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建立新的DAG-内酯化学库，继续在各种具有潜在治疗意义的领域产生重要的先导化合物：1)我们第一个具有RasGRP独有活性的C1域选择性DAG-内酯的结果最近公布。这种化合物使我们能够通过RasGRP剖析ERK的磷酸化途径，这一途径完全不依赖于PKCα。该DAG-内酯被鉴定为130C037，能够专门将RasGRP转移到细胞内的各个隔室，而其他PKC同工酶仍留在细胞质中；2)能够刺激α-分泌酶活性的DAG-内酯的优化和构效研究仍在继续；3)使用Balb/C小鼠JB6模型的研究已帮助鉴定出一些保留AP-1激活但不是促癌因子的DAG-内酯；4)另一套DAG-内酯与IL-12联合使用可刺激干扰素的产生，其水平高于PKC激活剂佛波酯(PMA)在没有促癌作用的情况下实现的水平。其中一些化合物的大规模合成正在进行中，以进行动物实验。B.通过Staudinger反应将一种新的叠氮功能化的黄酮乙酸类似物偶联到旗肽膦衍生物上。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。C.ZeBularine[2(1H)-嘧啶酮核苷]在过去3年中一直是许多研究的主题(主要期刊上发表了31篇论文)。不幸的是，它通往临床的道路因恒河猴身上一种不可预见的毒性而终止，当血浆水平达到25微摩尔时，这种毒性是致命的。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。在猴子身上，规避毒性和提高较低剂量的泽布拉林活性的努力，一直集中在防止代谢酶--乙醛氧化酶的高水平，这种酶中和了泽布拉林的作用。尽管发现了雷洛昔芬，一种临床批准的抑制该酶的药物，但我们的努力一直致力于开发2‘-脱氧泽布林的前体药物，该药物于去年启动，目的是改善2’-脱氧泽布林-5‘-三磷酸与DNA的结合。在研究了一系列导致非活性化合物的前体药物后，我们现在发现了两个类似物，它们在激活沉默的肿瘤抑制基因方面比ZeBularine强10倍，特别是在CF-Pac-1胰腺肿瘤细胞中。保护这些发现的知识产权的工作正在进行中。用DNA焦磷酸测序证实了与G相反的ZeBularine-5‘-Trips选择性掺入；当ZeBularine是模板碱基的一部分时，与G配对的选择性较低。D.使用LMC多年来合成的稳定的NAD类似物-β-亚甲基-TAD用于单-ADP-核糖化毒素(类似于白喉毒素)的高分辨晶体结构。这种结构对80年代核糖体的易位机制有一定的意义。

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.