hURAT1转运尿酸的活性位点识别及靶向hURAT1选择性抑制剂设计及降尿酸作用的分子机制

Hyperuricemia is mainly associated with reduced uric acid excretion. Among the renal organic anion transporter SLC22 subfamily, human uric acid transporter 1 (hURAT1) and human organic anion transporters 1 and 3 (hOAT1/3) are responsible for the reabsorption and secretion of uric acid in renal tubules, respectively. Up to now, It has been a hot topic to discover selective inhibitors of hURAT1. Our previous studies by homology modeling of inward- and outward-open hURAT1 3D structures and molecular docking between hURAT1 active sites and various inhibitors revealed that there are some putative residues contributing to the urate transporting and the selectivity of hUART1 on the surface of hURAT1 active pocket surrounding by some key residues, especially for those active sites such as G361、F364、F365、R487 on the hinge region controlling the hUART1 structure transformation . Based on our previous studies, we will do further work to map substrate and inhibitor binding sites and confirm their functions by mutational assay and high affinity inhibition of [14C] urate uptake different from hURAT1 and hOAT1/3. Besides, we will also evaluate the urate-lowering effects in vitro and vivo of more selective and potent compounds designed according to the active sites mentioned above. All these results might contribute to the design strategies for discovering selective inhibitors of hURAT1 and thus have important theoretical and practical values in the treatment of hyperuricemia and gout which are with high morbidity and lack of effective treatment approaches.

高尿酸血症主要与尿酸排泄减少有关。人尿酸转运体1（hURAT1）和人有机阴离子转运体1和3（hOAT1/3）分别负责尿酸在肾小管的重吸收和分泌。选择性抑制hURAT1是促尿酸排泄防治高尿酸血症和痛风的研究热点。前期我们通过同源模建hURAT1内、外开口结构和抑制剂相互作用发现，hURAT1活性口袋表面，存在与尿酸转运活性和hURAT1选择性相关的活性位点，尤其是控制hURAT1内、外开口变构的绞链区活性位点。本课题在前期研究的基础上，通过研究hURAT1和hOAT1/3突变载体或嵌合体对[14C]尿酸转运活性的影响，识别和证实这些活性位点及其功能，并针对这些活性位点设计合成新的hURAT1抑制剂，对其中选择性高、活性强的化合物进行体内外活性研究，为研发hURAT1选择性抑制剂提供靶向性设计策略，对目前高发病率和缺少有效治疗的高尿酸血症和痛风的防治具有重要的理论和应用价值。

选择性抑制hURAT1是促尿酸排泄防治高尿酸血症和痛风的研究热点。本课题首先通过同源模建、分子对接、序列比对以及定点突变技术构建了多个hURAT1突变载体或嵌合体，识别并验证了hURAT1转运尿酸以及抑制剂结合的关键氨基酸位点。主要发现包括：TMD7的W357-F365与TMD11的P484-R487是URAT1内外开口变构的关键柔性区域；F241、H245、W357、G361、T363、F365、R477和R487可能对尿酸具有很高的亲和力；带正电荷的R477与hURAT1的结构变化有关，有助于其与尿酸选择性结合。此外，针对这些活性位点我们提出了柔性理论，设计并合成了系列新的URAT1抑制剂，其中发现KPH2f体外抑制URAT1的IC50值为0.24 μM。在氧嗪酸钾联合次黄嘌呤建立的高尿酸血症小鼠模型中，KPH2f的降尿酸作用优于同等剂量阳性药RDEA3170（10 mg/kg）。本项目的完成为研发hURAT1选择性抑制剂提供了良好的设计策略，对目前高发病率和缺少有效治疗的高尿酸血症和痛风的防治具有重要的理论和应用价值。