ENZYME INHIBITORS AS POTENTIAL ANTICANCER AND ANTIVIRAL DRUGS

本工作的主要目的是设计蛋白激酶C(PK-C)的同工酶特异性激活剂/抑制物(PK-C)，能够选择性地与PK-C同工酶的调节C1域和其他具有相似C1域的转导酶家族结合。主要的合成工作是设计简单且化学上容易获得的化合物，这些化合物在结构上与天然的PK-C激动剂，第二信使二酰甘油(DAG)相关。利用4，4-二取代-γ-丁内酯模板作为构象刚性DAG支架(DAG-内酯)的组合方法，研究了烷基和酰基链的最佳组合，以改善与C1结构域边缘一组高度保守的疏水氨基酸(例如，PK-CDelta中的Phe-243、Leu-250、Trp-252和Leu-254)的疏水相互作用。直链和支链的组合产生了一个大的化合物文库，其中最优的配体达到了纳摩尔结合亲和力。已发现的最好的化合物为(Z)-{1-(羟甲基)-4-[4-甲基-3-(methylethyl)pentylidene]-3-oxo-2-oxolanyl}methyl 4-甲基-3-(甲乙基)戊酸]，是一种双支化的DAG-内酯，与PK-C的结合亲和力很低(约2 nm)。在体外筛选的NCI-60细胞株中，该化合物对特定的乳腺、结肠和白血病细胞株显示出强大的和选择性的抗肿瘤活性。DAG和佛波酯除了与PK-C同工酶经典成员和新成员的C1域结合外，还能与其他参与信号转导的蛋白家族中的C1域(S)结合，即PKD、嵌合体和RasGRP。上述化合物是第一个在效价(亚纳摩尔KI)和选择性(3倍)方面超过佛波酯的化合物。嵌合体不具有功能激活区，但它们是RAC的GTP激活蛋白(GAP)，RAC是RAS超家族的一个小的GTP结合蛋白。这里描述的组合化学方法，再加上对不同C1域的密集分子建模和对接研究，预示着未来将设计出具有良好抗肿瘤活性的特定C1域靶向配体。-分子模型受体，佛波酯，蛋白激酶C细胞信号，锌指，

The principal objective of this work is to design isozyme-specific activator/inhibitors of protein kinase C (PK-C) capable of binding selectively to the regulatory, C1 domain of PK-C isozymes and other families of transducing enzymes with structurally similar C1 domains. The main synthetic effort is to design, simple and chemically accessible compounds that are structurally related to the natural PK-C agonist, the second messenger diacylglycerol (DAG). A combinatorial approach that utilizes a 4,4-disubstituted-gamma-butyrolactone template as a conformationally rigid DAG scaffold (DAG-lactones) was implemented to investigate the best combination of alkyl and acyl chains to improve hydrophobic interactions with a group of highly conserved hydrophobic amino acids along the rim of the C1 domain (e.g., the Phe-243, Leu-250, Trp-252 and Leu-254 in PK-Cdelta). The combination of linear and branched chains produced a large library of compounds where optimal ligands reached namolar binding affinities. The best compound yet discovered, (Z)-{1-(Hydroxymethyl)-4-[4-methyl-3- (methylethyl)pentylidene]-3-oxo-2-oxolanyl}methyl 4-methyl-3- (methylethyl)pentanoate, is a doubly branched DAG-lactone with low namomolar (ca. 2 nM) binding affinity for PK-C. This compound showed potent and selective antitumor activity in the NCI 60-cell line in vitro screen against specific breast, colon and leukemia cell lines at low nanomolar concentrations. Besides binding to the C1 domains of the classical and novel members of PK-C isozymes, DAG and the phorbol esters are also able to bind to the C1 domain(s) in other families of proteins involved in signal transduction, namely PKD, the chimaerins and RasGRP. The compound described above is the first compound ever to surpass phorbol ester in potency (subnanomolar Ki) and selectivity (3- fold) for the C1 domain of beta2-chimaerin. Chimaerins do not possess a functional kinase domain but they are GTPase-activating proteins (GAP) for Rac, a small GTP binding protein of the Ras superfamily. The combinatorial chemical approach described here, coupled to intensive molecular modeling and docking studies of the different C1 domains, bodes well for the future design of specific C1-domain-targeted ligands with promising antitumor activity. - molecular models receptors, phorbol Esters, protein kinase C cell signalling, Zinc fingers,