ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase

<div><p>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 <i>in vitro</i> 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 <i>ENO1</i>-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.</p></div

Non-selective toxicity of ENOBlock to ENO1-deleted glioma cells.

<p>A representative plate of cancer cells treated with ENOblock is shown in panel <b>a</b>, with quantification shown in panel <b>b</b> A plate treated with SF2312 is shown in panel <b>c</b>, with quantification shown in panel <b>d</b>. Cell were treated for 7 days. <b>(b, d)</b> D423 <i>ENO1</i>-deleted (red diamonds), D423 <i>ENO1</i>-rescued (blue squares) and LN319 <i>ENO1</i> WT (grey circles) were treated with the indicated doses of ENOblock in panel <b>b</b> (N = 4 ± S.D) or SF2312 in panel <b>d</b> (N = 4 ± S.D). Cell density was quantified by crystal violet and expressed relative to vehicle control as a function of inhibitor concentration. At high concentrations, SF2312 selectively killed D423 <i>ENO1</i>-deleted cells as compared to D423 <i>ENO1</i>-rescued cells (p<0.05, Repeated Measures one-way ANOVA with Bonferroni correction). ENOblock failed to show such selectivity regardless of dose.</p

Additional file 1: of Metabolic activation of mitochondria in glioma stem cells promotes cancer development through a reactive oxygen species-mediated mechanism

Supplementary Figures. (PDF 628 kb