Research School of Biology, Australian National University, Canberra, Australia.
Department of Biochemistry, Key University Laboratory of Metabolism and Health of Guangdong, School of Medicine, Institute for Biological Electron Microscopy, Southern University of Science and Technology, Shenzhen, Guangdong, China.
J Biol Chem. 2024 Sep;300(9):107687. doi: 10.1016/j.jbc.2024.107687. Epub 2024 Aug 17.
The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.
与神经递质转运体相比，SLC6 家族中的氨基酸转运体的药理学研究还不够发达。为了鉴定脯氨酸转运体 SIT1（SLC6A20）的新抑制剂，优化了其在非洲爪蟾卵母细胞中的表达。SIT1 的转运通过在卵母细胞中共同表达血管紧张素转换酶 2（ACE2）得到增强，但 ACE2 的共表达不是必需的。基于药效团的筛选鉴定出替加滨是 SIT1 的一种有效非竞争性抑制剂。为了了解其结合模式，我们确定了与替加滨结合的 ACE2-SIT1 的冷冻电镜（cryo-EM）结构。抑制剂结合在正位脯氨酸结合位点附近，但由于其大小延伸到细胞溶质前庭。这导致转运体采用内向开放构象，其中细胞内门被阻断。这项研究首次提供了 SIT1 抑制的结构见解，并为更好地理解 ACE2-SIT1 复合物生成了工具。这些发现可能对 SARS-CoV-2 在人肺肺泡细胞中与其受体 ACE2 的结合具有重要意义，SIT1 和 ACE2 在这些细胞中具有功能表达。
