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

Abstract

33Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria 34 fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic 35 encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates 36 due to lack of effective therapeutics. In pursuit of repurposing drugs for chemotherapies, we conducted a high throughput 37 phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors 38 (IC 50 <1 M) against N. fowleri (n=19), A. castellanii (n=12), and B. mandrillaris (n=27) plus an additional 90 micromolar 39 inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or 40 Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit 41 N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, 42 solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, 43 acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the 44 drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for 45 discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many 46 of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for 47 structure-based drug design. 48 49 Author Summary 50 Free-living amoebae (FLA) are ubiquitous in soil and freshwater and most are non-pathogenic to people; 51 however, three different pathogenic FLA have been found to cause severe, most often fatal diseases in humans. Due to 52 poor detection and inadequate treatment options available for pathogenic FLA, the fatality rates are still > 90% for the 53 diseases caused by Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba spp. With hundreds of cases in the 54 United States and many more cases reported worldwide, there is still an urgent clinical need for effective diagnosis and 55 specific treatments discovered against these opportunistic parasites. Drug repurposing is a powerful approach for drug-56 discovery because it significantly improves the discovery time, reduces the amount of resources, and decreases costs 57 required to advance lead candidate drugs of interest into the clinic. This is extremely helpful for neglected diseases 58 including pathogenic FLA where there is a need for new active therapies with limited budgets. This report addresses the 59 discovery of new active drugs with potential for repurposing, multiple new drug classes that inhibit pathogenic FLA, and 60 numerous putative drug targets that can be used as tools for further investigation and structure-based drug design. 61 7 . Naegleria, being ther...

The transcriptome ofBalamuthia mandrillaristrophozoites for structure-based drug design

“…Although miltefosine has been reported to have moderate activity at concentrations of 63-100 µm 25,26 , another study 27 as well as ours suggest miltefosine activity may be less potent with a higher IC50 of >122 µm. As for pentamidine, our results are consistent with prior studies that obtained comparable IC50 values between 9 and 29 µm 24,28,29 . Thus, of the combination drug cocktail recommended by the CDC, only pentamidine and flucytosine appears to have in vitro activity against B. mandrillaris, though we were unable to test sulfadiazine.…”

“…We therefore cannot rule out activity for the recommended drugs based solely on our in vitro sensitivity screens. We previously identified statins, which target 3-hydroxy-3-methylglutaryl-coenzyme A reductase A (HMG-CoA), as active compounds against B. mandrillaris 29 . As part of this study, we tested three additional statins: fluvastatin, atorvastatin and simvastatin and observed that they were active against B. mandrillaris at 1.18 to 3.03 µM concentration.…”

“…These include screening the MMV Malaria and Pathogen boxes that identified 11 compounds with equipotency to nitroxolone (8-Hydroxy-5nitroquinoline) IC50 of 2.84 µM 25 , indicating these molecules could be used as an initial starting point for medicinal chemistry structure activity relationship (SAR) studies. More recently, we identified 63 compounds through screening the Calibr ReFRAME drug repurposing library, with activities ranging from 40 nM to 4.8 µM 29 . Chemical inference from our previous drug screening efforts and the results from this study identified a total of 52 potential protein targets in B. mandrillaris.…”

Discovery of Anti-Amoebic Inhibitors from Screening the MMV Pandemic Response Box on Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba castellanii

“…These are the epitome of neglected diseases with no major pharmaceutical companies and only a few academic labs working to discover new drugs. Phenotypic screening has significantly advanced the discovery and development of drugs targeting other parasitic protozoans and has been shown to be useful for the discovery of new drugs against pathogenic free-living amoebae as well [42][43][44]. In this study, we screened 400 bioactive compounds from the MMV Pandemic Response Box for activity against all three pathogenic free-living amoebae.…”

“…It is composed of 201 (50.25%) antibacterial compounds, 153 (38.25%) antiviral compounds, and 46 (11.5%) antifungal compounds. Previous high-throughput screening efforts on free-living amoebae have yielded promising leads for therapeutic development [40][41][42][43][44][45][46][47]. However, there is still a need for the discovery and development of novel chemical scaffolds potent against these amoebae.…”