High-Throughput Screen of Microbial Metabolites Identifies F₁F_O ATP Synthase Inhibitors as New Leads for Naegleria fowleri Infection

Primary amebic meningoencephalitis (PAM), a brain infection caused by a free-living ameba Naegleria fowleri, leads to an extensive inflammation of the brain and death within 1-18 (median 5) days after symptoms begin. Although natural products have played a significant role in the development of drugs for over a century, research focusing on identifying new natural product-based anti-N. fowleri agents is limited. We undertook a large-scale ATP bioluminescence-based screen of about 10,000 unique marine microbial metabolite mixtures against the trophozoites of N. fowleri. Our screen identified about 100 test materials with >90% inhibition at 50 mu g/mL and a dose-response study found 20 of these active test materials exhibiting an EC50 ranging from 0.2 to 2 mu g/mL. Examination of four of these potent metabolite mixtures, derived from our actinomycete strains CNT671, CNT756, and CNH301, resulted in the isolation of a pure metabolite identified as oligomycin D. Oligomycin D exhibited nanomolar potency on multiple genotypes of N. fowleri, and it was five- or 850-times more potent than the recommended drugs amphotericin B or miltefosine. Oligomycin D is fast-acting and reached its EC(50 i)n 10 h, and it was also able to inhibit the invasiveness of N. fowleri significantly when tested on a matrigel invasion assay. Since oligomycin is known to manifest inhibitory activity against F(1)F(O )ATP synthase, we tested different F1FO ATP synthase inhibitors and identified a natural peptide leucinostatin as a fast-acting amebicidal compound with nanomolar potency on multiple strains.