Fragment-based and structure-guided discovery and optimization of Rho kinase inhibitors

Using high concentration biochemical assays and fragment-based screening assisted by structure-guided design, we discovered a novel class of Rho-kinase inhibitors. Compound 18 was equipotent for ROCK1 (IC ₅₀ = 650 nM) and ROCK2 (IC ₅₀ = 670 nM), whereas compound 24 was more selective for ROCK2 (IC ₅₀ = 100 nM) over ROCK1 (IC ₅₀ = 1690 nM). The crystal structure of the compound 18-ROCK1 complex revealed that 18 is a type 1 inhibitor that binds the hinge region in the ATP binding site. Compounds 18 and 24 inhibited potently the phosphorylation of the ROCK substrate MLC2 in intact human breast cancer cells.