriptase Reversa/farmacologia
7.
Eur J Med Chem ; 220: 113498, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33933756

RESUMO

Upon the basis of both possible ligand-binding site interactions and the uniformity of key residues in active sites, a novel class of HIV-1 PR/RT dual inhibitors was designed and evaluated. Cinnamic acids or phenylpropionic acids with more flexible chain and smaller steric hindrance were introduced into the inhibitors, giving rise to significant improvement in HIV-1 RT inhibitory activity by one or two orders of magnitude, with comparable or even improved potency against PR at the same time, compared with coumarin anologues in our previous studies. Among these inhibitors, 38d displayed a 19-fold improvement in anti-PR activity with IC50 value of 0.081 nM compared to the control DRV. In addition, inhibitor 38c exhibited an excellent anti-RT IC50 value of 0.43 µM, only a 4.7-fold less potent activity than the control EFV. More significantly, the disparate ratio between HIV-1 PR and RT inhibition became more reasonable with ratio of 1: 10.4, just as 37b. Furthermore, the assays on HIV-1 late stage and early stage supported the rationality of designing dual inhibitors. The SAR data as well as molecular modeling studies provided new insight for further optimization of more potent HIV-1 PR/RT dual inhibitors.


Assuntos
Amidas/farmacologia , Fármacos Anti-HIV/farmacologia , Cinamatos/farmacologia , Inibidores da Protease de HIV/farmacologia , Fenilpropionatos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Amidas/síntese química , Amidas/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Cinamatos/síntese química , Cinamatos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Protease de HIV/metabolismo , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Fenilpropionatos/síntese química , Fenilpropionatos/química , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade
8.
Retrovirology ; 18(1): 11, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33952315

RESUMO

BACKGROUND: The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. RESULTS: Gag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa. CONCLUSIONS: Although prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.


Assuntos
Protease de HIV/genética , HIV-1/genética , HIV-1/fisiologia , Replicação Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Adulto , África Oriental , África Ocidental , Estudos Transversais , Feminino , Genótipo , Infecções por HIV/virologia , Protease de HIV/classificação , HIV-1/classificação , HIV-1/enzimologia , Humanos , Masculino , Filogenia , Recombinação Genética , Estudos Retrospectivos , Replicação Viral/fisiologia , Adulto Jovem , Produtos do Gene gag do Vírus da Imunodeficiência Humana/classificação
9.
BMC Infect Dis ; 21(1): 379, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33892628

RESUMO

BACKGROUND: The Integrase (IN) strand transfer inhibitor (INSTI), Dolutegravir (DTG), has been given the green light to form part of first-line combination antiretroviral therapy (cART) by the World Health Organization (WHO). DTG containing regimens have shown a high genetic barrier against HIV-1 isolates carrying specific resistance mutations when compared with other class of regimens. METHODS: We evaluated the HIV-1 CRF02_AG IN gene sequences from Cameroon for the presence of resistance-associated mutations (RAMs) against INSTIs and naturally occurring polymorphisms (NOPs), using study sequences (n = 20) and (n = 287) sequences data derived from HIV Los Alamos National Laboratory database. The possible impact of NOPs on protein structure caused by HIV-1 CRF02_AG variations was addressed within the context of a 3D model of the HIV-1 IN complex and interaction analysis was performed using PyMol to validate DTG binding to the Wild type and seven mutant structures. RESULTS: We observed 12.8% (37/287) sequences to contain RAMs, with only 1.0% (3/287) of the sequences having major INSTI RAMs: T66A, Q148H, R263K and N155H. Of these,11.8% (34/287) of the sequences contained five different IN accessory mutations; namely Q95K, T97A, G149A, E157Q and D232N. NOPs occurred at a frequency of 66% on the central core domain (CCD) position, 44% on the C-terminal domain (CTD) position and 35% of the N-terminal domain (NTD) position. The interaction analysis revealed that DTG bound to DNA, 2MG ions and DDE motif residues for T66A, T97A, Q148H, N155H and R263K comparable to the WT structure. Except for accessory mutant structure E157Q, only one MG contact was made with DTG, while DTG had no MG ion contacts and no DDE motif residue contacts for structure D232N. CONCLUSIONS: Our analysis indicated that all RAM's that resulted in a change in the number of interactions with encompassing residues does not affect DTG binding, while accessory mutations E157Q and D232N could affect DTG binding leading to possible DTG resistance. However, further experimental validation is required to validate the in silico findings of our study.


