Exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H

J Med Chem. 2013 Jul 11;56(13):5436-45. doi: 10.1021/jm400405z. Epub 2013 Jun 20.


The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3',4'-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology
  • Drug Resistance, Viral / drug effects*
  • Drug Resistance, Viral / genetics
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Enzyme Stability / drug effects
  • HIV Reverse Transcriptase / antagonists & inhibitors*
  • HIV Reverse Transcriptase / chemistry
  • HIV Reverse Transcriptase / genetics
  • HIV-1 / drug effects
  • HIV-1 / enzymology
  • HIV-1 / genetics
  • Humans
  • Models, Molecular
  • Molecular Structure
  • Mutation
  • Protein Binding
  • Protein Structure, Tertiary
  • Pyrimidinones / chemistry
  • Pyrimidinones / pharmacology*
  • Reverse Transcriptase Inhibitors / chemistry
  • Reverse Transcriptase Inhibitors / pharmacology
  • Ribonuclease H, Human Immunodeficiency Virus / antagonists & inhibitors*
  • Ribonuclease H, Human Immunodeficiency Virus / chemistry
  • Ribonuclease H, Human Immunodeficiency Virus / genetics
  • Temperature


  • Antiviral Agents
  • Enzyme Inhibitors
  • Pyrimidinones
  • Reverse Transcriptase Inhibitors
  • HIV Reverse Transcriptase
  • Ribonuclease H, Human Immunodeficiency Virus