Discovery of Novel Scaffolds for Rho Kinase 2 Inhibitor Through TRFRET- Based High Throughput Screening Assay

Recent advances in basic and clinical studies have identified Rho kinase (ROCK) as an important target potentially implicated in a variety of cardiovascular diseases and ROCK inhibitors were considered as a pharmacological strategy to prevent and treat cardiovascular diseases. To screen the small molecule compound library against ROCK, a high throughput screening (HTS) campaign was carried out using immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Z’ value and signal to background (S/B) ratio were achieved at 0.76 and 5.27 for the pilot library screening of the most diverse set consisting of 15,040 compounds with a reasonable reconfirmation rate. From this screening campaign, four novel scaffolds, such as 3- nitropyridine, 4-methoxy-1,3,5,-triazine, naphthalene-1,4-dione, and 2,3-dihydro-1H-pyrrolo[2,3-b]quinoxaline, were yielded. Particularly, we found that 3-nitropyridine derivatives possess potent inhibitory activity and selectivity for ROCK. Our findings provide important information for the design of novel ROCK inhibitor. Keywords: High throughput screening, Immobilized metal affinity for phosphochemicals, rho kinase, ROCK, ROCK2 inhibitor, time-resolved fluorescence resonance energy transfer, IMAP-TR-FRET assay, Dimethyl sulfoxide, Dithiothreitol, Protein kinase C, Serum and glucocorticoid-induced protein kinase.