Oxygen binding isotope effects of triazole-based HIV-1 reverse transcriptase inhibitors indicate the actual binding site

Abstract Binding isotope effects (BIEs) associated with binding of four triazole-based ligands to HIV-1 reverse transcriptase have been calculated at the QM/MM MD level of theory. Two main binding sites: allosteric cavity and RNase H active site, as well as three other sites reported in the literature (the Knuckles, the NNRTI Adjacent, and Incoming Nucleotide Binding) have been considered. The interactions between inhibitors and these protein sites have been quantified by binding free energies obtained from free energy perturbation (FEP) calculations, supported by interaction energy analysis. It has been shown that binding in the allosteric cavity can be distinguished from binding to other sites based on BIEs as it is associated with normal 18 O-BIEs of the carbonyl oxygen atom while binding to RNase H active site is characterized by inverse binding isotope effect ( 18 O-BIE  18 O-BIEs close to unity are predicted. This information points to oxygen binding isotope effects of carbonyl group as indicative of the actual binding site of studied inhibitors.