Homology Modeling and Screening of New 14α-Demethylase Inhibitor (DMI) Fungicides Based on Optimized Expression of CYP51 from Ustilago maydis in Escherichia coli

Abstract

Ustilago maydis infection is a serious disease affecting corn crops worldwide. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis and an effective target of antifungal drugs. To further study the interaction between CYP51 and drugs and exploit more specific 14α-demethylase inhibitor (DMI) fungicides for U. maydis, in this study homology modeling of CYP51 from U. maydis (UmCYP51) templated as the eukaryotic orthologues (the human CYP51) and screening of new DMI fungicides based on optimized expression were carried out for the first time. In addition, XF-113 and ZST-4 were screened by analyzing the spectral characteristics between the purified UmCYP51-35 and fungicides. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal drugs.

A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicillium digitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

“…Previously, we reported the cloning, expression, and characterization of cyp51 from P . digitatum and Ustilago maydis [ 52 – 55 ]. The structural characteristics of the interaction between heterologous CYP51 and commercial azoles were also analyzed by binding assays.…”

“…digitatum [ 54 ]. The screening of new DMI fungicides based on optimized expression has been carried out for the first time in Ustilago maydis [ 55 ]. Recently, a cell-based high-throughput screen has identified small-molecule inhibitors of the Upc2-dependent induction of sterol gene expression in response to azole drug treatment.…”

Targeting CYP51 for drug design by the contributions of molecular modeling

“…An appropriate selection of a suitable template and alignment of sequences is essential for the success of such tool [95,96]. Homology models have been proposed for CYP51 of several species such as Ustilago maydis, which was built using the human enzyme as template (PDB code 3LD6) to search for fungicides against infections in corn fields [97]. Models of CYP51 from C. albicans and A. fumigatus were constructed and used to investigate the mechanism of action of azoles [98].…”

Azole fungicides - understanding resistance mechanisms in agricultural fungal pathogens

“…More recently, in silico modelling of fungal CYP51 enzymes has been used to predict the affinity of azole antifungals for these enzymes, predominantly looking at how mutations within the structure affect azole docking. Fungal CYP51 proteins currently modelled include Magnaporthe grisea CYP51, Ustilago maydis CYP51, Aspergillus fumigatus CYP51, M. fijiensis CYP51, P. digitatum CYP51 and Z. tritici CYP51 …”

“…More recently, in silico modelling of fungal CYP51 enzymes has been used to predict the affinity of azole antifungals for these enzymes, predominantly looking at how mutations within the structure affect azole docking. Fungal CYP51 proteins currently modelled include Magnaporthe grisea CYP51, 43 Ustilago maydis CYP51, 44 Aspergillus fumigatus CYP51, 45 M. fijiensis CYP51, 16 P. digitatum CYP51 46 -48 and Z. tritici CYP51. 49 The 3D structures of fungal cytochromes P450 (CYPs) have primarily been based on the crystal structures of bacterial CYPs, which are readily soluble and relatively easy to crystallise, unlike eukaryotic CYPs, which tend to aggregate in free solution owing to their hydrophobic N-terminal membrane anchors.…”