Targeting CYP5122A1 and CYP51 for the Inhibition of Ergosterol Biosynthesis in Trypanosomatids

Trypanosomatid parasite infections are important public health threats affecting millions of people in tropical and subtropical areas. However, current treatment options are limited and have severe drawbacks, and there is still an unmet medical need for safe, effective, and affordable drugs. Targeting ergosterol biosynthesis has been considered a promising strategy for the development of new anti-trypanosomatid agents. Cytochrome P450 (CYP) 51 acts as a sterol C14α-demethylase during ergosterol biosynthesis and has been widely studied for its potential as a therapeutic target. CYP5122A1, another CYP enzyme found in both Leishmania and Trypanosoma species, has been demonstrated to be essential for L. donovani survival and plays an important but unknown role in leishmanial ergosterol biosynthesis.In this study, an N-terminal truncated construct of L. donovani CYP5122A1 was heterologously expressed, purified, and spectrally characterized. By reconstituting the catalytic activity of CYP5122A1 in vitro, its biochemical function in ergosterol biosynthesis was elucidated. CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential C4-oxidation of lanosterol to the hydroxy, aldehyde, and carboxylate/formyloxy metabolites.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. In the search for CYP5122A1-selective inhibitors, a series of benzyloxyphenyl-arylalkylpiperazine-1-carboxamide compounds were assessed for their CYP inhibitory potencies and binding modes. Compounds containing different terminal rings displayed different selectivity towards CYP enzymes, with the one having a terminal pyrrole group being the most selective inhibitor of CYP5122A1 (25-fold over CYP51). Results from sterol analysis of azole-treated parasites supported that, in L. donovani, lanosterol undergoes C4-demethylation catalyzed by CYP5122A1 and inhibition of both CYP5122A1- and CYP51-mediated reactions by dual inhibitors like clotrimazole blocked the ergosterol biosynthesis at earlier steps to a greater extent and produced better antileishmanial effects. To facilitate structure-based drug design, the crystal structure of CYP51 complexed with clotrimazole was obtained. The crystallization construct of CYP5122A1 requires further optimization.Furthermore, our study on the CYP5122A1 ortholog in T. cruzi showed that it acts as a sterol C4-methyl oxidase, displaying the highest activity at pH 6.2 – 6.6. Selected antifungal azoles were evaluated for their inhibitory potencies and binding modes. Clotrimazole had type II binding to both enzymes, indicating direct coordination of nitrogen with heme iron. It was identified as the strongest dual inhibitor with IC50 values of 0.052 μM and 0.27 μM against CYP51 and CYP5122A1, respectively, and therefore should be tested for its antiparasitic activity against T. cruzi in the future. Posaconazole was a strong inhibitor of CYP51 (IC50: 0.055 μM) and a moderate inhibitor of CYP5122A1 (IC50: 3.3 μM). It displayed type II binding to CYP51 and reversed type II binding to CYP5122A1. The different binding modes of posaconazole to CYP5122A1 from T. cruzi and L. donovani suggested the structural difference between the enzymes from the two species, which prompted us to perform crystallization screening experiments for T. cruzi CYP5122A1. Using posaconazole as the ligand, several interesting hits were identified, which need to be optimized to obtain high-quality protein crystals.In summary, the research presented in this dissertation elucidated the biochemical role of CYP5122A1 in two important trypanosomatid parasites and provided an important foundation for developing new anti-parasitic agents by targeting ergosterol biosynthesis.

Grantor dc:publisher

University of Kansas

Year dc:date.issued

2023

Author dc:creator

• Jin, Yiru

Advisor dc:contributor.advisor

• Wang, Michael Z

• Pharmaceutical sciences
• Biochemistry
• Azole antifungal
• CYP5122A1
• Leishmania
• Sterol C4-methyl oxidase
• Sterol demethylation
• Trypanosoma cruzi

Statement dc:rights

• Copyright held by the author.

Language dc:language.iso

en

Dc Identifier Other

http://dissertations.umi.com/ku:19155

OAI identifier oai:identifier

oai:kuscholarworks.ku.edu:1808/36568