Mechanism of Action of Novel Dual Actiing Pyrimidinediones

DESCRIPTION (provided by applicant): The goal of this project is to define the mechanism of anti-HIV activity of the small molecule pyrimidinedione HIV inhibitor IQP-0410. IQP-0410 inhibits HIV replication by two distinct mechanisms, acting as a nonnucleos ide reverse transcriptase inhibitor while also potently suppressing virus entry. Unlike other NNRTIs, IQP-0410 inhibits both HIV-1 and HIV-2. The ability of IQP-0410 to prevent HIV entry into target cells occurs through a unique mechanism and the compound has been found to be more potent against clinical strains of HIV than against laboratory-derived viruses. The proposed research will utilize in vitro assays for selection and genotypic and phenotypic characterization of viruses resistant to IQP-0410. The p rogram will focus on resistance engendering mutations within the viral gag, pol and envelope genes. A series of chimeric viruses containing wild type HIV-1 and HIV-2 sequences and sequences from resistant viruses at early, mid, and late stages of the selec tion process will be engineered to examine the contribution of the individual domains to IQP-0410 resistance and fitness of the virus. Pseudotype viruses possessing envelope glycoproteins from wild type and IQP-0410-resistant HIV-1 and HIV-2 viruses will b e evaluated for their ability to infect target cells in the presence and absence of IQP-0410 and to quantify the effects of env mutations on the antiviral activity of IQP-0410. Pseudotype viruses with site-specific mutations engineered into envelope gp120 and gp41 domains identified by resistance selection will be utilized to define the conformational epitope within the envelope structure targeted by IQP-0410 in entry inhibition. Additionally, experiments will be performed to further understand the role of mutations in the gag gene and evaluate potential effects of IQP-0410 on virus maturation from infected cells. The impact of resistance mutations on the activity of viral RT and protease will also be examined using biochemical analysis. PUBLIC HEALTH RELEVA NCE The goal of this grant proposal is to define the mechanism of action of a small molecule inhibitor of human immunodeficiency virus (HIV). The HIV inhibitor (IQP-0410) is known to inhibit two essential stages of virus infection and replication (reverse transcription and virus entry), and thus has the potential for providing a combination therapy with administration of a single drug. The proposed research will define the molecular targets of the compound within the virus, the mechanism by which the compou nd inhibits virus replication, and mutations that occur in the virus that make it resistant to the compound and the effects of these mutations on virus replication and infectivity.