Enzyme Inhibitors as Potential Anticancer and Antiviral Drugs

a .蛋白激酶C项目：使用IRORI技术固相法生成的二酰基甘油内酯（dag -内酯）文库产生了大量独特的生物活性。分子中两个区域化学多样性的细微差别，结合起来产生了我们所称的化学邮政编码，能够将相对较小的化学空间转化为更大的生物活动空间，因为细胞内含膜的细胞器似乎能够解码这些化学邮政编码。在与蛋白激酶C （PKC）同工酶或其他含有膜反应结构域（C1结构域）的非激酶靶蛋白结合后，所产生的复合物似乎被定向到精确的细胞内位点，在那里会遇到不同的底物。我们的结果与一般的假设相反，即相似的化学物质必须具有相似的生物活性。事实上，通过选择多种细胞生物测定的指标，我们能够证明一组相似的dag -内酯，它们都在体外不同程度地与pkc - α结合，将它们的生物学库扩展到更大的区域，靶向细胞环境中的其他蛋白质，从而产生潜在的治疗意义。我们去年发表的第一个具有C1结构域选择性的dag -内酯，对RasGRP具有完全选择性，已经被一个新的，最近的化合物所取代，该化合物对RasGRP的选择性比pkc - α高165倍。新的含有吲哚、吡啶、喹啉和异喹啉基团的dag -内酯文库最近被合成并正在进行评价。一个新的模板，如DAG-dioxolanone，被创建（J. Med. Chem. 2007, 50, 3465-3481）。利用这个模板，我们在PKC-delta的C1b结构域的结合中利用了一个额外的接触点（谷氨酰胺27）。与相应的dag -内酯相比，这个与谷氨酸接触点的突变选择性地破坏了dag -二恶酮的结合（分别为1200- 3000倍和35- 55倍）。这种突变的C1b结构域对dag -二氧基酮的不同反应与dag -内酯的不同反应提供了一种独特的工具来探索C1b结构域在pkc - δ功能中的作用，其中对dag -内酯的反应为保留功能提供了积极的控制。使用这种方法，我们发现PKC-delta的C1b结构域在DAG存在时酶向膜的易位中起主导作用。由乙烯取代的芳香间隔物[oligo-（对苯基乙烯），OPE]组成的高度刚性和几何定义良好的分子棒被纳入dag -内酯的酰基部分，并研究了它们与蛋白激酶C （PKC）结合并将PKC- α和δ同工异构体转运到血浆和细胞膜的能力。PKC易位动力学与ERK磷酸化、诱导IL-6分泌、抑制细胞增殖和诱导细胞附着等生物学反应相关。由于OPE棒通过非共价力组装并形成稳定的薄膜，它们可能影响dag -内酯膜结合位点周围的微域环境。对两种dag -内酯的比较，一种具有两个OPE单元，另一种具有相同的亲脂性的柔性酰基链，清楚地表明刚性OPE链在显著延长pkc - α和δ的易位状态方面的作用（J. Med. Chem. 2007, 50,962 -978）。B. Zebularine项目：由异常启动子DNA超甲基化引起的调控基因失活在各种类型的癌症中广泛存在，并且由于其可逆性而成为表观遗传治疗的一个有吸引力的靶点。Zebularine是一种基于机制的DNA甲基化抑制剂，由于其稳定性、对小鼠的相对无毒性质和抗肿瘤特性，它是癌症化疗的一个有希望的候选者。为了测试斑马碱是否可以预防肿瘤发生，MMTV-PyMT或ApcMin/+ (Min)小鼠在其饮用水中长时间服用斑马碱。通过这种方法，雌性MMTV-PyMT小鼠乳腺肿瘤的发展显著减少。此外，Min雌性的平均息肉数从58个减少到1个，而Min雄性的平均息肉数未受影响，这可能是由于雄性的醛氧化酶对药物的代谢相对于雌性增强。在处理后的Min雌性小肠和大肠中检测到B1 SINE元素的去甲基化。雌性小鼠的生长速度和结肠中整体基因表达均受到中度影响，慢性莪麻碱对Min小鼠的肠道和肝脏组织的组织学没有变化，表明对小鼠的毒性较低。新的数据表明，zebularine具有强大的抗癌特性，并且在大多数接受治疗的小鼠中毒性很小，因此可能是癌症化疗和化学预防的优秀候选药物。Zebularine [2(1H)-嘧啶核糖体]在恒河猴身上发生了意想不到的毒性，当血浆浓度达到25微摩尔时，这种毒性是致命的，这使Zebularine [2(1H)-嘧啶核糖体]的临床进展结束了。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。在雄性Min小鼠中缺乏效果和可能毒性（在猴子中）被认为与分解代谢酶，醛氧化酶（AO）的水平和类型有关。雌性小鼠的AO水平很低，而雄性小鼠的AO水平很高；高于猴子和人类。人类的性别差异非常小；然而，物种差异很大。雷洛昔芬对猴子酶的抑制需要500 μ m，而对人类酶的抑制只需8 μ m。AO代谢似乎是主要的分解代谢途径，产生尿苷作为主要代谢物。医学化学，2006,14,62-66)。许多2-脱氧西丙拉碱前药仍在研究中，但它们似乎只有在添加胸腺嘧啶的情况下才有效。最有可能的是，产生的dZMP抑制了两个关键酶，脱氧胞苷脱氨酶和胸腺苷酸合成酶，这两个酶对于维持正常的胸腺嘧啶水平都是必不可少的。C.胞苷脱氨酶项目：合成含七元二氮平酮苷元的双环[3.1.0]己烷核苷作为胞苷脱氨酶抑制剂。生物学研究正在进行中

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