The Na+-coupled citrate transporter (NaCT/SLC13A5/mINDY) in the liver delivers citrate from the blood into hepatocytes. As citrate is a key metabolite and regulator of multiple biochemical pathways, deletion of Slc13a5 in mice protects against diet-induced obesity, diabetes, and metabolic syndrome. Silencing the transporter suppresses hepatocellular carcinoma. Therefore, selective blockers of NaCT hold the potential to treat various diseases. Here we report on the characteristics of one such inhibitor, BI01383298. It is known that BI01383298 is a high-affinity inhibitor selective for human NaCT with no effect on mouse NaCT. Here we show that this compound is an irreversible and non-competitive inhibitor of human NaCT, thus describing the first irreversible inhibitor for this transporter. The mouse NaCT is not affected by this compound. The inhibition of human NaCT by BI01383298 is evident for the constitutively expressed transporter in HepG2 cells and for the ectopically expressed human NaCT in HEK293 cells. The IC50 is ∼100 nM, representing the highest potency among the NaCT inhibitors known to date. Exposure of HepG2 cells to this inhibitor results in decreased cell proliferation. We performed molecular modeling of the 3D-structures of human and mouse NaCTs using the crystal structure of a humanized variant of VcINDY as the template, and docking studies to identify the amino acid residues involved in the binding of citrate and BI01383298. These studies provide insight into the probable bases for the differential effects of the inhibitor on human NaCT versus mouse NaCT as well as for the marked species-specific difference in citrate affinity.

中文翻译：

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对人Na +偶联柠檬酸盐转运蛋白（NaCT / SLC13A5 / mINDY）有选择性的物种特异性抑制剂的功能分析

肝脏中的Na +耦合柠檬酸盐转运蛋白（NaCT / SLC13A5 / mINDY）将柠檬酸盐从血液输送到肝细胞。由于柠檬酸盐是多种生化途径的关键代谢产物和调节剂，因此小鼠中Slc13a5的缺失可防止饮食引起的肥胖症，糖尿病和代谢综合征。沉默转运蛋白可抑制肝细胞癌。因此，NaCT的选择性阻滞剂具有治疗各种疾病的潜力。在这里，我们报告了一种此类抑制剂BI01383298的特性。已知BI01383298是对人NaCT具有选择性的高亲和力抑制剂，对小鼠NaCT没有影响。在这里，我们显示该化合物是人类NaCT的不可逆和非竞争性抑制剂，因此描述了该转运蛋白的第一种不可逆抑制剂。小鼠NaCT不受此化合物的影响。BI01383298对HepG2细胞中组成性表达的转运蛋白和HEK293细胞中异位表达的人NaCT的抑制作用对BI01383298的抑制是明显的。IC50为〜100 nM，代表迄今为止已知的NaCT抑制剂中最高的效价。HepG2细胞暴露于该抑制剂会导致细胞增殖减少。我们使用人源化的VcINDY变体的晶体结构作为模板，对人和小鼠NaCT的3D结构进行了分子建模，并进行了对接研究以鉴定参与柠檬酸盐和BI01383298结合的氨基酸残基。这些研究为抑制剂对人NaCT与小鼠NaCT的差异作用以及柠檬酸盐亲和力的显着物种特异性差异提供了可能的依据。