Adenosine-oligoarginine conjugate, a novel bisubstrate inhibitor, effectively dissociates the actin cytoskeleton

Aberrant regulation of protein kinases impairs normal cellular functioning and may lead to disease. The protein kinase involved in the regulation of the dynamics of the actin cytoskeleton, Rho-kinase (ROCK), phosphorylates various substrates (e.g. myosin light chain, myosin phosphatase), causing the formation of actin fibers and tension inside cells. Hyperactivation of ROCK, for example, causes hypertension and cardiovascular disorders. Thus, the design of highly specific protein kinase inhibitors is of the utmost importance. To date, the majority of inhibitors investigated have been found to mimic and compete with ATP. However, in the present study we characterized the cellular effects of a novel bisubstrate inhibitor -- adenosine-oligoarginine conjugate (ARC) -- designed to interfere simultaneously with the ATP site and the substrate-binding pocket of basophilic kinases. ARC effectively pulled down ROCK from cell lysates, showed no cytotoxicity and suppressed the assembly of the actin cytoskeleton (especially central actin bundles) as the result of interference with the activity of the kinase. Combination of ARC with chloroquine yielded a stronger inhibitory effect and gave results similar to treatment with Y-27632. However, treatment with ARC produced more actin fragments and yielded a longer-lasting effect than treatment with Y-27632. Additionally, quantification of phosphorylated myosin light chain levels in ARC-treated or Y-27632-treated cells implies that ARC is more effective than Y-27632 in suppressing the phosphorylation of at least one of the substrates of ROCK. We believe that the described bisubstrate strategy could be a useful lead for designing novel, highly specific inhibitors for different protein kinases.