(M1530-10-56) Leishmania Donovani CYP51 and CYP5122A1 Have Different Biochemical Roles in the Ergosterol Biosynthesis and Display Distinct Inhibition by Antifungal Azole Drugs

Purpose: CYP51 (1) and CYP5122A1 (2) are two cytochrome P450 (CYP) enzymes essential for the survival of Leishmania donovani, a major causative agent of human visceral leishmaniasis. Leishmanial CYP51 acts as a sterol C14α-demethylase during ergosterol biosynthesis and is known to be the target of antifungal azoles (3). CYP5122A1 is a newly discovered leishmanial CYP enzyme which is also involved in the leishmanial ergosterol biosynthesis (2). However, the exact biochemical function of this enzyme and its interaction with antifungal azoles remain unknown. The purpose of this study is to elucidate the biochemical role of CYP5122A1 and compare inhibitory effects of antifungal azoles on the two CYP enzymes.

Methods: Recombinant CYP51, CYP5122A1 from L. donovani and NADPH-dependent CYP reductase (CPR) from Trypanosoma brucei were expressed in Escherichia coli and purified using nickel affinity, ion exchange, and size exclusion chromatography on a Bio-Rad NGC chromatography system. Purified CYP51 and CYP5122A1 were analyzed for their P450 content on a Cary 3500 UV-Vis spectrophotometer (Agilent). To reconstitute CYP activities in vitro, the CYP enzyme was incubated with CPR and lanosterol in the presence of NADPH at 37°C and pH 6.2. After 1 h incubation, hexane:isopropanol (3:2, v/v) was added to quench the reaction. The organic extract was then subjected to sterol analysis using a triple quadrupole mass spectrometer (Waters) in the ESCI positive ion mode. A fluorescence-based inhibition assay was used to evaluate inhibitory activities of antifungal azoles (a total of twenty) against the two CYP enzymes. In this assay, 7-benzoyloxy-4-trifluoromethyl coumarin (BFC) was the probe substrate and converted to the fluorescent metabolite 7-hydroxy-4-trifluoromethyl coumarin (HFC, λ_excitation 410 nm, λ_emission 538 nm) by either CYP51 or CYP5122A1 in the presence of cumene hydroperoxide.

Results: Purified CYP51 and CYP5122A1 exhibited characteristic UV-Vis properties of a CYP protein and could be reduced by CPR in the presence of NADPH and by sodium dithionite. LC-MS/MS-based sterol analysis confirmed that leishmanial CYP51 catalyzes oxidation of lanosterol at the methyl group attached to C14, resulting in C14α-demethylation to form FF-MAS. In contrast, CYP5122A1 catalyzes oxidation at a different methyl group than the C14-methyl. Moreover, when probed with several intermediate sterols (4,14-dimethylzymosterol, FF-MAS and T-MAS) in the ergosterol biosynthesis pathway, CYP51 and CYP5122A1 showed selectivity to 14-methylated and 4-methylated sterols, respectively. Results from the inhibition assay showed that all tested antifungal azoles were potent inhibitors of CYP51 (IC₅₀s: 0.031 – 0.96 μM) but exhibited weaker and more varied inhibition against CYP5122A1 (IC₅₀s ranging from 0.26 to over 100 μM). Voriconazole was the most selective inhibitor of CYP51 ( >1176-fold over CYP5122A1) and clotrimazole was the least selective one (3.8-fold).

Conclusion: CYP51 and CYP5122A1 have different biochemical roles in the ergosterol biosynthesis pathway in Leishmania. CYP5122A1 likely acts as a C4-methyl oxidase. The differential inhibition of the two CYP enzymes may underlie the varying antileishmanial activities of antifungal azole drugs. In conclusion, this study improved our understanding of the biological roles of CYP51 and CYP5122A1 in Leishmania parasites and provided new insights as to develop novel antileishmanial drugs by targeting ergosterol biosynthesis.

References: 1. McCall LI, El Aroussi A, Choi JY, Vieira DF, De Muylder G, Johnston JB, et al. Targeting Ergosterol biosynthesis in Leishmania donovani: essentiality of sterol 14 alpha-demethylase. PLoS Negl Trop Dis. 2015;9(3):e0003588. doi: 10.1371/journal.pntd.0003588.
2. Verma S, Mehta A, Shaha C. CYP5122A1, a novel cytochrome P450 is essential for survival of Leishmania donovani. PLoS One. 2011;6(9):e25273. doi: 10.1371/journal.pone.0025273.
3. Lepesheva GI, Friggeri L, Waterman MR. CYP51 as drug targets for fungi and protozoan parasites: past, present and future. Parasitology. 2018;145(14):1820-36. doi: 10.1017/S0031182018000562.

Acknowledgments: This research was supported by R01 AI139198 (MZW), P20 GM113117 - CBID Pilot Project (MZW), and P30 GM110761 - PSF Pilot Project (MZW). The authors declare no potential conflict of interest.

Figure 1 UV-Vis spectrophotometric analysis of CYP51 (A) and CYP5122A1 (B). The absolute absorbance spectra were measured for the resting CYPs, showing the Soret band at around 420 nm. The insets show the CO difference spectra of reduced CYPs which exhibited the characteristic peak at around 450 nm.



Figure 3 Inhibition of CYP51 and CYP5122A1 by two representative antifungal azoles. Clotrimazole is a dual inhibitor of two CYPs (selectivity index: 3.8) whereas voriconazole is a selective inhibitor which only inhibits CYP51 but not CYP5122A1 (selectivity index >1176).



Figure 2 LC-MS/MS analysis of the reconstitution reactions of CYP51 (A) and CYP5122A1 (B). 1: lanosterol; 2: the hydroxy metabolite generated by CYP51; 3: the aldehyde metabolite generated by CYP51; 4: FF-MAS; 5: the hydroxy and aldehyde metabolites generated by CYP5122A1; 6: the acid metabolite generated by CYP5122A1. The distinct biochemical roles of CYP51 and CYP5122A1 were indicated by the different retention times of their intermediate metabolites.