A comprehensive analysis of screening assays for identifying pyruvate kinase M2 modulators

Pyruvate kinase M2 (PKM2) is an essential enzyme in cellular metabolism, playing a key role in regulating glycolysis. It has garnered significant attention in cancer research and is also implicated in the development of various chronic inflammatory conditions. As a result, PKM2 has become a target for drug discovery, with efforts focused on developing inhibitors and activators to modulate its activity. This review discusses various assays used to identify and validate these compounds. Enzyme-based assays help to evaluate PKM2 activity by monitoring substrate conversion. Techniques like dynamic laser light scattering spectrometry and surface plasmon resonance assess the interactions and stability of PKM2 with potential drugs. Cell-based assays measure PKM2 expression levels across different cellular contexts. Additional, methods such as fluorescence titration and mass spectrometry (MS/MS) analyze binding affinity and structural changes of PKM2 upon drug interaction. Metabolic assays, including oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), are employed to evaluate the functional effects of PKM2 modulation on cellular metabolism. This review aims to provide a comprehensive overview of these methodologies to enhance the understanding of PKM2's role and facilitate the discovery of therapeutic agents targeting this important enzyme. • PKM2 is overexpressed in various cancers and plays a critical role in the Warburg effect. • Cell-based assays, focus on the expression levels of PKM2 in the cellular contexts. • SPR reveals PKM2's binding affinity and structural changes upon drug interaction. • Metabolic assays assess the impact of PKM2 modulation on cellular metabolism. • This review highlights assays used to identify and validate PKM2 modulators.