Enzyme Inhibitors as Potential Anticancer and Antiviral

a .蛋白激酶C项目：使用我们新的固相合成方法生成新的dag -内酯化学文库，继续在各种潜在的治疗重要性领域产生重要的先导化合物：1)我们的第一个具有RasGRP专属活性的C1结构域选择性dag -内酯的结果最近发表。该化合物使我们能够通过完全独立于PKC α的RasGRP解析ERK磷酸化途径。这种dag -内酯，鉴定为130C037，能够专门将RasGRP转运到细胞内区室，而其他PKC同工酶留在细胞质中；2)继续对刺激α -分泌酶活性的dag -内酯进行优化和构效研究；3)使用Balb/C小鼠JB6模型的研究有助于确定一些保留AP-1激活但不是肿瘤促进剂的dag -内酯；4)一组不同的dag -内酯能够刺激干扰素与IL-12联合产生的水平优于PKC激活剂，肉豆蔻酸酯（PMA），而不会诱导肿瘤促进。其中一些化合物的大规模合成正在进行中，以进行动物研究。通过Staudinger反应将一种新的叠氮官能化黄酮乙酸类似物与FLAG肽膦衍生物偶联。当结合物与小鼠巨噬细胞的细胞提取物混合时，FAA-FLAG结合蛋白可以使用抗flag抗体进行免疫沉淀。这项工作最近被选为CCR公报的重点内容。C.Zebularine [2(1H)-嘧啶核苷]是近3年来众多研究的主题（在主要期刊上发表了31篇论文）。不幸的是，它走向临床的道路被恒河猴的意外毒性所终结，当血浆浓度达到25微摩尔时是致命的。这与啮齿动物（大鼠和小鼠）完全没有毒性形成鲜明对比，即使在高剂量下也是如此。在猴子体内，低剂量的乙醛氧化酶能中和乙醛的作用，从而避免毒性和提高乙醛氧化酶活性的努力一直集中在防止高浓度的乙醛氧化酶上。尽管发现了雷洛昔芬，一种临床批准的抑制该酶的药物，但我们的努力一直是针对去年启动的2'-脱氧西布拉林的前药的开发，目的是改善2'-脱氧西布拉林-5'-三磷酸与DNA的结合。在研究了一系列广泛的前药后，我们发现了两种类似物，它们在激活沉默的肿瘤抑制基因方面比西布拉林强10倍，特别是在CF-Pac-1胰腺肿瘤细胞中。对这些发现的知识产权保护正在进行中。用DNA焦磷酸测序证实了与G in相反的斑马醛-5′-三磷酸选择性结合；d .一种稳定的NAD类似物，β -亚甲基- tad，多年前在LMC合成，用于单adp -核糖基化毒素（类似于白喉毒素）的高分辨率晶体结构。该结构提示了80S核糖体的易位机制。

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.