TY - JOUR T1 - Probing HIV-1 Integrase Inhibitor Binding Sites with Position-Specific Integrase-DNA Cross-Linking Assays JF - Molecular Pharmacology JO - Mol Pharmacol SP - 893 LP - 901 DO - 10.1124/mol.106.030817 VL - 71 IS - 3 AU - Allison A. Johnson AU - Christophe Marchand AU - Sachindra S. Patil AU - Roberta Costi AU - Roberto Di Santo AU - Terrence R. Burke, Jr. AU - Yves Pommier Y1 - 2007/03/01 UR - http://molpharm.aspetjournals.org/content/71/3/893.abstract N2 - HIV-1 integrase binds site-specifically to the ends of the viral cDNA. We used two HIV-1 integrase-DNA cross-linking assays to probe the binding sites of integrase inhibitors from different chemical families and with different strand transfer selectivities. The disulfide assay probes cross-linking between the integrase residue 148 and the 5′-terminal cytosine of the viral cDNA, and the Schiff base assay probes cross-linking between an integrase lysine residue and an abasic site placed at selected positions in the viral cDNA. Cross-linking interference by eight integrase inhibitors shows that the most potent cross-linking inhibitors are 3′-processing inhibitors, indicating that cross-linking assays probe the donor viral cDNA (donor binding site). In contrast, strand transfer-selective inhibitors provide weak cross-linking interference, consistent with their binding to a specific acceptor (cellular DNA) site. Docking and crystal structure studies illustrate specific integrase-inhibitor contacts that prevent cross-linking formation. Four inhibitors that prevented Schiff base cross-linking to the conserved 3′-terminal adenine position were examined for inhibition at various positions within the terminal 21 bases of the viral cDNA. Two of them selectively inhibited upper strand cross-linking, whereas the other two had a more global effect on integrase-DNA binding. These findings have implications for elucidating inhibitor binding sites and mechanisms of action. The cross-linking assays also provide clues to the molecular interactions between integrase and the viral cDNA. The American Society for Pharmacology and Experimental Therapeutics ER -