二甲基富马酸（DMF）被批准用于治疗多发性硬化症（MS），但其作用模式仍不清楚。一种假说提出DMF通过Michael加成到巯基（尤其是谷胱甘肽）的方式能够免疫调节。另一种假说认为DMF的水解产物单甲基富马酸（MMF）是免疫细胞溶酶体中脂肪酸受体GPR109A的配体。我们制备了MMF的酯类和由阿奇霉素衍生的大环内酯，由于溶酶体的困扰而对免疫细胞具有热带性。我们在新鲜分离的人类外周血单个核细胞（PBMCs）的脂多糖（LPS）反应测定中测试了这些物质的作用。在这个系统中，我们观察到MMF的4''酯类（化合物2和3）在1µM浓度下显著降低了胞外白介素（IL）-1β，IL-12和肿瘤坏死因子α（TNFα）水平，而DMF需要约25µM才能达到同样的效果。与MMF本身一样，MMF的2'酯类（化合物1和2）在体外是不活跃的。4''酯类迅速形成谷胱甘肽结合物，而2'结合物不与巯基反应，但会在这些细胞中缓慢水解释放出MMF。我们随后在使用牛奶蛋白磺酸酯/异硬脂酸模型诱导的牛皮癣中进行了体内测试，其中2'酯类在0.06-0.12mg/kg（约0.1µmol/kg）时最为活跃，改善了皮肤评分，体重和细胞因子水平（TNFα，IL-17A，IL-17F，IL-6，IL-1β，NLRP3和IL-23A）。相比之下，在该模型中对巯基反应性的4''酯类的活性低于2'酯类，而DMF的活性则低了约300倍。巯基反应性的4''酯类在血浆或器官中都不易回收，而2'酯类则表现出常规的吸收和清除。2'酯类还降低了急性单钠尿酸（MSU）诱导的炎症中IL-6的水平。这些数据表明，与MMF相关的机制在体内主要集中在MMF的释放上。鉴于GPR109A定位在溶酶体上，而溶酶体困扰增加了2'酯类的活性超过300倍，这些数据表明GPR109A可能是体内的主要靶标。相比之下，在体内与谷胱甘肽（GSH）结合的效果在体内不太可能像体外那么有效，因为使用的剂量明显较低，无法滴定更高浓度的巯基。这些数据支持在自身免疫疾病中调节GPR109A的观点。© 2023.作者，独家许可Springer Nature Switzerland AG。

Dimethyl fumarate (DMF) is approved as a treatment for multiple sclerosis (MS), however, its mode of action remains unclear. One hypothesis proposes that Michael addition to thiols by DMF, notably glutathione is immunomodulatory. The alternative proposes that monomethyl fumarate (MMF), the hydrolysis product of DMF, is a ligand to the fatty acid receptor GPR109A found in the lysosomes of immune cells. We prepared esters of MMF and macrolides derived from azithromycin, which were tropic to immune cells by virtue of lysosomal trapping. We tested the effects of these substances in an assay of response to Lipopolysaccharide (LPS) in freshly isolated human peripheral blood mononuclear cells (PBMCs). In this system, we observed that the 4'' ester of MMF (compound 2 and 3) reduced levels of Interleukins (IL)-1β, IL-12 and tumor necrosis factor alpha (TNFα) significantly at a concentration of 1 µM, while DMF required about 25 µM for the same effect. The 2' esters of MMF (compound 1 and 2) were, like MMF itself, inactive in vitro. The 4'' ester formed glutathione conjugates rapidly while the 2' conjugates did not react with thiols but did hydrolyze slowly to release MMF in these cells. We then tested the substances in vivo using the imiquimod/isostearate model of psoriasis where the 2' ester was the most active at 0.06-0.12 mg/kg (approximately 0.1 µmol/kg), improving skin score, body weight and cytokine levels (TNFα, IL-17A, IL-17F, IL-6, IL-1β, NLRP3 and IL-23A). In contrast, the thiol reactive 4'' ester was less active than the 2' ester while DMF was ca. 300-fold less active. The thiol reactive 4'' ester was not easily recovered from either plasma or organs while the 2' ester exhibited conventional uptake and elimination. The 2' ester also reduced levels of IL-6 in acute monosodium urate (MSU) induced inflammation. These data suggest that mechanisms that are relevant in vivo center on the release of MMF. Given that GPR109A is localized to the lysosome, and that lysosomal trapping increases 2' ester activity by > 300 fold, these data suggest that GPR109A may be the main target in vivo. In contrast, the effects associated with glutathione (GSH) conjugation in vitro are unlikely to be as effective in vivo due to the much lower dose in use which cannot titrate the more concentrated thiols. These data support the case for GPR109A modulation in autoimmune diseases.© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.