4.6 Article

Discovery of a novel prolyl-tRNA synthetase inhibitor and elucidation of its binding mode to the ATP site in complex with L-proline

Journal

Publisher

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2017.05.064

Keywords

Prolyl-tRNA synthetase; Pre-transfer editing activity; Affinity selection mass spectrometry; Molecular mechanism of action; Pyrazinamide PRS inhibitor

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Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family of enzymes and catalyzes the synthesis of prolyl-tRNA(Pro) using ATP, L-proline, and tRNA(Pro) as substrates. An ATP dependent PRS inhibitor, halofuginone, was shown to suppress autoimmune responses, suggesting that the inhibition of PRS is a potential therapeutic approach for inflammatory diseases. Although a few PRS inhibitors have been derivatized from natural sources or substrate mimetics, small-molecule human PRS inhibitors have not been reported. In this study, we discovered a novel series of pyrazinamide PRS inhibitors from a compound library using pre-transfer editing activity of human PRS enzyme. Steady-state biochemical analysis on the inhibitory mode revealed its distinctive characteristics of inhibition with proline uncompetition and ATP competition. The binding activity of a representative compound was time-dependently potentiated by the presence of L-proline with K-d of 0.76 nM. Thermal shift assays demonstrated the stabilization of PRS in complex with L-proline and pyrazinamide PRS inhibitors. The binding mode of the PRS inhibitor to the ATP site of PRS enzyme was elucidated using the ternary complex crystal structure with L-proline. The results demonstrated the different inhibitory and binding mode of pyrazinamide PRS inhibitors from preceding halofuginone. Furthermore, the PRS inhibitor inhibited intracellular protein synthesis via a different mode than halofuginone. In conclusion, we have identified a novel drug-like PRS inhibitor with a distinctive binding mode. This inhibitor was effective in a cellular context. Thus, the series of PRS inhibitors are considered to be applicable to further development with differentiation from preceding halofuginone. (C) 2017 Elsevier Inc. All rights reserved.

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