Mechanistic Insights into Cytochrome P450 Inactivation by Azole Drugs in Acanthamoeba castellanii
Authors: Sharma Vandna , Shing Brian , Debnath Anjan , Podust Larissa 
Published: April 2020

Cytochrome P450 (CYPs) are heme containing enzymes that oxidize substances using iron and are capable to metabolize wide variety of xenobiotics and play key role in the activation or inactivation of various therapeutic agents. Studies have described more than 50,000 CYPs in most forms of life i.e. archaea, viruses, protists, bacteria, animals, plants and fungi.One of the major advancements in pharmacology is the discovery of antifungal activity of azole compounds. These compounds showed a very significant advancement in the management of fungal infections. Major enzyme inhibited by these azoles is 14 alpha‐demethylase, a cytochrome P450 enzyme, which is involved in the conversion of lanosterol to ergosterol.As these CYPs descend from a common ancestor and share structural features, we tried to repurpose these antifungal drugs against Acanthamoeba castellanii, an opportunistic pathogen and an emerging cause of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis.In the present study, we structurally characterized recombinant CYP51 from Acanthamoeba castellanii (AcCYP51), which is also an important enzyme in the biosynthesis of ergosterol. Interestingly, structure‐based analysis of ligand free and ligand bound forms of AcCYP51 made very remarkable observations. Here, we observed that ligand free AcCYP51 tends to dimerize both in solution (as observed in size exclusion chromatography,SEC) and in the crystals (1.8 Å) via N‐terminal swapping forming a large homo‐dimerization interface exceeding 2000 Å2. Spectral analysis of dimeric and small monomeric fractions of AcCYP51, which we obtained after SEC, have shown that only dimeric form of AcCYP51 has spectral characteristics typical of functionally competent CYP enzymes. This is for the first time that a strong and functionally competent CYP dimer was defined structurally. Further structural analysis of inhibitor (azole) bound AcCYP51 have shown that binding of inhibitors promoted dimer dissociation and we obtained AcCYP51‐inhibitor complexes in a monomeric form (2.9 Å). We have used here different azole inhibitors (according to their potency against Acanthamoeba ) and observed that according to their in‐vivo potency, these inhibitors elucidated differences in their mode of action. With a less potent inhibitor AcCYP51 was capable to restore the monomer structure whereas with the most potent inhibitor AcCYP51failed to fully restore the monomer structure. This failure of CYP51 to fully refold upon dimer dissociation, in vivo may cause an irreversible loss of a structurally aberrant target enzyme through proteolytic degradation. This proposed inactivation mechanism is consistent with the superior potency of this molecule against A. castellaniicells.Repurposing of the antifungal azole drugs (known as conazoles) emerges as a promising strategy to combat Acanthamoeba infections. The drug‐target crystal structures determined in this work and the mechanistic insights thus gained may be further exploited for the medicinal chemistry efforts aimed at improving potency and bioavailability of these drugs for applications in human diseases caused by infection with Acanthamoeba.