CYP5122A1 encodes an essential sterol C4-methyl oxidase in Leishmania donovani and determines the antileishmanial activity of antifungal azoles

Visceral leishmaniasis, caused by Leishmania donovani, is a life-threatening parasitic disease, but current antileishmanial drugs are limited and have severe drawbacks. There have been efforts to repurpose antifungal azole drugs for the treatment of Leishmania infection. Antifungal azoles are known to potently inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets for these compounds. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation reactions to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. Overexpression of CYP5122A1 results in growth delay, differentiation defects, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1, e.g., clotrimazole and posaconazole, possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors, e.g., fluconazole and voriconazole, have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes.