The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signaling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behaviour that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signalling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signaling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents. Protein kinase C, the best studied downstream target for diacylglycerol, represents the classic system for tumor promotion and is a therapeutic target for cancer chemotherapy. The vanilloid receptor represents a promising therapeutic target for cancer pain, among other indications, and thus represents an important direction in palliative care for cancer patients. The C1 domain, the interaction domain of diacylglycerol in protein kinase C or RasGRP, forms a complex with ligand and lipid. Studies using combinatorial libraries of diacylglycerol lactones reveal that apparently minor changes in the nature of the lipid interacting groups on the diacylglycerol lactone have substantial effects of the pattern of response selectivity. In collaboration with Victor Marquez and Raz Jelinek, we have characterized the nature of the ligand interactions with lipid bilayers using a range of biophysical methods. We show that there is marked diversity in the how such ligands interact, with self association, surface binding, and bilayer penetration all contributing to variable degrees. These insights provide new guidance for ligand design. The Vav family of Rho-GEFs possess C1 domains which have a homologous 3-dimensional structure to that PKC or RasGRP but which fail to bind diacylglycerol or phorbol ester. Using site directed mutagenesis, we have identified the specific residues responsible for this lack of binding of Vav1, one of the members of the Vav family, and have designed a variant Vav C1 domain which now does bind. In further work, we have developed ligands which show enhanced selectivity for a C1 domain modified to more approach that of Vav. We conclude that lack of binding results from cumulative changes, none of which alone is sufficient to abrogate recognition. These changes principally alter lipid recognition. Identification of the nature of the critical changes provides a guide for the design of novel ligands targeted to Vav family members, which are protooncogenes and critical signaling regulators. We are extending our findings on Vav1 to the other members of the Vav family, which show more widespread tissue distribution than does Vav1. In collaboration with the chemistry group of Gary Keck, we have shown that a close analog of bryostatin 1 fails to show this antagonism on U937 leukemia cells although it retains comparable potency to bryostatin 1 on protein kinase C. Other derivatives retain the unique behavior of bryostatin 1, focusing attention on critical structural features of the molecule responsible for the bryostatin like behavior. A critical structural conclusion is that the A,B ring system in the bryostatin structure is NOT simply a linker region, as had previously been hypothesized. Our studies are further providing insights into which structural features do form the basis for bryostatin like behavior. We have identified derivatives that are synthetically more accessible but resemble bryostatin 1 in mechanism. We demonstrate that transient duration of action is the most prominent mechanistic difference between bryostatin and the phorbol esters. RasGRP3 is an activator of the Ras pathway directly stimulated by diacylglycerol and phorbol esters. We have shown that it plays an important role in the transformed phenotype of prostate cancer and melanoma cells. Substantial RasGRP3 expression is also found in a range of other tissue types relevant to cancer such as lung and we find that it makes important contributions to their phenotype as well. In the development of therapeutics targeted to TRPV1, a major problem is designing sufficient specificity of action. We are clarifying TRPV1 structure activity relations, both for capsaicin based structural leads as well as for resiniferatoxin based structures. We have further developed through homology modeling a model of the ligand binding site on TRPV1 and are currently validating that model through site directed mutagenesis and photoaffinity labeling.
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