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 vitroand suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3Ј 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 ␤4 -␣2 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.

Fueling HIV-1 integrase drug design with structural insights

“…Until 2010, their precise molecular mechanism of action was still far from clear. With the solution of the PFV intasome structure, Hare et al delineated with atomic resolution the binding mode, proteinligand interactions and resistance mechanisms for several INSTIs [6,7,9]. These significant strides forward opened up the field further and allow in silico design of second generation INSTIs with maximal confidence.…”

“…Next to optimized interactions with the active site, DTG features a longer linker between the metal-chelating and fluorobenzyl pharmacophores, probably allowing for small compensatory movements within the active site [9]. Recently, a comprehensive study of the INSTI metal-chelating motif was published, combining in vitro work and docking simulations, shedding more light on this vital pharmacophore feature [10].…”

“…INSTIs effectively do just this, but the class is faced with viral resistance issues (Stanford HIV Drug Resistance Database; http://hivdb.stanford.edu). Recently, INSTIblocked intasome crystal structures have been solved, providing support for in silico development of next generation compounds [6,7,9]. Another emerging strategy is to target IN protein-protein interactions, for example, with its crucial cellular cofactor LEDGF/p75 [16,20].…”

Retroviral Integrase Proteins and HIV-1 DNA Integration

“…The amino acid sequences of the HIV-1 and PFV IN CCDs are 22% identical, and crystal structures of PFV intasomes harboring wild-type or drug resistance changes, with or without bound drug, have helped explain the structural basis of HIV-1 drug resistance (18,68,69). RAL in particular is unlikely to directly contact the side chains of resistance substitutions that occur at position 148 or 155 (45).…”

“…The crystal structures also revealed the role of the halobenzyl groups, which is to supplant the adenosine ring of the terminal deoxyadenylate residue at the processed LTR end and concordantly eject the nucleotide with its associated 3Ј-OH strand transfer nucleophile from the enzyme active site (Fig. 3B) (18,68,69). INSTI engagement of the IN active site within the PFV CDC clashes with the position of the scissile dinucleotide at the uncleaved LTR end in the analogous SSC structure (Fig.…”