A Nonisotopic HTS Assay to Elucidate Choline Transporter (CHT) Modulators

描述（由申请人提供）：在大脑中，化学物质乙酰胆碱（ACh）对唤醒、运动和认知回路具有强大的调节控制作用，已发现阿尔茨海默病（AD）缺乏这种物质。目前可用于积极影响阿尔茨海默病（AD）和其他痴呆症认知缺陷的药物是胆碱酯酶抑制剂。这些可以防止神经递质乙酰胆碱（ACh）的分解，从而增强ACh的功能。由于胆碱酯酶抑制剂的有限效用，增加乙酰胆碱酶缺陷的替代疗法在我们的老龄化人口中至关重要。另一种重要的蛋白质，即对胆碱敏感的胆碱转运蛋白（CHT）被认为是神经元有效吸收胆碱以合成乙酰胆碱的原因。我们开发了一种高通量筛选的检测系统，以鉴定具有高选择性的CHT化合物。预计这些化合物可能会导致未来的胆碱能治疗阿尔茨海默病，以及其他多种由胆碱能信号调节的中枢神经系统疾病。我们的研究目的是利用电压敏感荧光测量实现高通量筛选，以发现由CHT介导的高亲和力胆碱转运体摄取的新型增强子；进行二级分析，旨在鉴定选择性作用于胆碱转运蛋白（CHT）的新型调节剂；并使用数据库挖掘和化学合成来优化用于生物分析的HTS命中值。通过高通量筛选（HTS）方法的发展，允许筛选大型化学文库，我们寻求识别CHT激活剂和变构调节剂。这些化合物可能能够增强胆碱的摄取并增加神经元中产生的乙酰胆碱的水平。这些试剂将允许更多的乙酰胆碱酯酶从存活的神经元中释放出来，而不是在疾病过程中丢失，并且在靶向前体而不是乙酰胆碱酯酶本身方面，可能比胆碱酯酶治疗更具优势。

DESCRIPTION (provided by applicant): In the brain, the chemical acetylcholine (ACh) exerts powerful modulatory control over arousal, motor and cognitive circuits, and has been found to be deficient in Alzheimer's Disease (AD). The current drugs available to positively impact cognitive deficits in Alzheimer's Disease (AD) and other dementias are the cholinesterase inhibitors. These prevent the breakdown of the neurotransmitter acetylcholine (ACh), and thus augment Ach function. Due to the limited utility of the cholinesterase inhibitors, alternative therapies to augment ACh deficits are critical in our aging population. Another vital protein, the hemicholinium-3 sensitive choline transporter (CHT) is believed to be responsible for the efficient uptake of choline by neurons to allow for ACh synthesis. We have developed an assay system for high throughput screening to identify compounds with high selectivity for CHT. It is anticipated that these compounds may lead to future cholinergic therapies in AD, and multiple other CNS diseases regulated by cholinergic signaling. Our research aims are to implement a high throughput screen for discovery of novel enhancers of high-affinity choline transporter uptake as mediated by CHT using voltage-sensitive fluorescent measurements; to perform secondary assays aimed at identifying novel modulators that act selectively at the choline transporter (CHT); and to use database mining and chemical synthesis to optimize HTS hits for use in biological assays. Through the development of high-throughput screening (HTS) methodologies that allow for the screening of large chemical libraries, we seek to identify CHT activators and allosteric modulators. These compounds may be able to enhance choline uptake and increase the levels of ACh produced in the neuron. Such reagents would allow greater ACh release from viable neurons not lost in the disease process and could offer advantages over cholinesterase therapy in targeting precursors rather than ACh itself. PUBLIC HEALTH RELEVANCE: Cognitive deficits associated with Alzheimer's Disease (AD) and other dementias are primarily caused by loss of the neurotransmitter acetylcholine (ACh) in the brain and treatments are currently limited to cholinesterase inhibitors. Additional and improved therapies to augment ACh deficits are critical in our aging population and may be accessed by targeting another vital protein, the hemicholinium-3 sensitive choline transporter (CHT). CHT is responsible for the efficient uptake of choline by neurons and we have developed an assay for high throughput screening to identify compounds with high selectivity for CHT for future cholinergic therapies in AD, and other diseases regulated by cholinergic signaling.