設計與合成光親和探針以開發人類胸苷酸激酶之抑制劑

Abstract

胸苷酸激酶可以催化將胸苷單磷酸轉成胸苷雙磷酸之反應，而此反應則是細 胞內合成及修復去氧核糖核酸所必須之步驟。由於核糖核苷酸還原酶的活性於癌 細胞被提高，因此為了平衡地提供四種核苷三磷酸，胸苷酸激酶扮演一個重要的 角色，抑制人類胸苷酸激酶可以弱化甚至殺死癌細胞。於先前的研究中我們找到 一個新類型的人類胸苷酸激酶抑制劑 YMU1(化合物 1a)，因此，本篇研究的目的 在於了解其分子機制，並且進一步開發更有效的人類胸苷酸激酶抑制劑。 在此篇研究中，等溫滴定量熱法之分析確認此類融合芳烴異噻唑酮抑制劑會 與人類胸苷酸激酶形成一比一的複合物。根據分子模擬，YMU1 傾向結合在催化 部位(B 位)然而化合物 2b 及 15 則可結合在 B 位及 D 位並展示更強的抑制能力。 光親和探針 24 被設計帶有一個烯丙基疊氮基的光反應性部分及一個生物素標記。 此光親和標示實驗順利的標記出人類胸苷酸激酶活性部位有反應的氨基酸殘基。 由質譜法鑑定得到探針修飾的胜肽大多數處於 D 位，此結果與我們分子模擬的實 驗預測一致。根據探針分析及分子模擬結果，我們設計及合成一系列 YMU1 的衍 生物以達到對人類胸苷酸激酶具有更好的抑制能力。其中，十五個化合物具有比 YMU1 更佳的抑制活性。

Thymidylate kinase (TMPK) catalyzes the reaction of dTMP to form dTDP, which is an essential step for cellular dTTP supply for DNA synthesis and repair. Because ribonucleotide reductase (RNR) activity is elevated in cancer cell, TMPK plays an important role to balance dNTPs supply. It is known that inhibition of human thymidylate kinase (hTMPK) will weaken and even kill cancer cells. We have previously identified an hTMPK inhibitor YMU1 (compound 1a) as a new class inhibitor of hTMPK. Therefore, the purpose of this study is to understand its molecular mechanism and develop advanced potent hTMPK inhibitors. In this study, the isothermal titration calorimetry analysis confirms that the arene-fused isothiazolone inhibitor forms a 1:1 complex with hTMPK. According to molecule simulation, YMU1 prefers to bind at the catalytic site (B-site) of hTMPK, whereas compounds 2b and 15 can bind at both the B-site and ATP-binding site (D-site) and show stronger inhibitory activity. The photoaffinity probe 24 is designed to bear a photoreactive moiety of arylazide and a biotin tag. The photoaffinity labeling experiment works well to tag the interacting amino acid residues in the active sites of hTMPK. The probe-modified peptides are identified by mass spectrometric method to show that most of the interacting peptides occur at the D-site as predicted by our molecular docking experiment. According to the probe study and MD result, we designed and synthesized series of YMU1 derivatives to achieve better inhibitory activity against hTMPK. Among them, 15 compounds demonstrated better inhibitory activity than YMU1.