In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

Abstract

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s' interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.

Keywords: CYP51; Cryptococcus neoformans; Tremellomycetes; cytochrome P450 monooxygenases; docking; drug-resistance; sterol 14α-demethylase; web-application.

Figure 1

Phylogenetic analysis of CYP51s from Tremellomycetes. The analysis involved 22 CYP51 protein sequences. Phylogenetic analysis was carried out as described in the Methods section and the percentage of trees in which the associated taxa clustered together is shown next to the branches.

Figure 2

Superimposition of 3D structural models of 11 CYP51s from Tremellomycetes. Helices and beta sheets are shown in salmon and sky blue. Heme is shown in green along with the Fe atom in red.

Figure 3

Active site cavities of 11 CYP51s from Tremellomycetes. The active site cavity surface is shown in pink and amino acids, part of the active site cavity, are represented using a ball and stick model. A detailed list of amino acids, part of the active site cavity, is presented in Table S2.

Figure 4

This graphical overview of docking results at online web application. There are 99 squares representing the 11 × 9 = 99 CYP51-ligand combinations considered in this work. CYP51s were indicated with their protein IDs. Each square displays up to four bars, corresponding to models classified as ‘clashes’, ‘correct’, ‘near’, and ‘pocket’ (blue, orange, red, and cyan, respectively). Bars taller than 60 observations were reduced to 60 to fit into a plotting area; actual values are provided in Table 1. Docking results and corresponding information can be accessed at our online web application (Http://Bioshell.Pl/Azoledocking/).

Figure 5

Graphical representation of results of hierarchical clustering. Each of the 99 squares displays up to five clusters, represented by circles. The circular area is proportional to the size of the respective cluster, while the color scale denotes the d_NFe distance, with darker colors representing shorter values. All clusters of at least 10 structures found by hierarchical clustering are marked on this figure.