依受質結合區探討MTH1與抑制劑之交互作用研究

Investigation of interaction between MTH1 and synthesized inhibitors based on substrate binding site

李昱璇(Yu-Hsuan Lee)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/生命科學系/碩士(2019年)

https://doi.org/10.6342/NTU201901016

摘要

Mut T homolog 1 (MTH1) 為Nudix水解酶超家族的成員，在癌細胞的增生中扮演關鍵的角色。先前研究指出MTH1適合作為抗癌藥物的標靶，並已有MTH1抑制劑的體外以及體內成效被發表。本論文為深入探討MTH1-抑制劑複合體的結合機制，以高通量藥物片段篩選具有抑制MTH1的小分子，以其中一組作為骨架，進一步設計修飾2-氨基嘧啶衍生物共14個，依嘧啶環上5位碳乙基的有無分類為兩個群組，並分別解析MTH1與11個2-氨基嘧啶衍生物複合體結構，輔以生化及熱力學分析，以探討MTH1與抑制物複合體結合作用之多方位資訊。
根據已解出之MTH1與受質複合體結構，可將MTH1之活性口袋區初步劃分為三個特定區域。MTH1之受質8-oxo dGTP (8-Oxo-2’-deoxyguanosine-5’-Triphosphate)具有三部分官能基分別為鹼基，去氧核糖以及三磷酸，在鹼基部分主要由Asp119，Asp120及Trp117的側鏈鍵結，此區域也是這些抑制劑在結合口袋中的主要穩定作用力，旁側存在由多個疏水性胺基酸Phe72，Phe74及Phe139側鏈形成之空間，本實驗中將抑制劑的官能基延伸至由Phe72，Phe74，Phe139形成的空腔時，並無法提升抑制劑結合能力。從受質的8號位置氧原子位於Phe27與Met81的側鏈附近，且設計抑制劑亦與Phe27及Met81可形成凡得瓦力交互作用，本區將可提供與MTH1專一性結合。根據本研究系列合成抑制物之抑制效率分析，當抑制物延伸至受質之三磷酸的區域時，其結合能力降低三個級數，由Lys23，Glu52，Glu55，Glu56及Glu100組成的三磷酸結合區在催化水解反應時，會與金屬離子結合並形成水分子網絡，本區可作為未來延伸區域，以提高專一性結合。最後，本研究以受質結合區結構、熱力學分析及酵素動力學探討設計抑制劑的方式，將可作為未來在設計新穎抑制劑時參考依據。

關鍵字

Nudix水解酶 ； 抑制物 ； 抗癌藥物

並列摘要

Mut T homolog 1 (MTH1) is a member of Nudix hydrolase superfamily and engages in proliferation of cancer cells as critical determinant. In previous study, it was proposed that MTH1 serves as ideal anticancer target for anticancer therapeutic development , and in vitro and in vivo efficacy were reported for several MTH1 inhibitors.
In this study, to elucidate the mechanism of MTH1-inhibitor complex formation extensively, high-throughput screening (HTS) was performed on fragment library intended to identify small molecule compound with moderate inhibitory activity against MTH1. From identified hits, one hit compound was utilized as scaffold and a series of 2-aminopyrimidine derivatives comprising 14 structurally-related compounds were subsequently synthesized based on HTS results. These 2-aminopyrimidine-based compounds are categorized into two groups based on the presence of ethyl substituent on pyrimidine ring. Explicitly, MTH1 complex structures bound with eleven 2-aminopyrimidine derivatives were obtained and supplemented with statistics from biochemical and thermodynamic analyses to complement the intricate binding interaction of MTH1-inhibitor complex from multiple aspects.
Based on MTH1-substrate complex structure, the substrate binding pocket can be divided into three subregions which are occupied by base, deoxyribose and triphosphate group of substrate 8-oxo dGTP, respectively. In the base binding region, the bonds are formed by side chains of Asp119, Asp120 and Trp117 which are essential in binding of synthesized inhibitors in the binding pocket. Adjacent to the base binding motif, a minor cavity formed by side chains of hydrophobic residues Phe72, Phe74, Phe139 allows further exploration with substituents. However, no enhanced binding affinity was observed in exploring this cavity. Additionally, in the analysis of MTH1-inhibitor complex structures, it was observed that residues Phe27 and Met81 form van der Waals interactions selectively with synthesized compounds upon formation of complex. These residues sit in the proximity of 8-oxo group from substrate which was inferred to contribute to specificity of MTH1 through formation of favorable interactions. Intrinsically, as implied in structure-activity relationship study, incorporation of ligand moiety which occupies triphosphate binding region of MTH1 reduced binding affinity by three orders of magnitude. Structurally, triphosphate binding region constituted of Lys23, Glu52, Glu55, Glu56 and Glu100 facilitates catalysis of hydrolytic reaction through formation of water molecule network coordinated by metal ions. Therefore, it is indicated that this subregion can be further exploited for enhancement of specificity of inhibitors. In conclusion, through investigation of MTH1 substrate binding pocket with structural analysis, thermodynamic studies and enzyme kinetic analysis in this study, directions are provided for design of novel inhibitors in the future.

並列關鍵字

Nudix hydrolase superfamily ； inhibitor ； anticancer therapeutic agent

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