Allostery at Work: Mapping, Modulating, and Monitoring PKL Function with Chemotype Diversity

Abstract

Understanding the processes of protein regulation and our ability to control enzyme activity in the human body lie at the core of drug development. The liver isoform of pyruvate kinase (PKL) is a metabolic enzyme crucial for energy production. Because of its metabolic role, PKL and other pyruvate kinase isoforms are interesting drug targets for metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). In this thesis, the allosteric regulation of PKL was explored using multiple approaches to expand our knowledge about this extensively regulated enzyme. Known allosteric ligands were derivatized into fluorescent reporter probes to monitor ligand engagement with the allosteric pocket of PKL. This approach centred around the direct incorporation of fluorescent dye molecules into the scaffolds of the allosteric ligands, exploiting structurally overlapping features. A set of probes containing the environment-sensitive dye 4-sulfamoyl-7-aminobenzoxadiazole (SBD) was developed and used to measure the occupancy of unlabelled ligands in indicator displacement assays. The concept was further developed by applying this design philosophy to a more potent ligand class, generating a cell-permeable fluorescent tracer. In combination with NanoBRET technology, this tracer enabled the development of a target-engagement assay capable of detecting allosteric ligand binding to PKL in living cells. This thesis also proposes a model for how the allosteric regulation of PKL works. By modulating the enzyme with compounds derived from the known drug mitapivat, minor modifications were identified that had significant effects on functional outcomes. Biophysical techniques and molecular dynamics simulations were employed to investigate a structurally similar activator/inhibitor pair, demonstrating that they differentially affect the stability of the protein-protein interfaces. Furthermore, a fragment-based drug design effort was undertaken to map the allosteric pocket and identify novel ligand scaffolds capable of allosterically inhibiting PKL. An iterative design process starting from simple fragments yielded moderate inhibitors with large structural diversity, providing valuable insights into key binding interactions and serving as a foundation for future optimization toward more potent modulators. These findings collectively deepen our understanding of PKL allosteric regulation and provide useful chemical tools for further study. They may also inform future efforts aimed at developing ligands to modulate PKL activity in a physiological context.