Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain.
Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain.
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DOI：
10.1016/j.biochi.2021.01.007
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发表时间：
2021-04
期刊：
影响因子：
3.9
通讯作者：
Yue WW
中科院分区：
文献类型：
--
作者：
Bezerra GA;Holenstein A;Foster WR;Xie B;Hicks KG;Bürer C;Lutz S;Mukherjee A;Sarkar D;Bhattacharya D;Rutter J;Talukdar A;Brown PJ;Luo M;Shi L;Froese DS;Yue WW
叶酸和蛋氨酸循环构成单碳代谢，是细胞存活的重要途径。与这两个循环相交的是5,10-亚甲基四氢叶酸还原酶（MTHFR），它引导叶酸到蛋氨酸循环的一个碳单元，专门用于蛋氨酸和s -腺苷蛋氨酸（AdoMet）的合成。MTHFR缺乏和上调导致多种疾病状态，使其成为一个有吸引力的药物靶点。MTHFR的活性被AdoMet与酶活性位点远端的变构调节结构域的结合所抑制，我们之前已经确定该结构域构成了一个具有药物口袋的新折叠。在这里，我们使用差示扫描荧光法筛选了162个AdoMet模拟物，并鉴定了4个稳定该调节结构域的化合物。3个化合物是sinfungin类似物，与AdoMet和s -腺苷型同型半胱氨酸（AdoHcy）密切相关。最强的热稳定性是由(S)-SKI-72提供的，这是一种最初为蛋白精氨酸甲基转移酶4 （PRMT4）开发的有效抑制剂。利用表面等离子体共振，我们证实(S)-SKI-72通过其变构结构域与MTHFR结合，具有纳米摩尔亲和力。(S)-SKI-72存在下的MTHFR活性测定表明，HEK293细胞裂解液中具有亚微摩尔效力的纯化酶和内源性MTHFR在低微摩尔范围内的抑制作用，两者都低于AdoMet。然而，与AdoMet不同的是，(S)-SKI-72即使在非常高的浓度下也不能完全消除MTHFR活性。结合结合实验、动力学表征和化合物对接，本研究表明MTHFR的调控结构域可以被小分子靶向，并提出(S)-SKI-72是开发MTHFR抑制剂的良好候选物。
The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme’s active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.
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影响因子：
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作者：
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