13th International HIV Drug Resistance Workshop


8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain


STRUCTURAL STUDIES OF BENZOPHENONE/HIV-1 RT COMPLEXES: INSIGHTS INTO THE POTENCY OF THE NEXT GENERATION NNRTIS AGAINST WT AND MUTANT HIV-1

Antiviral Therapy 2004; 9:S36

DK Stammers1, DI Stuart1, J Ren1, PP Chamberlain1, K Weaver2, S Short2, C Andrews2, K Romines2, L Boone2, L Schaller2, A Freeman2 and J Chan2
1University of Oxford, Oxford, UK; and 2GlaxoSmithKline, Research Triangle Park, NC, USA

BACKGROUND: Approximately 250 analogues of a benzophenone scaffold have been synthesized and assayed for anti-HIV activity. Lead compounds were selected based on approximately equivalent potency against WT, Y181C and K103N mutants. Optimized compounds maintain activity against many NNRTI-resistant strains (e.g. the IC50 for 678248 is <10 nM against 22 of 24 mutant viruses including nine double mutants). We have used crystallographic structures of RT-inhibitor complexes to aid in the design of compounds to define the binding mode of prototype compounds. A compound from this series, 678248X, is the active agent from the prodrug 695634G now in clinical trials.

METHODS: WT and site-directed mutant RTs were produced in E. coli and purified by ion-exchange procedures. RT/inhibitor complex crystals were obtained by co-crystallization and X-ray data collected at synchrotron sources. Data were indexed and integrated with DENZO, and data were merged with SCALEPACK. The molecular orientation and position in the unit cell were determined using rigid-body refinement with CNS. The structures were refined with CNS using positional, simulated annealing and individual B-factor refinement with bulk solvent correction and anisotropic B-factor scaling. Model rebuilding was done using O.

RESULTS: We have determined 14 structures of WT, K103N, L100I, and V106A/Y181C HIV-1 RT with various benzophenone NNRTIs, the best to 2.1 Å resolution. Novel features in the binding of these NNRTIs to RT include an intramolecular hydrogen bond in the inhibitor. Tyr181 is in a ‘down’ position which is unusual for such high affinity compounds. Also, there are minimal inhibitor interactions with Tyr181. The inhibitors extend out of the NNRTI pocket in the region of Pro236. There are subtle changes in inhibitor conformation as a result of different mutations.

CONCLUSIONS: The potency of the benzophenones against WT and mutant RTs is accomplished by a wide range of contacts with the protein as well as the stabilizing effect of hydrogen bond formation within the compound itself. The minimal contacts with Tyr181 and the relative flexibility of these compounds that allow subtle rearrangements are factors that contribute to the remarkable resilience of the benzophenone series to such a wide range of resistance mutations.

PRESENTING AUTHOR: DK Stammers

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2004-06-08

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