Naegleria fowleri is a pathogenic free-living amoeba (FLA) that causes an acute fatal disease known as primary amoebic meningoencephalitis (PAM). The major problem for infections with any pathogenic FLA is a lack of effective therapeutics, since PAM has a case mortality rate approaching 99%. Clearly, new drugs that are potent and have rapid onset of action are needed to enhance the treatment regimens for PAM. Diamidines have demonstrated potency against multiple pathogens, including FLA, and are known to cross the blood-brain barrier to cure other protozoan diseases of the central nervous system. Therefore, amidino derivatives serve as an important chemotype for discovery of new drugs. In this study, we validated two new in vitro assays suitable for medium-or high-throughput drug discovery and used these for N. fowleri. We next screened over 150 amidino derivatives of multiple structural classes and identified two hit series with nM potency that are suitable for further lead optimization as new drugs for this neglected disease. These include both mono-and diamidino derivatives, with the most potent compound (DB173) having a 50% inhibitory concentration (IC 50 ) of 177 nM. Similarly, we identified 10 additional analogues with IC 50 s of <1 M, with many of these having reasonable selectivity indices. The most potent hits were >500 times more potent than pentamidine. In summary, the mono-and diamidino derivatives offer potential for lead optimization to develop new drugs to treat central nervous system infections with N. fowleri. The first report of animal pathogenicity by free-living amoebae (FLA) was by Culbertson et al., who identified Acanthamoeba in necrotic lesions of monkeys and mice (1). He later suggested that FLA causes similar disease in humans. Fowler and Carter reported the first cases of an acute fatal disease caused by Naegleria fowleri in four victims in Australia in 1965 (2). Since the identification of primary amoebic meningoencephalitis (PAM), hundreds of cases have been reported worldwide, including 142 cases in the United States (3-5). Most infections with N. fowleri occur during the summer months, victims usually are symptomatic within 5 days, and the disease is almost always fatal. Infection usually occurs after the victim swam in warm, fresh, or brackish water or from exposure to contaminated tap water associated with the use of Neti pots for nasal irrigation (4-7). Amoebic invasion occurs via disruption of the olfactory mucosa, penetration of organisms into the submucosal nervous plexus, and ultimately passage through the cribriform plate to the frontal lobes of the brain (8, 9). Although most cases of PAM occur in warm climates, such as the southern tier states of the United States, pathogenic amoeba found in thermally polluted water sources, hot springs, and poorly chlorinated pools have been linked to infections (10, 11). Interestingly, most of the PAM cases have been diagnosed in developed countries, including the United States, Australia, and Europe. Although sporadic cases have been d...
The soil amoebae Acanthamoeba causes Acanthamoeba keratitis, a severe sight-threatening infection of the eye and the almost universally fatal granulomatous amoebic encephalitis. More effective treatments are required. Sterol biosynthesis has been effectively targeted in numerous fungi using azole compounds that inhibit the cytochrome P450 enzyme sterol 14α-demethylase. Herein, using Gas Chromatography Mass Spectrometry (GCMS), we demonstrate that the major sterol of Acanthamoeba castellanii is ergosterol and identify novel putative precursors and intermediate sterols in its production. Unlike previously reported, we find no evidence of 7-dehydrostigmasterol or any other phytosterol in Acanthamoeba. Of five azoles tested, we demonstrate that tioconazole and voriconazole have the greatest overall inhibition for all isolates of Acanthamoeba castellanii and Acanthamoeba polyphaga tested. While miconazole and sulconazole have intermediate activity econazole is least effective. Through GCMS, we demonstrate that voriconazole inhibits 14α-demethylase as treatment inhibits the production of ergosterol, but results in the accumulation of the lanosterol substrate. These data provide the most complete description of sterol metabolism in Acanthamoeba, provide a putative framework for their further study and validate 14α-demethylase as the target for azoles in Acanthamoeba.
Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration-approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 μM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri-infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.
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