Development of a High Throughput Screen for Antagonists of Vif Dimerization

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DESCRIPTION (provided by applicant): HIV-1 is the causative agent of AIDS, that presently infects approximately 33 million persons worldwide with 1.9 million infected persons in North America alone (http://www.unaids.org). Recent studies have shown that HIV/AIDS has become a global epidemic that is not under control in developing nations. The rapid emergence of drug-resistant strains of HIV throughout the world has placed a priority on innovative approaches for the identification of novel drug targets that may lead to a new class of anti-retroviral therapies. In this regard, the HIV protein known as Vif is essential for HIV infection because it binds to and induces the destruction of APOBEC3G; a broad antiviral hostdefense factor. Vif subunits interact to form multimers and this property has been shown to be necessary for HIV infectivity. The segment of Vif that mediates subunit interaction is known. A unique opportunity has presented itself in the finding this protein-protein interaction interface is accessible in living cells infected with wild type HIV and consequently, viral replication was markedly impaired by the transduction of peptide mimics of this domain. This proposal seeks to develop an HTS primary screen for small molecules that have Vif multimerization antagonist activity based of a live cell quenched FRET assay. This homogeneous assay is based on the expression of fluorescent protein chimeras of Vif in HEK 293T cells to achieve distance-dependent quenching through fluorescence resonance energy transfer (FRET) mediated by Vif multimerization. Compounds that disrupt Vif multimerization will yield an enhanced fluorescence signal. Hits from the primary screen will be subjected to an orthogonal secondary screen in E. coli. In this assay, the secretion of ¿-lactamase into the periplastic space of bacteria occurs through an energy-independent, twin-arginine translocation pathway and is essential for ampicilin-resistant growth. We have rendered ¿-lactamase transport dependent on Vif self-association. Hits from the secondary screen will be validated for their: (1) antiviral activity through infectivity assays, (2) ability to inhibit co-immunoprecipitation of differentially epitope tagged Vif and for their ability to protect APOBEC3G from Vif-dependent degradation. Compounds identified through this proposal will be important as lead compounds to address a mandate for novel therapeutics and also provide new research reagents to study the structure and function of Vif. PUBLIC HEALTH RELEVANCE: The rapid emergence of drug-resistant strains of HIV throughout the world has placed a priority on innovative approaches for the identification of novel drug targets that may lead to a new class of anti-retroviral therapies. The HIV protein known as Vif and its ability to self-associate is required for HIV infectivity. A unique opportunity has presented itself in the finding that the interface for Vif-Vif interaction is accessible in living cells infected with wild type HIV and that viral replication can be impaired in these cells by peptide mimics of the self-association domain. This proposal seeks to develop an HTS primary screen for small molecules that have Vif multimerization antagonist activity based of a live cell FRET assay and an orthogonal secondary screen in E. coli. Compounds identified by these assays will be validated for their antiviral activity and ability to rescue host cell defense. These compounds will satisfy a mandate for novel therapeutics and also provide new research reagents for studying the structure and function of Vif.