Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

Abstract

Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. In pursuit of repurposing drugs for chemotherapies, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 M) against N. fowleri (n=19), A. castellanii (n=12), and B. mandrillaris (n=27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design. Author SummaryFree-living amoebae (FLA) are ubiquitous in soil and freshwater and most are non-pathogenic to people; however, three different pathogenic FLA have been found to cause severe, most often fatal diseases in humans. Due to poor detection and inadequate treatment options available for pathogenic FLA, the fatality rates are still > 90% for the diseases caused by Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba spp. With hundreds of cases in the United States and many more cases reported worldwide, there is still an urgent clinical need for effective diagnosis and specific treatments discovered against these opportunistic parasites. Drug repurposing is a powerful approach for drug-discovery because it significantly improves the discovery time, reduces the amount of resources, and decreases costs required to advance lead candidate drugs of interest into the clinic. This is extremely helpful for neglected diseases including pathogenic FLA where there is a need for new active therapies with limited budgets. This report addresses the discovery of new active drugs with potential for repurposing, multiple new drug classes that inhibit pathogenic FLA, and numerous putative drug targets that can be used as tools for further investigation and structure-based drug design.