Extended Data Fig. 8. The synthetic cell permeable pyruvate pro-metabolite, methyl 2-oxopropanoate, significantly attenuates toxicity of POMHEX.

ENO1-deleted (D423, red), ENO1-isogenically rescued (D423 ENO1, blue), and ENO1-WT (LN319, grey) cells were treated with POMHEX at the doses indicated (x-axis) in media (DMEM) free of pyruvate with either (a) 2.5 mM methyl 2-oxopropanoate “methyl pyruvate,” (b) 5 mM lactate, or (c) 5 mM acetate (d). Cell density after 5 days exposure was determined by crystal violet staining and expressed relative to non-drug contain controls (n = 4 biological replicates as indicated, +/ S.E.M.). The IC₅₀ for pyruvate-free media is indicated by a dashed line, for comparison. Exogenous methyl pyruvate attenuates sensitivity to Enolase inhibitors especially in ENO1-homozygously deleted cells (IC₅₀ shifted from ~20 nM to ~150 nM), but supplementation with lactate or acetate had minimal effects. (e) Methyl pyruvate is a cell-permeable, synthetic pro-metabolite of pyruvate that can passively diffuse into the cell without requiring monocarboxylate transporter (SLC16) activity. It can then be hydrolyzed by intracellular carboxylesterases to release pyruvate. While lactate and acetate could serve a similar purpose, the present data (c, d) suggest that the rate of acetyl-CoA production by these carbon sources is insufficient to compensate for loss of ATP and pyruvate by POMHEX-mediated inhibition of glycolysis.