Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

PLoS One. 2016 Jan 22;11(1):e0147225. doi: 10.1371/journal.pone.0147225. eCollection 2016.

Abstract

The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.

Publication types

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

MeSH terms

  • Alkynes
  • Anti-HIV Agents / pharmacology*
  • Benzoxazines / antagonists & inhibitors
  • Benzoxazines / pharmacology
  • Binding Sites
  • Cyclopropanes
  • Enzyme Inhibitors / pharmacology*
  • HIV Reverse Transcriptase / antagonists & inhibitors*
  • HIV-1 / enzymology
  • Humans
  • Hydrazones / pharmacology*
  • Isatin / analogs & derivatives*
  • Isatin / pharmacology
  • Microbial Sensitivity Tests
  • Molecular Docking Simulation
  • Protein Structure, Tertiary
  • Reverse Transcriptase Inhibitors / pharmacology*
  • Ribonuclease H, Human Immunodeficiency Virus / antagonists & inhibitors*
  • Virus Replication / drug effects

Substances

  • 4-(2-(2-(2-oxoindolin-3-ylidene)hydrazinyl)thiazol-4-yl)benzonitrile
  • Alkynes
  • Anti-HIV Agents
  • Benzoxazines
  • Cyclopropanes
  • Enzyme Inhibitors
  • Hydrazones
  • Reverse Transcriptase Inhibitors
  • Isatin
  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase
  • Ribonuclease H, Human Immunodeficiency Virus
  • efavirenz