Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase

• Published in: Chembiochem : a European journal of chemical biology 2023-01, Vol.24 (1), p.e202200339-n/a
• Main Authors: Battisti, Umberto Maria, Gao, Chunxia, Nilsson, Oscar, Akladios, Fady, Lulla, Aleksei, Bogucka, Agnieszka, Nain‐Perez, Amalyn, Håversen, Liliana, Kim, Woonghee, Boren, Jan, Hyvönen, Marko, Uhlen, Mathias, Mardinoglu, Adil, Grøtli, Morten
• Format: Article
• Language: English
• Subjects: 1 dioxide; Allosteric properties; Allosteric Regulation; allosterism; aqueous solution; benzo[d]isothiazole 1; binding; Bioactive compounds; biocatalysis; biochemical analysis; Biochemistry & Molecular Biology; Biochemistry and Molecular Biology; Biokemi och molekylärbiologi; Biological activity; Biological effects; Catalysts; Catalytic Domain; chloro saccharin; computer model; controlled study; Covalence; covalent activators; discovery; Drug development; EC50; enzyme activation; enzyme active site; enzyme inhibitor; enzyme modification; enzyme structure; Enzymes; erythrocyte; glycolysis; human; Humans; in vitro study; inhibitors; isoenzyme; isoenzymes; ligand design; Liver; Lysine; mass spectrometry; Modulators; molecular docking; molecular dynamics; Molecular modelling; molecular stability; Pharmacology & Pharmacy; phosphoenolpyruvate; PKL; protein; protein analysis; protein expression; protein PKL; protein PKM1; protein PKM2; protein PKR; Pyruvate kinase; Pyruvate Kinase - chemistry; Pyruvate Kinase - metabolism; Pyruvic acid; relationships; Residues; RNA splicing; saccharin; site; structure-activity; structure-activity relationships; synthesis; Tandem Mass Spectrometry; unclassified drug
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202200339

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50=0.29 μM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators. MS/MS experiments and molecular modelling studies identified a mechanism by which a lysine residue in the active site of human liver pyruvate kinase (PKL) can be targeted by benzo[d]isothiazole‐1,1‐dioxide derivatives, resulting in the covalent activation of PKL. This previously unknown mechanism holds promise for further development of PKL activators.