Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein-Ligand Interactions of CYP51 of Candida krusei Azole-Resistant Fungal Strain

Abstract

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.

Keywords: CYP51; Candida krusei; antifungal drugs; azole inhibitors; cytochrome P450; drug resistance; heterocyclic analogues of steroids; lanosterol 14-alpha demethylase; marine steroids.

Figure 1

Classification of azole drugs. The nitrogen atom that forms a bond with the heme iron is marked with a red circle.

Figure 2

The multiple sequence alignment of CYP51 proteins from fungi of the genus Candida. Alignment was performed using the GeneDoc v.2.6 program. The assignment of secondary structure elements is based on the Candida albicans CYP51 structure (PDB: 5V5Z). White characters on the black background show 100% identity, white characters on the grey background show 75% similarity, and black characters on the grey background show 50% similarity. Mutations in azole-resistant C. albicans clinical isolates are highlighted and mutated residues are shown below the sequence (red colored—mutations attributed to resistance [27,28,41,42,43,44,45]; blue colored—resistance exhibited in combination with other mutations [27,28,44,46,47,48,49,50]; green colored—mutations we detected for the first time in azole-resistant C. albicans clinical isolate [51,52]). The numbers on top are shown for CYP51 Candida albicans. The red star shows a heme-bound cysteine. Blue frames indicate Gotoh’s SRSs. The amino acid sequences were taken from the NCBI: calb (Candida albicans—XP_716761); cgla (Candida glabrata—XP_445876); ctro (Candida tropicalis—XP_002550985); ckru (Candida krusei/Pichia kudriavzevii/—XP_029322955); cpara (Candida parapsilosis—ACT67904); cgui (Candida guilliermondii/Meyerozyma guilliermondii/—XP_001484034).

Figure 3

Three-dimensional alignment of CYP51 structures from Candida albicans (magenta) and Candida krusei (cyan). Overall folding (A), active site (B), and access to the substrate channel (C).

Figure 4

Map of CYP51-pCW-lic expression vector.

Figure 5

Absorption spectrum of the purified C.krCYP51 (a); difference spectrum of the carbonyl complex of reduced C.krCYP51 (b); results of electrophoretic analysis of purified C.krCYP51 in 12% PAGE under denaturing conditions. St (P7712, NEB)—molecular weight standard; 1—C.krCYP51 fraction after purification on HAP (c).

Figure 6

Results of mass spectrometric analysis of purified CkrCYP51.