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同工酶的调节结构域结合，以及与结构相似的其他酶结合。这些蛋白包括参与信号转导的蛋白家族，即PKD、嵌合体和RasGRP，所有这些蛋白都对佛波酯表现出很强的结合能力。主要的合成努力是设计简单的、化学上可访问的化合物，这些化合物在结构上与天然第二信使二酰甘油(DAG)相关，但具有与佛波酯相似的结合能力。以4，4-二取代-伽马丁内酯为模板的化合物文库的扩展作为刚性DAG支架(DAG-内酯)的目的是为了实现烷基和酰基链的最佳组合，该组合能够改善与C1结构域边缘一组高度保守的疏水氨基酸(如Phe-243、Leu-250、Trp-252和Leu-254)的疏水相互作用。已发现的最有效的化合物之一，(Z)-{1-(羟甲基)-4-[4-甲基-3-甲基-3-(甲基乙基)戊酸，4-甲基-3-甲基-3-(甲乙基)戊酸，对PK-C表现出较低的NAMOMOL(2 NM)结合亲和力，并被证明选择性地将PK-C(methylethyl)pentylidene]-3-oxo-2-oxolanyl}methyl转移到细胞内的高尔基体(J.Biol)。化学。1999年、274、37233、37239)。该化合物还在前列腺癌细胞中显示出很强的凋亡活性，并在低纳摩尔浓度下对乳腺、结肠和白血病细胞株进行体外筛选的NCI 60细胞株中显示出选择性抗肿瘤活性。在我们的DAG-内酯模板上引入了一个羟胺侧链，显示出与PK-C良好的结合亲和力(5 NM)。根据我们对PK-C三角洲C1域与佛波醇13-O-乙酸酯结合的X射线晶体结构的对接模拟，NH-OH药效团与佛波醇酯的4-β-OH结合在PK-C三角洲上的同一位置。这第四个药效团通过消除拥有三个药效团的化合物观察到的两种能量等效的结合模式的模糊性，帮助定义了这些分子的结合模式。一种类似于DAG-内酯的组合方法被应用于设计DAG类似物，该DAG类似物结合了最高的结合亲和力和最低的亲脂性(LogP)。本研究确定的最好的DAG类似物具有38 nM的结合亲和力和3.8的对数P。这些参数的组合使该化合物相当于有效的二辛酰甘油，但它的亲水性要高1.4个数量级。相对于常用的OAG(亲和力=50 nm)，新发现的化合物的效力是后者的1.3倍，疏水性降低了1.8个数量级。这些戏剧性变化的后果正在系统中进行研究，在这些系统中，结合所需的脂质不那么重要。蛋白激酶C同工酶。奇马林。激活/抑制，药物设计。锌指。细胞凋亡，细胞定位。

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, as well as to other enzymes with structurally similar C1 domains. These include families of proteins involved in signal transduction, namely PKD, the chimaerins and RasGRP, all of which show strong binding affinity for the phorbol esters. The main synthetic effort has been to design simple and chemically accessible compounds that are structurally related to the natural second messenger diacylglycerol (DAG), and yet have binding potencies similar to those shown by the phorbol esters. The expansion of a library of compounds based on a 4,4-disubstituted-gamma butyrolactone template as a conformationally rigid DAG scaffold (DAG-lactones) continues in order to reach the best possible combination of alkyl and acyl chains capable of improving 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). One of the most potent compounds yet discovered, (Z)-{1-(Hydroxymethyl)-4-[4-methyl-3 (methylethyl)pentylidene]-3-oxo-2-oxolanyl}methyl 4-methyl-3-(methylethyl)pentanoate, displayed low namomolar (2 nM) binding affinity for PK-C and was shown to selectively translocate PK-Cdelta to the Golghi apparatus inside the cell (J. Biol. Chem. 1999, 274, 37233 37239). This compound also showed potent apoptotic activity in prostate cancer cells and displayed selective antitumor activity in the NCI 60-cell line in vitro screen against breast, colon, and leukemia cell lines at low nanomolar concentrations. The incorporation of a hydroxamide side chain to our main DAG-lactone template showed excellent binding affinity (5 nM) for PK-C. According to our docking simulations with the X-ray crystal structure of the C1 domain of PK-C delta bound to phorbol 13-O-acetate, the NH-OH pharmacophore binds at the same site on PK-C delta that the 4-beta-OH of the phorbol esters binds to. This fourth pharmacophore has helped define the binding mode of these molecules by eliminating the ambiguity of two energetically equivalent binding modes observed with compounds possessing a three pharmacophore set. A similar combinatorial approach to the one used for the DAG-lactones was applied to the design a DAG analogue that combined the highest binding affinity with the lowest lipophilicity (log P). The best DAG analogue identified by this study has a binding affinity of 38 nM and a log P of 3.8. The combination of these parameters makes this compound equivalent to the potent dioctanoylgycerol, and yet it is 1.4 orders of magnitude more hydrophilic. Relative to the commonly used OAG (affinity = 50 nM), the newly discovered compound is 1.3 times more potent and 1.8 orders of magnitude less hydrophobic. The consequences of these dramatic changes are being investigated in systems where the lipid requirement for binding is less important. Protein kinase C isozymes. Chimaerins. Activation/inhibition, Drug design. Zinc finger. Apoptosis, cellular localization.