In an effort to further extend the number of targets for development of antiretroviral agents, we are investigating potential HIV integrase inhibitors using in vitro integrase assays. The active compounds are submitted for testing antiviral activity in the NCI Anti-HIV Drug Screen. Conversely, we are testing active compounds from the screen for HIV-1 integrase inhibition. The molecular interactions of drugs with HIV-1 integrase are investigated by comparing the drugs effects using several assays exploring different steps of the integration reaction: 1) DNA binding, 2) dinucleotide cleavage and differential use of attacking nucleophiles (water, 3'-DNA terminus, glycerol), 3) strand transfer (integration), 4) dis- integration (reverse of the integration reaction) by wild-type and truncated HIV-1 integrases. Our goals are to discover new antiviral agents, to determine whether drug screening of active agents will lead to the discovery of integrase inhibitors, to evaluate which step of the integration reaction can be inhibited by drugs (enzyme oligomerization, DNA binding, 3'-processing, DNA strand transfer, disintegration), and to determine the drug binding site(s) in the HIV-1 integrase. In order to test the hypothesis that some of the inhibiors interfere selectively with a specific functional domain of the enzyme (zinc finger, catalytic region, DNA binding domain), genetically altered HIV-1 integrases are used. Discovery of HIV integrase inhibitors may provide new strategies for antiretroviral therapy and basic knowledge for drug-enzyme interactions. During the past year, we have continued our studies with nucleotide inhibitors and identified a series of dinucleotides active in the |M range and water-soluble. Nucleotides are attractive as lead compounds for co-crystallization and photoaffinity labeling studies. We have also shown that guanosine quartet derivatives are active at nanomolar concentrations and exhibit antiviral activity. A novel assay has been developed to monitor integrase-DNA binding, that can be used to further investigate the enzyme-DNA interactions and the effects of inhibitors. In collaboration with the medicinal chemists, we have continued our structure- activity studies from CAPE derivatives and identified novel classes of inhibitors from the active compounds of the NCI screen. A novel hydrazide derivative was discovered from pharmacophore searches. Analogs are being made. An immediate goal is to obtain cocrystals with the most active inhibitors.
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