DEVELOPMENT OF CANCER THERAPEUTICS BASED ON REGULATION OF CHROMATIN STRUCTURE AND FUNCTION

我们已经确定组蛋白脱乙酰酶(HDAC)是曲古菌素A(TSA)和曲泊辛(TPX)的分子靶标，曲古菌素是抑制哺乳动物细胞周期的微生物代谢产物。最近，我们已经证明了FK228，一种在美国进行第二阶段临床试验的有效抗癌药物，也专门抑制HDAC。由于越来越多的证据表明HDAC是抗癌药物的一个新的分子靶点，我们在这个项目中开发了新型的HDAC抑制剂。X-射线结晶学研究表明，TSA通过将活性中心锌与其异羟肟酸基团螯合来抑制HDAC，而TPX则通过其环氧酮基团共价修饰活性中心残基。我们将TPX的环状四肽与TSA的异羟肟酸偶联，合成了一种杂化缓蚀剂，CHAP被证明是一种有效的HDAC缓蚀剂。与TSA或TPX相比，CHAP在血清中高度稳定，其衍生物(CHAP31)在异种移植模型中显示出强大的抗肿瘤活性。FK228具有分子内二硫键。我们证明FK228是一种天然前药，它可以通过细胞还原活性与活性中心锌相互作用而转化为还原形式。因此，我们设计了一种新型的抑制剂，将硫醇基团引入到类TPX环状四肽抑制剂中，命名为SCOP。ScoP在正常情况下被氧化和失活，但当它在细胞中被还原时变得活跃。HDAC家族包括十多种酶。TSA对几乎所有的酶都有抑制作用，但TPX不能抑制HDAC6。通过差示灵敏度分析，我们确定HDAC6为微管蛋白脱乙酰酶。由于HDAC家族的每个成员可能都有各自不同的功能，因此开发亚型特异性的抑制剂是很重要的。在筛选四种亚型特异性抑制剂的过程中，我们发现了一种能特异性抑制HDAC4的SCOP衍生物。

We have identified histone deacetylases (HDAC) as the molecular target of trichostatin A (TSA) and trapoxin (TPX), microbial metabolites that inhibit mammalian cell cycle. Recently, we have shown that FK228, a potent anti-cancer agent under the phase II clinical trials in the US, also specifically inhibits HDAC. Since accumulating evidence suggests that HDAC is a novel molecular target for anti-cancer drugs, we developed new types of HDAC inhibitors in this project The X-ray crystallographic studies suggested that TSA inhibits HDAC by chelating the active-site zinc with its hydroxamic acid group, while TPX covalently modifies an active site residue via its epoxiketone moiety. We synthesized a hybrid inhibitor by coupling the cyclic tetrapeptide of TPX with the hydroxamic acid of TSA, named CHAP CHAP was shown to be a potent HDAC inhibitor. In contrast to TSA or TPX, CHAP was highly stable in serum and a derivative (CHAP31) showed potent antitumor activity in the xenograft models. FK228 has an intramolecular disulfide. We showed that FK228 is a natural prodrug, which can be converted to the reduced form with a thiol group interacting with the active-site zinc by cellular reducing activity. We therefore designed a new type of inhibitors by introducing the thiol group into the TPX-like cyclic tetrapeptide inhibitors named SCOP. SCOP was normally oxidized and inactivated but became active when it is reduced in cells. The HDAC family includes more than ten species of enzymes. TSA inhibited almost all the enzymes but TPX failed to inhibit HDAC6. By using the differential sensitivity, we identified that HDAC6 is the tubulin deacetylase. Since each member of HDAC family may have each distinct function, it is important to develop subtype-specific inhibitors. During the course of screening forth subtype-specific inhibitors, we found a SCOP derivative that specifically inhibits HDAC4.