A cleavable chelation-assisted biotin probe enables the discovery of a celecoxib binding site on prostaglandin E synthase

The coxibs are a subset of non-steroidal anti-inflammatory drugs (NSAIDs) that selectively target cyclooxygenase-2 (COX-2) to inhibit prostaglandin signaling and reduce inflammation. However, only celecoxib remains in use, necessitating exploration of broader mechanisms of the coxibs. Here, we report a novel binding site for celecoxib on prostaglandin E synthase (PTGES), an enzyme downstream of COX-2 in the prostaglandin signaling pathway using an isotopically-coded cleavable chelation-assisted biotin probe. Evaluation of the multi-functional probe revealed significantly improved tagging efficiencies attributable to promotion of CuAAC chemistry by the embedded picolyl functional group. Application of the probe within the small molecule interactome mapping by photo-affinity labeling (SIM-PAL) platform using photo-celecoxib as a reporter for celecoxib identified known targets (e.g., carbonic anhydrase 12) and the significant enrichment of PTGES, along with six additional membrane proteins and 15 subunits of the cytochrome complex. In addition, four binding sites to celecoxib were mapped by the probe, including a direct interaction with PTGES. The interaction between celecoxib and PTGES was validated by competitive displacement and thermal shift assay. The binding site between celecoxib and PTGES enabled the development of a structural model of the interaction and will inform the further development of new selective inhibitors of the prostaglandin signaling pathway.

昔布类药物（coxibs）是非甾体抗炎药（non-steroidal anti-inflammatory drugs, NSAIDs）的子类，可选择性靶向环氧合酶-2（cyclooxygenase-2, COX-2），通过抑制前列腺素信号通路以减轻炎症反应。但目前仅有塞来昔布仍在临床应用，因此亟需对昔布类药物的更广泛作用机制展开研究。本研究利用同位素编码可裂解螯合辅助生物素探针（isotopically-coded cleavable chelation-assisted biotin probe），在前列腺素信号通路中位于COX-2下游的前列腺素E合酶（prostaglandin E synthase, PTGES）上，发现了塞来昔布的全新结合位点。对该多功能探针的评估结果显示，其标记效率得到显著提升，这源于探针内嵌的吡啶甲基官能团对铜催化叠氮-炔环加成（CuAAC）反应的催化促进作用。我们以光标记塞来昔布作为塞来昔布的报告分子，将该探针应用于光亲和标记小分子相互作用组图谱（small molecule interactome mapping by photo-affinity labeling, SIM-PAL）平台，不仅鉴定出了已知的靶点（如碳酸酐酶12），还实现了PTGES的显著富集，同时富集得到另外6种膜蛋白以及细胞色素复合物的15个亚基。此外，该探针还绘制出塞来昔布的4个结合位点，其中包含与PTGES的直接相互作用。研究人员通过竞争性置换实验与热位移测定法，验证了塞来昔布与PTGES之间的相互作用。塞来昔布与PTGES的结合位点助力构建了二者相互作用的结构模型，将为前列腺素信号通路新型选择性抑制剂的后续开发提供重要参考。