Human ion channel pharmacology in droplet bilayer membranes

描述(由申请人提供)：离子通道药物发现和安全筛选受到每个数据点的高成本、低吞吐量和复杂性的限制。为了直接解决这些行业范围的痛点，Librede Inc.正在开发一种替代的无细胞离子通道筛选技术，该技术基于对人造液滴膜中离子通道的测量。通过在测量点消除细胞，我们的技术实现了更低的运行成本和简化的分析开发。在前期工作中，我们使用我们的平台测量了TRPM8和HERG离子通道，并获得了与最先进的离子通道电生理平台一致的药物IC50和EC50值。在我们技术的第一阶段开发中，我们创建了一个自动化就绪的原型阵列板，并用它制造了1000多个人造薄膜，验证了其可靠性和高产率。在这里提出的第二阶段工作中，我们将使用四个离子通道来验证我们的平台：HERG、Kv7.1、TRPV1和Cav1.2，选择这些通道是因为它们与筛选的相关性和它们的复杂程度。我们将优化从商业上可用的宿主细胞制备这些离子通道的方法，以实现高度可重复和可靠的电导测量。然后，我们将使用这些制剂以剂量依赖的方式测量药物对通道电导的影响，生成可靠且可重复的IC50和EC50值，并将其与自动膜片钳筛选仪器获得的值进行比较。这些药物测量将使用我们在第一阶段开发的阵列板进行，使我们能够评估它们在模拟筛选应用中的性能。在拟议的工作中，我们将与加州大学洛杉矶分校的雅各布·施密特教授的研究小组合作，后者与PI一起最初开发了拟议的技术。施密特团队将通过最初处理和测量离子通道制剂，然后将这项技术转让给Librede进行复制、优化、放大，并最终实现商业化，从而为Librede提供帮助。在这项工作完成后，我们将在我们的系统中验证四个高度相关的离子通道，并在分析条件下演示其操作。这一验证将向潜在的最终用户展示我们系统的性能，并允许将仪器原型放置在已确定的测试员和早期采用者中。这里提出的第二阶段工作是我们新的无细胞电生理平台开发的关键一步，该平台可以解决主要的行业痛点，如成本、吞吐量和易用性(Librede的价值主张)，并有可能显著影响离子通道药物发现和安全筛选。

DESCRIPTION (provided by applicant): Ion channel drug discovery and safety screening are limited by high cost per data point, low throughput, and complexity. To directly address these industry-wide pain points, Librede Inc. is developing an alternative cell-free technology for ion channel screening, based on the measurement of ion channels in artificial droplet membranes. By eliminating cells at the point of measurement, our technology enables much lower running costs and simplified assay development. In preliminary work, we have used our platform to measure TRPM8 and hERG ion channels and obtain drug IC50 and EC50 values in agreement with state-of-the-art ion channel electrophysiology platforms. In Phase I development of our technology, we created an automation- ready prototype array plate, and verified its reliability and high yield by using it to create over 1000 artificial membranes. In the Phase II work proposed here, we will validate our platform with four ion channels: hERG, Kv7.1, TRPV1, and Cav1.2, selected for their relevance to screening and their degree of complexity. We will optimize the methods of preparing these ion channels from commercially available host cells to achieve highly repeatable and reliable conductance measurements. We will then use these preparations to measure the effects of drugs on the channels' conductance in a dose-dependent manner, generating reliable and repeatable IC50 and EC50 values that we will compare to those obtained from automated patch clamp screening instrumentation. These drug measurements will be performed using the array plates we developed in Phase I, allowing us to evaluate their performance for a simulated screening application. During the proposed work, we will collaborate with the research group of Prof. Jacob Schmidt at UCLA who, with the PI, initially developed the proposed technology. The Schmidt group will aid Librede by initially processing and measuring the ion channel preparations and then transferring this technology to Librede to replicate, optimize, scale-up, and ultimately commercialize. At the completion of this work, we will have validated four highly relevant ion channels pharmacologically in our system and demonstrated its operation in assay conditions. This validation will demonstrate our system's performance to potential end users and allow placement of instrument prototypes with identified beta testers and early adopters. The Phase II work proposed here is a critical step in the development of our new cell-free electrophysiology platform which can address the main industry pain points of cost, throughput, and ease of use (Librede's value proposition), and has the potential to significantly impact ion channel drug discovery and safety screening.