Enolase inhibitors as therapeutic leads forNaegleria fowleriinfection

Abstract

Infections with the pathogenic free-living amoebaeNaegleria fowlerican lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors ofN. fowleriENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50value of 0.14 ± 0.04 μM) that was toxic to trophozoites (EC50value of 0.21 ± 0.02 μM) while the reported CC50was >300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site ofNfENO with a binding affinity of −8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream ofNfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.