The ribonuclease H (RNH) activity of HIV-1 reverse transcriptase (RT) is essential for viral replication and can be a target for drug development. Yet, no RNH inhibitor to date has substantial antiviral activity to allow advancement into clinical development. Herein, we describe our characterization of the detailed binding mechanisms of RNH active-site inhibitors, YLC2-155 and ZW566, that bind to the RNH domain through divalent metal ions, using NMR, molecular docking, and quantum mechanical calculations. In the presence of Mg2+, NMR spectra of RNH exhibited split (two) resonances for some residues upon inhibitor binding, suggesting two binding modes, an observation consistent with the docking results. The relative populations of the two binding conformers were independent of inhibitor or Mg2+ concentration, with one conformation consistently more favored. In our docking study, one distinctive pose of ZW566 showed more interactions with surrounding residues of RNH compared to the analogous binding pose of YLC2-155. Inhibitor titration experiments revealed a lower dissociation constant for ZW566 compared to YLC2-155, in agreement with its higher inhibitory activity. Mg2+ titration data also indicated a stronger dependence on Mg2+ for the RNH interaction with ZW566 compared to YLC2-155. Combined docking and quantum mechanical calculation results suggest that stronger metal coordination as well as more protein-inhibitor interactions may account for the higher binding affinity of ZW566. These findings support the idea that strategies for the development of potent competitive active site RNH inhibitors should take into account not only metal-inhibitor coordination but also protein-inhibitor interaction and conformational selectivity.
Bibliographical noteFunding Information:
We especially acknowledge Michael A. Parniak, who is now deceased, for his long-term support and encouragement of the work. We thank Karen Kirby for discussion, Teresa Brosenitsch for critical reading of the manuscript, and Michael Delk for NMR support. This research was supported in part by Chatham University, University of Pittsburgh, and the National Institutes of Health (AI100890 to S.G.S. and Z.W. and GM105401 to R.I.).
Copyright © 2019 American Chemical Society.
- RNase H
- molecular docking
- quantum mechanical calculations