Assessment of the structure–activity relationship of analogs of the <i>Naegleria fowleri</i> enolase inhibitor HEX

The pathogenic free-living amoeba <i>Naegleria fowleri</i> causes primary amoebic meningoencephalitis (PAM), a highly fatal disease with limited treatment options, underscoring the urgent need for new therapeutics. Our previous studies identified (1-hydroxy-2-oxopiperidin-3-yl)phosphonic acid (HEX), an inhibitor of human enolase 2 (ENO2) involved in glucose metabolism, as a potent inhibitor of <i>N. fowleri</i> enolase (<i>Nf</i>ENO) with potent amoebicidal activity. In this study, we explored the structure-activity relationship (SAR) of HEX by modifying its hydroxamate and phosphonate functional groups, as well as introducing steric alterations to generate new analogs. Functional assays and computational-assisted SAR analysis provided insights into the impact of HEX modifications on <i>N. fowleri</i> agonism. Ultimately, the results of this study demonstrated that the activity of the HEX scaffold toward NfENO is rather sensitive to structural purturbations, confirming the necessity of both key functional groups - the hydroxamate and phosphonate - to maintain potency. Additionally, structural modifications of the parent compound into bicyclic analogs resulted in loss of biological activity ostensibly due to unfavorable steric interactions in the active site. These findings enhance our understanding of the activity of HEX's molecular architecture, and underscore potential limitations of further structural tuning efforts of the scaffold by means of SAR.