Microdroplet Transporter Protein Assays

 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药耐药性的可能性。早期筛选通常涉及诸如平行人工膜渗透性测定（PAMPA）或ATP酶测定的测定，其操作简单但不是体内药物转运的良好预测因子，因为PAMPA测定的双层的粗略描述和ATP酶测定中假阴性和假阳性结果的可能性。对于有前景的候选药物，使用Caco-2或Madin-Darby犬肾（MDCK）细胞进行更复杂的基于细胞的测定。然而，这些测定进行起来更加昂贵和耗时，并且结果的价值受到初始PAMPA和ATP酶筛选的准确性的限制。我们提出了基于使用液滴双层的替代检测策略。这些测定可提供与Caco-2或MDCK测定类似的结果，具有PAMPA或ATP酶测定的较低复杂性和较高通量。此外，这些测定避免了长时间的细胞生长，使用非常少量的转运蛋白和底物，允许研究多种转运蛋白类型， 避免与Caco-2或MDCK检测中发现的细胞融合间隙相关的问题。最后，该测定将允许容易地比较活性底物转运和简单扩散。该项目是法里斯博士和SRI国际（SRI）的Diplomat之间的合作， 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，他是药物开发转运活性测定的专家。为了了解液滴双层测定条件和结果如何转化为常规测定，为了便于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对相同的两种转运蛋白和相同的底物进行可比的测定类型，并且在进行测定时，每组将访问另一组。灵活和多用途的转运蛋白活性测定将提高我们的基础生物学和人类健康的知识。为了促进和加速药物发现和开发过程，需要更灵敏、特异、更快和更便宜的方法。我们拥有一种新型微滴技术，能够灵敏、准确地测量传输。将这项技术应用于转运蛋白，如P-糖蛋白和有机阴离子转运多肽，将降低研究成本，提高新药候选物的成功率。

 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.