Poster Sessions

Biochem-18

Michaela Wendeler

M. Wendeler, H. F. Lee, A. Bermingham, J. T. Miller, O. Chertov, M. K. Bona, N. S. Baichoo, J. Beutler, B. R. O'Keefe, M. Kvaratskhelia, S. F. J. Le Grice

Mechanistic and Structural Studies of Novel Inhibitors of HIV RNase H

The RNase H (RH) activity of HIV is indispensable for virus replication and has been recognized for 20 years as a target for antiretroviral therapy. However, development of potent and selective antagonists has made surprisingly slow progress, and so far no RH inhibitor is in clinical trial. We have characterized the natural products beta-hydroxytropolones, derived from the bark of the western red cedar, and the synthetic vinylogous ureas, obtained from NCI small molecule libraries, as two novel classes of potent RH inhibitors. beta-hydroxytropolones inhibit HIV RH at nanomolar concentrations, and vinylogous ureas at 2-4 micromolar, both being considerably less active against the human or bacterial enzyme. We combined biochemical analyses with kinetic studies and mass spectrometry to investigate how these compounds interact with the enzyme. To examine whether inhibitor binding is linked to the primer grip motif within the RH domain, residue Y501 was replaced with both natural and unnatural amino acid analogs, which permit precise changes in the steric and electronic properties of this binding pocket, while retaining full enzyme functionality. Most notably, a bulky benzoyl-phenylalanine at position 501 rendered the enzyme completely resistant to hydroxytropolone inhibition, suggesting exclusion of the inhibitor from its binding site. The same mutation induced 30-fold resistance to vinylogous ureas, indicating that the two compound classes bind to neighboring, but not identical pockets within the enzyme. For the vinylogous ureas, our kinetic analyses identified an irreversible mode of inhibition. To map the binding site of this inhibitor class on the viral enzyme, we performed mass spectrometric protein footprinting and identified a region outside the catalytic center as affected by antagonist binding. Our study illustrates that these new RH inhibitors inhibit the viral enzyme by a mode different from all previously reported antagonists, showing for the first time that pockets outside the RH catalytic center can be exploited as small molecule binding sites. Kinetic studies are in progress to investigate if these novel inhibitors can act in synergy with previously reported inhibitor classes, opening new possibilities for therapeutic intervention.