Towards Novel 3-Aminopyrazinamide-Based Prolyl-tRNA Synthetase Inhibitors: In Silico Modelling, Thermal Shift Assay and Structural Studies

Int J Mol Sci. 2021 Jul 21;22(15):7793. doi: 10.3390/ijms22157793.


Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

Keywords: X-ray crystallographic studies; in silico modelling; inhibitor; prolyl-tRNA synthetase; thermal shift assay.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Amino Acyl-tRNA Synthetases / antagonists & inhibitors*
  • Binding Sites
  • Biological Assay / methods*
  • Computer Simulation*
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / isolation & purification
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Ligands
  • Models, Molecular
  • Protein Conformation
  • Pyrazinamide / chemistry*


  • Enzyme Inhibitors
  • Ligands
  • Pyrazinamide
  • Adenosine Triphosphate
  • Amino Acyl-tRNA Synthetases
  • prolyl T RNA synthetase