Relaxed Substrate Requirements of Sterol 14α-Demethylase from Naegleria fowleri Are Accompanied by Resistance to Inhibition

Naegleria fowleri is the protozoan
pathogen that causes primary amoebic meningoencephalitis (PAM), with
the death rate exceeding 97%. The amoeba makes sterols and can be
targeted by sterol biosynthesis inhibitors. Here, we characterized N. fowleri sterol 14-demethylase, including catalytic
properties and inhibition by clinical antifungal drugs and experimental
substituted azoles with favorable pharmacokinetics and low toxicity.
None of them inhibited the enzyme stoichiometrically. The highest
potencies were displayed by posaconazole (IC50 = 0.69 μM)
and two of our compounds (IC50 = 1.3 and 0.35 μM).
Because both these compounds penetrate the brain with concentrations
reaching minimal inhibitory concentration (MIC) values in an N. fowleri cellular assay, we report them as potential
drug candidates for PAM. The 2.1 Å crystal structure, in complex
with the strongest inhibitor, provides an explanation connecting the
enzyme weaker substrate specificity with lower sensitivity to inhibition.
It also provides insight into the enzyme/ligand molecular recognition
process and suggests directions for the design of more potent inhibitors.