A Three-Dimensional Model of Lanosterol 14α-Demethylase of Candida albicans and Its Interaction with Azole Antifungals

Abstract

The three-dimensional structure of lanosterol 14alpha-demethylase (P450(14DM), CYP51) of Candida albicans was modeled on the basis of crystallographic coordinates of four prokaryotic P450s: P450BM3, P450cam, P450terp, and P450eryF. The P450(14DM) sequence was aligned to those of known proteins using a knowledge-based alignment method. The main chain coordinates of the core regions were transferred directly from the corresponding coordinates of P450BM3. The side chain conformations of the core regions were determined by the conformations of the equivalent residues with the highest homologous scores in four crystal structures. The model was then refined using molecular mechanics and molecular dynamics. The reliability of the resulting model was assessed by Ramachandran plots, Profile-3D, hydropathy plot analysis, and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel, and the substrate binding residues were identified from the model. These residues are candidates for further site-directed mutagenesis and site-specific antipeptide antibody binding experiments. The active analogue approach was employed to search the pharmacophoric conformations for 14 azole antifungals. The resulting bioactive conformations were docked into the active site of lanosterol 14alpha-demethylase of Candida albicans. All 14 azole antifungals are shown to have a similar docking mode in the active site. The halogenated phenyl group of azole inhibitors is deep in the same hydrophobic binding cleft as the 17-alkyl chain of substrate. The pi-pi stacking interaction might exist between halogenated phenyl ring of inhibitors and the aromatic ring of residue Y132. The long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel. To compare with mammalian enzymes, structurally selective residues of the active site of fungal lanosterol 14alpha-demethylase are distributed in the C terminus of F helix, beta6-1 sheet and beta6-2 sheet.

Biological spectra analysis: Linking biological activity profiles to molecular structure

“…Modular structure design within protein families by using conserved structure elements in the active site (11), for example, forms the basis for 3D models, which drive SAR studies within protein families. This strategy even succeeds in protein families whose members have low sequence homology such as the cytochrome P450 superfamily, which consists of Ͼ2,000 members distributed across different organisms (12)(13)(14)(15).…”

2D QSAR studies on a series of bifonazole derivatives with antifungal activity

“…5 Indeed, several three-dimensional quantitative structure-activity relationship studies (3D QSAR) have been reported for different datasets of azole derivatives 2,6,[9][10][11][12] however, this strategy afforded models with moderate robustness and local predictive ability only. For instance, Di Santo and co-workers 1 report that their CoMFA model was contradicted by the synthesis and evaluation of novel compounds.…”

“…An initial step to overcome this dilemma was the resolution of the crystallographic structure of Mycobacterium tuberculosis sterol 14α-demethylase (MTCYP51) in complex with two azole inhibitors (4-phenylimidazole and fluconazole). 9 Yet, the bacterial source of this enzyme still poses as a problem for the development of 3D QSAR (ex. : comparative molecular field analysis -CoMFA) and 3D pharmacophore models, as these methods are highly dependent on molecular alignment.…”

Altered expression profile of mycobacterial surface glycopeptidolipids following treatment with the antifungal azole inhibitors econazole and clotrimazole

“…cytochrome P450 14a-lanosterol demethylase, also termed CYP51. They are competitive inhibitors that bind reversibly to the haem iron moiety of CYP51 via a specific nitrogen atom in the azole ring (Hitchcock et al, 1990;Ji et al, 2000). The cytochromes P450 (P450s) are haem-thiolate enzymes that are able to oxygenate a large variety of substrates (Lewis, 1996).…”