Abstract
Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered “rare” (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug ‘repurposing’—a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially ‘druggable’ target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.
Highlights
The amphizoic amoeba, Naegleria fowleri is commonly found in water resources such as swimming pools having inadequate levels of chlorine, lakes and rivers
N. fowleri can act as an opportunistic pathogen causing severe brain injury called Primary Amebic Meningoencephalitis (PAM), often in healthy children and young adults
Carrying out biochemical studies and xray-crystallography of the target enzyme, we demonstrated that azole anti-fungal drugs, known as conazoles, disrupt sterol biosynthesis in amoebae by competing with the natural substrate for binding in the active site of the sterol 14-demethylase (CYP51)
Summary
The amphizoic amoeba (existing both in free-living and parasitic forms), Naegleria fowleri is commonly found in water resources such as swimming pools having inadequate levels of chlorine, lakes and rivers. It feeds mostly on bacteria, but can act as an opportunistic pathogen causing infection of the central nervous system (CNS) of humans and animals.[1] N. fowleri usually infects people when contaminated water enters the body through the nose. In the absence of data to estimate the true risk of PAM or to set up and reinforce the measurable standards to protect the human population, early diagnosis and aggressive antimicrobial treatment remain the only option to treat the disease
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