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Interaction of N-carbamoylglutamate, an orphan drug for the treatment of N-acetylglutamate deficiency with glutamate transporters of the SLC1 genfamily

N-氨基甲酰谷氨酸（一种治疗 N-乙酰谷氨酸缺乏症的孤儿药）与 SLC1 基因家族谷氨酸转运蛋白的相互作用

基本信息

批准号：
275317313
负责人：
Professorin Dr. Birgitta C. Burckhardt
金额：
--
依托单位：
Zentrum Physiologie und Pathophysiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/275317313?language=en
关键词：
Interaction carbamoylglutamate orphan drug treatment

项目摘要

Disturbances in urea metabolism are associated with hyperammonia, i.e. an increased plasma level of the ammonium ion (NH4+), leading to severe neurological symptoms if not treated properly. Besides restriction of dietary protein and hemodialysis, the orphan drug, N-carbamoylglutamate (NCG) or Carbaglu (trade name), has been approved to treat certain forms of hyperammonemia. NCG activates carbamoylphosphate synthase 1 (CPS-1), the key enzyme of urea synthesis. Physiologically, CPS-1 requires the allosteric activator N-acetylglutamate (NAG) that, in turn, is formed by the N-acetylglutamate synthase (NAGS). Inborn defects in NAGS severely impair urea synthesis. By activating CPS-1, the orally available NCG reduces plasma ammonium close to normal levels in these patients. Previously, we have shown that renal secretion of NCG is accomplished by the sodium-dependent dicarboxylate transporter 3 and the organic anion transporters 1 and 4. The intestinal absorption and hepatic uptake of NCG, however, remained ill-defined. The aim of this proposal is to identify transporters for NCG in small intestine and liver. We shall focus on sodium-dependent glutamate transporters belonging to the SLC-1 family, because NCG can be envisaged as a structural analog of glutamate and these transporters are expressed both in intestinal epithelium and hepatocytes. To this end Xenopus laevis oocytes will be injected with RNA coding for the respective high-affinity glutamate transporters SLC1A1, 2, and 3. After expression of the proteins in the plasma membrane, NCG-mediated currents can be detected and set in relation to glutamate-evoked currents. Additional uptake experiments using radio-labeled glutamate in the absence and presence of NCG and transporter-specific inhibitors in human enterocytes and hepatocytes supported by antibody studies will help to identify the transporters responsible for the uptake of NCG in enterocytes and hepatocytes.

尿素代谢紊乱与高氨血症有关，即血浆中铵离子 (NH4+) 水平升高，如果治疗不当，会导致严重的神经系统症状。除了限制膳食蛋白质和血液透析外，孤儿药 N-氨基甲酰谷氨酸 (NCG) 或 Carbaglu（商品名）已被批准用于治疗某些形式的高氨血症。 NCG 激活氨基甲酰磷酸合酶 1 (CPS-1)，这是尿素合成的关键酶。从生理学角度来说，CPS-1 需要变构激活剂 N-乙酰谷氨酸 (NAG)，而 N-乙酰谷氨酸合酶 (NAGS) 又可形成 N-乙酰谷氨酸。 NAGS 的先天缺陷严重损害尿素合成。通过激活 CPS-1，口服 NCG 可将这些患者的血浆铵降低至接近正常水平。此前，我们已经表明，NCG 的肾脏分泌是通过钠依赖性二羧酸转运蛋白 3 以及有机阴离子转运蛋白 1 和 4 完成的。然而，NCG 的肠道吸收和肝脏摄取仍然不明确。该提案的目的是确定小肠和肝脏中 NCG 的转运蛋白。我们将重点关注属于 SLC-1 家族的钠依赖性谷氨酸转运蛋白，因为 NCG 可以被设想为谷氨酸的结构类似物，并且这些转运蛋白在肠上皮和肝细胞中表达。为此，非洲爪蟾卵母细胞将被注射编码各自高亲和力谷氨酸转运蛋白 SLC1A1、2 和 3 的 RNA。在质膜中表达蛋白质后，可以检测到 NCG 介导的电流，并设置与谷氨酸诱发电流的关系。在抗体研究支持的人肠上皮细胞和肝细胞中，在不存在和存在 NCG 和转运蛋白特异性抑制剂的情况下，使用放射性标记的谷氨酸进行额外的摄取实验，将有助于确定负责肠上皮细胞和肝细胞中 NCG 摄取的转运蛋白。