Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

https://doi.org/10.1016/j.jmb.2010.01.075Get rights and content

Abstract

The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B′ helix and F–G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

Introduction

Sterol biosynthesis is an essential metabolic pathway in most organisms throughout the biological kingdoms and results in the production of cholesterol in animals, ergosterol in fungi, and a variety of 24-modified sterols in plants, algae, and protozoa.1 CYP51 (sterol 14-alpha demethylase) is involved in the postsqualene part of the pathway and catalyses the removal of the methyl group at the 14α-position of sterol precursors. The reaction consists of three sequential steps of successive conversion of methyl group to hydroxymethyl and then into carboxyaldehyde followed by the elimination of formic acid and concomitant formation of the Δ14,15 double bond in the sterol core. While some nematodes, insects, and other arthropods lack CYP51 and obtain sterols from their diet, most CYP51 genes show a high degree of structural and functional conservation irrespective of the sterol end product.

Sterols are required in most eukaryotic organisms as a membrane component, providing its fluidity, permeability, and asymmetry. In addition, sterols serve as the precursors to various types of hormones, vitamin D, and bile acids. The essential nature of sterol biosynthesis makes CYP51 an important drug target. Inhibition of CYP51 in fungi causes the accumulation of membrane-disrupting methylated sterol precursors of ergosterol, preventing fungal growth. Azole drugs have been leading agents used to treat fungal infections of plants, animals, and human.2, 3 These drugs also interfere with the sterol biosynthesis of some pathogenic protozoans.4, 5 Azole drugs bind to the CYP51 active site through coordination of a heme iron by the N-heterocycle nitrogen. However, use of azole drugs often results in the development of drug resistance, which involves, among others, mutations in CYP51 that alter drug–target interactions.6, 7, 8 The continued development of new azole agents is needed to address this problem as well as a growing number of fungal infections. The adverse effect of azole antifungal therapy is cross-over inhibition of human CYP51 and other P450s.

Human CYP51 is an important enzyme in mammalian cholesterol biosynthesis and a potential target for cholesterol-lowering drug design. Although the target of current clinical anticholesterol agents is HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase,9 inhibition of cholesterol synthesis at later stages of the pathway has been investigated by inhibition of human CYP51 by azalanstat10 derivatives of lanosterol,11, 12 dual-acting agents inhibiting glycogen phosphorylase and CYP51,13 and pyridylethanol(phenylethyl)amines.14 CYP51 in mammals is also responsible for production of the follicular fluid (FF) meiosis-activating sterol (MAS).15 Together with the testis MAS (T-MAS), the product of the downstream sterol Δ14-reductase reaction, these sterols are present at elevated levels in gonads16, 17 and are involved in oocyte maturation18, 19 and spermatogenesis.20, 21 While the importance of CYP51 activity in MAS-dependent gametogenesis and its mechanism is not fully understood, pharmacological applications for in vitro fertilization22 and possibly the control of reproduction have been suggested.

To gain insights into structural determinants of human CYP51 inhibition, we solved the structure of ligand-free CYP51, as well as CYP51 complexed with two antifungal drugs, ketoconazole and econazole. The structures revealed the binding mode of azole compounds and their interactions with the protein. Comparison of the azole-bound structures to the ligand-free structure showed that the ligands induced similar but dramatic changes in the B′ helix and F–G loop regions. The conformational flexibility of the human protein involves regions comprising the access channel, which is different from Mycobacterium tuberculosis (Mt) CYP51.23 Comparison of the human ketoconazole structure to the Mt fluconazole structure [Protein Data Bank (PDB) code 1EA1] provided structural evaluation of relative inhibitory potency of these azoles on the activities of human and Mt enzymes. The obtained data can be used for the rational design of more specific inhibitors/modulators of CYP51.

Section snippets

Results and Discussion

To determine the azole compounds with the strongest binding to CYP51, the purified N-term truncated human CYP51 was tested against a panel of azole inhibitors by monitoring the type II spectral shift in the Soret region caused by the coordination of azole nitrogen to the heme iron. The Kd values for the inhibitors are summarized in Table 1. Nanomolar affinities were obtained for miconazole, econazole, and bifonazole. Strong binding affinities were also observed for some fungicides used in

Protein purification and crystallization

The CYP51A1 cDNA was purchase from MGC collection (accession code BC032322) and subcloned into a modified pCW-LIC vector. The N-terminal transmembrane anchor domain (residues 1–53) was replaced with MAKKT sequence.47 The modified C-terminal His6-tagged protein was coexpressed with GroEL/ES in Escherichia coli. CYP51A1 was purified using metal-affinity chromatography on a NiHiTrap chelating column, followed by cation-exchange chromatography on a Source S column. The purified protein was stored

Acknowledgements

We thank Dr. A. Gilep for critical reading of the manuscript and T. Cherkesova, I. Grabovec, A. Yantsevich, and SGC members F. MacKenzie and Dr. W. Tempel for excellent technical assistance. The Structural Genomics Consortium is a registered charity (No. 1097737) and receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg

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