# Structural Requirements for Sterol 14alpha-Demethylases

> **NIH NIH R01** · VANDERBILT UNIVERSITY · 2022 · $24,286

## Abstract

PROJECT SUMMARY
Sterol 14α-demethylases are the cytochrome P450 enzymes found in all biological kingdoms and, regardless
of their low (22-35%) sequence identity across phylogeny, grouped into one family (CYP51) because of their
strict functional conservation. From bacteria to humans, they all catalyze the same unusual three-step reaction
of the oxidative removal of the 14α-methyl group from one or more of five cyclized sterol precursors (14α-
methyl →14α-alcohol→14α-aldehyde→14α-demethylated product plus formic acid). Eukaryotic microsomal
membrane-bound CYP51s use NADPH-cytochrome P450 reductase (CPR) as their redox partner, while
water-soluble bacterial orthologs accept electrons from ferredoxins and/or flavodoxins. The CYP51 reaction
is required for biosynthesis of sterols, which are essential for eukaryotic membrane biogenesis and also serve
as precursors for a variety of regulatory molecules that are involved in cellular growth, development, and
division processes (hormones, vitamins, nuclear receptors, etc.). For more than 50 years, the CYP51 reaction
has served as the target for clinical antifungal drugs and agricultural fungicides (imidazoles, triazoles, or
sometimes pyridines), yet the enzyme per se has not been included in the drug discovery paradigm because
of the difficulties of its handling.
Our long-term goal is to understand what makes/keeps a CYP51 a CYP51 and what structural features of this
P450 can be used to make rationally designed, potent, and functionally irreversible species-selective
inhibitors. We have found that while upon binding of exogenous ligands (azoles, pyridines, and even a
substrate analog) CYP51s remain in their resting, ligand-free-like state, accommodation of the physiological
substrate causes a large-scale conformational switch that involves the backbone of the active site and the
surface of interaction with the electron donor partner, preparing the enzyme for catalysis.
The aims of the current renewal application are 1) to determine, by combining cryo-electron microscopy and
X-ray crystallography, the structures of the complex of the substrate-bound CYP51/CPR and the substrate-
bound Methylococcus capsulatus CYP51/ferredoxin fusion; 2) to use computational structural biology to better
understand CYP51 molecular dynamics; 3) to evaluate the efficacy of our two VNI derivatives with optimized
pharmacokinetics in the mouse models of Chagas disease caused by drug resistant strains of
Trypanosoma cruzi, to analyze our in-house library of CYP51 inhibitors against a fungus Cryptococcus
neoformans (cryptococcal meningitis) and two free-living pathogenic amoebas, Acanthamoeba castellanii
(blinding keratitis) and Nagleria fowleri (primary amebic meningoencephalitis), and to test our two potent
functionally irreversible inhibitors of human CYP51 in cancer cell lines and in cytomegalovirus infected
human cells.

## Key facts

- **NIH application ID:** 10576563
- **Project number:** 3R01GM067871-19S1
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Galina I Lepesheva
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $24,286
- **Award type:** 3
- **Project period:** 2004-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10576563

## Citation

> US National Institutes of Health, RePORTER application 10576563, Structural Requirements for Sterol 14alpha-Demethylases (3R01GM067871-19S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10576563. Licensed CC0.

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