The Terminal (Catalytic) Adenosine of the HIV LTR Controls the Kinetics of Binding and Dissociation of HIV Integrase Strand Transfer Inhibitors

Abstract

Specific HIV integrase strand transfer inhibitors are thought to bind to the integrase active site, positioned to coordinate with two catalytic magnesium atoms in a pocket flanked by the end of the viral LTR. A structural role for the 3′ terminus of the viral LTR in the inhibitor-bound state has not previously been examined. This study describes the kinetics of binding of a specific strand transfer inhibitor to integrase variants assembled with systematic changes to the terminal 3′ adenosine. Kinetic experiments are consistent with a two-step binding model in which there are different functions for the terminal adenine base and the terminal deoxyribose sugar. Adenine seems to act as a "shield" which retards the rate of inhibitor association with the integrase active site, possibly by acting as an internal competitive inhibitor. The terminal deoxyribose is responsible for retarding the rate of inhibitor dissociation, either by sterically blocking inhibitor egress or by a direct interaction with the bound inhibitor. These findings further our understanding of the details of the inhibitor binding site of specific strand transfer inhibitors.

In VitroAntiviral Characteristics of HIV-1 Attachment Inhibitor BMS-626529, the Active Component of the Prodrug BMS-663068

“…Additionally, the dissociation curve for BMS-626529 fit best to a biphasic exponential dissociation equation in which the remainder of the compound was released more slowly, with a secondary half-life of ϳ23 h. Our interpretation is that the biphasic kinetics are a result of multistep binding kinetics, possibly involving an initial binding event, followed by conformational changes which lead to the final, more stable complex. This kind of complex binding has been described for small-molecule HIV integrase inhibitors (23). In addition, the binding of CD4 itself to gp120 is reported to be a slow complex process (30).…”

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

“…Dissociation studies. Dissociation experiments with INIs and IN-DNA complexes were performed by methods similar to published methods (32). Viral long terminal repeat DNA duplexes were prepared by heating 50 M 5Ј-biotinylated, 3Ј-processed plus-strand DNA and 50 M minus-strand DNA at 95°C for 5 min in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS [pH 7.2]), 50 mM NaCl, and 10 mM MgCl 2 , followed by cooling at room temperature for several hours.…”

“…The strand transfer assay was conducted as described previously (2), except that DMSO was omitted and the reaction was quenched after 30 min with 25 mM MOPS (pH 7), 50 mM EDTA, and 500 mM NaCl. (22,23,32). All experiments were performed at 37°C to approximate physiological conditions.…”

“…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).…”

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.…”