A. Protein Kinase C Projet. Protein kinase C comprises a family of more than 10 isozymes involved in the regulation of cell signalling. They have been grouped into 3 subclasses according to their structure and regulation. Classical or calcium-dependent (cPKC), novel or calcium-independent (nPKC) and atypical or diacylglycerol (DAG)/phorbol ester-independent (aPKC). The first two types respond to the release of the second messenger DAG, which recruits the enzymes from their inactive cytosolic location to the membrane where they become allosterically activated. A similar effect can be achieved pharmacologically with the phorbol esters, which have substantially higher binding affinity for PKC than the DAGs. It is for that reason, that the phorbol esters have become the preferred agents to study the effects of PKC activation. On the other hand, we have synthesized a set of rationally designed DAG-lactones, which despite their structural simplicity, behave as highly potent PKC ligands capable of displaying comparable, albeit distinct activities to that of the phorbol esters, sometimes even surpassing them in potency. Last year we reported the design of the first isozyme-specific PKC activator capable of inducing the exclusive tranlocation of PKC-alpha to the cellular membrane and PKC-delta to the nuclear membrane. This compound was also able to induce strong apoptosis in human prostate cancer cells, and in the 60-cell line NCI screen potent and selective inhibition of small cell carcinoma and melanoma cells was discovered. Toxicological studies with this compound are underway under the auspices of DTP. B. DNA Methyl Transferase Project (Zebularine). We have demonstrated the ability of zebularine [2(1H)-pyrimidinone riboside] to induce the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. We also have provided evidence that zebularine can reactivate a silenced p16 gene and demethylate the promoter region in the T24 bladder carcinoma cell line in vitro as well as in tumors grown in BALB/c nu/nu mice. As a result an intensive synthetic program has started aimed at understanding the structure-activity relationship of zebularine and its analogues. Surprisingly, 2'-deoxyzebularine is inactive despite the fact that 2'-deoxyzebularine-5'-triphosphate is suspected of being the key metabolite. Synthesis of several analogues with increased lipophilicity to enhance bioavailability and facilitate formation of 2'-deoxyzebularine-5'-triphosphate is in progress. Protein kinase C isozymes. Chimaerins. Activation/inhibition, Drug design. Zinc finger. DNA methyation, Tumor suppressor genes. Apoptosis.
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