The Tetrazole VT-1161 Is a Potent Inhibitor of Trichophyton rubrum through Its Inhibition of T. rubrum CYP51

Abstract

Prior to characterization of antifungal inhibitors that target CYP51, Trichophyton rubrum CYP51 was expressed in Escherichia coli, purified, and characterized. T. rubrum CYP51 bound lanosterol, obtusifoliol, and eburicol with similar affinities (dissociation constant [K_d ] values, 22.7, 20.3, and 20.9 μM, respectively) but displayed substrate specificity, insofar as only eburicol was demethylated in CYP51 reconstitution assays (turnover number, 1.55 min^-1; K_m value, 2 μM). The investigational agent VT-1161 bound tightly to T. rubrum CYP51 (K_d = 242 nM) with an affinity similar to that of clotrimazole, fluconazole, ketoconazole, and voriconazole (K_d values, 179, 173, 312, and 304 nM, respectively) and with an affinity lower than that of itraconazole (K_d = 53 nM). Determinations of 50% inhibitory concentrations (IC₅₀s) using 0.5 μM CYP51 showed that VT-1161 was a tight-binding inhibitor of T. rubrum CYP51 activity, yielding an IC₅₀ of 0.14 μM, whereas itraconazole, fluconazole, and ketoconazole had IC₅₀s of 0.26, 0.4, and 0.6 μM, respectively. When the activity of VT-1161 was tested against 34 clinical isolates, VT-1161 was a potent inhibitor of T. rubrum growth, with MIC₅₀, MIC₉₀, and geometric mean MIC values of ≤0.03, 0.06, and 0.033 μg ml^-1, respectively. With its selectivity versus human CYP51 and drug-metabolizing cytochrome P450s having already been established, VT-1161 should prove to be safe and effective in combating T. rubrum infections in patients.

Keywords: CYP51; Trichophyton rubrum; VT-1161; azole resistance; substrate specificity.

FIG 1

Spectral characteristics of Trub51. (A) Absolute spectra were determined using 3 μM purified Trub51 in the oxidized resting state. (B) Reduced carbon monoxide difference spectra were determined using 3 μM purified Trub51, with sequential measurements being made every 45 s.

FIG 2

Sterol binding properties of Trub51. (A) Absorbance difference spectra were measured during the progressive titration of 5 μM Trub51 with lanosterol, eburicol, and obtusifoliol. (B) Saturation curves for lanosterol (filled circles), eburicol (hollow circles), and obtusifoliol (crosses) were constructed from the absorbance difference (ΔA_388–421) of the type I difference spectra observed. Sterol binding data were fitted using the Michaelis-Menten equation.

FIG 3

GC/MS analysis of Trub51 reconstitution assay metabolites. (A) GC traces for Trub51 reconstitution assays (37°C, 15 min) using lanosterol and eburicol as the substrates are shown. (B) In addition, the mass fragmentation pattern for the TMS-derivatized C-14-demethylated eburicol product (M⁺ 496; product P) is shown. Abundance is expressed in thousands (K).

FIG 4

K_m determination for eburicol. A velocity curve was constructed for eburicol with Trub51 using the CYP51 reconstitution assay (34, 55). The single substrate inhibition equation v = (V_max · [S])/{K_m + [S] · (1 + [S]/K_i)} (57) was used to fit the velocity curve. Mean values from three replicates and the associated standard deviation bars are shown.

FIG 5

Type II azole binding spectra for Trub51. Clotrimazole, fluconazole, voriconazole, itraconazole, ketoconazole, and VT-1161 were progressively titrated against 2 μM CYP51 protein, with the difference spectra being determined after each addition of azole. The resultant type II difference spectra obtained for each azole are shown. Each experiment was performed in triplicate, although only one replicate is shown.

FIG 6

Azole binding saturation curves for Trub51. Saturation curves were constructed from the absorbance difference (ΔA_peak-trough) of the type II difference spectra (Fig. 5) for clotrimazole (circles), fluconazole (squares), voriconazole (triangles), itraconazole (diamonds), ketoconazole (asterisks), and VT-1161 (crosses). A rearrangement of the Morrison equation (25) was used to fit the tight ligand binding observed. Each experiment was performed in triplicate, although only one replicate is shown.

FIG 7

IC₅₀ determinations for antifungal agents. CYP51 reconstitution assays were performed using a CYP51/AfCPR ratio of 1:2 for 0.5 μM Trub51 with 25 μM eburicol as the substrate at various fluconazole (filled circles), itraconazole (hollow circles), ketoconazole (bullets), and VT-1161 (crosses) concentrations ranging from 0 and 8 μM. Mean relative velocity values are shown along with the associated standard deviation values. Relative velocities of 1.00 were equivalent to velocities of 1.55 min⁻¹.