Microdroplet Transporter Protein Assays

微滴转运蛋白检测

基本信息

批准号：
9270031
负责人：
GREGORY W FARIS
金额：
$ 25.31万
依托单位：
SRI INTERNATIONAL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-15 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9270031
关键词：
ABCB1 gene AQP1 gene ATP phosphohydrolase ATP-Binding Cassette Transporters Active Biological Transport Adverse drug effect Adverse effects Binding Biological Assay Biological Availability Biology Blood - brain barrier anatomy Blood Circulation Brain Canis familiaris Carrier Proteins Cell Membrane Permeability Cells Collaborations Cyclic AMP Deposition Detection Development Diffusion Digoxin Dipyridamole Distal Dose Drug Interactions Drug Kinetics Drug Transport Endothelial Cells Equilibrium Excision Exposure to Family Family member Fluorescein Fluorescence Fluorescent Probes Future Glycine decarboxylase Health Hepatocyte Human Human body Hydroxymethylglutaryl-CoA Reductase Inhibitors International Kidney Knowledge Lasers Liver MDCK cell Measurement Mediating Membrane Methods Monitor Motion Movement Multi-Drug Resistance Muscle Fibers Normal tissue morphology OAT Transport Proteins OATP Transporters Oral Organic Anion Transporters Osmosis P-Glycoprotein Penetration Pharmaceutical Preparations Physiological Preclinical Drug Evaluation Process Pump Reagent Research Rhodamine 123 Side Skeletal Muscle Speed Structure of choroid plexus Technology Testing Time Translating Tubular formation Universities Vesicle Virginia Visit Water Work antiport base calcein AM cancer cell cancer therapy cell growth cell type chemotherapeutic agent cost drug candidate drug development drug discovery experimental study flexibility fluorescence imaging fluorexon improved innovation novel novel therapeutics overexpression passive transport polypeptide protein transport public health relevance reconstitution screening solute success uptake water channel

项目摘要

DESCRIPTION (provided by applicant): A critical early step in drug discovery and development is understanding the likely pharmacokinetics in the human body and the potential for multi-drug resistance. Early screening typically involves assays such as the parallel artificia membrane permeability assay (PAMPA) or the ATPase assay, which are simple to perform yet are not good predictors for drug transport in the body because of the crude description of the bilayers for the PAMPA assay and potential for false-negative and false-positive results in the ATPase assay. For promising drug candidates, more sophisticated cell-based assays using Caco-2 or Madin-Darby canine kidney (MDCK) cells are performed. However, these assays are much more costly and time-consuming to perform, and the value of the results is limited by the accuracy of the initial PAMPA and ATPase screens. We propose alternate assay strategies based on using droplet bilayers. These assays can provide results similar to the Caco-2 or MDCK assays with the lower complexity of the PAMPA or ATPase assays and higher throughput. Furthermore, these assays avoid long periods for cell growth, use very low quantities of transport protein and substrate, allow study of multiple transport protein types, and avoid issues associated with gaps in confluence of cells found in the Caco-2 or MDCK assays. Finally, the assays will allow ready comparison of active substrate transport and simple diffusion. This project is a collaboration between Drs. Faris and Dixit of SRI International (SRI), who developed the direct-deposition and laser-mediated droplet bilayer methods, and Dr. Gerk of Virginia Commonwealth University (VCU), who is an expert on transporter activity assays for drug development. To understand how the droplet bilayer assays conditions and results translate to conventional assays, to facilitate troubleshooting of the droplet assays, confirm reliability of the reagents, and transfer useful knowledge between SRI and VCU, comparable assay types will be performed at both SRI and VCU for the same two transporters and same substrates, and each group will visit the other while the assays are being performed. Flexible and multi-purpose assays for the activity of transporter proteins will improve our knowledge of basic biology and human health. To facilitate and accelerate the drug discovery and development processes, more sensitive, specific, faster, and cheaper methods are desired. We have a novel microdroplet technology capable of sensitive and accurate measurement of transport. Applying this technology to transporter proteins such as P-glycoprotein and Organic Anion Transporting Polypeptides would result in lowering research costs and increasing the success rate of new drug candidates.

描述（由申请人提供）：药物发现和开发的关键早期步骤是了解人体内可能的药代动力学和多重耐药性的潜力，早期筛选通常涉及平行人工膜渗透性测定（PAMPA）等测定。或 ATP 酶测定，该测定操作简单，但由于 PAMPA 测定双层的粗略描述以及潜在的假阴性和假阳性结果，因此不能很好地预测体内药物转运对于有前途的候选药物，可以使用 Caco-2 或 Madin-Darby 犬肾 (MDCK) 细胞进行更复杂的细胞测定，但是，这些测定的成本更高、耗时更长，而且价值也更高。结果的准确性受到初始 PAMPA 和 ATPase 筛选的准确性的限制，我们交替提出基于使用液滴双层的测定策略，这些测定可以提供与 Caco-2 或 MDCK 测定相似的结果，但复杂性较低。 PAMPA 或 ATP 酶测定和更高的通量此外，这些测定避免了长时间的细胞生长，使用极少量的转运蛋白和底物，允许研究多种转运蛋白类型，并且避免与 Caco-2 或 MDCK 检测中发现的细胞汇合间隙相关的问题，该检测可以方便地比较活性底物转运和简单扩散。社会责任研究所），开发了直接沉积和激光介导的液滴双层方法，以及弗吉尼亚联邦大学 (VCU) 的 Gerk 博士，他是药物开发转运蛋白活性测定的专家，以了解液滴双层测定条件和结果如何转化为。传统测定，为了促进液滴测定的故障排除，确认试剂的可靠性，并在 SRI 和 VCU 之间转移有用的知识，将在 SRI 和 VCU 上对相同的两个转运蛋白和相同的相同的测定类型进行类似的测定类型转运蛋白活性的灵活且多用途的检测将提高我们对基础生物学和人类健康的了解，以促进和加速药物发现和开发过程。我们需要一种新的微滴技术，能够灵敏、准确地测量转运蛋白，例如 P-糖蛋白和有机阴离子转运多肽，从而降低研究成本。并增加新候选药物的成功率。