Our laboratory has been among the first to recognize and study HIV integrase as a therapeutic target. Using recombinant integrase and various enzymatic assays, we have identified and made available to the international scientific community a large chemical database of integrase inhibitors. Our current studies are aimed at: 1/ identifying novel integrase inhibitors using a high throughput assay developed in our laboratory; 2/ elucidating the molecular mechanisms of action of integrase inhibitors; 3/ studying the molecular interactions of established inhibitors with integrase by developing novel assays; and 4/ understanding the enzymology of HIV integrase and the detailed mechanisms by which integrase processes its DNA substrates.Integrase (the 3rd retroviral enzyme) is a rationale drug target because it is required for viral replication and does not have a known cellular equivalent. Two integrase inhibitors have recently started clinical trials. Integrase is a rationale target for drug development because it is essential for viral replication. It is encoded by HIV and does not have a cellular equivalent. Our laboratory has pioneered the integrase inhibitors research field, discovered several families of lead inhibitors and patented some with the aim of therapeutic development. Recently, the first class of selective HIV-1 integrase inhibitors with demonstrated antiviral activity related to integrase inhibition has been introduced in clinical trials. We are investigating the molecular mechanism of action of diketo acid (DKA) derivatives in collaboration with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, CCR, NCI) and more specifically their binding site in the integrase-DNA complex. We found that azido derivatives of diketo acids are potent and selective anti-integrase inhibitors and are antiviral. A patent application has been filed for our derivatives. Our current hypothesis is that DKA inhibitors bind at the interface of the integrase-viral DNA complex by forming a coordination complex with the divalent metal at the enzyme active catalytic site. We have now extended this concept of chelation to a two new families of drugs, conocurvones (isolated from an Australian plant) and tropolones (isolated from Canadian trees). In addition, we recently found in collaboration with Dr. Peter Roller (Laboratory of Medicinal Chemistry, CCR, NCI) that cationic peptides derived from indolicidin are integrase inhibitors. Their mechanism of action is novel as they bind to DNA, and thereby interfere with the formation of competent integrase-DNA complexes. In parallel, we have developed new integrase-DNA crosslinking assays using modified DNA substrates and mutant enzymes to determine the drug binding sites.Our recent studies supported by an IATAP grant led to the implementation of a novel high-throughput assay based on electrochemiluminescence (BioVeris). Using recombinant HIV-1 integrase, we have now screened the 3161 compounds from the three NCI Developmental Therapeutics Program (DTP) chemical libraries. A total of 123 compounds are inhibitory at 10 M (3.9% hit ratio). These 123 lead compounds are being examined in gel-based assays to evaluate their potency and selectivity for particular steps of the integrase reactions. The compounds are prioritized in collaboration with our colleagues from the NCI-CCR Laboratory of Medicinal Chemistry, the HIV Drug Resistance Program and the HIV and AIDS Malignancy Branch. Highest priority compounds are examined for mechanism of action, antiviral activity and structure-activity relationship with the aim of optimizing the drugs for medicinal development. We have filed a patent application for tropolones as anti-integrase inhibitors and anti-HIV drugs. We also discovered several tetracyclines active against integrase and with anti-HIV activity (recent patent from The Johns Hopkins University).
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