靶向谷氨酸脱氢酶NADP+结合口袋的小分子抑制剂及其作用机制研究

Highly active glutaminolysis provides tumor cells with energy and material compensation to fuel their rapid proliferation. Glutamate dehydrogenase (GDH1) is a key enzyme in downstream of glutaminolysis, which is abnormally high expressed in many malignant tumors. The known GDH1 inhibitors are limited to polyphenols obtained by high-throughput screening, while rationally designed GDH1 inhibitors has not been reported. Our recent work showed that NADP+ had high affinity (Kd of 77 nM) for GDH1, which is 1000-fold higher affinity than its substrate and other coenzymes. Therefore, we concluded that NADP+ competitive inhibitors may block the binding of NADP+ to GDH1, thereby increasing the inhibitory effect and anti-tumor activity of GDH1 inhibitors. Given all this, this project intends to design and synthesize a series of competitive inhibitors targeting GDH1 at the NADP+ binding site based on its crystal structure with the non-phenolic compound ebselen as lead compound. After that, we will select 1-2 active lead compounds through multiple activity evaluation, and then focus on the study of their binding mode and cell-level targeting. So this project will lay a good experimental foundation for the reasonable design of GDH1 inhibitors, and provide a theoretical basis for the in-depth research and application of GDH1 inhibitors.

异常活跃的谷氨酰胺代谢为肿瘤细胞的快速增殖提供能量和物质补偿，谷氨酸脱氢酶(GDH1)是该通路下游的关键酶，在许多恶性肿瘤中异常高表达。现有的GDH1抑制剂局限于高通量筛选得到的多酚类化合物，针对GDH1的合理药物设计尚未见文献报道。我们的前期研究发现辅酶NADP+与GDH1具有高亲和力(Kd=77nM)，比底物和其他辅酶的亲和力高1000倍以上，因此我们推断“NADP+竞争性抑制剂可能阻断NADP+与GDH1的结合，从而增加化合物对GDH1的抑制作用和抗肿瘤活性”。据此本项目拟以非酚类化合物依布硒啉为先导，基于GDH1的晶体结构设计并合成一系列靶向GDH1-NADP+结合口袋的竞争性抑制剂。通过多重活性评价得到1-2个活性先导化合物，并重点研究其与GDH1的结合模式和细胞水平的靶向性。本项目的研究将为GDH1抑制剂的合理设计奠定良好的实验基础，为GDH1抑制剂的深入研究和应用提供理论依据

在本项目中，我们探索了以非酚的依布硒啉为先导，基于GDH1的晶体结构，设计并合成一系列旨在靶向NADP+结合口袋的小分子抑制剂。首先，我们合成得到了70个具有多样性结构的苯并异硒唑酮化合物；其次，由于苯并异硒唑酮分子中N-Se键易被半胱残基进攻，可能引起非特异性结合。因此，我们以苯并异硒唑酮作为硒源，开发了适于多孔板组合合成的二芳基硒醚合成方法，原位构建了库容量为1350的吲哚-3-硒醚类化合物，这类化合物具有良好的代谢稳定性，也不含具有反应性的基团。通过对以上的苯并异硒唑酮和二芳基硒醚化合物进行活性研究，我们获得了对GDH1酶IC50为1 μM，对人乳腺癌MCF-7细胞和人卵巢癌SKOV-3细胞的IC50分别为0.82 μM和0.95 μM的小分子探针，其酶、细胞水平的抑制活性有较好的相关性，同时表现出化合物浓度依赖性。它对GDH1酶的抑制活性可被高浓度的NADP+阻断，说明它是一个可逆抑制剂，并且作用于NADP+结合口袋。本项目的研究将为GDH1抑制剂的合理设计奠定良好的实验基础，为GDH1抑制剂的深入研究和应用提供理论依据。