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 behavior 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 signaling 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. DAG-lactones represent a synthetically accessible platform for probing the structure activity relations of protein kinase C and the other targets downstream of the second messenger diacylglycerol. We have continued to evaluate DAG-lactones designed to have selectivity for the RasGRP subclass of diacylglycerol targets. This class is of particular importance because it functions as an activator of Ras, which is many tumors shows enhanced activity without being mutated. The atypical protein kinase C isoforms zeta and iota play important role in cellular polarity and cellular invasion. We have previously identified the specific structural differences between their DAG recognition domain and that of typical DAG responsive signaling proteins such as the classical and novel PKC isoforms. We have described a first generation of DAG lactones with selectivity for the specific differences in the recognition domains of PKC zeta and iota. We have evaluated the role of aliphatic side chains on the DAG lactone for providing selectivity for protein kinase C epsilon, a pro-tumorigenic protein kinase C isoform. Bryostatin is an agent in clinical trials with a unique mechanism of action. It binds to protein kinase C with high affinity and activates the enzyme but paradoxically antagonizes many protein kinase C mediated responses. We find, using microarray analysis of gene expression along with detailed examination of the time and dose dependence of genes representative of the differences in expression revealed by the microarray analysis, that transient duration is the predominant difference in the mode of action of bryostatin as compared to typical protein kinase C activators such as the phorbol esters. In collaboration with the groups of Gary Keck and Michael Krische, we seek to define the critical structural elements in bryostatin conferring its unique pattern of activity, with the goal of developing the next generation of bryostatin analogues. One recent insight is our identification of the B ring structure as critical for the bryostatin like activity. Protein kinase C is subject to post-translational modification, in particular phosphorylation. In collaboration with the CCR Collaborative Protein Technology Resource, we have shown that the extent of protein kinase C modification is much more extensive than had been recognized. Moreover, the pattern of modification was different for different ligands such as phorbol ester or bryostatin. We suggest that such modification signatures may be of particular value for the structure activity evaluation of ligands with complex effects where, as in the case of protein kinase C, ligand binding causes both activation and change in subcellular localization. For protein kinase C or other targets of diacylglycerol, ligand binding drives membrane association. We show that the charge on the DAG recognition domain plays a critical role in the selectivity for membrane phospholipids. Further, this selectivity also depends on the specific ligand, with more hydrophilic ligands showing greater sensitivity to membrane composition. On our TRPV1 project, we have made considerable progress in defining the optimal structures for binding of antagonists to human TRPV1, as part of our effort which seeks to advance the development of drug candidates for this target. We further are continuing our efforts to use photoaffinity labeling to confirm directly the ligand binding site on TRPV1 as well as to use mutational analysis to refine computer modeling of the putative binding site.
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