Allosteric inhibitors of the NS3 protease from the hepatitis C virus

PLoS One. 2013 Jul 30;8(7):e69773. doi: 10.1371/journal.pone.0069773. Print 2013.

Abstract

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn(+2)-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn(+2), the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn(+2)-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation / drug effects
  • Antiviral Agents / chemistry
  • Antiviral Agents / pharmacology*
  • Cell Line
  • Drug Evaluation, Preclinical
  • Drug Resistance, Viral / genetics
  • Enzyme Activation / drug effects
  • Hepacivirus / drug effects*
  • Hepacivirus / enzymology*
  • Hepacivirus / genetics
  • Humans
  • Ligands
  • Models, Molecular
  • Molecular Conformation
  • Protease Inhibitors / chemistry
  • Protease Inhibitors / pharmacology*
  • Protein Binding
  • Viral Nonstructural Proteins / antagonists & inhibitors*
  • Viral Nonstructural Proteins / chemistry
  • Virus Replication / drug effects

Substances

  • Antiviral Agents
  • Ligands
  • NS3 protein, hepatitis C virus
  • Protease Inhibitors
  • Viral Nonstructural Proteins

Grant support

This work was supported by Spanish Ministerio de Ciencia e Innovación (BFU2010-19451 to AVC, PTA2009-2341-I to SV, and BFU2010-16297 to JS), Miguel Servet Program from Instituto de Salud Carlos III (CP07/00289 to OA), Fondo de Investigaciones Sanitarias (PI10/00186 to OA), Diputación General de Aragón (PI044/09 to AVC, Protein Targets Group B89-2011/2012 to JS, and Digestive Pathology Group B01-2011/2012 to OA), and Universidad de Zaragoza - Spain (UZ2009-BIO-05 to AVC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.