Selectivity for strand-transfer over 3′-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme–DNA interactions in the active site

Abstract

Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3′-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and −1 bases, which flank the 3′-processing site, play a critical role for 3′-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3′-processing activity, becomes more active and more resistant to inhibition of 3′-processing by RAL and DTG in the absence of the −1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3′-processing and ST reactions. The potency of integrase inhibitors against 3′-processing and their ability to overcome resistance is discussed.

Non-Cryogenic Structure and Dynamics of HIV-1 Integrase Catalytic Core Domain by X-ray Free-Electron Lasers

“…In particular, the hydrogen bond network mediated by the water molecules around the Asn120 located near the active site changed. Since water molecules are used for nucleophilic attack in the 3′-processing reaction, these changes in the water molecules located near the active site may affect integrase (IN) catalysis [29,30,31]. This suggests that the changes in enzymatic activity due to temperature are related to this series of structural changes.…”

Probing Resistance Mutations in Retroviral Integrases by Direct Measurement of Dolutegravir Fluorescence

“…S7 ). Moreover, the intrinsic ability of INSTI to also inhibit 3′P activity has been recently described but this occurs only above micromolar drug concentrations 49 , 50 . All considerations mentioned above explain why the fluorescence-based DTG-binding assay remains robust and predictive of both DNA-binding properties and resistance mutations of IN and could be extended to the study of other resistance mutations against DTG or EVG, the anisotropy mode being more suitable in the latter case since EVG fluorescence emission is not strongly dependent on Mg 2+ chelation.…”

Discovery of Novel Integrase Inhibitors Acting outside the Active Site Through High-Throughput Screening

“…Inhibition of IN by NSC34931 exhibits some remarkable characteristics that set NSC34931 apart from current IN active site inhibitors. First, NSC34931 inhibits both 3′-P and ST with similar efficiencies, which is in contrast with the high ST-selectivity of active site inhibitors [21]. Second, NSC34931 inhibits RAL-resistant IN mutants G140S/Q148H, N155H, and Y143R, which are involved in the three major clinical resistance pathways to RAL (for review see [3]).…”

“…Recombinant enzymes were expressed in E. coli RosettaII (IPTG induction) and purified on nickel chelating column as described in [21]. Integrity and purity of wild type (WT) and mutant enzymes were checked by direct coomassie coloration of elution fractions on SDS-PAGE.…”