Discovery of Novel ROCK1 Inhibitors via Integrated Virtual Screening Strategy and Bioassays

Abstract

Rho-associated kinases (ROCKs) have been regarded as promising drug targets for the treatment of cardiovascular diseases, nervous system diseases and cancers. In this study, a novel integrated virtual screening protocol by combining molecular docking and pharmacophore mapping based on multiple ROCK1 crystal structures was utilized to screen the ChemBridge database for discovering potential inhibitors of ROCK1. Among the 38 tested compounds, seven of them exhibited significant inhibitory activities of ROCK1 (IC50 < 10 μM) and the most potent one (compound TS-f22) with the novel scaffold of 4-Phenyl-1H-pyrrolo [2,3-b] pyridine had an IC50 of 480 nM. Then, the structure-activity relationships of 41 analogues of TS-f22 were examined. Two potent inhibitors were proven effective in inhibiting the phosphorylation of the downstream target in the ROCK signaling pathway in vitro and protecting atorvastatin-induced cerebral hemorrhage in vivo. The high hit rate (28.95%) suggested that the integrated virtual screening strategy was quite reliable and could be used as a powerful tool for identifying promising active compounds for targets of interest.

Figure 1. The workflow of the integrated VS protocol by combining molecular docking and pharmacophore mapping based on multiple crystal structures of ROCK1.

Figure 2. The similarity scores of Assemblies for the top 500 molecules ranked by the Glide docking scores based on eight crystal structures of ROCK1-ligand complexes.

Figure 3. The concentration-dependent inhibition of ROCK1 activities for Y27632 and TS-f22.

Figure 4. The chemical structures of eleven inhibitors of ROCK1 identified by integrated VS strategy and enzyme-based assay.

Figure 5. The concentration-dependent inhibition of ROCK1 activities for TS-13, TS-15 and TS-40.

Figure 6. The binding pocket of ROCK1 represented by solvent-accessible surface for (a) TS-13 and (b) TS-20.

Figure 7. The averaged structures from the MD simulations for the (a) TS-15–ROCK1 complex and (b) TS-11–ROCK1 complex (carbon atoms of the key residues were colored in gray, and carbon atoms of the TS-15 and TS-11 were colored in cyan and green, respectively).

The contributions of the key residues to the binding of (c) TS-15 and (d) TS-11.

Figure 8. The down-regulations of TS-15 and TS-40 in the phosphorylations of cofilin in human umbilical vein endothelial cells.

HUVEC was treated with Y27632, TS-15 or TS-40 for 2 hours. Then, the ratio of p-conflin to T-conflin of each group was normalized by the value the control. Data represented the mean ± RSD (n = 4). Statistical comparison of the data was performed by one-way ANOVA followed by Dunnett’s test. *P < 0.05 and **P < 0.01 compared to the control group.

Figure 9. The protective effects of TS-15 and TS-40 against atorvastatin-induced cerebral hemorrhage in zebrafish.

(A,a and Aa) the embryos treated with 0.2% DMSO served as the normal control group; (B,b and Bb; C,c and Cc; D,d and Dd; E,e and Ee) the embryos were pretreated with either 0.2% DMSO, Y27632 (10 mM), TS-15 (20 mM) or TS-40 (20 mM) for 2.0 hours and replaced with 1 mM atorvastatin for 24 hours. Homozygous double transgenic zebrafish, the red fluorescence was the Tg (gata1: dsRed) sd2 (A–E), the green fluorescence was Tg (fli1a: EGFP) y1 (a–e), and the third column was the overlapping photos of the first two columns (Aa, Bb, Cc, Dd and Ee). The arrows indicated the erythrocyte accumulation in cerebral hemorrhage region in zebrafish head.