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Updated: Jul 11, 2025
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
High-Throughput Screen of Microbial Metabolites Identifies F1FO ATP Synthase Inhibitors as New Leads for Naegleria
Alice Yuan1, Hayley Fong1, Jennifer V Nguyen1
1Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States.
Abstract:
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 μg/mL and a dose-response study found 20 of these active test materials exhibiting an EC50 ranging from 0.2 to 2 μg/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 EC50 in 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 F1FO 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.
Insights
Researchers screened marine microbial metabolites to find new treatments for primary amebic meningoencephalitis (PAM). They discovered oligomycin D, a potent natural compound effective against Naegleria fowleri, offering a promising new therapeutic avenue.
Area of Science:
- Marine microbiology
- Drug discovery
- Infectious diseases
Background:
- Primary amebic meningoencephalitis (PAM) is a fatal brain infection caused by Naegleria fowleri.
- Natural products are a vital source for drug development, but research on anti-Naegleria fowleri agents is limited.
- Existing treatments for PAM have significant limitations.
Purpose of the Study:
- To identify novel natural product-based compounds with anti-Naegleria fowleri activity.
- To evaluate the efficacy of isolated compounds against Naegleria fowleri trophozoites.
Main Methods:
- A large-scale ATP bioluminescence-based screen of approximately 10,000 marine microbial metabolite mixtures was performed.
- Hit compounds underwent dose-response studies to determine EC50 values.
- Potent metabolite mixtures were analyzed to isolate pure compounds, including oligomycin D.
Main Results:
- The screen identified about 100 test materials with >90% inhibition, and 20 showed EC50 values between 0.2-2 μg/mL.
- Oligomycin D was isolated and demonstrated nanomolar potency against multiple Naegleria fowleri genotypes, outperforming current drugs.
- Oligomycin D was fast-acting, inhibited Naegleria fowleri invasiveness, and leucinostatin was also identified as a potent amebicidal compound.
Conclusions:
- Oligomycin D is a highly potent and fast-acting natural compound effective against Naegleria fowleri.
- Targeting F1F0 ATP synthase is a viable strategy for developing anti-Naegleria fowleri therapies.
- Natural products, like oligomycin D and leucinostatin, represent promising candidates for new PAM treatments.
