Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572)

Abstract

Raltegravir (RAL) and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) efficiently block viral replication in vitro and suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3' end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV-1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL-resistant INs, we determined crystal structures of wild-type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the β4-α2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared with those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.

Fig. 1.

A, wall-eye stereo view of DTG bound to the active site of the WT PFV intasome. Carbon atoms of the INSTI are colored magenta, the protein green, and the DNA orange, whereas other atoms follow standard coloration: blue for nitrogen, red for oxygen, and pale blue for fluorine. Magnesium atoms are represented as gray spheres. Viral DNA, DTG, and selected protein residues are shown as sticks; for clarity, the 3′-nucleotide is displayed in semitransparent mode. Pale green arrowheads indicate alternative conformations for Gln215. B, comparison of the structures of RAL (yellow), MK-2048 (cyan), and DTG (magenta) when bound to the WT PFV intasome.

Fig. 2.

Activities of RAL and DTG against HIV-1 and PFV INs in in vitro strand transfer assays. IC₅₀ values of RAL and DTG for the different INs are shown as dark and light gray bars, respectively. Error bars represent the S.D. of five (HIV-1) and three (PFV) independent measurements. Mean IC₅₀ values are given atop the bars.

Fig. 3.

Single-cycle infectivity assays. EC₅₀ values of RAL are shown as dark gray bars and those of DTG as light gray bars. S.D. values are calculated from four independent measurements are indicated. Mean EC₅₀ values are given atop the bars.

Fig. 4.

Comparisons of DTG binding to WT and mutant PFV intasomes. A, superposition of WT (magenta) and S217H (cyan) structures. Ribbon representations are used for the protein backbones, residues mentioned in the text, and DTG shown as sticks, and Mg²⁺ ions as gray spheres. Black labels indicate the PFV residue numbers and mutations; with the corresponding HIV-1 residues/mutations in gray. B, superposition of WT (magenta) and N224H (green) structures. Black arrowheads indicate significant positional shifts (more than 0.35 and 0.31 Å for WT/DTG versus S217H/DTG and N224H/DTG, respectively); gray arrowheads indicate atomic displacements that may be important yet fall short of reaching significance based on the diffraction-component precision indices of the refined models.