Potent Fluorescent Probe for Target‐Engagement Studies of Allosteric Pyruvate Kinase Modulators

Abstract Pyruvate kinases (PKs) are highly allosterically regulated enzymes that play a central role in cellular metabolism and are increasingly recognized as valuable therapeutic targets in cancer, metabolic diseases, and diabetes. Despite their biological and clinical significance, methods to directly assess allosteric ligand engagement of PK isoforms remain limited. Here, we report the development of LumiPK , a novel, environment‐sensitive fluorescent tracer designed to monitor allosteric binding to the liver isoform of pyruvate kinase (PKL). LumiPK integrates an environment‐sensitive 4‐sulfamonyl‐7‐aminobenzoxadiazole fluorophore into a potent allosteric modulator scaffold. It emerged as the lead compound from a small ligand series, showing high affinity for PKL ( K D = 37 ± 5 nM) in recombinant assays; the most potent fluorescent PK reporter reported to date. A NanoBRET assay using a PKL‐Nluc fusion (PKL Nluc ) enabled intracellular monitoring of unlabeled ligand engagement. LumiPK maintained high potency (EC 50 = 18.4 nM) in cellular experiments. Competitive NanoBRET and fluorescence titration assays confirmed binding of known PKL activators (mitapivat, TEPP‐46, DASA‐58) in both cellular and recombinant settings, with K D values remaining consistent across these methods. LumiPK thus provides a robust tool for probing PKL allosteric modulation and fills a key gap in target engagement technologies for PKL.