Enzyme Inhibitors as Potential Anticancer and Antiviral

蛋白激酶C包括一个超过10个同工酶的家族，这些同工酶参与细胞信号的调节。根据它们的结构和规律可分为3个亚类。经典或Ca2+ (cPKC)，新型或Ca2+独立（nPKC）和非典型或二酰基甘油(DAG)/磷酯独立（aPKC）。前两种类型对第二信使DAG的释放作出反应，DAG将酶从无活性的细胞质位置招募到膜上，在那里它们被变构激活。在药理学上，佛波酯也可以达到类似的效果，它对PKC的结合亲和力比dag高得多。正因如此，佛波酯成为研究PKC活化作用的首选试剂。另一方面，我们已经合成了一组合理设计的dag -内酯，尽管它们结构简单，但它们表现为高效的PKC配体，能够显示出与磷酯相当的活性，尽管它们的活性不同，有时甚至在效力上超过它们。从超过300种化合物的文库中，我们选择了一种候选的dag -内酯，它在体外结合和激活方面表现出α同工酶（cPKC）和δ同工酶（nPKC）之间的有效但非歧视性的活性。然而，在整个细胞的生理条件下，我们已经能够证明该化合物具有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。先前的研究表明pkc - α和pkc - δ在LNCaP细胞中对PMA的凋亡作用中存在重叠作用。LNCaP细胞的独特之处在于它们只表达经典的pkc - α、新型pkc - δ和非典型pkc zeta和lambda。本研究表明，PMA和dag -内酯激活PKC同工酶的不同亚群，促进LNCaP细胞凋亡。具体来说，新的dag -内酯诱导pkc - α专一易位到细胞膜，pkc - δ专一易位到核膜。虽然多种PKC同工酶可能参与凋亡效应，但dag -内酯似乎只通过α同工酶起作用。事实上，特异性cPKC抑制剂G 6976完全阻断了dag -内酯的凋亡作用，仅部分阻断了PMA诱导的凋亡作用，特异性pkc - δ抑制剂rottlerin对dag -内酯诱导的细胞凋亡无效，但能够限制PMA诱导的细胞凋亡。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例子。对其他dag -内酯的进一步研究有望有助于设计新的工具来解剖细胞中同工酶的特异性功能。最后，鉴于一些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。

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.