MODELING CHEMISTRY, AND BIOLOGY OF THE BENZOLACTAM ANALOGS OF INDOLACTAM-V (ILV) .2. IDENTIFICATION OF THE BINDING-SITE OF THE BENZOLACTAMSIN THE CRD2 ACTIVATOR-BINDING DOMAIN OF PKC-DELTA AND DISCOVERY OF ANILV ANALOG OF IMPROVED ISOZYME SELECTIVITY

Protein kinase C (PKC) is a complex enzyme system comprised of at leas t II isozymes that serves to mediate numerous extracellular signals wh ich generate lipid second messengers. The discovery of isozyme-selecti ve activators and inhibitors (modulators) of PKC is crucial to ascerta ining the role of the individual isozymes in physiological and pathoph ysiological processes and to manipulating their function. The discover y of such small molecule modulators of PKC is at present a largely unm et pharmacological need. Herein we detail our modeling studies which r eveal how the natural product indolactam V (ILV) and its 8-membered ri ng analogue, the benzolactam 15, bind to the CRD2 activator domain of PKC. These modeling studies reveal that not all PKC ligands possess a common pharmacophore, and further suggest an important role of specifi c hydrophobic contacts in the PKC-ligand interaction, The modeling stu dies find strong experimental support from mutagenesis studies on PKC alpha that reveal the crucial role played by the residues proline 11, leucine 20, leucine 24, and glycine 27. Next, we describe the synthesi s of two 8-substituted benzolactams starting from L-phenylalanine and characterize their isozyme selectivity; one of the two benzolactams ex hibits improved isozyme selectivity relative to the n-octyl-ILV, Lastl y, we report inhibition of cellular proliferation of two different bre ast carcinoma cell lines by the benzolactam 5 and show that the compou nd preferentially down-regulates PKC beta in both cell lines.