Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri

Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti- N . fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti- Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N . fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ 8 −Δ 7 -isomerase (ERG2), as potential therapeutic drug targets in N . fowleri . The sterol biosynthetic cascade in N . fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo -synthesized sterols by feeding N . gruberi trophozoites on the U 13 C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13 C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a “switch” between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N . fowleri . The similarity of the Naegleria sterol Δ 8 −Δ 7 -isomerase to the human non-opioid σ 1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti- Naegleria activity.