Kurosawa Toshiki, Tega Yuma, Uchida Yasuo, Higuchi Kei, Tabata Hidetsugu, Sumiyoshi Takaaki, Kubo Yoshiyuki, Terasaki Tetsuya, Deguchi Yoshiharu
Laboratory of Drug Disposition and Pharmacokinetics, Faculty of Pharma-Sciences, Teikyo University, Tokyo 173-8605, Japan.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA.
Pharmaceutics. 2022 Aug 12;14(8):1683. doi: 10.3390/pharmaceutics14081683.
A proton-coupled organic cation (H/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the roteomics-based dentification of transporter by rosslinking substrate in eyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H/OC antiporter. We synthesized a novel H/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H/OC antiporter.
在人类和啮齿动物的血脑屏障（BBB）中发挥作用的质子偶联有机阳离子（H/OC）反向转运体，被认为是将阳离子药物有效递送至大脑的一个有前景的候选者。因此，识别表现出这种活性的分子实体很重要。在此，为了这个目的，我们建立了基于蛋白质组学的通过小孔交联底物鉴定转运体（PICK）方法，该方法将光亲和标记与使用SWATH-MS的综合蛋白质组学分析相结合。使用预先选定的标准，PICK方法产生了16种候选蛋白质。从这些蛋白质中，在体外血脑屏障模型hCMEC/D3细胞中进行的敲低筛选，确定了两种蛋白质TM7SF3和LHFPL6作为H/OC反向转运体的候选者。我们合成了一种新型的H/OC反向转运体底物，用于在hCMEC/D3细胞和HEK293细胞中对TM7SF3和LHFPL6进行功能分析。结果表明，TM7SF3和LHFPL6都是H/OC反向转运体的组成部分。
