小分子激动OATP分子机制的研究

Organic anion transporting polypeptides (OATP) are a family of important drug transporters. They have great effects on the pharamcokinetics of drugs. OATP can transport anticancer drugs into cancer cells and some OATP have higher expression levels in cancer cells than in normal cells. Meanwhile, OATP can be stimulated by small molecules. Thus, by coadministration of OATP agonists with anticancer drugs, we can improve the selectivity of anticancer drugs against cancer cells. The stimulating effect of small molecules on OATP is transporter- and substrate-dependent. Some evidence indicates that agonists regulate OATP function by allosteric effect, but their molecular mechanisms are not clear yet. However, elucidation of their molecular mechanisms can help us utilize the property of OATP activation better to improve anticancer drug targeting and bioavailability and to reduce their side effects. Therefore, in this project we plan to identify and characterize the interacting proteins of OATP, the binding sites of OATP substrates and agonists, and the key domains and residues of OATP for their activation. Based on this information, we will put forward an interaction model between OATP, their interacting proteins, substrates and agonists, and eventually elucidate the molecular mechanisms of OATP activation by small molecules.

有机阴离子转运多肽（OATP）是一类重要的药物转运蛋白，它们对药物的代谢动力学有很大的影响。OATP可以转运抗癌药物进入细胞内，而不少OATP在癌细胞中的表达比在正常细胞中高；同时OATP还可以被小分子激动。因此我们可以通过OATP激动剂和抗癌药物联合用药的办法，来提高抗癌药物对癌细胞的选择性。小分子对OATP的激动作用呈蛋白和底物依赖性。有迹象表明，激动剂可能通过别构效应来调节OATP的功能，但其具体的分子机制目前并不清楚。而对其分子机制的阐明可以帮助我们更好地利用OATP的这一性质，提高抗癌药物的靶向运输和生物利用率、降低抗癌药物的毒副作用。因此在本项目中，我们将鉴定和表征OATP的相互作用蛋白、OATP底物和激动剂的结合位点、对OATP激动起重要作用的结构域和氨基酸残基；并基于上述信息提出OATP与结合蛋白、底物和激动剂的相互作用模型，最终阐明小分子激动OATP的分子机制。

本项目开展了有机阴离子转运多肽（OATPs）功能调节机制的研究。我们构建了OATPs的细胞表达体系，开发了检测OATPs底物雌酮-3-硫酸酯（E3S）和他汀类药物阿托伐他汀、氟伐他汀和罗伐他汀的液质联用（LC-MS/MS）方法，并以OATP2B1和1B1为靶标对天然产物库进行了筛选，得到了一些对上述OATPs功能具有调节作用的活性分子，并测定了它们的IC50值。根据天然化合物的IC50值和它们在临床上的血药浓度，该结果可为临床预测OATPs介导的药物-药物和药物-食物相互作用提供实验基础。对活性较好的分子，我们还进行了底物依赖性研究。因我国拥有丰富的中草药资源，这项研究可进一步应用到更多天然产物的筛选，可为OATPs功能研究提供更多的分子工具。此外我们还研究了色氨酸残基对OATP2B1功能和表达的影响，鉴定了该蛋白中关键的色氨酸残基。