Discovery of novel highly selective Rho kinase (ROCK) inhibitory peptides and evaluation of their biological activities



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Rho/Rho kinase pathway is a major regulator of actin cytoskeletal dynamics generation of actin-myosin contractility and has been linked to many human diseases including heart failure, hypertension, PAH, atherosclerosis, renal failure, glaucoma, and cancer. This makes ROCK as promising potential target to limit the pathogenesis of these disorders. Therefore, there are several molecules developed or being under development to inhibit ROCK activity. However, most of these small molecular inhibitors of ROCK are type I which compete with ATP to bind to the ATP binding site on catalytic domain of ROCK in its active conformation and since the ATP binding site is highly conserved in kinases, these inhibitors can cause many side effects associated with their nonspecific binding and inhibition of other kinases such as PKA and PKC. In order to overcome this poor-selectivity limitation, targeting allosteric pockets of kinases outside the ATP pocket can increase inhibitor selectivity. We used phage display libraries to identify inhibitory polypeptides that were capable of blocking ROCK1 activity in the presence of a high ATP concentration (1mM) which avoids any inhibitor bind to the ATP binding pocket. The binding affinity of Peptide25 to ROCK isoforms was measured using SPR and the binding epitope on ROCK1 was mapped using chemical cross-linking and computation modeling. It was identified to be on the activation loop, a novel locus for designing a new class of inhibitory drugs. Furthermore, alanine scanning and synthesized truncated mutants of Peptide25 were used to identify amino acids critical for ROCK1’s activation loop binding. This binding was furtherly confirmed by STD-NMR. Furthermore, the high selectivity of Peptide25 was evaluated on a panel of 182 protein kinases selected from various protein kinase families. Finally, the activities of newly developed ROCK1 inhibitory peptide and its derivatives were evaluated in tissue- and organ bath- based models of heart failure, hypertension, PAH, myogenesis, and corneal neovascularization. Peptide25 and its derivative (Pep25-P2) were shown to be as effective as the current widely used ROCK inhibitor, Y-27632, in attenuating the pathogenesis of heart failure, hypertension, PAH, and corneal neovascularization on cellular level.



Drug design, Rho kinase inhibitor, Phage display, Highly selective, Activation loop,