1. Discovery of a novel prolyl-tRNA synthetase inhibitor and elucidation of its binding mode to the ATP site in complex with l -proline
- Author
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Kazumasa Ogawa, Tomohiro Kawamoto, Kazuhiro Tsuchinaga, Kengo Okada, Tsutomu Henta, Masanori Miwa, Shoichi Ohkubo, Ryutaro Adachi, and Robert J. Skene
- Subjects
0301 basic medicine ,Proline ,Biophysics ,Context (language use) ,Prolyl tRNA synthetase ,Biology ,Inhibitory postsynaptic potential ,Biochemistry ,Amino Acyl-tRNA Synthetases ,Structure-Activity Relationship ,03 medical and health sciences ,Adenosine Triphosphate ,0302 clinical medicine ,Drug Discovery ,medicine ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Ternary complex ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,Halofuginone ,Substrate (chemistry) ,Cell Biology ,Pyrazinamide ,HEK293 Cells ,030104 developmental biology ,Enzyme ,chemistry ,030220 oncology & carcinogenesis ,medicine.drug - Abstract
Prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase family of enzymes and catalyzes the synthesis of prolyl-tRNAPro using ATP, l-proline, and tRNAPro 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 Kd 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.
- Published
- 2017