Data_Sheet_1_New Quinolinone O-GlcNAc Transferase Inhibitors Based on Fragment Growth.PDF

O-GlcNAcylation is an important post-translational and metabolic process in cells that must be carefully regulated. O-GlcNAc transferase (OGT) is ubiquitously present in cells and is the only enzyme that catalyzes the transfer of O-GlcNAc to proteins. OGT is a promising target in various pathologies such as cancer, immune system diseases, or nervous impairment. In our previous work we identified the 2-oxo-1,2-dihydroquinoline-4-carboxamide derivatives as promising compounds by a fragment-based drug design approach. Herein, we report the extension of this first series with several new fragments. As the most potent fragment, we identified 3b with an IC50 value of 116.0 μM. If compared with the most potent inhibitor of the first series, F20 (IC50 = 117.6 μM), we can conclude that the new fragments did not improve OGT inhibition remarkably. Therefore, F20 was used as the basis for the design of a series of compounds with the elongation toward the O-GlcNAc binding pocket as the free carboxylate allows easy conjugation. Compound 6b with an IC50 value of 144.5 μM showed the most potent OGT inhibition among the elongated compounds, but it loses inhibition potency when compared to the UDP mimetic F20. We therefore assume that the binding of the compounds in the O-GlcNAc binding pocket is likely not crucial for OGT inhibition. Furthermore, evaluation of the compounds with two different assays revealed that some inhibitors most likely interfere with the commercially available UDP-Glo™ glycosyltransferase assay, leading to false positive results. This observation calls for caution, when evaluating UDP mimetic as OGT inhibitors with the UDP-Glo™ glycosyltransferase assay, as misinterpretations can occur.

O-连接N-乙酰葡糖胺糖基化修饰（O-GlcNAcylation）是细胞内一类重要的翻译后修饰与代谢调控过程，其调控需受到严格把控。O-连接N-乙酰葡糖胺转移酶（O-GlcNAc transferase，OGT）广泛分布于各类细胞中，是唯一能够催化O-GlcNAc基团向蛋白质转移的酶。OGT是癌症、免疫系统疾病、神经损伤等多种病理状态下极具潜力的药物靶点。我们团队此前的研究通过基于片段的药物设计策略，筛选得到了2-氧代-1,2-二氢喹啉-4-甲酰胺类衍生物作为候选化合物。本文中，我们报道了对该首代化合物系列的拓展，新增了多个片段类化合物。其中活性最强的片段化合物为3b，其半抑制浓度（IC50）为116.0 μM。将其与首代系列中活性最强的抑制剂F20（IC50=117.6 μM）相比，新片段化合物并未显著提升OGT的抑制活性。因此，我们以F20为骨架开展后续系列化合物的设计，通过向O-GlcNAc结合口袋延伸修饰优化化合物，因游离的羧基可实现便捷的偶联反应。在延伸修饰后的化合物中，6b的OGT抑制活性最强，其IC50值为144.5 μM，但相较于UDP模拟物F20，其抑制活性有所下降。据此我们推测，化合物在O-GlcNAc结合口袋中的结合对于OGT的抑制作用可能并非关键。此外，通过两种不同检测方法对化合物进行评估后发现，部分抑制剂极有可能干扰市售的UDP-Glo™糖基转移酶检测试剂盒，进而导致假阳性结果。该发现提示我们，在采用UDP-Glo™糖基转移酶检测试剂盒评估UDP模拟物作为OGT抑制剂时需格外谨慎，以免出现解读偏差。