RESUMO
HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N'-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.
Assuntos
Fármacos Anti-HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Farmacorresistência Viral , HIV-1/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Ribonuclease HRESUMO
The bulbs and aerial parts of Zephyranthes concolor (Lindl.) Benth. & Hook. f. (Amaryllidaceae), an endemic species to Mexico, were found to contain the alkaloids chlidanthine, galanthamine, galanthamine N-oxide, lycorine, galwesine, and epinorgalanthamine. Since currently only partial and low resolution (1)H-NMR data for chlidanthine acetate are available, and none for chlidanthine, its 1D and 2D high resolution (1)H- and (13)C-NMR spectra were recorded. Unambiguous assignations were achieved with HMBC, and HSQC experiments, and its structure was corroborated by X-ray diffraction. Minimum energy conformation for structures of chlidanthine, and its positional isomer galanthamine, were calculated by molecular modelling. Galanthamine is a well known acetylcholinesterase inhibitor; therefore, the isolated alkaloids were tested for this activity. Chlidanthine and galanthamine N-oxide inhibited electric eel acetylcholinesterase (2.4 and 2.6 × 10(-5) M, respectively), indicating they are about five times less potent than galanthamine, while galwesine was inactive at 10(-3) M. Inhibitory activity of HIV-1 replication, and cytotoxicity of the isolated alkaloids were evaluated in human MT-4 cells; however, the alkaloids showed poor activity as compared with standard anti-HIV drugs, but most of them were not cytotoxic.
Assuntos
Acetilcolinesterase/metabolismo , Alcaloides/química , Inibidores da Colinesterase/química , Liliaceae/química , Extratos Vegetais/química , Alcaloides/metabolismo , Alcaloides/farmacologia , Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/metabolismo , Alcaloides de Amaryllidaceae/farmacologia , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Electrophorus , Galantamina/química , Galantamina/metabolismo , Galantamina/farmacologia , HIV-1/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Liliaceae/anatomia & histologia , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Difração de Raios XRESUMO
A hit optimization protocol applied to the first nonnucleoside inhibitor of the ATPase activity of human DEAD-box RNA helicase DDX3 led to the design and synthesis of second-generation rhodanine derivatives with better inhibitory activity toward cellular DDX3 and HIV-1 replication. Additional DDX3 inhibitors were identified among triazine compounds. Biological data were rationalized in terms of structure-activity relationships and docking simulations. Antiviral activity and cytotoxicity of selected DDX3 inhibitors are reported and discussed. A thorough analysis confirmed human DDX3 as a valid anti-HIV target. The compounds described herein represent a significant advance in the pursuit of novel drugs that target HIV-1 host cofactors.
Assuntos
Fármacos Anti-HIV/síntese química , RNA Helicases DEAD-box/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/toxicidade , Linhagem Celular Tumoral , Simulação por Computador , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Técnicas de Silenciamento de Genes , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , MicroRNAs/metabolismo , Rodanina/síntese química , Rodanina/química , Rodanina/toxicidade , Relação Estrutura-Atividade , Triazinas/síntese química , Triazinas/química , Triazinas/toxicidade , Replicação Viral/efeitos dos fármacosRESUMO
As a continuation of our previous work, which resulted in the identification of a new hit compound as an HIV-1 integrase inhibitor, three novel series of salicylic acid derivatives were synthesized using three versatile and practical synthetic strategies and were assayed for their capacity to inhibit the catalytic activity of HIV-1 integrase. Biological evaluations revealed that some of the synthesized compounds possess good inhibitory potency in enzymatic assays and are able to inhibit viral replication in MT-4 cells at low micromolar concentrations. Finally, docking studies were conducted to analyze the binding mode of the synthesized compounds within the DNA binding site of integrase in order to refine their structure-activity relationships.
Assuntos
Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The alpha4beta7 integrin has been shown to serve as a coreceptor for HIV. One anti-alpha4 integrin agent (natalizumab) has been approved for the treatment of multiple sclerosis and Crohn's disease. We found that activation of CD4+ T cells with retinoic acid induced the upregulation of alpha4 and beta7 integrins. However, natalizumab failed to block the replication of HIV-1 strains in lymphoid MT-4 cells or CD4+ T cells at concentrations up to 125microg/ml. Our results suggest that alpha4 integrins are not essential cofactors for HIV replication.
