Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes

Abstract

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3' end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg(2+). The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Figure 1

Binding of radiolabeled DKAs to IN assembled on donor DNA ends. IN assembled onto U5 viral end DNA that was immobilized on SPA beads was equilibrated with ³H-I (●) or ³H-II (○).

Figure 2

Competitive binding assays at two concentrations of tritiated ligand. (A) Compounds I and II were assayed in competitive binding assays using either 5 nM or 50 nM ³H-II: compound I vs. 5 nM ³H-II (○); compound I vs. 50 nM ³H-II (▿); compound II vs. 5 nM ³H-II (●); compound II vs. 50 nM ³H-II (▾). (B) 5CITEP titrated vs. 5 nM (●) or 50 nM (○) ³H-II. (C) l-chicoric acid titrated vs. 5 nM (●)or 50 nM (○) ³H-II.

Figure 3

DKA binding requires divalent metal. IN was assembled onto the beads in the presence of 27 mM MnCl₂. The beads were washed in buffer lacking divalent metal, and the complex was incubated with 5 nM of ³H-II in MnCl₂ (●) or MgCl₂ (□) as indicated. cpm recorded were normalized to maximal binding calculated for each curve (%B_max) The average of at least three separate determinations is shown.

Figure 4

Binding of DKA to wild-type and DKA-resistant IN. After assembly onto biotinylated U5 donor DNA, binding of ³H-II to IN was assessed as follows: wild type-IN, 25 mM MnCl₂ (●); T66I,M154I-IN, 25 mM MnCl₂ (○); wild type-IN, 25 mM MgCl₂ (▾); T66I,M154I-IN, 25 mM MgCl₂ (▿).

Figure 5

Model for the binding of two divalent metals by DKA inhibitors.