ENOblock Does Not Inhibit the Activity of the Glycolytic Enzyme Enolase.

Nikunj Satani,Florian L Muller,Dimitra K Georgiou,Sudhir Raghavan,Naima Hammoudi,Yu-Hsi Lin,Pankaj K Singh

doi:10.1371/journal.pone.0168739

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.

Highlights

The inhibition of glycolytic/gluconeogenic enzymes is of interest in diverse area of medicine, including treatment of diabetes [1], the treatment of cancer [2] as well as the development of novel antimicrobials [3]
Transition state analogue active-site inhibitors of Enolase, and several other glycolytic enzymes have been described [4, 5], their utility is limited by poor cell permeability and otherwise poor pharmacological properties [6]
A recent report by Jung et al described an altogether different glycolysis inhibitor: a heterocyclic, cell permeable inhibitor of Enolase that apparently binds outside the active site [7]

Summary

Introduction

The inhibition of glycolytic/gluconeogenic enzymes is of interest in diverse area of medicine, including treatment of diabetes [1], the treatment of cancer [2] as well as the development of novel antimicrobials [3]. Transition state analogue active-site inhibitors of Enolase, and several other glycolytic enzymes have been described [4, 5], their utility is limited by poor cell permeability and otherwise poor pharmacological properties [6]. A recent report by Jung et al described an altogether different glycolysis inhibitor: a heterocyclic, cell permeable inhibitor of Enolase that apparently binds outside the active site [7]. Jung et al identified (N-2-2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl] amino]1,3,5-triazin-2-yl]amino]benzeneacetamide hydrochloride) known as AP-III-a4 as PLOS ONE | DOI:10.1371/journal.pone.0168739. Jung et al identified (N-2-2-2-aminoethoxy)ethoxy]ethyl]4-4-cyclohexylmethyl)amino]6-4-fluorophenyl)methyl] amino]1,3,5-triazin-2-yl]amino]benzeneacetamide hydrochloride) known as AP-III-a4 as PLOS ONE | DOI:10.1371/journal.pone.0168739 December 28, 2016

Objectives

Methods

Results