Microdroplet Transporter Protein Assays

微滴转运蛋白检测

• 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
• 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

 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（由应用提供）：药物发现和发育的关键早期步骤是了解人体中可能的药代动力学以及多药耐药性的潜力。早期筛选通常涉及测定法，例如平行艺术膜渗透性测定法（PAMPA）或ATPase测定法，由于对PAMPA分析的双层和假性和假阳性结果的潜在的biLayers对BiLayers的粗略描述，它们对体内的药物运输却不是很好的预测指标。对于承诺的候选药物，进行了更复杂的基于CACO-2或MADIN-DARBY犬肾（MDCK）细胞的细胞测定。但是，这些测定的成本要高得多，并且可以执行时间耗时，并且结果的价值受到初始PAMPA和ATPase屏幕的准确性的限制。我们建议基于使用液滴双层的替代测定策略。这些测定方法可以提供类似于CACO-2或MDCK分析的结果，其复杂性较低，ATPase分析和较高的吞吐量。此外，这些测定法避免了长时间的细胞生长，使用非常低的运输蛋白和底物，允许研究多种转运蛋白类型，以及 避免与CACO-2或MDCK测定中发现的细胞汇合的间隙相关的问题。最后，这些测定将允许现有的主动底物传输和简单扩散的比较。该项目是Drs之间的合作。 SRI International（SRI）的Faris和Dixit， 他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，后者是药物开发的运输蛋白活动评估专家。要了解液滴双层测定条件和结果如何转化为常规测定，以促进液滴测定的故障排除，确认试剂的可靠性以及在SRI和VCU之间转移有用的知识，同一两个转运蛋白和相同基本的SRI和VCU将执行可比较的测定类型，而其他组将访问其他组合。用于转运蛋白活性的灵活和多功能测定将提高我们对基本生物学和人类健康的了解。为了促进和加速药物发现和开发过程，需要更敏感，更具体，更快和廉价的方法。我们有一种能够敏感和准确测量运输的新型微螺旋杆技术。将该技术应用于转运蛋白，例如P-糖蛋白和有机阴离子运输多肽，将降低研究成本并提高新药候选者的成功率。