Discovery of PKM2 activators from Aconiti Lateralis Radix Praeparata via computer-aided drug design and experimental validation

Computer-aided drug design (CADD) provides unique benefits for discovering and optimizing lead compounds. Aconiti Lateralis Radix Praeparata (Fuzi in Chinese) is a traditional herbal medicine widely used in China and Other Asian countries. This study employed virtual screening, molecular dynamics simulations, and experimental validation to identify activators of Pyruvate Kinase M2 (PKM2) from Fuzi Alkaloids. The process involved molecular docking and ADME analyses, which led to the selection of 17 Alkaloids for further investigation. The results from microscale thermophoresis showed that 14 Alkaloids exhibited strong binding affinities to the PKM2 protein. The kinase activity assays showed that mesaconine, benzoylaconine, and hypaconitine were effective in activating the PKM2 recombinant protein. Western blotting, glutaraldehyde cross-linking assay and immunofluorescence experiments revealed that hypaconitine inhibited PKM2 phosphorylation, obstructed its nuclear translocation, increased the tetrameric form, and accordingly decreased the dimeric form. The stability of the hypaconitine-PKM2 complex was confirmed through molecular dynamics simulations. This study is the first to report that hypaconitine activated PKM2 significantly, which would provide references for the clinical use of Fuzi and lay foundation for the discovery of PKM2 activators.

Aconiti Lateralis Radix Praeparata; Alkaloids; Computer-aided drug design; Molecular docking; Molecular dynamics simulations; Pyruvate kinase M2.