title: ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase.
abstract: Inhibition of glycolysis is of great potential for the treatment of cancer. However, inhibitors of glycolytic enzymes with favorable pharmacological profiles have not been forthcoming. Due to the nature of their active sites, most high-affinity transition-state analogue inhibitors of glycolysis enzymes are highly polar with poor cell permeability. A recent publication reported a novel, non-active site inhibitor of the glycolytic enzyme Enolase, termed ENOblock (N-[2-[2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl]amino]1,3,5-triazin-2-yl]amino]benzeneacetamide). This would present a major advance, as this is heterocyclic and fully cell permeable molecule. Here, we present evidence that ENOblock does not inhibit Enolase enzymatic activity in vitro as measured by three different assays, including a novel 31P NMR based method which avoids complications associated with optical interferences in the UV range. Indeed, we note that due to strong UV absorbance, ENOblock interferes with the direct spectrophotometric detection of the product of Enolase, phosphoenolpyruvate. Unlike established Enolase inhibitors, ENOblock does not show selective toxicity to ENO1-deleted glioma cells in culture. While our data do not dispute the biological effects previously attributed to ENOblock, they indicate that such effects must be caused by mechanisms other than direct inhibition of Enolase enzymatic activity.
authors.affiliation: Department of Neurology, McGovern Medical School, UTHealth, Houston, TX, United States of America.|Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, United States of America.
authors.forename: Nikunj
authors.identifier: http://orcid.org/0000-0003-3908-3731
authors.initials: N
authors.lastname: Satani
authors.affiliation: Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, United States of America.
authors.forename: Yu-Hsi
authors.initials: YH
authors.lastname: Lin
authors.affiliation: Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, United States of America.
authors.forename: Naima
authors.initials: N
authors.lastname: Hammoudi
authors.affiliation: Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, United States of America.
authors.forename: Sudhir
authors.initials: S
authors.lastname: Raghavan
authors.affiliation: Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, United States of America.
authors.forename: Dimitra K
authors.initials: DK
authors.lastname: Georgiou
authors.affiliation: Department of Cancer Systems Imaging, University of Texas MD Anderson Cancer Center, Houston, TX, United States of America.
authors.forename: Florian L
authors.initials: FL
authors.lastname: Muller
mesh_terms: D001549:Benzamides / Q000494:pharmacology*; D045744:Cell Line, Tumor; D006019:Glycolysis; D006801:Humans; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014227:Triazines / Q000494:pharmacology*
chemical_list: D001549:Benzamides; C000621265:ENOblock; D014227:Triazines; D010751:Phosphopyruvate Hydratase