Symmetry-guided fusion converts weak liver pyruvate kinase inhibitors into potent allosteric activators.

Structure-guided exploitation of symmetry within multimeric protein interfaces offers an opportunity to convert partial ligand-binding poses into higher-affinity modulators. Here, we report a class of symmetrical "Z-shaped" allosteric activators of human liver pyruvate kinase (PKL), generated by symmetry-guided fusion of binding poses identified in inhibitor-bound crystal structures. The resulting ligands span the inter-subunit pocket at the tetrameric dimer-dimer interface and display substantially enhanced activity, with several analogues showing nanomolar activation potency. In HepG2 PKM2-knockout cells, the compounds increased PKL activity, maximal mitochondrial respiratory capacity and glycolytic proton efflux without altering PKL expression, Ser113 phosphorylation-dependent localisation, cell proliferation or triglyceride accumulation under the conditions examined. These findings establish symmetry-guided ligand fusion as a productive design strategy in this PKL system and provide structurally defined chemical tools for investigating the metabolic consequences of direct PKL activation.