A.Protein kinase C project: The generation of novel chemical libraries of DAG-lactones using our new solid-phase method of synthesis continues to yield important lead compounds in various areas of potential therapeutic importance: 1) The results on our first C1 domain-selective DAG-lactone with exclusive activity for RasGRP was published recently. This compound has allowed us to dissect an ERK phosphorylation pathway via RasGRP, which is totally independent of PKC alpha. This DAG-lactone, identified as 130C037, is capable of exclusively translocating RasGRP to intracellular compartments while other PKC isozymes remain in the cytoplasm; 2) Optimization and structure-activity studies of DAG-lactones capable of stimulating alpha-secretase activity continues; 3) Research using the Balb/C mouse JB6 model has helped identify some DAG-lactones that retain AP-1 activation but are not tumor promoting agents; 4) A different set of DAG-lactones is capable of stimulating interferon production in combination with IL-12 to levels superior to those achieved with the paradigm PKC activator, phorbol myristate acetate (PMA) without inducing tumor promotion. Large-scale syntheses of some of these compounds are in progress to perform animal studies. B.A new azido-functionalized flavone acetic acid analogue was conjugated to a FLAG peptide phosphine derivative via the Staudinger reaction. When the conjugate was mixed with cell extracts from mouse macrophage cells, the FAA-FLAG conjugated proteins could be immunoprecipiated using an anti-FLAG antibody. This work was recently selected to be highlighted in the CCR bulletin. C.Zebularine [2(1H)-pyrimidinone riboside] has been the subject of numerous studies in the last 3 years (31 papers in major journals). Unfortunately its path towards the clinic 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. Efforts to circumvent toxicity and improve the activity of zebularine at lower doses in monkeys have been centered on preventing the high levels of a metabolizing enzyme, aldehyde oxidase, which neutralizes the action of zebularine. Despite the discovery of raloxifene, a clinically approved agent to inhibit this enzyme, our efforts have been directed to the development of pro-drugs of 2'-deoxyzebularine which were initiated last year with the intent to improve the incorporation of 2'-deoxyzebularine-5'-triphosphate into DNA. After studying an extensive series of pro-drugs, which resulted in inactive compounds, we have now identified two analogues that are 10-fold more potent than zebularine in the activation of silenced tumor suppressor genes, particularly in CF-Pac-1 pancreatic tumor cells. Securing intellectual protection of these findings is in progress. The selective incorporation of zebularine-5'-triphosphate opposite to G in was demonstrated using DNA pyrosequencing; whe zebularine is part of the template base paring to G is less selective.D.A stable NAD analogue, beta-methylene-TAD, synthesized at the LMC in years past was used for the high-resolution crystal structure of a mono-ADP -ribosylating toxin (similar to diphteria toxin). The structure has implications for the mechanism of translocation on the 80S ribosome.
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