Probing the Allosteric Landscape of Phosphoglycerate Kinase: Development and Application of a 19F NMR Fragment Screening Assay

The discovery of allosteric modulators for protein kinases is a major frontier in drug discovery, offering higher selectivity but lacking general screening methods. This thesis addresses this by developing and validating a novel, integrated nuclear magnetic resonance (NMR)-based platform to discover allosteric modulators of the atypical kinase, phosphoglycerate kinase (PGK), a crucial metabolic enzyme that also exhibits non-canonical functions implicated in cancer.
The platform uses a stable, 19F-containing transition state analogue (TSA) complex as a direct probe of allostery, leading to the identification of a validated set of novel fragment hits. Subsequent biophysical characterisation using 19F and TROSY NMR revealed that these fragments act through complex, multi-step induced-fit mechanisms, which were classified into three distinct allosteric "fingerprints".
Furthermore, this thesis presents the first structural hypotheses for protein-protein interaction networks that regulate PGK's non-canonical functions, including pro-tumorigenic kinase cascades that control mitochondrial respiration and autophagy. A key discovery was the identification of pyruvate as a natural allosteric regulator of PGK, thereby establishing a metabolic feedback loop that coordinates glycolysis and the tricarboxylic acid (TCA) cycle.
A robust synthesis of all experimental and computational data demonstrates that these synthetic fragments do not bind to random pockets but instead engage pre-existing, functionally critical allosteric pathways that nature already exploits. This work has therefore moved from method development to hit discovery and the elucidation of novel biological pathways. It delivers a validated set of starting points for drug design against PGK and a powerful, transferable platform for discovering allosteric modulators for other phosphoryl transfer enzymes.