Probing HIV-1 Integrase Inhibitor Binding Sites with Position-Specific Integrase-DNA Cross-Linking Assays

Abstract

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 5Ј-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 3Ј-processing inhibitors, indicating that crosslinking 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 3Ј-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.

In-Silico docking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate

“…Although some attributed the position of 5CITEP to physical entrapment during crystallization (crystal packing), recent biochemical data confirmed some of the contacts observed by Goldgur et al . [10], but showed that 5CITEP, though presenting some structural features of INSTIs, resembles more a 3'P inhibitor [10], in line with enzyme inhibition data in the presence of Mg ++ ( i.e . the metal thought to act as a cofactor in vivo ) [18].…”

“…A credible theory sees 3'P inhibitors as docking at the HIV-1 DNA-binding site, and INSTIs as occupying the position of acceptor DNA [1,10]. This theory is supported by biochemical evidence [10,11]. IN inhibitors currently in clinical trials belong to the INSTI group.…”

Impact of Y143 HIV-1 Integrase Mutations on Resistance to Raltegravir In Vitro and In Vivo

“…5A, right panel). RAL did not significantly inhibit the 3Ј-P reaction in vitro, as expected for an INSTI compound (14,21,29). This assay, which used short blunt ODNs, also revealed that the ST reaction was impaired for both mutants.…”

A Versatile and Practical Synthesis toward the Development of Novel HIV‐1 Integrase Inhibitors

“…[13,18] Through mutagenesis and photo-crosslinking studies, several amino acids that are involved in the DNA binding of IN, in particular Tyr143, have been identified, although these do not belong directly to the catalytic site of the enzyme. [19,20] In this work, pursuing our goal to identify novel HIV-1 IN inhibitors capable of blocking the IN-viral DNA interaction, and taking into account structure-activity relationship studies performed on the previous set of compounds, [17] we synthesized three new series of second-generation salicylic acid derivatives (A, B, and C in Figure 2). These compounds were designed to investigate the influence of synthetic derivatizations at position 5 of the furan ring, keeping the 2-salicylic furan moiety fixed.…”