NF-56-EJ40, a succinate receptor (SUCNR1) antagonist, blocks succinate's effect on promoting Treg numbers in CD4+ T cells isolated from healthy donor PBMCs with an IC50 value of 0.025μM[1-2]. NF-56-EJ40 enters SUCNR1 between TM-I and -II and does not unlock the extracellular loop (ECL)-2b[3].
In vitro, after 24 hours of treatment with NF-56-EJ40 (5μM) in lipopolysaccharide (LPS)-stimulated intestinal epithelial cells (IECs), the expression of IL-1β, IL-6, IL-10, and TNF-α in IECs was decreased[4]. The enteroids were pre-treated with 5μM NF-56-EJ40 for 30 minutes, and then cultured with 1mM succinate for 48 hours, resulting in a significant decrease in the promoting effects of succinate on the protein levels of leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) and proliferating cell nuclear antigen protein (PCNA)[5]. NF-56-EJ40 (4μM; 24h) treatment could significantly interrupt succinate/IL-1β signal in human umbilical vein endothelial cells (HUVECs) and macrophages[6].
References:
[1] Sinéad Kinsella, Colton W Smith, Kelly McKenna, et al. Elevated Metabolite Secretion Facilitates a Treg-Mediated Immunosuppressive Microenvironment in the Solid Tumor. BLOOD. 2023; 142 (Supplement 1): 3458.
[2] Haffke M, Fehlmann D, Rummel G, et al. Structural basis of species-selective antagonist binding to the succinate receptor. Nature. 2019;574(7779):581-585.
[3] Shenol A, Lückmann M, Trauelsen M, et al. Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91. Mol Cell. 2024;84(5):955-966.e4.
[4] Huo L, Chen Q, Jia S, et al. Gut microbiome promotes succinate-induced ulcerative colitis by enhancing glycolysis through SUCNR1/NF-κB signaling pathway. Am J Physiol Cell Physiol. Published online June 23, 2025.
[5] Luo D, Zou M, Rao X, et al. Lactobacillus salivarius metabolite succinate enhances chicken intestinal stem cell activities via the SUCNR1-mitochondria axis. Poult Sci. 2025;104(2):104754.
[6] Xu J, Zheng Y, Zhao Y, et al. Succinate/IL-1β Signaling Axis Promotes the Inflammatory Progression of Endothelial and Exacerbates Atherosclerosis. Front Immunol. 2022;13:817572.
NF-56-EJ40是一种琥珀酸受体（SUCNR1）拮抗剂，能够阻断琥珀酸对从健康供体PBMCs中分离的CD4+ T细胞中Treg细胞数量的促进作用，其半数抑制浓度（IC50）值为0.025μM[1-2]。NF-56-EJ40从SUCNR1的跨膜区I（TM-I）和跨膜区II（TM-II）之间进入，且不会解锁胞外环2b（ECL-2b）[3]。
在体外，脂多糖刺激的肠上皮细胞（IECs）经过24小时的NF-56-EJ40（5μM）处理后，能够降低IECs中IL-1β、IL-6、IL-10和TNF-α的表达[4]。肠类器官预先用5μM的NF-56-EJ40处理30分钟，然后与1mM的琥珀酸共同培养48小时，导致琥珀酸对富含亮氨酸重复序列的G蛋白偶联受体5（LGR5）和增殖细胞核抗原蛋白（PCNA）蛋白水平的促进作用显著降低[5]。在人脐静脉内皮细胞（HUVECs）和巨噬细胞中，NF-56-EJ40（4μM；24小时）处理能够显著阻断琥珀酸/IL-1β信号[6]。