Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes

Abstract

The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile.

Fig. 1.

HIV-1 integrase inhibitors. (A) Dolutegravir (DTG; S/GSK1349572); (B) raltegravir (RAL); (C) elvitegravir (ELV).

Fig. 2.

DTG dissociation from wild-type IN-DNA-bead complex at 37°C. Signal in the absence (●) or presence (○) of excess unlabeled DTG following subtraction of the appropriate nonspecific binding control. Bkgr, background; ADU, analogue to digital units.

Fig. 3.

INI dissociation from selected IN-DNA complexes. Symbols are the same for all graphs and represent DTG (●), RAL (○), and ELV (▼). IN-DNA complexes contained wild type (WT) IN (A), Y143R IN (B), Q148H IN (C), or N155H IN (D).

Fig. 4.

Statistical analysis of INI off-rates from IN-DNA complexes. An analysis of variance model was fit to relate the log-transformed k_off values to the inhibitors and proteins. Statistical comparisons were performed, and the resulting P values were calculated using this model. The symbols represent the k_off estimate and 95% confidence intervals for each IN-DNA complex containing the indicated IN protein (3 to 8 independent k_off values were generated for each INI with each IN-DNA complex). For each INI, the k_off values for complexes with the IN substitutions were different (P < 0.05) from the wild type (WT) except where noted (*).

Fig. 5.

Comparison of dissociative half-life and in vitro antiviral activity. The fold change data were previously reported (30). Symbols represent DTG (●), RAL (○), and ELV (▾). The horizontal line indicates the threshold for resistance that was determined in the cell-based study (FC ≥ 3).