Identification of a Putative Binding Site for [2‘,5‘-Bis-O-(tert-butyldimethylsilyl)-β-d-ribofuranosyl]-3‘-spiro-5‘ ‘-(4‘ ‘-amino-1‘ ‘,2‘ ‘-oxathiole-2‘ ‘,2‘ ‘-dioxide)thymine (TSAO) Derivatives at the p51−p66 Interface of HIV-1 Reverse Transcriptase

Abstract

A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.

TSAO Compounds: The Comprehensive Story of a Unique Family of HIV- 1 Specific Inhibitors of Reverse Transcriptase

“…However the TSAO compound (23) "Fig. (9)", resulting from the removal of the oxygen atom from position 4 of the thymine ring in the prototype compound, resulted in a partial loss of activity rather than in the expected increment (EC 50, 23 : 0.23 µM vs EC 50,TSAO-T : 0.05 µM) [83]. On the other hand, the model suggested that mutation of Arg172 to Ala would decrease the affinity for TSAO compounds but would not affect the binding of other NNRTIs, because the negative electrostatic potential generated by the solvent-exposed sulfone group of TSAO could give rise to a favorable interaction energy with the positively charged side chain of Arg172 (or Lys172 in other HIV-1 strains).…”

“…However, again, the experimental results did not corroborate the molecular model. Instead, replacement of the arginine at position 172 by an alanine in RT led to an unanticipated improvement in the inhibitory activity of TSAO analogues [83]. Undoubtedly, our early model of interaction was strongly biased by attempts to make use of the NNRTI binding pocket, as seen in many complexes solved by X-ray crystallography.…”

“…Undoubtedly, our early model of interaction was strongly biased by attempts to make use of the NNRTI binding pocket, as seen in many complexes solved by X-ray crystallography. This led us to explore alternative binding modes and to study the energetics of dimerization and the rationale for the effects of TSAO-T on RT dimer stabilization [83]. The docking strategy consisted of extending the putative binding site further away from the standard NNRTI binding pocket and using all the reported X-ray crystal structures of the enzyme, both complexed with other inhibitors and in the apo form, as input for an automated docking program.…”

“…The docking strategy consisted of extending the putative binding site further away from the standard NNRTI binding pocket and using all the reported X-ray crystal structures of the enzyme, both complexed with other inhibitors and in the apo form, as input for an automated docking program. This led us to propose a novel binding mode for TSAO-m 3 T that is quite distinct from that of "classical" NNRTIs [83,84]. In the updated model, TSAO-m 3 T straddles between subunits binding at the p66/p51 interface but it does not make use of the NNRTI binding pocket.…”

“…In fact, it has been demonstrated that the heterodimeric enzyme shows a reduced DNA binding ability in the presence of 5-fold molar excess of TSAO-T and also that TSAO-e 3 T can induce the dissociation of RT into inactive monomers under certain experimental conditions. By studying the residues that are involved in dimer formation, we have identified the β7-β8 loop in the p51 subunit as an important component of the dimerization interface [83]. Interestingly, Glu138, the residue that is found mutated to Lys in TSAO-resistant viruses, is located in this loop, and our results with the Glu138Lys mutant enzyme are consistent with the experimental results that this substitution drastically reduces the affinity of TSAO derivatives and at the same time does not lead to gross changes in dimerization binding energy.…”