Assuntos
Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/farmacologia , HIV-1/efeitos dos fármacos , Integrina alfa4/imunologia , Anticorpos Monoclonais Humanizados , Linfócitos T CD4-Positivos/virologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , HIV-1/fisiologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Natalizumab , Células U937 , Replicação Viral/efeitos dos fármacosRESUMO
Antiretroviral treatment has modified the course of human immunodeficiency virus (HIV) infection transforming it into a chronic disease. However, as treatment is conceived "for life", more effective and safety drugs, overcoming the growing resistance of the virus are required. New molecules may block the known viral targets or other new ones. The mechanism of the virus union and entrance to the cell includes the new therapeutic targets that are studied more frequently. Although studies with substances that efficiently block the virus-CD4 receptors union are in very early phases, other studies of molecules capable to block the entrance co-receptors are in more advanced phases (II or III), and enfuvirtide, a substance that blocks membrane fusion, the last phase of virus entrance, has been recently marketed. Another very promising pharmacological target is the integration of the proviral DNA as we know some substances that in vitro block HIV integrase. Besides this, new drugs are increasing the three classic antiretroviral families. Among nucleoside analogs emtricitabine (recently marketed) and amdoxovir are the more prominent. Capravirine and TMC-125 are the non-nucleoside analogs whose studies are more advanced. And atazanavir, fos-amprenavir, tipranavir and TMC-114 are the new protease inhibitors recently marketed or near to be.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Drogas em Investigação/uso terapêutico , Infecções por HIV/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores da Fusão de HIV/farmacologia , Inibidores da Fusão de HIV/uso terapêutico , Inibidores de Integrase de HIV/farmacologia , Inibidores de Integrase de HIV/uso terapêutico , Inibidores da Protease de HIV/farmacologia , Inibidores da Protease de HIV/uso terapêutico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Humanos , Receptores Virais/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/uso terapêutico , Fenômenos Fisiológicos Virais/efeitos dos fármacosRESUMO
A flow cytometry-based assay was used to simultaneously quantify X4 and R5 human immunodeficiency virus (HIV) envelope-mediated cell-to-cell viral transfer, cell death, and cell-to-cell fusion. In this assay, different anti-HIV envelope drugs showed characteristic inhibitory profiles for each measured parameter, allowing for the rapid identification of the mode of action of active compounds.
Assuntos
Fármacos Anti-HIV/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Infecções por HIV/transmissão , HIV-1/efeitos dos fármacos , Proteínas do Envelope Viral/antagonistas & inibidores , Contagem de Células , Morte Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Sais de Tetrazólio , Tiazóis , Proteínas do Envelope Viral/efeitos dos fármacosRESUMO
OBJECTIVE: To evaluate the predictive value and evolution of immunological and virological parameters related to HIV entry and pathogenesis in patients receiving enfuvirtide (ENF) plus an optimized regimen. METHODS: A phase III clinical trial substudy of ENF in 22 patients measured virus coreceptor use and sensitivity to ENF, levels of chemokines, cytokines and chemokine receptors, CD38 and HLA-DR expression as markers of T cell activation and ex vivo cell death at baseline and at week 32. RESULTS: Treatment including ENF reduced HIV viral load (P < 0.001) and increased the CD4 cell count in patients that responded (RP) to treatment (n = 14). Significant (P < 0.05) increases were noted in the RP group in CXCR4 and CCR5 expression in CD4 cells without major differences in chemokine and interleukin-7 levels. A decrease in CD38 expression in the absence of HLA-DR changes was observed in CD4 cells. Apoptosis of peripheral blood mononuclear cells was significantly reduced in the RP group. Coreceptor use or ENF sensitivity of virus isolated at baseline was not associated with virus resistance or response to treatment, which appeared to be related to the activation state (HLA-DR expression) of CD4 cells at baseline. CONCLUSION: The outcome of ENF-containing treatment could not be associated with HIV coreceptor use at baseline. CD4 cell activation and viral drug resistance were the only markers of treatment response. Changes induced by ENF-containing regimen were seen in HIV coreceptor expression, including an increase in CCR5+CD4+ cells, a decrease in CD38 T cells and a concomitant reduction of T cell apoptosis.
Assuntos
Proteína gp41 do Envelope de HIV/uso terapêutico , Inibidores da Fusão de HIV/uso terapêutico , Soropositividade para HIV/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , ADP-Ribosil Ciclase/análise , ADP-Ribosil Ciclase 1 , Adulto , Antígenos CD/análise , Apoptose/efeitos dos fármacos , Biomarcadores/análise , Contagem de Linfócito CD4 , Quimiocinas/análise , Citocinas/análise , Farmacorresistência Viral/imunologia , Enfuvirtida , Proteína gp41 do Envelope de HIV/imunologia , Inibidores da Fusão de HIV/imunologia , Soropositividade para HIV/imunologia , Soropositividade para HIV/transmissão , Antígenos HLA-DR/análise , Humanos , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/imunologia , Receptores CCR5/análise , Receptores CXCR4/análise , Resultado do Tratamento , Carga ViralRESUMO
Mannose-specific lectins (MSLs) were isolated from bulbs of fifteen wild Narcissus species growing in Spain and assayed for their HIV-1 infection inhibitory activity in MT-4 cells and compared to the Narcissus pseudonarcissus agglutinin (NPA), the commercially available MSL obtained from daffodils. Almost all the tested MSLs were found to be active, showing EC50 values (microg/mL) similar to that of NPA, with some being comparable to those obtained with dextran sulfate without significant cytotoxicity. However, on a molar basis almost all of the MSLs tested exhibited lower EC50 values than dextran sulfate whilst six MSLs had values lower than AZT. The most efficacious anti-HIV-1 activity was exhibited by the Narcissus tortifolious MSL, which was 10- (microg/mL) and 100- (molarity) fold more potent than dextran sulfate. Significantly, although this MSL was 15-fold less potent than AZT in terms of quantity (microg/mL), it was 68-fold more potent on a molar basis. The antiviral indices, a ratio of the concentrations that produce cytotoxicity and HIV-1 replication, were calculated and three of the MSLs, N. confusus, N. leonensis and N. tortifolius reported 1.5-, 2- and 8.5-fold greater AI values than dextran sulfate or AZT. Comparison of MSL haemagglutination activities (HAA) to their anti-HIV-1 activities showed that there was no significant correlation. It was suggested that this may be due to a dissociation between both activities as a consequence of multiple isolectin composition.