Inhibition of the ribonuclease H activity of HIV-1 reverse transcriptase by GSK5750 correlates with slow enzyme-inhibitor dissociation

J Biol Chem. 2014 Jun 6;289(23):16270-7. doi: 10.1074/jbc.M114.569707. Epub 2014 Apr 9.

Abstract

Compounds that efficiently inhibit the ribonuclease (RNase) H activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have yet to be developed. Here, we demonstrate that GSK5750, a 1-hydroxy-pyridopyrimidinone analog, binds to the enzyme with an equilibrium dissociation constant (K(d)) of ~400 nM. Inhibition of HIV-1 RNase H is specific, as DNA synthesis is not affected. Moreover, GSK5750 does not inhibit the activity of Escherichia coli RNase H. Order-of-addition experiments show that GSK5750 binds to the free enzyme in an Mg(2+)-dependent fashion. However, as reported for other active site inhibitors, binding of GSK5750 to a preformed enzyme-substrate complex is severely compromised. The bound nucleic acid prevents access to the RNase H active site, which represents a possible biochemical hurdle in the development of potent RNase H inhibitors. Previous studies suggested that formation of a complex with the prototypic RNase H inhibitor β-thujaplicinol is slow, and, once formed, it dissociates rapidly. This unfavorable kinetic behavior can limit the potency of RNase H active site inhibitors. Although the association kinetics of GSK5750 remains slow, our data show that this compound forms a long lasting complex with HIV-1 RT. We conclude that slow dissociation of the inhibitor and HIV-1 RT improves RNase H active site inhibitors and may circumvent the obstacle posed by the inability of these compounds to bind to a preformed enzyme-substrate complex.

Keywords: Drug Development; HIV-1; Retrovirus; Reverse Transcription; Ribonuclease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • HIV Reverse Transcriptase / antagonists & inhibitors*
  • Kinetics
  • Oligodeoxyribonucleotides
  • Pyridines / pharmacology*
  • Pyrimidinones / pharmacology*
  • Reverse Transcriptase Inhibitors / pharmacology*
  • Ribonuclease H / antagonists & inhibitors*

Substances

  • GSK5750
  • Oligodeoxyribonucleotides
  • Pyridines
  • Pyrimidinones
  • Reverse Transcriptase Inhibitors
  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase
  • Ribonuclease H