RESUMO
Small-molecule inhibitors of HIV integrase (HIV IN) have emerged as a promising new class of antivirals for the treatment of HIV/AIDS. The compounds currently approved or in clinical development specifically target HIV DNA integration and were identified using strand-transfer assays targeting the HIV IN/viral DNA complex. The authors have developed a second biochemical assay for identification of HIV integrase inhibitors, targeting the interaction between HIV IN and the cellular cofactor LEDGF/p75. They developed a luminescent proximity assay (AlphaScreen) designed to measure the association of the 80-amino-acid integrase binding domain of LEDGF/p75 with the 163-amino-acid catalytic core domain of HIV IN. This assay proved to be quite robust (with a Z' factor of 0.84 in screening libraries arrayed as orthogonal mixtures) and successfully identified several compounds specific for this protein-protein interaction.
Assuntos
Inibidores de Integrase de HIV/isolamento & purificação , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sequência de Bases , Primers do DNA , Avaliação Pré-Clínica de Medicamentos , Integrase de HIV/metabolismo , HumanosRESUMO
A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 3,4-dihydro-1H-[1]-benzothieno[2,3-c]pyran and 3,4-dihydro-1H-pyrano[3,4-b]benzofuran scaffolds were designed and synthesized. Optimization of the alkyl substituent in the pyran ring showed preference for an n-propyl group, while 5,8-disubstitution pattern is preferred for the aromatic region. Analog 19 displayed potent activity with an IC(50) of 50 nM against HCV NS5B enzyme and was selective over a panel of polymerases.
Assuntos
Benzofuranos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Piranos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Benzofuranos/síntese química , Benzofuranos/química , Benzofuranos/farmacologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Piranos/síntese química , Piranos/química , Piranos/farmacologia , RNA Polimerase Dependente de RNA/química , Relação Estrutura-Atividade , Células Vero , Proteínas não Estruturais Virais/químicaRESUMO
A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1R)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 microM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log10 reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log10 reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.