Mannich Base Derived from Lawsone Inhibits PKM2 and Induces Neoplastic Cell Death

<b>Background/Objectives:</b> Pyruvate kinase M2, a central regulator of cancer cell metabolism, has garnered significant attention as a promising target for disrupting the metabolic adaptability of tumor cells. This study explores the potential of the Mannich base derived from lawsone (<b>MB-6a</b>) to interfere with PKM2 enzymatic activity both in vitro and in silico. <b>Methods:</b> The antiproliferative potential of <b>MB-6a</b> was tested using MTT assay in various cell lines, including SCC-9, Hep-G2, HT-29, B16-F10, and normal human gingival fibroblast (HGF). The inhibition of PKM2 mediated by <b>MB-6a</b> was assessed using an LDH-coupled assay and by measuring ATP production. Docking studies and molecular dynamics calculations were performed using Autodock 4 and GROMACS, respectively, on the tetrameric PKM2 crystallographic structure. <b>Results:</b> The Mannich base <b>6a</b> demonstrated selective cytotoxicity against all cancer cell lines tested without affecting cell migration, with the highest selectivity index (SI) of 4.63 in SCC-9, followed by B16-F10 (SI = 3.9), Hep-G2 (SI = 3.4), and HT-29 (SI = 2.03). The compound effectively inhibited PKM2 glycolytic activity, leading to a reduction of ATP production both in the enzymatic reaction and in cells treated with this naphthoquinone derivative. <b>MB-6a</b> showed favorable binding to PKM2 in the ATP-bound monomers through docking studies (PDB ID: 4FXF; binding affinity scores ranging from -6.94 to -9.79 kcal/mol) and MD simulations, revealing binding affinities stabilized by key interactions including hydrogen bonds, halogen bonds, and hydrophobic contacts. <b>Conclusions:</b> The findings suggest that <b>MB-6a</b> exerts its antiproliferative activity by disrupting cell glucose metabolism, consequently reducing ATP production and triggering energetic collapse in cancer cells. This study highlights the potential of <b>MB-6a</b> as a lead compound targeting PKM2 and warrants further investigation into its mechanism of action and potential clinical applications.