RESUMO
Novel ethyl 2-(5-aryl-1H-imidazol-1-yl)-acetates 17 and propionates 18, together with their acetic acid 19 and acetohydrazide 20 derivatives, were designed and synthesized using TosMIC chemistry. Biological evaluation of these newly synthesized scaffolds in the HIV-1 Vpu- Host BST-2 ELISA assay identified seven hits (17a, 17b, 17c, 17g, 18a, 20f and 20g) with greater than 50% inhibitory activity. These hits were validated in the HIV-1 Vpu- Host BST-2 AlphaScreen™ and six of the seven compounds were found to have comparable percentage inhibitory activities to those of the ELISA assay. Compounds 17b and 20g, with consistent percentage inhibitory activities across the two assays, had IC50 values of 11.6 ± 1.1 µM and 17.6 ± 0.9 µM in a dose response AlphaScreen™ assay. In a cell-based HIV-1 antiviral assay, compound 17b exhibited an EC50 = 6.3 ± 0.7 µM at non-toxic concentrations (CC50 = 184.5 ± 0.8 µM), whereas compound 20g displayed antiviral activity roughly equivalent to its toxicity (CC50 = 159.5 ± 0.9 µM). This data suggests that compound 17b, active in both cell-based and biochemical assays, provides a good starting point for the design of possible lead compounds for prevention of HIV-1 Vpu and host BST-2 protein binding in new anti-HIV therapeutics.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Proteínas do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Imidazóis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Fármacos Anti-HIV/síntese química , Antígenos CD , Linhagem Celular , Desenho de Fármacos , Proteínas Ligadas por GPI/antagonistas & inibidores , HIV-1/química , Humanos , Imidazóis/síntese química , Testes de Sensibilidade Microbiana , Replicação Viral/efeitos dos fármacosRESUMO
Vpu is one of four accessory proteins encoded by human immunodeficiency virus type I (HIV-1). Vpu modulates the expression of several cellular restriction factors within the HIV-1 infected cell including CD4, CD74, the bone marrow stromal antigen 2 (BST-2) and NK-T-and-B antigen. The interaction of HIV-1 Vpu with these proteins interferes with the innate immune response directed against HIV-1; thereby promoting viral persistence. The involvement of HIV-1 Vpu in manipulating the cellular environment in ways that favor viral replication makes it an attractive target for anti-HIV drug intervention. This paper describes the over-expression and purification of a soluble HIV-1 Vpu from inclusion bodies by ion-exchange chromatography, allowing production of 6 mg of highly purified protein (>95% purity) per 10 mg of pelleted cells obtained from 1 L of bacterial culture. Far-UV circular dichroism showed that the recombinant protein is folded and retained its secondary structure. Moreover, using ELISA, known HIV-1 Vpu binding partners, BST-2 and CD74, showed that the refolded purified protein is functional or at least assumes a conformation that is capable of binding these putative binding partners. To our knowledge, this is the first report of the purification and successful solubilization of full-length, wild-type HIV-1 Vpu from inclusion bodies in Escherichia coli.
Assuntos
Antígenos CD/química , Antígenos de Diferenciação de Linfócitos B/química , HIV-1/genética , Antígenos de Histocompatibilidade Classe II/química , Proteínas do Vírus da Imunodeficiência Humana , Proteínas Virais Reguladoras e Acessórias , Escherichia coli , Proteínas Ligadas por GPI/química , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/biossíntese , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/isolamento & purificação , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/biossíntese , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificaçãoRESUMO
Mitochondria are key to eukaryotic cell survival and their activity is linked to generation of reactive oxygen species (ROS) which in turn acts as both an intracellular signal and an effective executioner of cells with regards to cellular senescence. The mitochondrial molecular chaperone tumor necrosis factor receptor associated protein 1 (TRAP1) is often termed the cytoprotective chaperone for its role in cancer cell survival and protection from apoptosis. Here, we hypothesize that TRAP1 serves to modulate mitochondrial activity in stem cell maintenance, survival and differentiation.
