RKI-18

The Rho-associated protein kinase (ROCK) is an essential regulator of Rho signaling pathways in a variety of cellular processes, which has been established as a potential therapeutic target to promote neuroregeneration after brain surgery.The kinase family contains two highly homologous, tissue-specific isoforms ROCK1 and ROCK2, and selective inhibition of them would result in distinct biol. effects in neurotherapy.The two kinase isoforms have only a slight difference across their active sites, but structural components out of the sites seem to confer exquisite specificity for selective binding of inhibitor ligands to ROCK1 and ROCK2.Here, a quant. structure-selectivity relationship model is developed to characterize the relative contribution of each kinase residue to inhibitor selectivity.The model was then used to screen against a panel of known kinase inhibitors, from which five compounds with high theor. selectivity are tested in vitro using a kinase assay protocol to determine their biol. activity and selectivity.Consequently, the compound CAY10576 is found to have moderate and high inhibitory potencies against ROCK1 and ROCK2 (IC₅₀ = 1.8 and 0.04 μM, resp.) as well as strong selectivity between the two isoforms (S = -1.65), which could be considered as a good lead entity to develop potent, selective ROCK inhibitors to promote neurogenesis and neuroregeneration.

Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH.

In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products.

In the pipeline, the stepwise high-throughput virtual screening, quantitative structure-activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol.

With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC50 = 0.22 and 0.95 μM, respectively.

Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.