HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase

Abstract

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.

Figure 1

Schematic of the FLAG-SPA. Biotinylated anti-FLAG antibody (striped bars) is bound to SPA beads. FLAG-integrase (circles with flags) associates with the SPA beads via interaction with the anti-FLAG antibody; the immobilized enzyme binds [³H]L-731,988 (✷) and/or DNA (solid bar). [³H]L-731,988 stimulates scintillation specifically when brought into close proximity to the SPA beads (arrows) by association with integrase.

Figure 2

Activity of immobilized FLAG-integrase and inhibition by L-731,988. FLAG-integrase was assembled on the SPA beads and incubated with ³²P-labeled U5 viral ends in the absence or presence of L-731,988. The beads were pelleted and resuspended in gel loading buffer. Lane 1, no FLAG-integrase; lane 2, SPA beads with FLAG-integrase; lanes 3–14, FLAG-integrase with a titration of L-731,988; lane 3, 100 μM; lane 4, 33 μM; lane 5, 11 μM; lane 6, 3.7 μM; lane 7, 1.23 μM; lane 8, 0.41 μM; lane 9, 0.14 μM; lane 10, 0.045 μM; lane 11, 0.015 μM; lane 12, 0.005 μM; lane 13, 0.0016 μM; lane 14, 0.0005 μM. (A) Inhibition of strand transfer (1-h exposure). (B) Inhibition of 3′ processing. N-2 processing product is indicated (arrowhead) (15-min exposure). (C) Inhibition of disintegration. The cleaved (arrow) and uncleaved (arrowhead) disintegration products are indicated. Lane 1, dumbbell probe; lane 2, FLAG-integrase in solution; lane 3, FLAG-integrase associated with SPA beads; lanes 4–7, FLAG-integrase associated with SPA beads with a titration of L-731,988; lane 4, 100 μM; lane 5, 10 μM; lane 6, 1 μM; lane 7, 0.1 μM.

Figure 3

Stimulation of binding of L-731,988 to FLAG-integrase by HIV-1 donor DNA. (A) FLAG-integrase associated with SPA beads was incubated with labeled L-731,988 titrated in the absence (●) or presence (■) of 100 nM U5 DNA. (B) FLAG-integrase associated with SPA beads was incubated with labeled L-731,988 (35 nM) and unlabeled L-731,988 titrated in the absence (●) or presence (■) of 100 nM U5 DNA. FLAG-Asp116Ala associated with SPA beads was incubated with labeled L-731,988 and a titration of unlabeled L-731,988 in the presence of 100 nM U5 DNA (×). (C) FLAG-integrase associated with SPA beads was incubated with L-731,988 with a titration of U5 viral ends (■, n = 7), a nonspecific DNA containing a 2-bp 3′ overhang (×, n = 4), or a disintegration dumbbell (●, n = 2). Averaged results of multiple experiments are shown and the standard error is indicated.

Figure 4

Competition between L-731,988 and target DNA substrates in strand transfer. (A) Effect of target concentration on inhibition. In a staged microtiter plate assay (18), integrase was assembled on donor DNA and washed. After assembly, the reactions were incubated with L-731,988 (0–0.4 μM) and a titration of target DNA ranging from 0 to 125 nM. The effect of increasing amounts of target DNA on the IC₅₀ for the inhibition of strand transfer by L-731,988 is shown. (B) The effect of target preincubation on inhibition. Integrase was assembled on donor DNA in the presence of MnCl₂. Unbound integrase and MnCl₂ were removed, and target DNA was added in the absence of divalent cation. Target DNA was incubated for 0–30 min. At the specified time, L-731,988 was titrated into the reactions, and strand transfer was initiated with MnCl₂. The IC₅₀ for the inhibition of strand transfer activity by L-731,988 as a function of time of preincubation with the target is shown.

Figure 5

Direct competition of target DNA for the L-731,988 binding site. (A) Competition of target DNA for L-731,988. L-731,988 was incubated with NL4–3 integrase assembled on SPA beads coated with 2′,3′-dideoxy donor DNA. The ability of two nonspecific target DNA sequences to compete for inhibitor binding was determined. A representative experiment done with duplicate samples is shown with bars indicating standard error. ●, ▴, target DNAs 1 and 2 as described; ■, unlabeled L-731,988; ○ and ×, single-stranded G-rich and T-rich 20-mer. (B) Exonuclease protection of donor DNA in the presence of target DNA. NL4–3 integrase was assembled on SPA beads coated with ³²P-labeled, 2′,3′- dideoxy donor DNA. The assembled beads were incubated overnight with target DNA, washed, and treated with exonuclease III. Lane 1, no integrase; lanes 2–9, beads with assembled integrase incubated with a titration of target DNA; lane 2, 1,000 nM; lane 3, 333 nM; lane 4, 111 nM; lane 5, 37 nM; lane 6, 12.3 nM; lane 7, 4.1 nM; lane 8, 1.4 nM; lane 9, 0.45 nM; lane 10, no target DNA added; lane 11, no exonuclease III treatment.