Microdroplet Transporter Protein Assays

微滴转运蛋白检测

• 批准号: 9092415
• 负责人: GREGORY W FARIS
• 金额: $ 30.61万
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9092415
• 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; 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; Health; Hepatocyte; Human; Human Biology; Human body; Hydroxymethylglutaryl-CoA Reductase Inhibitors; International; Kidney; Knowledge; Lasers; Liver; MDCK cell; Malignant Neoplasms; Measurement; Mediating; Membrane; Methods; Monitor; Motion; Movement; Multi-Drug Resistance; Normal tissue morphology; 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; aquaporin 3; base; brain cell; calcein AM; cancer cell; cell growth; cell type; chemotherapeutic agent; cost; drug candidate; drug development; drug discovery; flexibility; fluorescence imaging; fluorexon; high throughput screening; improved; innovation; novel; novel therapeutics; overexpression; passive transport; public health relevance; reconstitution; research study; screening; skeletal; solute; success; uptake

 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)：药物发现和开发的一个关键早期步骤是了解可能在人体内的药代动力学和多药耐药的可能性。早期筛查通常涉及平行人工膜通透性试验(PAMPA)或ATPase试验等分析，这些方法操作简单，但不能很好地预测药物在体内的转运，因为PAMPA试验对双层的描述很粗糙，而且ATPase试验可能出现假阴性和假阳性结果。对于有希望的候选药物，使用Caco-2或Madin-Darby犬肾(MDCK)细胞进行更复杂的基于细胞的分析。然而，这些检测的成本和时间要高得多，而且结果的价值受到初始PAMPA和ATPase筛查准确性的限制。我们提出了基于液滴双层的替代检测策略。这些检测方法可以提供与Caco-2或MDCK检测类似的结果，而PAMPA或ATPase检测的复杂性较低，并且吞吐量较高。此外，这些分析避免了细胞生长的长时间，使用了非常少的运输蛋白和底物，允许研究多种运输蛋白类型，以及 避免与Caco-2或MDCK检测中发现的细胞汇合间隙相关的问题。最后，这些分析方法可以比较活性底物的传输和简单的扩散。这个项目是法里斯博士和SRI国际(SRI)的Dixit合作完成的， 世卫组织开发了直接沉积和激光介导的液滴双层方法，弗吉尼亚联邦大学(VCU)的GERK博士是药物开发转运蛋白活性分析方面的专家。为了了解液滴双层分析的条件和结果如何转化为常规分析，为了促进液滴分析的故障排除，确认试剂的可靠性，并在SRI和VCU之间传递有用的知识，将在SRI和VCU对相同的两个转运体和相同的底物进行类似的分析类型，并且在进行分析时，每个小组将互访对方。对转运蛋白活性的灵活和多用途分析将提高我们对基础生物学和人类健康的知识。为了促进和加速药物的发现和开发过程，需要更敏感、更特异、更快和更便宜的方法。我们有一种新的微滴技术，能够灵敏而准确地测量运输。将这项技术应用于P-糖蛋白和有机阴离子转运多肽等转运蛋白，将降低研究成本，提高新药候选的成功率。