The Antifungal Drug Isavuconazole Is both Amebicidal and Cysticidal against Acanthamoeba castellanii

Abstract

Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba. The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii. Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.

Domain-Swap Dimerization ofAcanthamoeba castellaniiCYP51 and a Unique Mechanism of Inactivation by Isavuconazole

“…CYP51 from A. castellanii is a dimer 6 excystation of viable Acanthamoeba cysts (Shing et al, 2020). Potency against both trophozoite and cyst makes isavuconazole a promising drug candidate to block the propagation of trophozoite-cyst cycling of Acanthamoeba in Acanthamoeba keratitis.…”

“…CYP51 in A. castellanii is an essential enzyme in the biosynthesis of ergosterol, a functional analog of cholesterol in mammalian cells (Lamb et al, 2015;Thomson et al, 2017). CYP51 is a validated drug target in fungi and emerging drug target in the eukaryotic human pathogens (Choi et al, 2014b), including Trypanosoma cruzi (Calvet et al, 2014;Choi et al, 2013;Choi et al, 2014a;Choi et al, 2014b;Vieira et al, 2014a;Vieira et al, 2014b), Naegleria fowleri (Debnath et al, 2017;Zhou et al, 2018), and Acanthamoeba (Shing et al, 2020;Thomson et al, 2017;Zhou et al, 2019). Repurposing of the antifungal azole drugs targeting CYP51 (known as conazoles) is a promising strategy to combat Acanthamoeba infections (Shing et al, 2020;Thomson et al, 2017;Zhou et al, 2019).…”

“…The majority of anti-fungal conazoles have sub-micromolar or low micromolar potency against Acanthamoeba in cell-based assays (Lamb et al, 2015;Martin-Navarro et al, 2015;Thomson et al, 2017). However, the activity of isavuconazole against proliferating trophozoites is superior to both standard anti-Acanthamoeba therapy and other conazole drugs (Shing et al, 2020). Depending on the A. castellanii strain, isavuconazole potency varies in different strains from 26 nanomolar (MEEI 0184) to 4.6 nanomolar (Ma) to < 1 nM (CDC:V240) (Shing et al, 2020).…”

“…However, the activity of isavuconazole against proliferating trophozoites is superior to both standard anti-Acanthamoeba therapy and other conazole drugs (Shing et al, 2020). Depending on the A. castellanii strain, isavuconazole potency varies in different strains from 26 nanomolar (MEEI 0184) to 4.6 nanomolar (Ma) to < 1 nM (CDC:V240) (Shing et al, 2020). Against the A. castellanii Ma strain, isavuconazole potency (EC 50 of 4.6 nM) was one order of magnitude higher than that of posaconazole (EC 50 of 44.5 nM) or clotrimazole (EC 50 of 200 nM) (Shing et al, 2020).…”

“…Depending on the A. castellanii strain, isavuconazole potency varies in different strains from 26 nanomolar (MEEI 0184) to 4.6 nanomolar (Ma) to < 1 nM (CDC:V240) (Shing et al, 2020). Against the A. castellanii Ma strain, isavuconazole potency (EC 50 of 4.6 nM) was one order of magnitude higher than that of posaconazole (EC 50 of 44.5 nM) or clotrimazole (EC 50 of 200 nM) (Shing et al, 2020). Furthermore, isavuconazole at 70 M completely prevented This article has not been copyedited and formatted.…”