# Assessment of the structure–activity relationship of analogs of the Naegleria fowleri enolase inhibitor HEX

## TL;DR

This study examines how changes to the structure of a compound that inhibits a key enzyme in a deadly amoeba affect its effectiveness.

## Contribution

The study reveals the sensitivity of the HEX scaffold's activity to structural changes and identifies essential functional groups for potency.

## Key findings

- HEX's activity against N. fowleri enolase is highly sensitive to structural modifications.
- Both hydroxamate and phosphonate groups are necessary to maintain potency.
- Bicyclic analogs of HEX lose biological activity due to steric hindrance.

## Abstract

The pathogenic free-living amoeba Naegleria fowleri 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 N. fowleri enolase (NfENO) 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 N. fowleri 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.

The SAR of the N. fowleri enolase inhibitor HEX revealed that the activity of the HEX scaffold toward NfENO is rather sensitive to structural perturbations, confirming the necessity of both the hydroxamate and phosphonate groups to maintain potency.