Assuntos
Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , Integrase de HIV/genética , HIV-1/enzimologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Modelos Moleculares , Mutação , Oxazinas/uso terapêutico , Piperazinas/uso terapêutico , Piridonas/uso terapêutico , Camarões/epidemiologia , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Integrase de HIV/química , Inibidores de Integrase de HIV/química , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Oxazinas/química , Filogenia , Piperazinas/química , Polimorfismo Genético , Piridonas/química
10.
Eur J Med Chem ; 220: 113450, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33906049

RESUMO

A novel class of HIV-1 protease inhibitors with flexible piperidine as the P2 ligand was designed with the aim of improving extensive interactions with the active subsites. Many inhibitors exhibited good to excellent inhibitory effect on enzymatic activity and viral infectivity. In particular, inhibitor 3a with (R)-piperidine-3-carboxamide as the P2 ligand and 4-methoxybenzenesulfonamide as the P2' ligand showed an enzyme Ki value of 29 pM and antiviral IC50 value of 0.13 nM, more than six-fold enhancement of activity compared to DRV. Furthermore, there was no significant change in potency against DRV-resistant mutations and HIV-1NL4-3 variant for 3a. Besides, inhibitor 3a exhibited potent antiviral activity against subtype C variants with low nanomole EC50 values. In addition, the molecular modeling revealed important hydrogen bonds and other favorable van der Waals interactions with the backbone atoms of the protease and provided insight for designing and optimizing more potent HIV-1 protease inhibitors.


Assuntos
Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Inibidores da Protease de HIV/farmacologia , Protease de HIV/metabolismo , HIV-1/efeitos dos fármacos , Piperidinas/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Darunavir/farmacologia , Relação Dose-Resposta a Droga , Farmacorresistência Viral/efeitos dos fármacos , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/química , HIV-1/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Relação Estrutura-Atividade
11.
Comput Math Methods Med ; 2021: 5559338, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868450

RESUMO

A key enzyme in human immunodeficiency virus type 1 (HIV-1) life cycle, integrase (IN) aids the integration of viral DNA into the host DNA, which has become an ideal target for the development of anti-HIV drugs. A total of 1785 potential HIV-1 IN inhibitors were collected from the databases of ChEMBL, Binding Database, DrugBank, and PubMed, as well as from 40 references. The database was divided into the training set and test set by random sampling. By exploring the correlation between molecular descriptors and inhibitory activity, it is found that the classification and specific activity data of inhibitors can be more accurately predicted by the combination of molecular descriptors and molecular fingerprints. The calculation of molecular fingerprint descriptor provides the additional substructure information to improve the prediction ability. Based on the training set, two machine learning methods, the recursive partition (RP) and naive Bayes (NB) models, were used to build the classifiers of HIV-1 IN inhibitors. Through the test set verification, the RP technique accurately predicted 82.5% inhibitors and 86.3% noninhibitors. The NB model predicted 88.3% inhibitors and 87.2% noninhibitors with correlation coefficient of 85.2%. The results show that the prediction performance of NB model is slightly better than that of RP, and the key molecular segments are also obtained. Additionally, CoMFA and CoMSIA models with good activity prediction ability both were constructed by exploring the structure-activity relationship, which is helpful for the design and optimization of HIV-1 IN inhibitors.


Assuntos
Desenho de Fármacos , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/classificação , Integrase de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Aprendizado de Máquina , Teorema de Bayes , Biologia Computacional , Bases de Dados de Produtos Farmacêuticos/estatística & dados numéricos , Árvores de Decisões , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores de Integrase de HIV/farmacologia , HIV-1/enzimologia , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
12.
Elife ; 102021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33904396

