High-Throughput Assay for the Intestinal Peptide Transporter

描述（由申请方提供）：本提案旨在进一步开发用于快速测定转运蛋白亲和力的生物测定法。 我们将研究重点放在与小肠相关的转运系统，小肠肽转运蛋白（PepT1）上。 PepT1在二肽和三肽的肠道吸收中起关键作用，并介导广泛的治疗化合物的吸收，例如氨基青霉素、头孢菌素和“血管紧张素转换酶”（ACE）抑制剂。我们已经开发了新型的，选择性的，拟肽荧光化合物与PEPT1的亲和力，可用于高通量生物测定。 稳定表达人PepT1的上皮细胞系将用于验证和优化试验。 成功实施该测定将显著增加我们对驱动肠肽转运的结构相互作用的理解，并进一步提高我们对SLC蛋白的结构知识。 此外，它可能有助于未来开发策略，通过特异性靶向PepT1优化药物的口服生物利用度，并合理开发靶向过表达PepT1的肿瘤（如胰腺癌）的化疗药物。我们的具体目标是：1。 为了合成和评价新型的PepT1的荧光肽模拟物底物，在这个目标中，我们将设计和合成新型的荧光PepT1底物，并选择那些表现出高相对荧光量子产率，在细胞培养系统中的代谢稳定性和高hPepTI亲和力的组合，用于在高通量环境中进一步测试。2. 分析验证和优化。在这一特定目标中，我们将通过确定测定准确度、精密度、检测限以及已知标准品的定量限、专属性、线性和范围以及耐用性来验证我们的生物测定法。本研究的长期目标是开发一种可靠的，成本效益高，高通量的肽转运蛋白系统的生物测定。 这将有助于我们了解通过该系统控制肽和药物转运的结构特征，并将显著支持旨在靶向该转运蛋白的研究。

DESCRIPTION (provided by applicant): This proposal aims to further develop a bioassay for the rapid determination of transporter affinity. We focus our proposed research on a pharmacologically relevant transport system, the small intestinal peptide transporter (PepT1). PepT1 plays a key role in intestinal absorption of di- and tripeptides, and mediates the absorption of a wide range of therapeutic compounds, such as aminopenicillins, cephalosporins, and 'angiotensin converting enzyme' (ACE) inhibitors. We have developed novel, selective, peptidomimetic fluorescent compounds with affinity for PEPT1 that can be used in high-throughput bioassays. An epithelial cell line stably expressing human PepT1 will be used to validate and optimize the assay. Successful implementation of this assay will significantly increase our understanding of the structural interactions that drive intestinal peptide transport and further our structural knowledge of SLC proteins in general. Additionally, it may aid future development of strategies to optimize the oral bioavailability of drugs by targeting specifically to PepT1 and the rational development of chemotherapeutics that are targeted to tumors overexpressing PepT1, such as pancreatic adenocarcinoma. Ou specific aims are: 1. To synthesize and evaluate novel fluorescent peptidomimetic substrates for PepT1In this aim we will design and synthesize novel fluorescent PepT1 substrates and select those that display a combination of high relative fluorescence quantum yield, metabolic stability in cell culture system, and high hPepTI affinity for further testing in a high-throughput setting. 2. Assay validation and optimization. In this specific aim we will validate our bioassay by determining assay accuracy, precision, limit of detection as well as limit of quantitation for known standards, specificity, linearity and range, and robustness. The long-term goal of this study is to develop a reliable, cost efficient, high-throughput bioassay for the peptide transporter system. This will aid in our understanding of the structural features that govern peptide and drug transport via this system and will significantly support studies aiming to target this transporter.