Development of new agents that regulate cellular signal transduction

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Protein kinase C (PKC) isozymes are major receptors of tumor-promoting phorbol esters. They contain two cysteine-rich C1 domains (C1A, C1B), both of which are candidates for phorbol 12,13-dibutyrate (PDBu) binding sites. To investigate the mechanism of tumor promotion in molecular level, the C1 peptides corresponding to the C1 domains of all PKC isozymes were synthesized, and their dissociation constants for PDBu were measured. The resultant C1 peptide library was used to screen for new ligands with PKC isozyme and C1 domain selectivity to find that indolactam-V (IL-V) is a promising lead compound.We verified that the indole ring of IL-V could be involved in the CH/πinteraction with Pro-11 of the C1B domain of PKCδ using its mutant peptide, in which the CH/π interaction was inhibited by substitution of the hydrogen atom with a fluorine atom at position 4 of Pro-11. IL-V showed about a 10 times lower binding affinity to the mutant peptide compared to the wild-type peptide, suggesting th … More at the CH/π interaction could play a pivotal role on the binding of IL-V to the PKCδ C1B domain. On the other hand, benzolactam-V8 (BL-V8), with the benzene ring instead of the indole ring of IL-V, might lack the CH/π interaction. The low binding affinity of BL-V8 could be enhanced by the effective formation of the CH/n interaction as exemplified by the synthesis of naphtholactam-V8. Based on the difference among the CH/π interaction in PKC isozymes, 1-hexyl-indolactma-V10 and 8-octyl-benzolactam-V9 with potent selectivity for novel PKC isozymes (δ,ε,η,θ) that play significant roles in tumor promotion were developed. Moreover, a simplified analogue of tumor-promoting aplysiatoxin with less hydrophobicity was design-synthesized and shown to translocate PKCδ from cytosol to nuclear membrane like bryostatin 1 with anti-neoplastic activity.Recently, new phorbol ester receptors other than PKC (n-chimaerins, Unc-13s, and RasGRPs) have been reported. We unambiguously demonstrated that diacylglycerol kinase (DGK) γ and β are new targets of tumor-promoting phorbol esters by synthesizing the C1 peptides of all DGK isozymes. Less