The 3S Enantiomer Drives Enolase Inhibitory Activity in SF2312 and Its Analogues.

Pisaneschi Pisaneschi,Raghavan Raghavan,Satani Satani,Yan Yan,Georgiou Georgiou,Muller Muller,Link Link,Czako Czako,Leonard Leonard,Lin Lin,Hammoudi Hammoudi

doi:10.3390/molecules24132510

Pisaneschi Pisaneschi, Raghavan Raghavan + Show 9 more

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https://doi.org/10.3390/molecules24132510

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Abstract

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

Highlights

Glycolysis is a conserved catabolic pathway [1], with a set of essential glycolysis genes and corresponding enzymes present in most organisms [2,3]
We show that MethylSF2312 is similar to SF2312 for inhibiting Enolase both vitro and in cell-based systems
Our previous work showed that SF2312 is a high potency Enolase inhibitor with potential utility for the treatment of glioblastoma with Enolase 1 (ENO1) deletions [14]

Summary

Introduction

Glycolysis is a conserved catabolic pathway [1], with a set of essential glycolysis genes and corresponding enzymes present in most organisms [2,3]. Given that glycolytic deregulation has been implicated in a number of diseases, such as cancer [4,5,6,7], malaria [8,9], and Trypanosoma [10,11], makes the rarity of natural product inhibitors of glycolysis striking. Molecules 2019, 24, 2510 of cancer, many tumors exhibit a shift in metabolism, favoring glycolysis, in what is known as the Warburg Effect. 2 of 18 few high-affinity glycolysis inhibitors have been described, with most existing as tool compounds with of cancer, manyutility tumors[13]. Exhibit a shift inismetabolism, glycolysis, in what is known as pathway. Even fewer the numberfavoring of natural antibiotic inhibitors of this Warburg cancer Effect [12]

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