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

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.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

“…While SF2312 exists as a racemic mixture in solution due to stereocenters at the 3- and 5-positions, it is noteworthy that in our complex structure with E. coli enolase, only the 3 S,5 S configuration is observed. This is further confirmed in the recent work which describes the human ENO2 enolase in complex with the active S,S-enantiomer form of SF2312 14 .…”

ENO2 Promotes Colorectal Cancer Metastasis by Interacting with the LncRNA CYTOR and Activating YAP1-Induced EMT

“…ENO1 is the major isoform of enolase in cancer cells, accounting for 75–90% of the cellular enolase activity [ 29 ]. In a series of studies by Muller, ENO2-specific inhibitors did not significantly affect enolase activity in ENO1 wild-type cells [ 38 , 39 ]. All the above information suggests that ENO2 promotes CRC metastasis through non-glycolytic pathways.…”

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers

“…Human ENO1 and ENO2 share 84% sequence identitybearing even higher resemblance in the active site. , Maximizing inhibitor specificity for ENO2 was an important design goal to avoid inhibition of erythrocytic ENO1 the sole isoform present in erythrocyteswhich can result in anemia. , With these constraints, we identified a cyclic derivative of PhAH, deoxySF2312 ( 2 ), which bore close resemblance to the natural product antibiotic, SF2312 ( 3 ; PDB: 4ZCW). We clarified the stereochemical requirements for active site binding by SF2312 by synthesizing a Cα-methyl derivative of SF2312 (mSF2312, 4 ; PDB: 5EU9), which showed that ENO2 binding was specific to the 3 S , 5 S enantiomer . Further structure–activity relationship (SAR) studies aimed at improving ENO2 specificity led to the identification of 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX, 5 ; Figure , PDB: 5IDZ).…”

“… 8 We clarified the stereochemical requirements for active site binding by SF2312 by synthesizing a Cα-methyl derivative of SF2312 (mSF2312, 4 ; PDB: 5EU9 ), which showed that ENO2 binding was specific to the 3 S , 5 S enantiomer. 14 Further structure–activity relationship (SAR) studies aimed at improving ENO2 specificity led to the identification of 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX, 5 ; Figure 1 , PDB: 5IDZ ). 12 Of these four candidates ( Figure 1 c), we focused on the development of 5 due to its clear preference for ENO2 at lower concentrations and lower molecular complexity, which eases the synthesis of prodrug derivatives ( Figure 1 d).…”