Objective

Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools.

Methods and results

Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase.

Conclusions

These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy.

中文翻译：

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基于受体结构的琥珀酸酯受体GPR91的非代谢激动剂的发现。

目的

琥珀酸除了起细胞内代谢物的作用外，还通过激活GPR91（SUCNR1）充当应激诱导的细胞外信号，而我们缺乏合适的药理工具。

方法与结果

在这里，我们首先确定琥珀酸骨架的顺式构象是优选的，并且某些骨架修饰可用于GPR91激活。通过对密切相关的P2Y1受体的X射线结构进行受体建模，我们发现结合口袋部分被细胞外环的一部分占据，因此琥珀酸以与通常认为的非常不同的方式结合。重要的是，在琥珀酸结合位点旁边发现了一个空的侧袋。所有这些信息构成了基于子结构的搜索查询的基础，该查询与分子对接相结合，用于ZINC数据库的虚拟筛选，以选择总共仅245种化合物的两个连续微型文库，其中亚微摩尔，鉴定出具有独特结构的选择性GPR91激动剂。最好的化合物是骨架修饰的琥珀酸酯类似物，其中酰胺连接的疏水部分与琥珀酸酯相邻接入侧链，如功能缺失和功能获得诱变所示。这些化合物在改变人类M2巨噬细胞中与琥珀酸相似的细胞因子表达中具有GPR91依赖性活性，重要的是对主要的细胞内靶标琥珀酸脱氢酶没有任何影响。

结论

这些新颖的，合成的非代谢GPR91激动剂，不仅可以作为药理学工具来描述GPR91介导的琥珀酸功能，而且可以作为开发GPR91靶向药物的先导，以潜在地治疗低度代谢性炎症和糖尿病并发症，例如视网膜病变和肾病。