Potent Inhibitors of HIV-1 Integrase Display a Two-Step, Slow-Binding Inhibition Mechanism Which Is Absent in a Drug-Resistant T66I/M154I Mutant

Abstract

Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs.

Dolutegravir (S/GSK1349572) Exhibits Significantly Slower Dissociation than Raltegravir and Elvitegravir from Wild-Type and Integrase Inhibitor-Resistant HIV-1 Integrase-DNA Complexes

“…Streptavidin-coated scintillation proximity assay (SPA) imaging beads were purchased from PerkinElmer (Boston, MA). Full-length HIV-1 BH10 wild-type and mutant IN enzymes were expressed and purified as described previously (19) with or without 2-mercaptoethanol in the lysis and extraction buffers. Site-directed mutagenesis was used to make mutations in the wild type HIV-1 BH10 IN sequence (41).…”

“…Of note, biochemical studies have suggested that binding of INIs to HIV-1 IN proceeds by a two-step mechanism with a slow second step, and mutations that increase resistance may alter this mechanism (19,32). In addition, fast INI dissociation has been proposed to contribute to INI resistance with the N155H, T66I, and Q148R IN substitutions (14,23).…”

Rous Sarcoma Virus Synaptic Complex Capable of Concerted Integration Is Kinetically Trapped by Human Immunodeficiency Virus Integrase Strand Transfer Inhibitors

“…Localization of these STIs within the active site of the surrogate PFV intasome (6,19) has provided significant structural insights into their mechanisms for inhibiting strand transfer and the development of drug resistance. STIs also have varying capabilities to kinetically "trap" the HIV SC (10,20,21), apparently due to slow dissociation properties of the STI (22)(23)(24)(25)(26). The HIV IN STIs appear to trap SC by making contacts with both IN and DNA, as shown for the PFV intasome (6,19).…”

Molecular Dynamics Approaches Estimate the Binding Energy of HIV-1 Integrase Inhibitors and Correlate with In Vitro Activity

“…INSTIs interact with the intasome in two distinct ways; they bind the two Mg 2ϩ ions in the active site and they displace the terminal adenosine of the integrating DNA strand, allowing a halobenzyl moiety to stack with the base of the penultimate cytosine (28). Kinetic data indicate a two-step binding mechanism (22,37), suggesting that Mg 2ϩ binding and cytosine stacking could be decoupled and proceed at different rates. These structures are particularly exciting because they reveal that INSTIs interact extensively with highly conserved elements in and around the active site and that INSTI binding may require less direct protein contact than do those of other types of anti-HIV drugs.…”