Synthesis and functional analysis of the cysteine-rich domains of protein kinase C (PKC) for development of new medicinal leads with PKC isozyme and C1 domain selectivity

摘要

Protein kinase C (PKC) isozymes play a critical role in many signal transduction pathways and are also main targets tumor-promoting phorbol esters. Conventional and novel PKC isozymes contain two cysteine-rich C1 domains (C 1 A and C 1 B), both of which are candidates for phorbol ester binding sites. These C 1 domains of about 50-70 amino acids were synthesized by an Fmoc solid phase strategy, and were successfully folded by zinc treatment. We measured the dissociation constants (K_d's) of [~3H]phorbol-12,13-dibutyrate for all PKC C 1 peptides. Most of the C 1 peptides showed strong PDBu binding affinities with K_d's in the nanomolar range (0.45-7.4 nM) comparable to the respective whole PKC isozymes. The resultant C 1 peptide library can be used to screen for new ligands with PKC isozyme and importantly C 1 domain selectivity. We here recently developed a new lactone analogue of benzolactams (6) which shows significant selectivity in the PKC_(eta) - C 1 B binding on the basis of the structure-activity relationship of teleocidin-type tumor promoters.