Coumarin-sulfonamide hybrids as PKM2 activators induce metabolic reprogramming and suppress ovarian cancer cell...

Coumarin-piperazine sulfonamides (CPS series) were rationally designed as hybrid modulators of pyruvate kinase M2 (PKM2) to exploit metabolic vulnerabilities in ovarian cancer. Phenotypic screening across cancer cell lines guided hit identification, and molecular docking revealed favorable binding of CPS analogues within the PKM2 regulatory pocket, stabilized by interactions with tyrosine residues, similar to DASA-58 (3-(4-(2,3 Dihydrobenzo[b][1,4]dioxin 6 ylsulfonyl)-1,4 diazepan 1 ylsulfonyl)aniline), the standard PKM2 activator. Among 20 synthesized molecules, CPS8s emerged as an early-stage lead candidate, demonstrating significant cytotoxicity (A2780 IC₅₀: 36.63 ± 0.13 μM) with 3-fold selectivity in HEK293T cells. Dynamic Light Scattering (DLS) analysis showed increased PKM2 particle size upon CPS8s binding compared to standalone PKM2 as well as PKM2 treated with DASA-58, corroborating activation. Enzymatic verification by indirect cell free lactate dehydrogenase-coupled enzyme assay further established that CPS8s activates PKM2 activity. Consistent with metabolic engagement, pyruvate release increased while lactate levels remained similar to control, which was further supported by qRT-PCR analysis showing modulation of PKM2-associated mRNA expression profiles when treated with CPS8s, consistent with metabolic reprogramming. Functionally, CPS8s displayed marked anti-proliferative effects, inducing apoptosis (Annexin V/PI staining) and G1 phase arrest with S-phase depletion, indicative of disrupted G1-S transition. PKM2 activation likely contributed to impaired glycolytic flux and reduced anabolic biosynthesis, establishing metabolic constraints that mediate cytostatic (G1 arrest) and cytotoxic (apoptotic) outcomes through ROS induction. Finally, isothermal calorimetry unambiguously confirms the binding of CPS8s with PKM2 (K_d = 198 ± 67.4 μM). Collectively, CPS8s represents a promising, structurally distinct PKM2-activating hybrid scaffold that integrates metabolic activation with cell cycle arrest and apoptosis, providing a mechanistically unique avenue for ovarian cancer therapy.