标题: 天然抗癌药物K-252a醣基生合成的化学分子机制研究与其衍生物的合成应用
Molecular Mechanism of Glycosyl Biosynthesis for Natural Anticancer K-252a and Combinatorial Synthesis of Its Derivatives
作者: 邱显泰
Chiu Hsien-Tai
国立交通大学生物科技学系(所)
关键字: 天然物;???唑化合物;K-252a;五圆环醣;功能性鉴定;生物合成;Natural Products;Indolocarbazole;K-252a;Furanose;Functional Characterization;Biosynthesis
公开日期: 2010
摘要: K-252a 是一种结构特殊的含醣天然药物,对多种生物体中的重要蛋白质激酶有强效的抑制作
用,因此能对各种癌症有广泛且有效的抑制药性,另外,K-252a 也具有抑制神经退化衰竭而达
到神经保护的作用。此一天然有机分子是由吲哚醣苷K-252c 与特異的双氢链霉醣
(dihydrostreptose),经由兩个碳-氮醣苷键連结而成,其一为醣基转移酵素摧化形成。最近,这个
重要天然物的完整生物合成基因群组已由我们所发表,我们发现K-252a 的生合成可能利用二磷
酸核苷双氢链霉醣作为醣的提供者,而藉由醣基转移酵素而連结至醣苷。至今,科学家对二磷酸
核苷链霉醣的生物合成途径所知甚少,其所參与酵素群的化学分子机制亦不明瞭。
本研究将针对K-252a 生合成途径与酵素群,进行以下探讨:(一)于異体物种中选殖、表现与
大量纯化二磷酸核苷双氢链霉醣的酵素群蛋白质,并合成所有可能的醣類前驱物与中间产物,以
功能性活性验证、终止反应法、及中间产物喂食方法,來厘清并证明其化学反应途径与催化机制。
并运用定点突变法,配合酵素动力学,來探讨链霉醣合成酵素活性中心的重要保留胺基酸,以发
现其功能与分子机制。(二)利用四个K-252c 醣苷生合成酵素群,将一群色胺酸類似物,使用前
趋物导向生合成的方式,合成出众多组合的醣苷衍生物。(三)应用K-252a 醣基转移酵素,将生
合成、化学酵素法及化学合成的五元及六元环醣連接至醣苷上,而获得一群(近千种)K-252b 及
K-252d 的類似物分子库,并进而探讨醣转酵素对这些醣基的分子认知与专一性。(四)进行上述分
子库類似物的蛋白质激酶的抑制效果与亲和力,而解析其药物结构与标的活性的关系。
总之,本研究藉由完成重要抗癌药物K-252a 生合成酵素的分子机制探讨,应用组合式生合
成的方式,进行含醣天然药物之衍生物合成,以提高药物的选择性与效能,达到药物研发而解决
或治療癌症与神经退化的病症。
K-252a is a structurally unique natural product glycoside exhibiting neuroprotective activity and
displaying potent cytotoxic activities against numerous cancer cells by inhibiting various protein
kinases. Due to its potent antitumor, anticancer and neuroprotective activities, K-252a has recently been
of intense focus. Structurally, K-252a is characterized by a special dihydrostreptose moiety and two
C–N covalent linkages, one generated by a catalytic action of N-glycosyltransferase and the other by an
oxidative coupling enzyme. We have recently cloned and identified the gene cluster for biosynthesis of
K-252a, revealing key enzymes responsible for the formation of TDP-2-deoxy-dihydrostreptose
(TDP-dStp), K-252c aglycone and N-glycosylation. To date, little is known about the catalytic
mechanism and pathways leading to formation of TDP-dStp.
This study aims to study the K-252a biosynthetic pathway and enzymes, as well as their
applications in combinatorial biosynthesis, described as follows. (1) By use of TDP-deoxysugars as
substrates or intermediates, the TDP-dStp enzymes, cloned, expressed and purified from heterologous
hosts, will be functionally and kinetically characterized. The molecular mechanism and pathways will
be resolved by enzymatic assays, quench experiments and intermediate-feeding methods. Site-directed
mutagenesis coupled with steady-state kinetics will be applied to reveal functional roles of conserved
amino acids in TDP-dStp synthase. (2) In a fashion of precursor-directed biosynthesis, the four
aglycone biosynthesis enzymes will be used to convert a group of L-tryptophan analogs to a library of
K-252c analogs. (3) Taking advantage of TDP-rhamnose and TDP-furanose analogs made from
biosynthesis, chemical synthesis and chemo-enzymatic methods, N-glycosyltransferase will decorate
K-252c and its analogs with their sugar moieties to give a molecular library of K-252d and K-252b
analogs. The substrate specificity of K-252a glycosyltransferase will also be characterized on both of
aglycone and sugar analogs. (4) The inhibitory effects and binding affinity of above K-252 analogs will
be evaluated to reveal their activities against protein kinases and structure-activity relationship.
In conclusion, by means of combinatorial biosynthesis and resolving molecular mechanism of the
key K-252a biosynthetic enzymes, this study would lead to generation of K-252 analogs with higher
selectivity and potency for drug development and possible therapeutic applications in cancers and
neurodegenerative disorders.
官方说明文件#: NSC99-2113-M006-010-MY3
URI: http://hdl.handle.net/11536/100293
https://www.grb.gov.tw/search/planDetail?id=2123350&docId=340083
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