Ennis Elizabeth A, Wright Jane, Retzlaff Cassandra L, McManus Owen B, Lin Zhinong, Huang Xiaofang, Wu Meng, Li Min, Daniels J Scott, Lindsley Craig W, Hopkins Corey R, Blakely Randy D
#Johns Hopkins University Ion Channel Center, Baltimore, Maryland 21205, United States.
ACS Chem Neurosci. 2015 Mar 18;6(3):417-27. doi: 10.1021/cn5001809. Epub 2015 Feb 2.
The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents.
高亲和力胆碱转运体（CHT）是乙酰胆碱（ACh）合成的限速决定因素，但该转运体在突触胆碱能信号检测和调控方面仍是一个很大程度上未被开发的靶点。为了拓展CHT药理学，我们基于转运体介导的胆碱转运的电生特性，对新型CHT靶向小分子进行了高通量筛选。在此过程中，我们鉴定出了五类新型的CHT特异性抑制剂。对其中一类抑制剂进行化学多样化和功能分析，确定ML352为一种高亲和力（Ki = 92 nM）且具有选择性的CHT抑制剂。在转染细胞和神经末梢制剂中完全拮抗CHT的浓度下，ML352对乙酰胆碱酯酶（AChE）或胆碱乙酰转移酶（ChAT）无抑制作用，并且在多巴胺、5-羟色胺和去甲肾上腺素转运体以及许多受体和离子通道上也没有活性。ML352在完整细胞和突触体中表现出非竞争性胆碱摄取抑制作用，并在膜分析中降低了半胱氨酸-3（HC-3）结合位点的表观密度，提示其与转运体存在变构相互作用。药代动力学研究表明其体外代谢有限且具有显著的中枢神经系统渗透性，其特征预示着快速清除。ML352代表了一种用于调控CHT的新型、强效且特异性的工具，为胆碱能成像和治疗药物的开发提供了一个可能的平台。
