CYP51 as drug targets for fungi and protozoan parasites: past, present and future

Abstract

The efficiency of treatment of human infections with the unicellular eukaryotic pathogens such as fungi and protozoa remains deeply unsatisfactory. For example, the mortality rates from nosocomial fungemia in critically ill, immunosuppressed or post-cancer patients often exceed 50%. A set of six systemic clinical azoles [sterol 14α-demethylase (CYP51) inhibitors] represents the first-line antifungal treatment. All these drugs were discovered empirically, by monitoring their effects on fungal cell growth, though it had been proven that they kill fungal cells by blocking the biosynthesis of ergosterol in fungi at the stage of 14α-demethylation of the sterol nucleus. This review briefs the history of antifungal azoles, outlines the situation with the current clinical azole-based drugs, describes the attempts of their repurposing for treatment of human infections with the protozoan parasites that, similar to fungi, also produce endogenous sterols, and discusses the most recently acquired knowledge on the CYP51 structure/function and inhibition. It is our belief that this information should be helpful in shifting from the traditional phenotypic screening to the actual target-driven drug discovery paradigm, which will rationalize and substantially accelerate the development of new, more efficient and pathogen-oriented CYP51 inhibitors.

Keywords: Amoeba; CYP51; Leishmania; Trypanosoma brucei; Trypanosoma cruzi; antifungal azoles; crystal structure; inhibition; sterol 14α-demethylase; sterol biosynthesis.

Figure 1. CYP51 reaction is an essential step upon sterol biosynthesis

The pathway involves multiple (>30) steps, beginning with the condensation of acetyl-CoA molecules that serve as initial building blocks, and proceeds to squalene, which then forms epoxide and cyclizes into the triterpene sterol skeleton (cycloartenol or lanosterol). These precursors are further modified to produce cholesterol, ergosterol, or sitosterol, which are the major membrane sterols in humans, fungi/protozoa, and plants, respectively. The CYP51 reaction occurs either immediately or soon after squalene cyclization. The 14α-methyl group of the substrate (lanosterol, 24,25-dihydrolanosterol, eburicol, obtusifoliol, and/or C4-norlanosterol) is converted into the alcohol, then into the aldehyde derivative and finally is removed as formic acid with the introduction of the Δ14–15 double bond into the sterol core. The CYP51 reaction includes three consecutive cytochrome P450 catalytic cycles, consuming three molecules of oxygen, 6 electrons and 6 protons. Detailed description of other reactions of the pathway can be found in Nes, 2011. Inset: Sterol molecules are incorporated into membranes with the 3β-OH facing the water interface and the side chain extending into the hydrophobic core to interact with fatty acyl chains of phospholipids and proteins.

Figure 2. Clinical antifungal azoles used for treatment of systemic human infections

The whole set is represented by six derivatives of two basic scaffolds, fluconazole and ketoconazole. Ketoconazole is an imidazole, the others are 1,2,4-triazoles.

Figure 3. Amino acid sequence alignment of eukaryotic CYP51

The alignment was performed using >200 proteins. The sequences of two fungal (C. albicans and A. fumigatus), three protozoan: two Trypanosomatidae (T. cruzi and L. infantum) and amoeba (A. polyphaga), and human CYP51s are displayed as examples. The residues conserved in >99% CYP51 family members are in black, the phyla-specific residues that form the surface of the substrate binding cavity are in gray. The residue that defines the CYP51 substrate preferences is marked with black circle (●): F- C4-monomethylated sterols, L/I – C4-dimethylated sterols). Two CYP51 family signatures are underlined, the P450 signature, involving the heme-coordinating cysteine, is marked with the dashed line.

Figure 4. CYP51 binding ligands can be identified by spectral titration

A. Absolute absorbance spectra of water-bound (black), obtusifoliol-bound (blue, type 1 response), and azole-bound (red, type 2 response) T. brucei CYP51.The Soret band maxima are marked. Inset: the water-bound heme iron. B, C. Type 2 response of T. cruzi CYP51 to the binding of imidazole-based VNI [PDB code 3gw9] (B) and, pyridine-based UDO [PDB code 3zg3] (C). Absolute (top) and difference (bottom) absorption spectra. The P450 concentration ~0.4 µM, the optical path length 5 cm. Insets: the titration curves, prepared in Prism.

Figure 5. Low spectral K_ds do not necessarily mean strong inhibition of the CYP51 activity

While both VNF and it’s α-phenyl isomer display high spectral binding affinity and comparable inhibitory effects on the initial rate of reaction (5 min), VNF is not replaced in the CYP51 active site with the substrate overtime (60 min). The reaction mixture contained 1 µM T. cruzi CYP51, 1 µM inhibitor, and 50 µM substrate.

Figure 6

Inhibitory effects of systemic clinical antifungal azoles and experimental inhibitors on the activity of (A) T. cruzi, (B) T. brucei, (C) L. infantum, and (D) human CYP51 orthologs; 60 min reaction. The results are presented as means ± SEM. In all experiments the P450 concentration was 0.5 µM, the concentration of the sterol substrates (A, eburicol; B, C, obtusifoliol; D, lanosterol) was 50 µM. The values of the apparent spectral K_ds for human CYP51 are given in µM.

Figure 7. Binding of inhibitors does not cause any large-scale rearrangements in the backbone of the CYP51 molecule

A, B. Superimposed protozoan CYP51 structures, A: protein chains of ligand-free T. brucei (black) and inhibitor-bound T. brucei, T. cruzi and L. infantum CYP51. The substrate entry is circled. B: inhibitors bound in the protozoan CYP51 active site. C. Inhibitors bound in the fungal CYP51 active site. The heme is depicted in grey. D. Formulas of the inhibitors (posaconazole, voriconazole and fluconazole are shown in Fig. 2). The color code of each crystal structure corresponds to the color of the inhibitor name (PDB ID) beneath the formulas. The correspondent PDB codes of the crystal structures are shown in brackets, the codes of fungal structures are italicized.

Figure 8. VFV bound in the CYP51 active site

The 2F_o-F_c electron density map around the heme and the inhibitor is contoured at 2.0 σ and shown as grey mesh. The carbon atoms of VFV are green, the carbon atoms of 22 CYP51 residues that form Van der Waals contacts with the inhibitor are light grey, and the carbon atoms of the heme are dark grey. The atoms of oxygen, nitrogen, and sulfur are read, blue, and yellow, respectively. The heme iron is presented as an orange sphere. The active site-defining secondary structural elements (semitransparent cartoon) are labeled for clarity. The H-bonds between the enzyme and inhibitor are displayed as red dashes.

Figure 9. Surface representation of T. cruzi CYP51 bound to posaconazole

The long arm of the inhibitor protrudes above the entrance into the substrate access channel.

Figure 10. Pharmacokinetics of VNI and derivatives

A. Plasma concentration curves after a single oral dose of 25 mg/kg. B. VNI and VFV tissue distribution 4 hours after administration (single dose) and 16 hours after administration (2 doses).