Genotype‐Dependent Functional and Pharmacological Profiling of Rare GCK Variants Reveals Implications for Precision Management of Monogenic Diabetes

ABSTRACT Aims Rare GCK variants contribute to heterogeneous forms of monogenic diabetes, yet the molecular basis underlying their diverse functional consequences and pharmacological responsiveness remains incompletely understood. As glucokinase (GCK) serves as a central glucose sensor in pancreatic β‐cells and hepatocytes, defining the functional properties of individual variants may improve molecular diagnosis and inform the interpretation of potential therapeutic responses in GCK‐maturity‐onset diabetes of the young (GCK‐MODY, also called MODY2). Materials and Methods We established a recombinant protein‐based functional platform to investigate two rare GCK variants, p.Leu88Pro and p.Val374Glu, identified in Chinese families with GCK‐MODY. Wild‐type and mutant GCK proteins were expressed and purified using a GST‐tagged prokaryotic system. Functional consequences were assessed by glucose‐response kinetics, pharmacological modulation with dorzagliatin, thermal stability analysis and structural stability prediction. Results The p.Leu88Pro variant demonstrated impaired glucose sensing, with an increased S 0.5 value, together with reduced stability and a marked predicted destabilizing effect. The p.Val374Glu variant retained near‐normal glucose‐response kinetics under basal conditions but exhibited reduced thermal stability. Dorzagliatin markedly reduced the S 0.5 of p.Val374Glu with limited effects on maximal catalytic activity. Structural stability analysis further indicated reduced protein stability for both variants, with a greater predicted destabilizing effect for p.Leu88Pro than for p.Val374Glu. Conclusions Rare GCK variants can cause heterogeneous biochemical defects and show differential responses to pharmacological activation. Integrating glucose‐response kinetics, protein stability assessment and drug‐response profiling provides complementary functional evidence for variant interpretation and highlights the molecular heterogeneity of GCK‐MODY and the potential for genotype‐dependent pharmacological responses.