The clinical candidate VT-1161 is a highly potent inhibitor of Candida albicans CYP51 but fails to bind the human enzyme

Abstract

The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [Kd], ≤ 39 nM) and similar to that of the pharmaceutical azole antifungals (Kd, ≤ 50 nM). In stark contrast, VT-1161 at concentrations up to 86 μM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 μM). In addition, VT-1161 (MIC = 0.002 μg ml(-1)) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 μg ml(-1)). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.

FIG 1

Chemical structures of azole antifungal agents. The chemical structures of clotrimazole (molecular weight, 345), fluconazole (molecular weight, 306), itraconazole (molecular weight, 706), voriconazole (molecular weight, 349), and VT-1161 (molecular weight, 527) are shown.

FIG 2

Azole binding properties of CaCYP51 (Ca) and Δ60HsCYP51 (Δ60Hs). VT-1161 and itraconazole were progressively titrated against 5 μM CaCYP51 and 5 μM Δ60HsCYP51, and binding saturation curves were constructed from the change in absorbance (ΔA_430-412) against the antifungal concentration for CaCYP51 (filled circles) and Δ60HsCYP51 (hollow circles). All spectral determinations were performed in triplicate, although only one replicate is shown.

FIG 3

Red shift of the heme Soret peak induced by azole ligand binding. Absolute spectra of 5 μM CaCYP51 and 5 μM Δ60HsCYP51 were measured in the absence (solid lines) and presence (dashed lines) of 10 μM antifungal compound. The 380- to 490-nm region of the spectra is shown to highlight any red shift of the Soret peak in response to ligand binding. The difference spectra for CaCYP51 (solid lines) and Δ60HsCYP51 (dashed lines), obtained by subtracting the resting-state absolute CYP51 spectra from the +10 μM azole-treated absolute CYP51 spectra, are also shown.

FIG 4

IC₅₀ determinations for antifungal agents with CaCYP51 and Δ60HsCYP51. CYP51 reconstitution assays containing either 2.5 μM CaCYP51 (A) or 2.5 μM Δ60HsCYP51 (B) were performed. For CaCYP51, the concentrations of fluconazole (filled circles), itraconazole (hollow circles), voriconazole (crosses), and VT-1161 (bullets) were varied from 0 to 4 μM. For Δ60HsCYP51, the concentrations of clotrimazole (filled squares) and VT-1161 (bullets) were varied from 0 to 50 μM. The DMSO concentration was kept constant at 0.5% in all assays. Mean relative enzyme velocities from three replicate data points along with the associated standard error bars are shown. Relative velocities of 1.0 correspond to actual velocities of 0.32 ± 0.04 nmol min⁻¹ for CaCYP51 and 0.69 ± 0.18 nmol min⁻¹ for Δ60HsCYP51.

FIG 5

Gas chromatograms of the sterol fraction isolated from untreated and antifungal-treated C. albicans. Wild-type C. albicans strain SC5134 (26) was treated with 0.004 μg ml⁻¹ antifungal agents and an untreated control received DMSO alone, followed by 18 h growth at 37°C and 180 rpm. The sterol content was extracted and analyzed as previously described (27). Gas chromatograms of the extracted sterols are shown for the untreated C. albicans cells (A) and the cells treated with the antifungals fluconazole (B), voriconazole (C), and VT-1161 (D). The four most abundant sterols present were ergosterol (peak 1), lanosterol (peak 2), 14α-methylergosta-8,24(28)-dien-3β,6α-diol (peak 3), and eburicol (peak 4). The data on the abundance axis are expressed in units of 1,000 (e.g., 600k = 600,000).