【作者】 孙蕾；

【导师】 李晓晖；

【作者基本信息】 大连理工大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 组蛋白去乙酰化酶(histone deacetylase, HDACs)和组蛋白乙酰转移酶(histone acetyltransferase, HAT)控制着组蛋白末端赖氨酸上e-氨基的乙酰化程度。表观遗传学研究表明,HDACs的过度表达,导致组蛋白与DNA高度亲和,染色质凝聚,阻碍转录蛋白质复合物进入启动子结合位点,使相应基因表达受到抑制,导致恶性肿瘤发生。组蛋白去乙酰化酶抑制剂(HDACi)可导致肿瘤细胞的生长停滞、诱导肿瘤细胞分化和凋亡,是一类预防与治疗兼具的抗肿瘤药物。Chlamydocin是从Diheterospora chlamydosporia菌中分离出的一种天然环四肽,由L-Phe,Aib,D-Pro和L-Aoe四个氨基酸残基组成骨架结构,其中L-Aoe的环氧酮结构构成与酶作用的结合区,对HDACs表现出良好的体外抑制活性(IC50=1.3 nmol/L),但抑制作用不可逆。为实现抑制剂与酶的高效、可逆抑制作用,我们以chlamydocin为骨架结构,用巯基吡啶和羟肟酸结构替代环氧酮,在抑制剂的金属结合区引入巯基和羟肟酸基团。同时为了增强抑制剂与HDACs活性口袋边缘氨基酸形成的疏水作用,提高对酶的抑制活性,在抑制剂骨架结构苯丙氨酸的侧链引入甲基,以4-甲基苯丙氨酸替换大环骨架的苯丙氨酸。设计合成了三个环四肽类HDACi,其序列为：cyclo(-L-Am7(S2Py)-Aib-L-Phe-D-Pr o-)、cyclo(-L-Am7(S2Py)-Aib-L-Phe(4-Me)-D-Pro-)和cyclo(-L-Asu(NHOH)-Aib-L-Phe(4-Me)-D-Pro-)。对抑制剂的合成采用传统的液相合成法,氨基酸的保护采用Boc/Bzl和Z／tBu策略,缩合试剂为DCC/HOBt,所得产物经ESI-MS测定均与目标分子量一致,HPLC测定其纯度为95%以上,NMR对其结构进行表征。考察HDACi体外抗肿瘤细胞(MCF-7、Hela和7721)活性,结果表明HDACi对三种肿瘤细胞系均显示良好的生长抑制作用,IC50在10微摩尔级以下,其中cyclo(-L-Asu(NHOH)-Aib-L-Phe(4-Me)-D-Pro-)对MCF-7的IC50最小,为43 nmol/L。HDACi作用3种肿瘤细胞24 h后,细胞形态发生明显改变。采用分子对接方法,初步分析了所合成的环四肽类HDACi与组蛋白去乙酰化酶类似蛋白(HDLP)的相互作用方式。结果表明,巯基结构与羟肟酸结构均能很好的与酶活性中心的Zn2+螯合；作为表面识别区的环四肽的骨架结构能与酶活性位点口袋处氨基酸形成疏水作用,该作用对酶与抑制剂的结合起到了稳定作用。更多还原

【Abstract】 Reversible acetylation and deacetylation of lysine residus on histone tails by histone acetyl transferase(HAT) and histone deacetylase(HDAC) enzymes play a fundamental role in the regulation of gene expression by changing the chromatin structure and transcriptional activity. High HDAC activity due to abnormal recruitment of HDACs is associated with a number of malignant diseases. Inhibitors of HDACs have demonstrated efficacy against cancer cell lines.Naturally occurring cyclic tetrapeptide chlamydocin which is isolated from fungus Diheterosporia Chlamdosphoria containing Aib, L-Phe, D-Pro, and L-Aoe shows a highly HDACs inhibition with an IC50 of 1.3 nmol/L in vitro. L-Aoe provides an epoxyketone moiety in the side chain which irreversible inhibits HDACs.To expect reversible and high active HDAC inhibitors, we replaced epoxykeone moiety of Aoe with sulfhydryl group and hydroxamic acid. To improve the hydrophobic interaction of the cap groups with HDACs, we replaced L-Phe to L-Phe(4-Me) in chlamydocin framework. In this paper we designed and synthesized three novel cyclic tetrapeptide HDAC inhibitors:cyclo(-L-Am7(S2Py)-Aib-L-Phe-D-Pro-)、cyclo(-L-Am7(S2Py)-Aib-L-Phe(4-Me)-D-Pro-) and cyclo(-L-Asu(NHOH)-Aib-L-Phe(4-Me)-D-Pro-).The conventional liquid phase method was used to synthesize the cyclic tetrapeptides. The amino acids were protected by Boc/Bzl and Z/tBu. The coupling regent was DCC/HOBt. Their final chemical structures were characterized by 1H and 13C NMR and ESI-MS.The cyclic peptides were tested for cytotoxic activity against three cancer cell lines, including MCF-7、Hela and 7721 cells. All the compounds demonstrated exciting antitumor abilities. The cellular shapes changed obviously with HDACi-treated for 24 h.Molecular docking method was used to analysis the interactions between the cyclic tetrapeptides HDAC inhibitors and histone-like protein (HDLP). The results showed that the thiol and hydroxamic acid could well chelating with Zn2+ at the active site of HDLP. Surface recognition cap groups of the cyclic tetrapeptides contact with the amino acid residues around the pocket of HDLP which stabilize the binding of enzyme and cyclic tetrapeptides.更多还原