RESUMEN
BMS-232632 is an azapeptide human immunodeficiency virus type 1 (HIV-1) protease (Prt) inhibitor that exhibits potent anti-HIV activity with a 50% effective concentration (EC(50)) of 2.6 to 5.3 nM and an EC(90) of 9 to 15 nM in cell culture. Proof-of-principle studies indicate that BMS-232632 blocks the cleavage of viral precursor proteins in HIV-infected cells, proving that it functions as an HIV Prt inhibitor. Comparative studies showed that BMS-232632 is generally more potent than the five currently approved HIV-1 Prt inhibitors. Furthermore, BMS-232632 is highly selective for HIV-1 Prt and exhibits cytotoxicity only at concentrations 6,500- to 23, 000-fold higher than that required for anti-HIV activity. To assess the potential of this inhibitor when used in combination with other antiretrovirals, BMS-232632 was evaluated for anti-HIV activity in two-drug combination studies. Combinations of BMS-232632 with either stavudine, didanosine, lamivudine, zidovudine, nelfinavir, indinavir, ritonavir, saquinavir, or amprenavir in HIV-infected peripheral blood mononuclear cells yielded additive to moderately synergistic antiviral effects. Importantly, combinations of drug pairs did not result in antagonistic anti-HIV activity or enhanced cytotoxic effects at the highest concentrations used for antiviral evaluation. Our results suggest that BMS-232632 may be an effective HIV-1 inhibitor that may be utilized in a variety of different drug combinations.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Oligopéptidos/farmacología , Piridinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Sulfato de Atazanavir , Proteínas Sanguíneas , Células Cultivadas , Combinación de Medicamentos , Interacciones Farmacológicas , Productos del Gen gag/metabolismo , Humanos , Técnicas In Vitro , Pruebas de Sensibilidad Microbiana , Precursores de Proteínas/metabolismoAsunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Estavudina/uso terapéutico , Fármacos Anti-VIH/farmacología , Recuento de Linfocito CD4 , Células Cultivadas , ADN Viral/genética , Farmacorresistencia Microbiana/genética , Proteína p24 del Núcleo del VIH/inmunología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , Humanos , Leucocitos Mononucleares/virología , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Recombinación Genética , Estavudina/farmacologíaRESUMEN
Current treatments for human immunodeficiency virus (HIV) include both reverse transcriptase and protease inhibitors. Results from in vitro and clinical studies suggest that combination therapy can be more effective than single drugs in reducing viral burden. To evaluate compounds for combination therapy, stavudine (d4T), didanosine (ddI), or BMS-186,318, an HIV protease inhibitor, were combined with other clinically relevant compounds and tested in a T-cell line (CEM-SS) that was infected with HIV-RF or in peripheral blood mononuclear cells infected with a clinical HIV isolate. The combined drug effects were analyzed by the methods described by Chou and Talalay (Adv. Enzyme Regul. 22:27-55, 1984) as well as by Prichard et al. (Antimicrob. Agents Chemother. 37:540-545, 1993). The results showed that combining two nucleoside analogs (d4T-ddI, d4T-zidovudine [AZT], and d4T-zalcitabine [ddC]), two HIV protease inhibitors (BMS-186,318-saquinavir, BMS-186,318-SC-52151, and BMS-186,318-MK-639) or a reverse transcriptase and a protease inhibitor (BMS-186,318-d4T, BMS-186,318-ddI, BMS-186,318-AZT, d4T-saquinavir, d4T-MK-639, and ddI-MK-639) yielded additive to synergistic antiviral effects. In general, analysis of data by either method gave consistent results. In addition, combined antiviral treatments involving nucleoside analogs gave slightly different outcomes in the two cell types, presumably because of a difference in phosphorylation patterns. Importantly, no strong antagonism was observed with the drug combinations studied. These data should provide useful information for the design of clinical trials of combined chemotherapy.
Asunto(s)
VIH/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , Línea Celular , Combinación de Medicamentos , Interacciones Farmacológicas , Estudios de Evaluación como Asunto , Humanos , Replicación Viral/fisiologíaRESUMEN
The human immunodeficiency virus (HIV) fusion inhibitor siamycin I, a 21-residue tricyclic peptide, was identified from a Streptomyces culture by using a cell fusion assay involving cocultivation of HeLa-CD4+ cells and monkey kidney (BSC-1) cells expressing the HIV envelope gp160. Siamycin I is effective against acute HIV type 1 (HIV-1) and HIV-2 infections, with 50% effective doses ranging from 0.05 to 5.7 microM, and the concentration resulting in a 50% decrease in cell viability in the absence of viral infection is 150 microM in CEM-SS cells. Siamycin I inhibits fusion between C8166 cells and CEM-SS cells chronically infected with HIV (50% effective dose of 0.08 microM) but has no effect on Sendai virus-induced fusion or murine myoblast fusion. Siamycin I does not inhibit gp120 binding to CD4 in either gp120- or CD4-based capture enzyme-linked immunosorbent assays. Inhibition of HIV-induced fusion by this compound is reversible, suggesting that siamycin I binds noncovalently. An HIV-1 resistant variant was selected by in vitro passage of virus in the presence of increasing concentrations of siamycin I. Drug susceptibility studies on a chimeric virus containing the envelope gene from the siamycin I-resistant variant indicate that resistance maps to the gp160 gene. Envelope-deficient HIV complemented with gp160 from siamycin I-resistant HIV also displayed a resistant phenotype upon infection of HeLa-CD4-LTR-beta-gal cells. A comparison of the DNA sequences of the envelope genes from the resistant and parent viruses revealed a total of six amino acid changes. Together these results indicate that siamycin I interacts with the HIV envelope protein.
