9rft
Structure of liver pyruvate kinase in complex with Liver pyruvate kinase in complex with fluorescent probe II
Structural highlights
DiseaseKPYR_HUMAN Defects in PKLR are the cause of pyruvate kinase hyperactivity (PKHYP) [MIM:102900; also known as high red cell ATP syndrome. This autosomal dominant phenotype is characterized by increase of red blood cell ATP.[1] Defects in PKLR are the cause of pyruvate kinase deficiency of red cells (PKRD) [MIM:266200. A frequent cause of hereditary non-spherocytic hemolytic anemia. Clinically, pyruvate kinase-deficient patients suffer from a highly variable degree of chronic hemolysis, ranging from severe neonatal jaundice and fatal anemia at birth, severe transfusion-dependent chronic hemolysis, moderate hemolysis with exacerbation during infection, to a fully compensated hemolysis without apparent anemia. FunctionKPYR_HUMAN Plays a key role in glycolysis (By similarity). Publication Abstract from PubMedPyruvate 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. Potent Fluorescent Probe for Target-Engagement Studies of Allosteric Pyruvate Kinase Modulators.,Nilsson O, Valaka AP, Haversen L, Bogucka A, Koteles I, Brear P, Rutberg M, Gunnarsson A, Hyvonen M, Grotli M Angew Chem Int Ed Engl. 2025 Oct 13;64(42):e202513969. doi: , 10.1002/anie.202513969. Epub 2025 Aug 29. PMID:40884041[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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