Probing HIV-1 integrase inhibitor binding sites with position-specific integrase-DNA cross-linking assays.
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.
Johnson
Allison A
AA
Laboratory of Molecular Pharmacology, Building 37, Room 5068, National Institutes of Health, Bethesda, MD 20892, USA.
Marchand
Christophe
C
Patil
Sachindra S
SS
Costi
Roberta
R
Di Santo
Roberto
R
Burke
Terrence R
TR
Pommier
Yves
Y
D001665:Binding Sites; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D019427:HIV Integrase / Q000737:chemistry* / Q000378:metabolism; D019428:HIV Integrase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D007211:Indoles / Q000737:chemistry; D009287:Naphthyridines / Q000494:pharmacology; D012545:Schiff Bases; D013777:Tetrazoles / Q000737:chemistry
C400757:1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)propenone; D004279:DNA, Viral; D004220:Disulfides; D019428:HIV Integrase Inhibitors; D007211:Indoles; C489415:L870810; D009287:Naphthyridines; D012545:Schiff Bases; D013777:Tetrazoles; D019427:HIV Integrase