Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We recently demonstrated that SF2312, a natural product phosphonate antibiotic, is a potent inhibitor of the glycolytic enzyme Enolase with potential utility for the collateral lethality-based treatment of Enolase-deficient glioblastoma (GBM). However, phosphonates are anionic at physiological pH, limiting cell and tissue permeability. Here, we show that addition of pivaloyloxymethyl (POM) groups to SF2312 (POMSF) dramatically increases potency, leading to inhibition of glycolysis and killing of ENO1-deleted glioma cells in the low nM range. But the utility of POMSF in vivo is dose-limited by severe hemolytic anemia. A derivative, POMHEX, shows equipotency to POMSF without inducing hemolytic anemia. POMHEX can eradicate intracranial orthotopic ENO1-deleted tumors, despite sub-optimal pharmacokinetic properties. Taken together, our data provide in vivo proof-of-principal for collateral lethality in precision oncology and showcase POMHEX as a useful molecule for the study of glycolysis in cancer metabolism.

Eradication of ENO1-deleted Glioblastoma through Collateral Lethality