恆溫滴定測焓儀應用於G-四股結構穩定劑之研究

Abstract

人類端粒以及基因啟動子等區域存在富含鳥糞嘌呤的基因序列，能夠形成G-四股結構。研究已經顯示這些基因序列和癌症等疾病具有密切關係，因此若是能夠設計穩定G-四股結構之小分子，對G-四股結構進行調控，便有潛力應用在相關疾病的治療。然而G-四股結構的多樣性增加了藥物設計上的困難度，因此探討不同的G-四股結構與不同小分子之間的結合，找出其中的關聯性，對於藥物開發具有相當大的幫助。 本篇論文主要探討小分子的結構與不同G-四股結構之交互作用。我們利用恆溫滴定測焓儀 (ITC) 提供的熱力學結合參數，探討本實驗室設計的BMVC系列衍生物的小分子結構對於G-四股結構熱力學結合模式。此外，我們也以ITC互補競爭法比較小分子之穩定效力。傳統上評估G-四股結構穩定劑之方法為測量解旋溫度，然而其在過程中會有大幅的溫度變化，未必符合實際人體的生理條件，因此我們利用G-四股結構之互補序列作為解旋驅動力，搭配ITC在37C下進行測定，探討BMVC衍生物之不同結構對於穩定效力的影響。 BMVC衍生物之結構主要可以分為Core和Side Chain兩部分，Core為對位以及鄰位，Side Chain則有較為疏水性的碳鏈8C，以及含有較為親水性的ethylene glycol的8C3O。結果顯示BMVC衍生物對於混合型G-四股結構，在不同Core之間的分子熱力學結合模式及穩定效力存在差異，然而對於其它之G-四股結構，不同Core的分子結合模式大致接近，而Side Chain整體與結合模式之關聯較不顯著。 此外，我們發現不同BMVC衍生物對於平行型和混合型G-四股結構之親和力雖無明顯差異，但ITC互補競爭法結果顯示不同衍生物對於這些G-四股結構的穩定效力並不相同，且穩定效力與結合焓的大小相關。進一步以膠體電泳實驗分析，也顯示BMVC衍生物傾向進行焓驅動反應與G-四股結構作用。表示小分子與G-四股結構的交互作用，結合焓可能在穩定G-四股結構上扮演重要角色。 本論文以恆溫滴定測焓儀得到的熱力學結合參數，探討G-四股結構與G-四股結構穩定劑之結構，以及結合反應三者之關聯，並且以不同的實驗方法印證熱力學資訊對於研究小分子與G-四股結構交互作用之重要性，以期利於將來相關的藥物開發。

G-rich sequences that are capable of forming G-quadruplex (G4) structures are present in many biological gene regions such as gene promoter and telomeres, which are related to cancer and other diseases. Thus, designing ligands to stabilize and to regulate G-quadruplex has become promising for cancer therapy. However, the variety of G4 structures makes the ligand design challenging. Better understanding of the relationship between G4 structures and ligand binding is important to improve G4 ligands. In this work, we investigate the binding features of different G-quadruplexes and BMVC derivatives, which are fluorescent G4 ligands synthesized in our laboratory, by isothermal titration calorimetry (ITC). In addition, we develop hybridization competition ITC assay to screen better G4 stabilizers. In general, melting temperature (Tm) analysis is used to evaluate the ability to stabilize G4 structure by comparing Tm before and after adding G4 ligands to G-quadruplex. However, the method requires large temperature change and may not be consistent with physiological condition. Here we use hybridization competition ITC assay to compare different ligands under isothermal condition. BMVC derivatives can be compared by two aspects: Core and Side Chain. We selected BMVC derivatives with different cores, BMVC Core and o-BMVC Core. Also, hydrophobic carbon chain, 8C, and hydrophilic ethylene glycol chain, 8C3O, are used for side chain. Our results show that the binding mode and the stability efficiency are core-dependent for hybrid form G-quadruplexes. BMVC Core is entropy driven and o-BMVC Core is enthalpy driven. Ligands with o-BMVC Core have better binding stability efficiency. However, the binding modes are similar between different cores for parallel and anti-parallel form G-quadruplexes. The side chain shows minor effect on thermodynamic binding features. Although ITC and interferometry results show that the binding affinities of BMVC derivatives binding to hybrid and parallel form of G4 are similar. However, the results of hybridization competition assay show that the abilities of BMVC derivatives to stabilize G4 are different, which are related to the binding enthalpy. Furthermore, the gel electrophoresis assay shows that BMVC derivatives tend to undergo enthalpy driven binding reaction more. Thus, it is suggested that the binding enthalpy, instead binding affinity, of G4 ligands plays an important role in stabilizing G4. By using ITC, we investigate the linkage among the structure of ligands, G-quadruplexes, and the binding features. Moreover, results of different methods show the significance of thermodynamic information. These results provide more information of structure–activity relationship, and can be further applied to G-quadruplex ligand and for anticancer drug design.