天然抗癌藥物K-252a醣基生合成的化學分子機制研究與其衍生物的合成應用

Abstract

K-252a 是一種結構特殊的含醣天然藥物，對多種生物體中的重要蛋白質激酶有強效的抑制作 用，因此能對各種癌症有廣泛且有效的抑制藥性，另外，K-252a 也具有抑制神經退化衰竭而達 到神經保護的作用。此一天然有機分子是由吲哚醣苷K-252c 與特異的雙氫鏈黴醣 (dihydrostreptose)，經由兩個碳-氮醣苷鍵連結而成，其一為醣基轉移酵素摧化形成。最近，這個 重要天然物的完整生物合成基因群組已由我們所發表，我們發現K-252a 的生合成可能利用二磷 酸核苷雙氫鏈黴醣作為醣的提供者，而藉由醣基轉移酵素而連結至醣苷。至今，科學家對二磷酸 核苷鏈黴醣的生物合成途徑所知甚少，其所參與酵素群的化學分子機制亦不明瞭。 本研究將針對K-252a 生合成途徑與酵素群，進行以下探討：(一)於異體物種中選殖、表現與 大量純化二磷酸核苷雙氫鏈黴醣的酵素群蛋白質，並合成所有可能的醣類前驅物與中間產物，以 功能性活性驗證、終止反應法、及中間產物餵食方法，來釐清並證明其化學反應途徑與催化機制。 並運用定點突變法，配合酵素動力學，來探討鏈黴醣合成酵素活性中心的重要保留胺基酸，以發 現其功能與分子機制。(二)利用四個K-252c 醣苷生合成酵素群，將一群色胺酸類似物，使用前 趨物導向生合成的方式，合成出眾多組合的醣苷衍生物。(三)應用K-252a 醣基轉移酵素，將生 合成、化學酵素法及化學合成的五元及六元環醣連接至醣苷上，而獲得一群(近千種)K-252b 及 K-252d 的類似物分子庫，並進而探討醣轉酵素對這些醣基的分子認知與專一性。(四)進行上述分 子庫類似物的蛋白質激酶的抑制效果與親和力，而解析其藥物結構與標的活性的關係。 總之，本研究藉由完成重要抗癌藥物K-252a 生合成酵素的分子機制探討，應用組合式生合 成的方式，進行含醣天然藥物之衍生物合成，以提高藥物的選擇性與效能，達到藥物研發而解決 或治療癌症與神經退化的病症。

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.