Enzyme Inhibitors as Potential Anticancer and Antiviral Drugs

这项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/抑制剂，能够选择性地结合到PK-C同工酶的调节C1结构域，以及其他具有结构相似的C1结构域的酶。这些包括参与信号转导的蛋白质家族，即PKD、嵌合蛋白和RasGRP，所有这些都显示出对佛波醇酯的强结合亲和力。主要的合成努力是设计简单的和化学上可获得的化合物，其在结构上与天然第二信使甘油二酯（DAG）相关，并且还具有与佛波醇酯所示的结合效力相似的结合效力。 基于作为构象刚性DAG支架（DAG-内酯）的4，4-二取代-γ丁内酯模板的化合物文库的扩展继续进行，以达到能够改善与沿着C1结构域边缘的一组高度保守的疏水氨基酸（例如，PK-C δ中Phe-243、Leu-250、Trp-252和Leu-254）。迄今发现的最有效的化合物之一，（Z）-{1-（羟甲基）-4-[4-甲基-3-（三氟甲基）苯基]-4-甲基-3-氧代-1，2，4-二氢呋喃4-甲基-3-氧代-2-氧代戊环基}甲基（甲基乙基）戊酸，显示出低纳摩尔（2nM）的PK-C结合亲和力，并显示出选择性地将PK-C δ转运到细胞内的高尔基体（J.Biol.Chem.1999，274，37233 - 37239）。 该化合物还在前列腺癌细胞中显示出有效的凋亡活性，并在低纳摩尔浓度下在NCI 60细胞系体外筛选中对乳腺癌、结肠癌和白血病细胞系显示出选择性抗肿瘤活性。 将羟酰胺侧链并入我们的主要DAG-内酯模板显示出对PK-C的优异结合亲和力（5 nM）。根据我们对与佛波醇13-O-乙酸酯结合的PK-C δ的C1结构域的X射线晶体结构的对接模拟，NH-OH药效团结合在与佛波醇酯的4-β-OH结合的PK-C δ上的相同位点。 这第四药效团有助于定义这些分子的结合模式，通过消除具有三药效团集的化合物观察到的两种能量等效结合模式的模糊性。 将与用于DAG-内酯的方法类似的组合方法应用于设计将最高结合亲和力与最低亲脂性（log P）组合的DAG类似物。通过本研究鉴定的最佳DAG类似物具有38 nM的结合亲和力和3.8的log P。 这些参数的组合使得该化合物等同于有效的二辛酰甘油，但其亲水性高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高1.3倍，疏水性低1.8个数量级。 正在结合所需的脂质不太重要的系统中研究这些巨大变化的后果。蛋白激酶C同工酶。银鲛激活/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.