Dimethyl Fumarate and Monoethyl Fumarate Exhibit Differential Effects on KEAP1, NRF2 Activation, and Glutathione Depletion <i>In Vitro</i>

Delayed-release dimethyl fumarate (also known as gastro-resistant dimethyl fumarate), an oral therapeutic containing dimethyl fumarate (DMF) as the active ingredient, is currently approved for the treatment of relapsing multiple sclerosis. DMF is also a component in a distinct mixture product with 3 different salts of monoethyl fumarate (MEF), which is marketed for the treatment of psoriasis. Previous studies have provided insight into the pharmacologic properties of DMF, including modulation of kelch-like ECH-associated protein 1 (KEAP1), activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway, and glutathione (GSH) modulation; however, those of MEF remain largely unexplored. Therefore, the aim of this study was to evaluate the in vitro effects of DMF and MEF on KEAP1 modification, activation of the NRF2 pathway, and GSH conjugation. Using mass spectrometry, DMF treatment resulted in a robust modification of specific cysteine residues on KEAP1. In comparison, the overall degree of KEAP1 modification following MEF treatment was significantly less or undetectable. Consistent with KEAP1 cysteine modification, DMF treatment resulted in nuclear translocation of NRF2 and a robust transcriptional response in treated cells, as did MEF; however, the responses to MEF were of a lower magnitude or distinct compared to DMF. DMF was also shown to produce an acute concentration-dependent depletion of GSH; however, GSH levels eventually recovered and rose above baseline by 24 hours. In contrast, MEF did not cause acute reductions in GSH, but did produce an increase by 24 hours. Overall, these studies demonstrate that DMF and MEF are both pharmacologically active, but have differing degrees of activity as well as unique actions. These differences would be expected to result in divergent effects on downstream biology.缓释富马酸二甲酯（delayed-release dimethyl fumarate，又称胃耐受性富马酸二甲酯）是一种以富马酸二甲酯（dimethyl fumarate，DMF）为活性成分的口服治疗药物，目前已获批用于复发型多发性硬化症的治疗。DMF同时也是一种特殊复方制剂的组分之一，该复方包含3种不同的单乙基富马酸酯（monoethyl fumarate，MEF）盐类，该复方制剂获批上市用于银屑病的治疗。既往研究已阐明DMF的多项药理学特性，包括对类Kelch ECH结合蛋白1（kelch-like ECH-associated protein 1，KEAP1）的调控、核因子（红细胞衍生2样2）通路（nuclear factor (erythroid-derived 2)-like 2，NRF2）的激活，以及对谷胱甘肽（glutathione，GSH）的调控；但针对MEF的药理学特性目前仍未得到充分探究。因此本研究旨在评估DMF与MEF在体外对KEAP1修饰、NRF2通路激活及GSH结合反应的影响。 通过质谱分析法检测发现，DMF处理可显著修饰KEAP1上的特定半胱氨酸残基。与之相比，MEF处理后KEAP1的整体修饰程度显著更低，甚至无法被检测到。与KEAP1半胱氨酸修饰的结果一致，DMF处理可诱导NRF2发生核转位，并使处理细胞产生强烈的转录应答，MEF处理亦产生了类似效应，但MEF引发的应答强度更低，或与DMF的应答特征存在差异。 研究同时显示，DMF可引发急性浓度依赖性的GSH耗竭；但GSH水平最终会在24小时内恢复并升至基线水平以上。与之相反，MEF不会引发GSH的急性降低，反而会在24小时时使GSH水平升高。 总体而言，本研究证实DMF与MEF均具有药理学活性，但二者的活性强度存在差异，且具备独特的作用模式。上述差异预计会对下游生物学过程产生截然不同的影响。