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

Area of Science:

• Microbiology
• Biochemistry
• Drug Discovery

Background:

• * *Naegleria fowleri* is a protozoan pathogen causing primary amoebic meningoencephalitis (PAM), a rapidly fatal brain infection with a mortality rate over 97%.
• * *N. fowleri* synthesizes sterols, making its sterol biosynthesis pathway a potential target for therapeutic intervention.

Purpose of the Study:

• * To characterize the catalytic properties of *N. fowleri* sterol 14-demethylase.
• * To evaluate the inhibitory potential of clinical antifungal drugs and novel substituted azoles against this enzyme.
• * To identify potential drug candidates for treating PAM.

Main Methods:

• * Enzyme inhibition assays were performed using clinical antifungal drugs and experimental azole compounds.
• * The crystal structure of *N. fowleri* sterol 14-demethylase complexed with an inhibitor was determined at 2.1 Å resolution.
• * Cellular assays were used to determine minimal inhibitory concentration (MIC) values and brain penetration of compounds.

Main Results:

• * Posaconazole and two novel compounds exhibited potent inhibition of *N. fowleri* sterol 14-demethylase, with IC50 values as low as 0.35 μM.
• * The determined crystal structure revealed insights into enzyme-ligand interactions and substrate specificity.
• * The identified potent inhibitors demonstrated brain penetration and achieved effective MIC values in cellular assays.

Conclusions:

• * *N. fowleri* sterol 14-demethylase is a viable drug target for PAM.
• * Novel azole compounds show significant potential as drug candidates for treating PAM due to their potency and brain penetrability.
• * Structural information provides a basis for designing more effective inhibitors against *N. fowleri*.