Identification of new LAT-1 transporter substrates for drug delivery

血脑屏障（BBB）阻止大多数药物分子进入大脑，这一障碍会增加时间 以及发现和开发中枢神经系统（CNS）药物的成本。 BBB拥有 转运蛋白，允许氨基酸、葡萄糖和激素等生物分子交叉 需要。 L 型氨基酸转运蛋白 (LAT-1) 是这些转运蛋白之一，可转运特定的氨基酸 酸（例如苯丙氨酸）以及类似的化合物（例如药物左旋多巴）。这个运输机 提供了一种将靶向药物输送到大脑的潜在途径；此外，已经证明，药物 与氨基酸化学连接可以通过 LAT-1 穿过 BBB。已知有些分子会被运输 （底物），而另一些仅与转运蛋白表面结合（抑制剂）；然而，结构要求 对于这两种类型的活动都知之甚少。我们建议绘制结构-活性关系（SAR） 与 LAT-1 相互作用的分子，希望使这种转运蛋白成为实用且广泛使用的转运蛋白 药物输送车辆。我们的方法涉及使用 LAT-1 结合位点的计算模型来指导 结构与天然氨基酸相似但具有独特化学性质的化合物的合成 可用于设计改进的药物输送剂。我们的具体目标有三个。首先我们要综合一下 用我们的模型预测的基团进行化学取代的氨基酸将与 运输者。其次，我们将使用基于细胞的测定来测试这些化合物。来确定它们是否是 运输时，我们将使用已成功鉴定的顺式抑制和反式刺激细胞测定 以前未知的底物。这些实验的 SAR 将用于完善我们的计算模型 为了产生假设来指导进一步的底物优化。选定的细胞内浓度 将通过 LC-MS/MS 分析化合物，以验证我们的假设，即 LAT-1 活性会增加细胞摄取。一个 该项目的第三个目标将涉及拓扑异构酶 I 抑制剂的氨基酸前药的合成和测试 用于癌症 (SN-38)，以评估 SAR 是否源自特定目标 1 中 LAT-1 底物的优化 2可用于改善前药细胞摄取。此外，连接子连接 SN-38 和的效果 氨基酸前体可能对前药药代动力学和功效产生重大影响， 被检查。该项目在治疗大脑和其他领域的许多不同疾病方面具有广泛的应用 LAT-1 大量表达的组织，包括癌症。

The blood-brain barrier (BBB) prevents entry of most drug molecules into the brain, an obstacle which adds time and cost to the discovery and development of central nervous system (CNS) drugs. The BBB possesses transporter proteins that allow biological molecules including amino acids, glucose, and hormones to cross as needed. One of these transporter proteins, the L-type amino acid transporter (LAT-1) transports specific amino acids (e.g. phenylalanine) as well as compounds that resemble them (e.g. the drug L-DOPA). This transporter offers a potential route for targeted drug delivery to the brain; moreover, it has been demonstrated that drugs chemically linked to amino acids can cross the BBB via LAT-1. It is known that some molecules are transported (substrates), while others only bind to the transporter surface (inhibitors); however, the structural requirements for either type of activity is poorly understood. We propose mapping the structure-activity relationship (SAR) for molecules that interact with LAT-1 with the aspiration of making this transporter a practical and widely used vehicle for drug delivery. Our approach involves using a computational model of the LAT-1 binding site to guide the synthesis of compounds similar in structure to natural amino acids but with unique chemical properties that could be used to design improved drug delivery agents. Our specific aims are three-fold. First, we will synthesize amino acids chemically substituted with groups that our model predicts will form beneficial interactions with the transporter. Second, we will use cell-based assays to test these compounds. To determine whether they are transported, we will use cis-inhibition and trans-stimulation cell assays which have successfully identified previously unknown substrates. SAR from these experiments will be used to refine our computational model in order to generate hypotheses to guide further substrate optimization. The intracellular concentration of selected compounds will be analyzed by LC-MS/MS to verify our hypothesis that LAT-1 activity increases cell uptake. A third aim of this project will involve the synthesis and testing of amino acid prodrugs of a topoisomerase I inhibitor for cancer (SN-38) to assess whether the SAR derived from optimization of LAT-1 substrates in specific aims 1 and 2 can be applied to improve prodrug cellular uptake. Additionally, the effect of the linker joining SN-38 and the amino acid promoiety, which may have a significant impact on prodrug pharmacokinetics and efficacy, will be examined. This project has broad applications for treating many different diseases both in the brain and other tissues where LAT-1 is heavily expressed, including cancer.