RESUMO

HIV-1 replication commences inside the cone-shaped viral capsid, but timing, localization, and mechanism of uncoating are under debate. We adapted a strategy to visualize individual reverse-transcribed HIV-1 cDNA molecules and their association with viral and cellular proteins using fluorescence and correlative-light-and-electron-microscopy (CLEM). We specifically detected HIV-1 cDNA inside nuclei, but not in the cytoplasm. Nuclear cDNA initially co-localized with a fluorescent integrase fusion (IN-FP) and the viral CA (capsid) protein, but cDNA-punctae separated from IN-FP/CA over time. This phenotype was conserved in primary HIV-1 target cells, with nuclear HIV-1 complexes exhibiting strong CA-signals in all cell types. CLEM revealed cone-shaped HIV-1 capsid-like structures and apparently broken capsid-remnants at the position of IN-FP signals and elongated chromatin-like structures in the position of viral cDNA punctae lacking IN-FP. Our data argue for nuclear uncoating by physical disruption rather than cooperative disassembly of the CA-lattice, followed by physical separation from the pre-integration complex.


Assuntos
Núcleo Celular/virologia , Replicação do DNA , DNA Viral/biossíntese , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , Internalização do Vírus , Replicação Viral , Desenvelopamento do Vírus , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , Proteínas do Capsídeo/metabolismo , Núcleo Celular/ultraestrutura , DNA Viral/genética , DNA Viral/ultraestrutura , Células HEK293 , Infecções por HIV/patologia , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/ultraestrutura , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Macrófagos/ultraestrutura , Macrófagos/virologia , Microscopia Eletrônica , Microscopia de Fluorescência , Fatores de Tempo
13.
Int J Biol Macromol ; 174: 309-318, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33524481

RESUMO

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is the key enzyme for the virus gene replication and the most important target for antiviral therapy. Toxicity, drug resistance and side effects have led to search for new antiviral agents. Farnesiferol C (FC) is a well-known biologically active sesquiterpene coumarin derivative from genus Ferula. The current study was designed to examine the impacts of FC on the structure and function of HIV-1 RT, using some theoretical and experimental methods. FC inhibited HIV-1RT activity via mixed inhibition mechanism (IC50 = 30 µM). Spectroscopic data showed some conformational changes in the secondary as well as tertiary structure of HIV-1RT following the interaction with FC. Results showed that FC could quench the intrinsic fluorescence emission of HIV-1RT through static quenching mechanism. Thermodynamic parameters revealed that hydrogen bondings and van der Waals forces are the major forces in the binding reaction and the low equilibrium constants (KD) value obtained from surface plasmon resonance data, confirmed the high affinity of FC for HIV-1RT. Molecular docking studies indicated that FC interacts with enzyme through hydrophobic pocket. Taken together, the outcomes of this research revealed that, sesquiterpene coumarines can be used to design natural remedies as anti-HIV agents.


Assuntos
Cumarínicos/farmacologia , Ferula/química , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Cumarínicos/química , Transcriptase Reversa do HIV/química , HIV-1/efeitos dos fármacos , Ligação de Hidrogênio , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Inibidores da Transcriptase Reversa/química , Ressonância de Plasmônio de Superfície
14.
Biochem Pharmacol ; 186: 114462, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33577894

RESUMO

While combination antiretroviral therapy (cART) durably suppresses HIV replication, virus persists in CD4+ T-cells that harbor latent but spontaneously inducible and replication-competent provirus. One strategy to inactivate these viral reservoirs involves the use of agents that continue to reinforce HIV latency even after their withdrawal. To identify new chemical leads with such properties, we investigated a series of naturally-occurring flavones (chrysin, apigenin, luteolin, and luteolin-7-glucoside (L7G)) and functionally-related cyclin dependent kinase 9 (CDK9) inhibitors (flavopiridol and atuveciclib) which are reported or presumed to suppress HIV replication in vitro. We found that, while all compounds inhibit provirus expression induced by latency-reversing agents in vitro, only aglycone flavonoids (chrysin, apigenin, luteolin, flavopiridol) and atuveciclib, but not the glycosylated flavonoid L7G, inhibit spontaneous latency reversal. Aglycone flavonoids and atuveciclib, but not L7G, also inhibit CDK9 and the HIV Tat protein. Aglycone flavonoids do not reinforce HIV latency following their in vitro withdrawal, which corresponds with their ability to also inhibit class I/II histone deacetylases (HDAC), a well-established mechanism of latency reversal. In contrast, atuveciclib and flavopiridol, which exhibit little or no HDAC inhibition, continue to reinforce latency for 9 to 14+ days, respectively, following their withdrawal in vitro. Finally, we show that flavopiridol also inhibits spontaneous ex vivo viral RNA production in CD4+ T cells from donors with HIV. These results implicate CDK9 inhibition (in the absence of HDAC inhibition) as a potentially favorable property in the search for compounds that durably reinforce HIV latency.


Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Flavonoides/farmacologia , HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Latência Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Quinase 9 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Flavonoides/uso terapêutico , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , HIV-1/enzimologia , Histona Desacetilases/metabolismo , Humanos , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Latência Viral/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
15.
Viruses ; 13(2)2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572956

RESUMO

Integrase strand transfer inhibitors (INSTIs) are currently recommended for the first line treatment of human immunodeficiency virus type one (HIV-1) infection. The first-generation INSTIs are effective but can select for resistant viruses. Recent advances have led to several potent second-generation INSTIs that are effective against both wild-type (WT) HIV-1 integrase and many of the first-generation INSTI-resistant mutants. The emergence of resistance to these new second-generation INSTIs has been minimal, which has resulted in alternative treatment strategies for HIV-1 patients. Moreover, because of their high antiviral potencies and, in some cases, their bioavailability profiles, INSTIs will probably have prominent roles in pre-exposure prophylaxis (PrEP). Herein, we review the current state of the clinically relevant INSTIs and discuss the future outlook for this class of antiretrovirals.


Assuntos
Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , DNA Viral/metabolismo , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Integrase de HIV/química , Integrase de HIV/genética , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/enzimologia , HIV-1/genética , Humanos , Mutação , Profilaxia Pré-Exposição , Replicação Viral
16.
Jpn J Infect Dis ; 74(4): 299-306, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-33390426

RESUMO

The dimeric form of HIV-1 protease (PR) is required for its full proteolytic activity. The stability of the dimer primarily depends on the termini interface, with N-terminal residues 1-4 of one monomer encountering C-terminal residues 96-99 of another. We made an alanine substitution for valine 3 (V3) or leucine 97 (L97) at the termini dimer interface and tested their proteolytic activity. We found that an alanine substitution for L97 (PRL97A) completely inhibited the proteolytic activity of the PR. However, an alanine substitution for V3 (PRV3A) partially impaired the proteolytic activity. We then introduced two forced-dimerization systems involving nucleocapsid (NC) replacement or the addition of 1-2 leucine zippers to determine whether the proteolytic activity of dimer-defective PRs could be restored. We found that two forced-dimerization systems compensated for the defect in PRV3A, but not in PRL97A. This implies that PRV3A and PRL97A potentially impair the PR via different mechanisms or cause defects in PR activity to different extents. These novel findings will likely serve as a foundation for developing new PR inhibitors for treating drug-resistant HIV-1 infections in the future.


Assuntos
Protease de HIV/química , HIV-1/genética , HIV-1/metabolismo , Replicação Viral/genética , Replicação Viral/fisiologia , Substituição de Aminoácidos , Regulação Viral da Expressão Gênica , Células HEK293 , Protease de HIV/genética , Protease de HIV/metabolismo , HIV-1/enzimologia , Humanos , Vírion/genética , Vírion/fisiologia
17.
Future Med Chem ; 13(3): 269-286, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33399497

RESUMO

Reverse transcriptase and integrase are key enzymes that play a pivotal role in HIV-1 viral maturation and replication. Reverse transcriptase consists of two active sites: RNA-dependent DNA polymerase and RNase H. The catalytic domains of integrase and RNase H share striking similarity, comprising two aspartates and one glutamate residue, also known as the catalytic DDE triad, and a Mg2+ pair. The simultaneous inhibition of reverse transcriptase and integrase can be a rational drug discovery approach for combating the emerging drug resistance problem. In the present review, the dual inhibition of RNase H and integrase is systematically discussed, including rationality of design, journey of development, advancement and future perspective.


