Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge

A discovery approach based on an intramolecular inhibitory mechanism was ap plied to a prototype calmodulin (CaM)-regulated protein kinase in order to demonstrate a proof-of-principle for the development of selective inhibitor s. The overall, approach used functional genomics analysis of myosin light chain kinase (MLCK) to identify short autoinhibitory sequences that lack Ca M recognition activity, followed by recursive combinatorial peptide library production and comparative activity screens. Peptide 18 (Arg-Lys-Lys-Tyr-L ys-Tyr-Arg-Arg-Lys-NH2 one of several selective inhibitors discovered, has an IC50 = 50 nM for MLCK, inhibits CaM kinase II only at 4000-fold higher c oncentrations, and does not inhibit cyclic AMP-dependent protein kinase. An alogues of peptide 18 containing conformationally constrained cis-4-aminocy clohexanecarboxylic acid retained affinity and selectivity. The inhibitors add to the armamentarium available for the deconvolution of complex signal transduction pathways and their relationship to homeostasis and disease, an d the approach is potentially applicable to enzymes in which the catalytic and regulatory domains are found within the same open reading frame of a cD NA.