The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signalling 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 signalling 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 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, a protooncogene and a critical signaling regulator. 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. In further studies, we have shown that, for different responses, the same bryostatin analog may give variable proportions on antagonism, ranging from virtually none to partial. We can thus conclude that the antagonistic behavior is not an all-or-none phenomenon. Our results imply that bryostatin analogs can be designed to antagonize a desired subset of protein kinase C responses. Mechanistic studies are zeroing in on the signaling changes that correlate with the bryostatin like behavior in the LNCaP cell, highlighting the role of PKC delta activity in a nuclear enriched subcompartment. We also find that transient duration of response is a critical aspect of bryostatin action in these cells. RasGRP3 is an activator of the Ras pathway directly stimulated by diacylglycerol and phorbol esters. We find that it is expressed at markedly enhanced levels in melanoma. Inhibition of the expression of RasGRP3 with siRNA in melanoma cell lines inhibits proliferation, growth in soft agar, and tumor formation in mouse xenografts. Reciprocally, overexpression causes enhanced tumor growth. Substantial RasGRP3 expression is also found in a range of other tissue types relevant to cancer such as lung and its role in these systems is currently being investigated. In the development of therapeutics targeted to TRPV1, a major problem is designing sufficient specificity of action. We are currently evaluating resiniferatoxin based ligands which show partial agonism / partial antagonism as a potential strategy for separating the side effect of initial pain induction from the therapeutic effect of desensitization. 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005270-30
Application #
8348872
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
30
Fiscal Year
2011
Total Cost
$1,963,725
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Cooke, Mariana; Zhou, Xiaoling; Casado-Medrano, Victoria et al. (2018) Characterization of AJH-836, a diacylglycerol-lactone with selectivity for novel PKC isozymes. J Biol Chem 293:8330-8341
Das, Joydip; Kedei, Noemi; Kelsey, Jessica S et al. (2018) Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation. Biochemistry 57:732-741
Czikora, Agnes; Pany, Satyabrata; You, Youngki et al. (2018) Structural determinants of phorbol ester binding activity of the C1a and C1b domains of protein kinase C theta. Biochim Biophys Acta Biomembr 1860:1046-1056
Cummins, Thomas J; Kedei, Noemi; Czikora, Agnes et al. (2018) Synthesis and Biological Evaluation of Fluorescent Bryostatin Analogues. Chembiochem 19:877-889
Zhao, Xiguang; Kedei, Noemi; Michalowski, Alexandra et al. (2018) Deletion of the C26 Methyl Substituent from the Bryostatin Analogue Merle?23 Has Negligible Impact on Its Biological Profile and Potency. Chembiochem 19:1049-1059
Kelsey, Jessica S; Géczy, Tamás; Kaler, Christopher J et al. (2017) The C1 domain of Vav3, a novel potential therapeutic target. Cell Signal 40:133-142
Ohashi, Nami; Kobayashi, Ryosuke; Nomura, Wataru et al. (2017) Synthesis and Evaluation of Dimeric Derivatives of Diacylglycerol-Lactones as Protein Kinase C Ligands. Bioconjug Chem 28:2135-2144
Pearce, Larry V; Ann, Jihyae; Jung, Aeran et al. (2017) Novel Radiolabeled Vanilloid with Enhanced Specificity for Human Transient Receptor Potential Vanilloid 1 (TRPV1). J Med Chem 60:8246-8252
Lee, Sunho; Kang, Dong Wook; Ryu, HyungChul et al. (2017) t-Butyl pyridine and phenyl C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists. Bioorg Med Chem 25:2451-2462
Elhalem, Eleonora; Donadío, Lucía Gandolfi; Zhou, Xiaoling et al. (2017) Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKC?, PKC?, and RasGRP. Bioorg Med Chem 25:2971-2980

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