Assuntos
Fármacos Anti-HIV/química , Integrase de HIV/metabolismo , HIV-1/enzimologia , Ribonuclease H/metabolismo , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/uso terapêutico , Domínio Catalítico , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Integrase de HIV/química , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Humanos , Ribonuclease H/antagonistas & inibidores , Relação Estrutura-Atividade
18.
Chembiochem ; 22(5): 915-923, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33095511

RESUMO

HIV-1 RT is a necessary enzyme for retroviral replication, which is the main target for antiviral therapy against AIDS. Effective anti-HIV-1 RT drugs are divided into two groups; nucleoside inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI), which inhibit DNA polymerase. In this study, new DNA aptamers were isolated as anti-HIV-1 RT inhibitors. The selected DNA aptamer (WT62) presented with high affinity and inhibition against wild-type (WT) HIV-1 RT and gave a KD value of 75.10±0.29 nM and an IC50 value of 84.81±8.54 nM. Moreover, WT62 decreased the DNA polymerase function of K103 N/Y181 C double mutant (KY) HIV-1 RT by around 80 %. Furthermore, the ITC results showed that this aptamer has small binding enthalpies with both WT and KY HIV-1 RTs through which the complex might form a hydrophobic interaction or noncovalent bonding. The NMR result also suggested that the WT62 aptamer could bind with both WT and KY mutant HIV-1 RTs at the connection domain.


Assuntos
Fármacos Anti-HIV/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos
19.
Chem Biol Drug Des ; 97(1): 157-166, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32757477

RESUMO

The HIV-1 reverse transcriptase (HIV-1 RT), which is responsible for transcription of viral RNA genomes into DNA genomes, has become an important target for the treatment of patients with HIV infection. Hydrolyzed peptides from plants are considered a new source of potential drugs. In order to develop new effective inhibitors, peptides extracted from 111 Asian medicinal plants were screened against the HIV-1 RT. The crude hydrolyzed peptides from the fruit peel of Quercus infectoria were selected for purification and peptide sequence determination by HPLC and LC-MS. Two peptides of interest were synthesized, and an IC50 test was performed to determine their ability to inhibit the HIV-1 RT. The IC50 values of the peptides AIHIILI and LIAVSTNIIFIVV were determined to be 274 ± 5.10 nm and 236.4 ± 7.07 nm, respectively. This indicated that these peptides could be further developed as potential HIV-1 RT inhibitors.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Peptídeos/química , Proteínas de Plantas/metabolismo , Quercus/química , Inibidores da Transcriptase Reversa/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Frutas/química , Frutas/metabolismo , Transcriptase Reversa do HIV/metabolismo , Hidrólise , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Extratos Vegetais/metabolismo , Proteínas de Plantas/química , Plantas Medicinais/química , Plantas Medicinais/metabolismo , Quercus/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Espectrometria de Massas em Tandem
20.
FEBS J ; 288(2): 427-433, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32506843

RESUMO

Integrase strand transfer inhibitors (INSTIs) are important components of drug formulations that are used to treat people living with HIV, and second-generation INSTIs dolutegravir and bictegravir impart high barriers to the development of drug resistance. Reported 10 years ago, X-ray crystal structures of prototype foamy virus (PFV) intasome complexes explained how INSTIs bind integrase to inhibit strand transfer activity and provided initial glimpses into mechanisms of drug resistance. However, comparatively low sequence identity between PFV and HIV-1 integrases limited the depth of information that could be gleaned from the surrogate model system. Recent high-resolution structures of HIV-1 intasomes as well as intasomes from a closely related strain of simian immunodeficiency virus (SIV), which were determined using single-particle cryogenic electron microscopy, have overcome this limitation. The new structures reveal the binding modes of several advanced INSTI compounds to the HIV/SIV integrase active site and critically inform the structural basis of drug resistance. These findings will help guide the continued development of this important class of antiretroviral therapeutics.


Assuntos
Inibidores de Integrase de HIV/química , Integrase de HIV/química , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Oxazinas/química , Piperazinas/química , Piridonas/química , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Animais , Domínio Catalítico , Microscopia Crioeletrônica , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Integrase de HIV/genética , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/farmacologia , HIV-1/química , HIV-1/enzimologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Oxazinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Piridonas/farmacologia , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/enzimologia , Spumavirus/química , Spumavirus/efeitos dos fármacos , Spumavirus/enzimologia
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