We have previously shown that PFV IN is sensitive to HIV-1 IN InSTIs17
. These compounds are thought to engage metal ion cofactors in the IN active site through interactions with uniquely positioned oxygen atoms of the pharmacophore31
. The role of the remaining common InSTI feature, a fluorobenzyl group, is enigmatic. Intasome structure refinement using diffraction data collected on crystals soaked in the presence of Mg2+
and the clinical InSTI MK0518 (also known as raltegravir)14
or GS9137 (elvitegravir)15
revealed strong additional electron density within the active sites of the inner IN subunits. Structures of MK0518 or GS9137 with pairs of Mg atoms could be easily fitted into the maps and refined to 2.85 and 3.15 Å resolution, respectively (, Supplementary Fig. 4d, f
and Supplementary Table 1
). In addition, soaking crystals in the presence of the drugs and Mn2+
produced similar results, with manganese atoms and drug molecules refining at almost precisely the same positions (Supplementary Fig. 4b, c, e, g
). Concordantly, InSTIs inhibited both the Mg2+
-dependent activities of the PFV intasome (Supplementary Fig. 1d
Based on the structures, the two InSTIs appear to have very similar modes of binding and action, involving an induced fit mechanism. Their metal chelating oxygen atoms orient towards the metal cofactors of the active site, while their halobenzyl groups fit within a tight pocket created by displacement of the 3′ adenosine (A17). Within it, the drugs make intimate Van der Waals interactions with the bases of the invariant CA dinucleotide, guanine 4 from the non-transferred strand and conserved residues Pro214 and Gln215 (). In addition, the isopropyl and methyl-oxadiazole groups of MK0518 are involved in hydrophobic and stacking interactions with the side chains of Pro214 and Tyr212, respectively (), further stabilizing this drug in the active site. Through its quinolone base and isopropyl group, GS9137 interacts with Pro214 (). Crucially, this mode of drug binding results in displacement of the reactive 3′ viral DNA end from the active site (), which can only result in deactivation of the intasome. Thus, upon binding of MK0518, the reactive 3′ hydroxyl group moves away from the active site by more than 6 Å, compared to its positions in the Mg2+ or Mn2+-containing, or apo crystals.
Because the core contact points consisting of invariant nucleotide bases and amino acid residues are conserved in HIV-1, the mode of InSTI binding and action are unlikely to significantly differ. The extensive contacts with the viral DNA end observed in our structures elucidate why the InSTIs preferentially interact with and inhibit the DNA-bound form of HIV-1 IN16
. Moreover, the induced fit caused by displacement of the 3′ adenosine by the halobenzyl groups of these compounds explains why the deletion of this base dramatically increased InSTI on- and off- rates for binding to HIV-1 IN-DNA complexes32
. Furthermore, mutations of HIV-1 IN residue Tyr143, which, based on our structure, is expected to interact with the methyl-oxadiazole group of MK0518 (, Supplementary Fig. 2
), are known to confer resistance to this drug33
. Common InSTI resistance pathways involve mutations of HIV-1 IN Gln148 or Asn15533
, which correspond to PFV IN residues Ser217 and Asn224, respectively (Supplementary Fig. 2
). Mutations at these positions are likely to interfere with coordination of metal cofactors by the active site carboxylates, as proposed recently34
. Conceivably, a slight shift in metal ion cofactor positions might suffice to abrogate drug binding, which relies on its spatially constrained metal chelating groups, albeit at a steep price of impaired viral replication fitness due to detuning of the IN active site structure.
Our findings will allow the generation of reliable HIV-1 IN and InSTI pharamacophore models, which will be invaluable for the development of next generation strand transfer inhibitors. Their design should take advantage of the most conserved elements of the IN active site elucidated here, such as the bases of the invariant CA dinucleotide, positions of the metal co-factors and the main chain atoms of the protein.