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1.
Antimicrob Agents Chemother ; 44(9): 2319-26, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10952574

ABSTRACT

BMS-232632 is an azapeptide human immunodeficiency virus (HIV) type 1 (HIV-1) protease inhibitor that displays potent anti-HIV-1 activity (50% effective concentration [EC(50)], 2.6 to 5.3 nM; EC(90), 9 to 15 nM). In vitro passage of HIV-1 RF in the presence of inhibitors showed that BMS-232632 selected for resistant variants more slowly than nelfinavir or ritonavir did. Genotypic and phenotypic analysis of three different HIV strains resistant to BMS-232632 indicated that an N88S substitution in the viral protease appeared first during the selection process in two of the three strains. An I84V change appeared to be an important substitution in the third strain used. Mutations were also observed at the protease cleavage sites following drug selection. The evolution to resistance seemed distinct for each of the three strains used, suggesting multiple pathways to resistance and the importance of the viral genetic background. A cross-resistance study involving five other protease inhibitors indicated that BMS-232632-resistant virus remained sensitive to saquinavir, while it showed various levels (0. 1- to 71-fold decrease in sensitivity)-of cross-resistance to nelfinavir, indinavir, ritonavir, and amprenavir. In reciprocal experiments, the BMS-232632 susceptibility of HIV-1 variants selected in the presence of each of the other HIV-1 protease inhibitors showed that the nelfinavir-, saquinavir-, and amprenavir-resistant strains of HIV-1 remained sensitive to BMS-232632, while indinavir- and ritonavir-resistant viruses displayed six- to ninefold changes in BMS-232632 sensitivity. Taken together, our data suggest that BMS-232632 may be a valuable protease inhibitor for use in combination therapy.


Subject(s)
HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/drug effects , Oligopeptides/pharmacology , Pyridines/pharmacology , Amino Acid Sequence , Atazanavir Sulfate , Drug Resistance, Microbial/physiology , Drug Resistance, Multiple/physiology , HIV Protease/genetics , HIV-1/enzymology , HIV-1/genetics , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Mutation , Sequence Homology, Amino Acid , Substrate Specificity
2.
Antimicrob Agents Chemother ; 44(8): 2093-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10898681

ABSTRACT

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.


Subject(s)
HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , Oligopeptides/pharmacology , Pyridines/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Atazanavir Sulfate , Blood Proteins , Cells, Cultured , Drug Combinations , Drug Interactions , Gene Products, gag/metabolism , Humans , In Vitro Techniques , Microbial Sensitivity Tests , Protein Precursors/metabolism
4.
Antimicrob Agents Chemother ; 40(6): 1346-51, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8725999

ABSTRACT

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.


Subject(s)
HIV/drug effects , Protease Inhibitors/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Virus Replication/drug effects , Cell Line , Drug Combinations , Drug Interactions , Evaluation Studies as Topic , Humans , Virus Replication/physiology
5.
Antimicrob Agents Chemother ; 40(1): 133-8, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8787894

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antiviral Agents/pharmacology , HIV/drug effects , Membrane Fusion/drug effects , Peptides , Base Sequence , CD4 Antigens/drug effects , CD4 Antigens/metabolism , Cell Line , Drug Resistance, Microbial , HIV/isolation & purification , HIV Envelope Protein gp160/drug effects , HIV Envelope Protein gp160/genetics , HeLa Cells , Humans , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Mutation , Reassortant Viruses/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/virology
6.
J Virol ; 68(7): 4442-9, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8207817

ABSTRACT

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.


Subject(s)
Gene Products, gag/physiology , HIV Antigens/physiology , HIV-1/physiology , Leukemia Virus, Murine/physiology , Viral Matrix Proteins/physiology , Viral Proteins , 3T3 Cells , Animals , Cell Division , Cell Line , Chimera , Gene Products, gag/genetics , Gene Products, gag/metabolism , HIV Antigens/genetics , HIV-1/genetics , HIV-1/metabolism , Half-Life , HeLa Cells , Humans , Kinetics , Leukemia Virus, Murine/genetics , Mice , Protein Precursors/metabolism , Tumor Cells, Cultured , Viral Matrix Proteins/genetics , Virion , Virus Replication , gag Gene Products, Human Immunodeficiency Virus
7.
J Virol ; 67(11): 6499-506, 1993 Nov.
Article in English | MEDLINE | ID: mdl-8411353

ABSTRACT

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.


Subject(s)
Gene Products, gag/metabolism , HIV-1/metabolism , Leukemia Virus, Murine/metabolism , 3T3 Cells , Animals , Base Sequence , Capsid/metabolism , HIV-1/growth & development , Helper Viruses/metabolism , Leukemia Virus, Murine/growth & development , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , RNA, Viral/metabolism , Recombinant Fusion Proteins/metabolism , Transfection , Viral Matrix Proteins/metabolism , Virus Replication
8.
J Virol ; 64(8): 3760-9, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2164596

ABSTRACT

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.


Subject(s)
Antigens, Polyomavirus Transforming/genetics , DNA Replication , Simian virus 40/genetics , Animals , Antibodies, Monoclonal , Capsid/genetics , Cell Line , DNA Probes , DNA, Viral/genetics , Glioma , Humans , Mutation , Nucleic Acid Hybridization , Simian virus 40/immunology
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