The discovery of selective modulators of the DAG superfamily, the major phorbel ester receptors in cells, which presently includes PKC, the chimaerins, and unc-13 is crucial to ascertaining the role of the individual proteins in physiological processes. The discovery of such small molecule modulators of the DAG superfamily is largely an unmet pharmacological need. The major objective of this proposal is to create novel, non-tumor promoting ligands for specific DAG family members. Presently, we know how ligands bind to the DAG site, and we know how ligands might be modified to achieve selectivity among the DAG superfamily members. The discovery of DAG-site modulators of improve selectivity will not only aid the elucidation of the roles of the individual proteins in biological processes, but should eventually foster the discovery of novel therapeutics for the treatment of disease states such as cancer and Alzheimer's disease. Bryostatin provides a compelling example of a DAG superfamily modulator that has found use clinically in the treatment of cancer. During the course of this study, we will undertake the following aims: .1. Molecular modeling studies of the interactions of ILV, its benzolactam mimic, and selected synthetic targets within the PKC CRDs, the activator-binding domains, as well as the DAG binding site of the chimaerins and unc-13 will be conducted. These studies will guide the design and selection of new synthetic targets likely to exhibit DAG-site selectivity. Structure-activity relationship studies will be carried out to uncover structural features important to both binding affinity and selectivity, thus guiding the design of novel structures of synthesis. .2. The synthesis of additional benzolactam analogues bearing substituents that may confer enhanced isozyme selectivity will be carried out using chemical methods already developed by us. Simultaneously, we will explore the activity of novel fire-membered ring analogues [2-pyrrolidones] of ILV; preliminary experiments reveal these compounds to bind with good affinity to PKCalpha. .3. To determine the DAG family selectivity of the newly synthesized materials, in vitro binding studies will be carried out using the relevant recombinant PKC isozymes including mutants as well as the chimaerins and unc-13. To assess the effect of alterations in the ligand's hydrophobic side chain on its tumor promoting activity, studies of skin hyperplasia will be conducted.
