A. Protein kinase C project: Diacylglycerol-lactone (DAG-lactone) libraries generated by a solid-phase approach using IRORI technology produced a plethora of unique biological activities. Subtle differences in chemical diversity in two areas of the molecule, the combination of which generates what we have dubbed chemical zip codes, are able to transform a relatively small universe of chemical space into a larger universe of biological activities, as membrane-containing organelles within the cell appear to be able to decode these chemical zip codes. After binding to protein kinase C (PKC) isozymes or other non-kinase target proteins that contain membrane responsive domains (C1 domains), the resulting complexes appear to be directed to precise intracellular sites where different sets of substrates are come upon. Our results are counter to the general assumption that similar chemicals must have similar biological activities. Indeed, by choosing the metrics of multiple cellular bioassays, we are able to show that a group of similar DAG-lactones, all of which bind in vitro to PKC-alpha to varying degrees, expand their biological repertoire into a larger domain, targeting other proteins in a cellular milieu that result in activities of potential therapeutic importance. Our first C1 domain-selective DAG-lactone that was published last year, showing exclusive selectivity for RasGRP, has been superseded by a new, recent compound that displays a 165-fold selectivity for RasGRP over PKC-alpha. New DAG-lactone libraries bearing indole, pyridine, quinoline, and isoquinoline moieties have been recently synthesized and are being evaluated. A novel template, such as the DAG-dioxolanone, was created (J. Med. Chem. 2007, 50, 3465-3481). With this template we exploit an additional point of contact (glutamine 27) in the binding of the C1b domain of PKC-delta. Mutation of this point of contact to glutamate selectively impaired binding of the DAG-dioxolanones compared to that of the corresponding DAG-lactones (1200- to 3000-fold versus 35- to 55-fold, respectively). The differential response of this mutated C1b domain to the DAG-dioxolanones relative to the DAG-lactones provides a unique tool to probe the role of the C1b domain in PKC-delta function, where the response to the DAG-lactones affords a positive control for retained function. Using this approach, we showed that the C1b domain of PKC-delta plays the predominant role in the translocation of the enzyme to the membrane in the presence of DAG. Highly rigid and geometrically well-defined molecular rods composed of ethynylene-substituted aromatic spacers [oligo-(p-phenyleneethynylene), OPE] were incorporated as acyl moieties on DAG-lactones and investigated for their ability to bind to protein kinase C (PKC) and translocate PKC-alpha and delta isoforms to plasma and internal membranes. The kinetics of PKC translocation was correlated with biological responses: ERK phosphorylation, induction of IL-6 secretion, inhibition of cell proliferation, and induction of cellular attachment. Because OPE rods assemble through non-covalent forces and form stable films, they may influence the microdomain environment around the DAG-lactone membrane-binding site. A comparison of two DAG-lactones, one with two OPE units and the other with an equivalent flexible acyl chain of matching lipophilicity, clearly demonstrated the effect of the rigid OPE chain in substantially prolonging the translocated state of both PKC-alpha and delta (J. Med. Chem. 2007, 50, 962-978). B. Zebularine project: Inactivation of regulatory genes by aberrant promoter DNA hypermethylation is widely seen in various types of cancers and makes an attractive target for epigenetic therapy because of its reversible nature. Zebularine is a mechanism-based inhibitor of DNA methylation and a promising candidate for a cancer chemotherapy due to its stability, relatively non-toxic nature in mice, and anti-tumor properties in mice. To test whether prevention of tumorigenesis can be achieved with zebularine, MMTV-PyMT or ApcMin/+ (Min) mice were treated with zebularine in their drinking water over extended time periods. The development of mammary tumors in female MMTV-PyMT mice was reduced dramatically by this approach. Additionally, the average number of polyps in Min females decreased from 58 to 1, while the average polyp number remained unaffected in Min males, probably due to enhanced metabolism of the drug by aldehyde oxidase in the males relative to the females. Demethylation of B1 SINE elements was detected in the small and large intestines of treated Min females. The rate of growth of all mice and global gene expression in the colon in females were moderately affected, and the histology of intestinal and hepatic tissues of the Min mice was unchanged after chronic zebularine treatment, indicating low toxicity in mice. The new data demonstrate that zebularine possesses potent anticancer properties and causes little toxicity in the majority of mice undergoing therapy, and therefore, may be an excellent lead candidate for chemotherapy and chemoprevention of cancer. The progression toward the clinic for Zebularine [2(1H)-pyrimidinone riboside] was brought to an end by an unforeseen toxicity in rhesus monkeys, which was lethal when plasma levels reached 25 micromolar. This is in total contrast to the complete lack of toxicity in rodents (rats and mice), even at high doses. The lack of effect (in male Min mice) and perhaps toxicity of zebularine (in monkeys) is thought to correlate with the levels and type of the catabolic enzyme, aldehyde oxidase (AO). In female mice, AO levels are very low while in males it is very high; higher than in monkeys and humans. Gender differences in human are very small; however, species differences are large. Inhibition of the monkey enzyme by raloxifene required 500 uM compared to just 8 uM to inhibit the human enzyme. Metabolism by AO appears to be the major catabolic route, yielding uridine as the primary metabolite (Bioorg. Med. Chem. 2006, 14, 62-66). A number of 2-deoxyzebularine prodrugs continue to be studied but they seem to work only in the presence of added thymidine. Most likely, the dZMP generated inhibits two key enzymes, deoxycytidine deaminase and thymidylate synthase, and both are essential in maintaining normal thymidine levels. C. Cytidine deaminase project: syntheses of bicyclo[3.1.0]hexane nucleosides bearing a seven-member diazepinone aglycon as inhibitors of cytidine deaminase were synthesized. Biological studies are ongoing
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