Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen

Abstract

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. The drug of choice in treating PAM is the antifungal antibiotic amphotericin B, but its use is associated with severe adverse effects. Moreover, few patients treated with amphotericin B have survived PAM. Therefore, fast-acting and efficient drugs are urgently needed for the treatment of PAM. To facilitate drug screening for this pathogen, an automated, high-throughput screening methodology was developed and validated for the closely related species Naegleria gruberi. Five kinase inhibitors and an NF-kappaB inhibitor were hits identified in primary screens of three compound libraries. Most importantly for a preclinical drug discovery pipeline, we identified corifungin, a water-soluble polyene macrolide with a higher activity against Naegleria than that of amphotericin B. Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin showed disruption of cytoplasmic and plasma membranes and alterations in mitochondria, followed by complete lysis of amebae. In vivo efficacy of corifungin in a mouse model of PAM was confirmed by an absence of detectable amebae in the brain and 100% survival of mice for 17 days postinfection for a single daily intraperitoneal dose of 9 mg/kg of body weight given for 10 days. The same dose of amphotericin B did not reduce ameba growth, and mouse survival was compromised. Based on these results, the U.S. FDA has approved orphan drug status for corifungin for the treatment of PAM.

Fig 1

Correlation between the number of viable N. gruberi trophozoites and ATP bioluminescence in 96-well (A) and 384-well (B) microtiter plates. Values plotted are means and standard deviations for triplicate wells. Each line represents the linear regression for plotted data.

Fig 2

Scatterplot of percent inhibition of each well from 19 96-well plates for the Asinex library. Ten compounds yielded both 50% inhibition and a value 3 standard deviations above the mean for the population of compounds tested in the primary screen (at 5 μM). Positive controls included amphotericin B-treated N. gruberi, and negative controls included 0.5% DMSO-treated N. gruberi.

Fig 3

Chemical structure of corifungin.

Fig 4

Effect of corifungin on N. fowleri growth. Trophozoites were incubated with different concentrations of corifungin for 72 h, and cell numbers were calculated at the end of incubation. Values plotted are means and standard deviations for three independent experiments. The cell viability was evaluated using trypan blue stain.

Fig 5

N. fowleri viability determination by the trypan blue exclusion method. Trophozoites were incubated with different concentrations of corifungin for 72 h. (A) Amebae with fresh medium only. (B) Amebae with 3.12 μM corifungin. (C) Amebae with 6.25 μM corifungin. (D) Amebae with 12.5 μM corifungin. (E) Amebae with 18.75 μM corifungin. (F) Amebae with 25 μM corifungin. Cells stained blue were considered nonviable. Magnification, ×40.

Fig 6

Transmission electron microscopy of N. fowleri trophozoites incubated with different concentrations of corifungin for 72 h. (A) Trophozoite treated with fresh medium only. (B) Higher-magnification view of panel A. (C) Trophozoite treated with 12.5 μM corifungin. The alteration of the plasma membrane is evident (arrows), and also the mitochondria are dilated and cristae are lost (arrowheads). (D) Trophozoite treated with 18.75 μM corifungin. The plasma membrane of the ameba is fragmented (arrows). Arrowheads indicate mitochondria. (E and F) Trophozoites treated with 25 μM corifungin. The amebae show complete lysis. Bars, 2 μm.

Fig 7

Light microscopy of olfactory bulbs in mice infected with N. fowleri and treated with PBS. (A) Olfactory bulbs show a mixed-cell inflammatory reaction with many neutrophils. Necrotic brain tissue is evident. Magnification, ×10. (B) Higher-magnification view of the same area, showing trophozoites (arrowheads) and polymorphonuclear cells (arrows). Magnification, ×60. The tissues were stained with hematoxylin and eosin.

Fig 8

Light microscopy of olfactory bulbs in mice infected with N. fowleri and treated intraperitoneally with 9 mg/kg/day amphotericin B for a total of 10 days. (A) Olfactory bulbs show several amebae (arrowheads) and a diffuse mixed-cell inflammatory infiltrate associated with widespread tissue necrosis (arrows). Magnification, ×10. (B) Higher-magnification view of the same area. Magnification, ×60. The tissues were stained with hematoxylin and eosin.

Fig 9

Light microscopy of olfactory bulbs in mice infected with N. fowleri and treated intraperitoneally with 9 mg/kg/day corifungin for a total of 10 days. (A) Olfactory bulbs show multiple spherical inflammatory foci (arrows) without trophozoites. Some areas of the brain have a discrete vacuolization adjacent to the inflammation, but the parenchyma is well preserved, with no evidence of tissue necrosis. Magnification, ×10. (B) Higher-magnification view of the same zone. Magnification, ×60. The tissues were stained with hematoxylin and eosin.

Fig 10

Survival of N. fowleri-infected mice treated with amphotericin B (ampho) or corifungin (cfn). At 24 h postinoculation, mice (n = 10) were treated once daily with 9 mg/kg of amphotericin B or corifungin for 10 days. Control mice received PBS.