Asunto(s)
Antibacterianos/farmacología , Antivirales/farmacología , VIH/efectos de los fármacos , Fusión de Membrana/efectos de los fármacos , Péptidos , Secuencia de Bases , Antígenos CD4/efectos de los fármacos , Antígenos CD4/metabolismo , Línea Celular , Farmacorresistencia Microbiana , VIH/aislamiento & purificación , Proteínas gp160 de Envoltorio del VIH/efectos de los fármacos , Proteínas gp160 de Envoltorio del VIH/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular , Datos de Secuencia Molecular , Mutación , Virus Reordenados/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Linfocitos T/virologíaRESUMEN
To map functional domains in the retroviral Gag protein we have constructed chimeric viruses where regions of the murine leukemia virus (MuLV) Gag protein have been replaced with analogous sequences from human immunodeficiency virus type 1 (HIV-1). Here we describe the chimeric virus MuLV(MAHIV) which contains the HIV-1 matrix (MA) protein in place of the MuLV MA. MuLV(MAHIV) is infectious but grows at a reduced rate compared with wild-type MuLV. We found that the partial defect in replication of the chimeric virus is at a late stage in the viral life cycle. The MuLV(MAHIV) Gag proteins are distributed aberrantly within cells and are not associated with cellular membranes. Unlike MuLV, HIV-1 is able to integrate into growth-arrested cells. Incorporation of the HIV-1 MA, which is known to play a role in infection of nondividing cells, does not enable MuLV(MAHIV) to be expressed in growth-arrested cells. While it possesses no amino acid homology, we found that the HIV-1 MA can efficiently replace the MuLV matrix protein in infection.
Asunto(s)
Productos del Gen gag/fisiología , Antígenos VIH/fisiología , VIH-1/fisiología , Virus de la Leucemia Murina/fisiología , Proteínas de la Matriz Viral/fisiología , Proteínas Virales , Células 3T3 , Animales , División Celular , Línea Celular , Quimera , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Antígenos VIH/genética , VIH-1/genética , VIH-1/metabolismo , Semivida , Células HeLa , Humanos , Cinética , Virus de la Leucemia Murina/genética , Ratones , Precursores de Proteínas/metabolismo , Células Tumorales Cultivadas , Proteínas de la Matriz Viral/genética , Virión , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The retroviral Gag polyprotein is necessary and sufficient for assembly and budding of viral particles. However, the exact inter- and intramolecular interactions of the Gag polyproteins during this process are not known. To locate functional domains within Gag, we generated chimeric proviruses between human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MuLV). In these chimeric proviruses, the matrix or capsid proteins of MuLV were precisely replaced with the matrix or capsid proteins of HIV-1. Although the chimeric proviruses were unable to efficiently assemble into mature viral particles by themselves, coexpression of wild-type MuLV Gag rescued the HIV proteins into virions. The specificity of the rescue of HIV proteins into MuLV virions shows that specific interactions involving homologous matrix or capsid regions of Gag are necessary for retroviral particle formation.
Asunto(s)
Productos del Gen gag/metabolismo , VIH-1/metabolismo , Virus de la Leucemia Murina/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Cápside/metabolismo , VIH-1/crecimiento & desarrollo , Virus Helper/metabolismo , Virus de la Leucemia Murina/crecimiento & desarrollo , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteínas de la Matriz Viral/metabolismo , Replicación ViralRESUMEN
Immunocytochemistry and in situ hybridization were used to identify simian virus 40 (SV40) large T-antigen expression and viral DNA replication in individual cells of infected semipermissive human cell lines. SV40 infection aborts before T-antigen expression in many cells of each of the human cell lines examined. In all but one of the human cell lines, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Since different structural and functional classes of T antigen can be recognized with anti-T monoclonal antibodies, we examined infected A172 cells with a panel of 10 anti-T monoclonal antibodies to determine whether viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal antibody, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentage of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Finally, in situ hybridization was used to show that the percentage of human cells expressing T antigen was similar to the percentage transcribing early SV40 mRNA. Thus, the block to T-antigen expression in human cells is at a stage prior to transcription of early SV40 mRNA.