{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T03:55:45Z","timestamp":1792036545223,"version":"4.4.0"},"reference-count":66,"publisher":"Wiley","issue":"42","license":[{"start":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T00:00:00Z","timestamp":1756425600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T00:00:00Z","timestamp":1756425600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Angew Chem Int Ed"],"published-print":{"date-parts":[[2025,10,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    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\n                    <jats:bold>LumiPK<\/jats:bold>\n                    , a novel, environment\u2010sensitive fluorescent tracer designed to monitor allosteric binding to the liver isoform of pyruvate kinase (PKL).\n                    <jats:bold>LumiPK<\/jats:bold>\n                    integrates an environment\u2010sensitive 4\u2010sulfamonyl\u20107\u2010aminobenzoxadiazole fluorophore into a potent allosteric modulator scaffold. It emerged as the lead compound from a small ligand series, showing high affinity for PKL (\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>D<\/jats:sub>\n                    \u00a0 = \u00a037\u00a0\u00b1\u00a05\u00a0nM) in recombinant assays; the most potent fluorescent PK reporter reported to date. A NanoBRET assay using a PKL\u2010Nluc fusion (PKL\n                    <jats:sub>Nluc<\/jats:sub>\n                    ) enabled intracellular monitoring of unlabeled ligand engagement.\n                    <jats:bold>LumiPK<\/jats:bold>\n                    maintained high potency (EC\n                    <jats:sub>50<\/jats:sub>\n                    \u00a0 = \u00a018.4\u00a0nM) in cellular experiments. Competitive NanoBRET and fluorescence titration assays confirmed binding of known PKL activators (mitapivat, TEPP\u201046, DASA\u201058) in both cellular and recombinant settings, with\n                    <jats:italic>K<\/jats:italic>\n                    <jats:sub>D<\/jats:sub>\n                    values remaining consistent across these methods.\n                    <jats:bold>LumiPK<\/jats:bold>\n                    thus provides a robust tool for probing PKL allosteric modulation and fills a key gap in target engagement technologies for PKL.\n                  <\/jats:p>","DOI":"10.1002\/anie.202513969","type":"journal-article","created":{"date-parts":[[2025,8,30]],"date-time":"2025-08-30T06:29:53Z","timestamp":1756535393000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Potent Fluorescent Probe for Target\u2010Engagement Studies of Allosteric Pyruvate Kinase Modulators"],"prefix":"10.1002","volume":"64","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2716-2563","authenticated-orcid":false,"given":"Oscar","family":"Nilsson","sequence":"first","affiliation":[{"name":"Department of Chemistry and Molecular Biology University of Gothenburg  Medicinaregatan 7B Gothenburg SE\u2010413 90 Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-7007-6971","authenticated-orcid":false,"given":"Anna P.","family":"Valaka","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Molecular Biology University of Gothenburg  Medicinaregatan 7B Gothenburg SE\u2010413 90 Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5180-7830","authenticated-orcid":false,"given":"Liliana","family":"H\u00e5versen","sequence":"additional","affiliation":[{"name":"Department of Molecular and Clinical Medicine University of Gothenburg and Sahlgrenska University Hospital  Gothenburg SE\u2010413 45 Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5317-4344","authenticated-orcid":false,"given":"Agnieszka","family":"Bogucka","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Cambridge  80 Tennis Court Road Cambridge CB2 1GA UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1678-233X","authenticated-orcid":false,"given":"Istv\u00e1n","family":"K\u00f6teles","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Molecular Biology University of Gothenburg  Medicinaregatan 7B Gothenburg SE\u2010413 90 Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Brear","sequence":"additional","affiliation":[{"name":"Department of Biochemistry University of Cambridge  80 Tennis Court Road Cambridge CB2 1GA 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