Discovery of novel anti- Prototheca compounds with diverse modes of action through screening of a chemical library

ABSTRACT Prototheca , a genus of unpigmented yeast-like microscopic algae, is an emerging pathogen of humans and animals. Treatment of Prototheca infections is often unsuccessful because the unusual algal biology of these pathogens limits the availability of effective therapeutics. Here, we screened the Pandemic Response Box, a library of 400 compounds with potential anti-infective activity that are either clinically approved or in preclinical development, and identified five promising compounds with anti- Prototheca activity: tafenoquine, OSU-03012, alexidine, ciclopirox, and eberconazole. Characterized alongside amphotericin B, which is currently often used for treatment, these identified compounds displayed potent activity against multiple Prototheca strains and species by inhibiting growth, killing the algae, and disrupting biofilms. Their diverse mechanisms of action included effects on cell walls and membranes, mitochondrial dysfunction, ROS overproduction, metabolic alterations, and induction of apoptosis. Most of the identified compounds were also effective against in vivo Prototheca infection in larval zebrafish, highlighting their translational potential and the possibility of future introduction into clinical practice. IMPORTANCE This study addresses the emerging threat of infections caused by Prototheca , a group of unusual algae that can cause difficult-to-treat infections in both humans and animals. Current treatment options are limited and often ineffective, highlighting the need to develop new therapeutic strategies, especially as reported cases of Prototheca infections quickly increase. Here, we identified several compounds with strong activity against Prototheca . These agents not only blocked growth but also killed the microorganisms, damaged their biofilms, and reduced infection in an animal model. Importantly, many of the identified compounds are already used clinically for other indications, which raises the possibility of rapid repurposing against Prototheca . Moreover, we identified multiple mechanisms responsible for these compounds’ activity, providing a framework for the future development of even more potent anti- Prototheca therapies.