Enzyme Inhibitors as Potential Anticancer and Antiviral

蛋白激酶C包括参与细胞信号调节的10多种同工酶的家族。根据它们的结构和调节，它们被分为3个亚类。经典或Ca 2+（cPKC）、新型或Ca 2+非依赖性（nPKC）和非典型或二酰基甘油（DAG）/佛波酯非依赖性（aPKC）。前两种类型响应于第二信使DAG的释放，第二信使DAG将酶从它们的非活性胞质位置募集到膜，在膜中它们变得变构激活。用佛波醇酯也可以达到类似的效果，佛波醇酯对PKC的结合亲和力比DAG高得多。正是由于这个原因，佛波醇酯已成为研究PKC激活作用的优选试剂。另一方面，我们已经合成了一组合理设计的DAG-内酯，尽管它们的结构简单，表现为高度有效的PKC配体，能够显示相当的，虽然不同的活动，佛波醇酯，有时甚至超过他们的效力。从超过300种化合物的库组中，我们选择了一种候选DAG-内酯，其在体外结合和活化方面在同工酶α（cPKC）和δ（nPKC）之间显示出有效但非区分活性。然而，在全细胞中的更多生理条件下，我们已经能够证明该化合物显示出独特的同工酶特异性，这是由特异性同工酶选择性靶向不同的细胞内区室决定的。以前的工作表明，在LNCaP细胞中，PKC-α和PKC-δ在PMA的凋亡作用中具有重叠作用。LNCaP细胞的独特之处在于它们仅表达经典的PKC-α、新型的PKC-δ和非典型的PKC ζ和λ。在这里，我们表明，PMA和DAG-内酯激活不同的PKC同工酶的子集，以促进LNCaP细胞凋亡。具体而言，新的DAG-内酯诱导PKC-α向细胞膜和PKC-δ向核膜的排他性转运。虽然多种PKC同工酶可能有助于凋亡效应，但DAG-内酯似乎仅通过α同工酶起作用。事实上，虽然特异性cPKC抑制剂G 6976完全阻断了DAG-内酯的凋亡作用，并且仅部分阻断了PMA诱导的凋亡作用，但特异性PKC-δ抑制剂rottlerin在对抗DAG-内酯诱导的凋亡方面无效，而它能够限制PMA诱导的凋亡。这种新的DAG-内酯是细胞模型中经典PK-C同工酶的选择性激活剂的第一个已知实例。与其他DAG-内酯的进一步研究有望在设计新工具以剖析细胞中同工酶特异性功能方面是有用的。最后，鉴于某些PKC同工酶的生长抑制特性及其参与细胞凋亡，一个新兴的主题是PKC激活而不是PKC抑制可能具有治疗价值。 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。

Protein kinase C comprises a family of more than 10 isozymes involved in the regulation of cell signalling. They have been grouped into 3 subclasses according to their structure and regulation. Classical or Ca2+ (cPKC), novel or Ca2+-independent (nPKC) and atypical or diacylglycerol (DAG)/phorbol ester-independent (aPKC). The first two types respond to the release of the second messenger DAG, which recruits the enzymes from their inactive cytosolic location to the membrane where they become allosterically activated. A similar effect can be achieved pharmacologically with the phorbol esters, which have substantially higher binding affinity for PKC than the DAGs. It is for that reason, that the phorbol esters have become the preferred agents to study the effects of PKC activation. On the other hand, we have synthesized a set of rationally designed DAG-lactones, which despite their structural simplicity, behave as highly potent PKC ligands capable of displaying comparable, albeit distinct activities to that of the phorbol esters, sometimes even surpassing them in potency. From a library set of more than 300 compounds we have selected a candidate DAG-lactone that showed potent but non-discriminating activity between isozymes alpha (cPKC) and delta (nPKC) in terms of in vitro binding and activation. However, under more physiological conditions in whole cells, we have been able to demonstrate that this compound shows unique isozyme specificity, which is determined by the selective targeting of specific isozymes to different intracellular compartments. Previous work has shown that there is an overlapping role for PKC-alpha and PKC-delta in the apoptotic effect on PMA in LNCaP cells. LNCaP cells are unique in that they only express the classical PKC-alpha, the novel PKC-delta and the atypical PKCs zeta and lambda. Here we show that PMA and the DAG-lactone activate a different subset of PKC isozymes to promote apoptosis in LNCaP cells. Specifically, the new DAG-lactone induced exclusive tranlocation of PKC-alpha to the cellular membrane and PKC-delta to the nuclear membrane. Although multiple PKC isozymes may contribute to the apoptotic effect, the DAG-lactone appears to operates exclusively through the alpha isozyme. Indeed, while the specific cPKC inhibitor G 6976 completely blocked the apoptotic effect of the DAG-lactone, and only partially that induced by PMA, the specific PKC-delta inhibitor rottlerin was ineffective in countering the apoptosis induced by the DAG-lactones while it was able to limit PMA-induced apoptosis. This novel DAG-lactone is the first known example of a selective activator of a classical PK-C isozyme in a cellular model. Further studies with other DAG-lactones promise to be useful in designing new tools to dissect isozyme-specific functions in cells. Finally, given the growth inhibitory properties of some PKC isozymes and their involvement in apoptosis, an emerging theme is that PKC activation rather than PKC inhibition may be of therapeutic value. Protein kinase C isozymes. Chimaerins. Activation/inhibition, Drug design. Zinc finger. Apoptosis, cellular localization.