Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy

Abstract

We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

Figure 1. Structures of LOX inhibitors.

Figure 2. Representative analogues evaluated for pseudoperoxidase activity and IC₅₀ potency (µM), with errors in brackets.

The UV-based manual inhibition data (3 replicates) were fit as described in the Materials and Methods section.

Figure 3. 5-LOX IC₅₀ values of representative analogues (µM), with errors in brackets.

The UV-based manual inhibition data (3 replicates) were fit as described in the Materials and Methods section.

Figure 4. IC₅₀ values of dual anti-fungal, anti-inflammatory inhibitors (µM), with error in parentheses.

The UV-based manual inhibition data (3 replicates) were fit as described in the Materials and Methods section. N/D = Not determined.

Figure 5. Docking ketoconazole (A) and ketaminazole (B) to the crystal structure of the Stable-5-LOX (PDB ID: 3O8Y).

Glide docking scores and poses were similar to other high-ranking docked inhibitors.

Figure 6. Binding properties of ketoconazole and ketaminazole with CaCYP51 and HsCYP51.

Azole antifungals were progressively titrated against 5 µM CaCYP51 (filled circles) and 5 µM HsCYP51 (hollow circles). The resultant type II difference spectra are shown for ketoconazole (A) and ketaminazole (B). Saturation curves for ketoconazole (C) and ketaminazole (D) were constructed and a rearrangement of the Morrison equation (45) was used to fit the data. The data shown represent one replicate of the three performed.

Figure 7. Determination of IC₅₀ values for ketoconazole and ketaminazole with CaCYP51 and HsCYP51.

CYP51 reconstitution assays (0.5-ml total volume) containing 1 µM CaCYP51 (A) or 0.3 µM HsCYP51 (B) were performed as detailed in Materials and Methods. Ketoconazole (solid circles) and ketaminazole (hollow circles) concentrations were varied from 0 to 4 µM for CaCYP51 and up to 190 µM for HsCYP51 with the DMSO concentration kept constant at 1% (vol/vol). Mean values from two replicates are shown along with associated standard deviation bars. Relative velocities of 1.0 were equivalent to 1.04 and 2.69 nmoles 14α-demetylated lanosterol produced per minute per nmole CYP51 (min⁻¹) for CaCYP51 and HsCYP51, respectively.