Clinical Candidate VT-1161's Antiparasitic Effect In Vitro, Activity in a Murine Model of Chagas Disease, and Structural Characterization in Complex with the Target Enzyme CYP51 from Trypanosoma cruzi

Abstract

A novel antifungal drug candidate, the 1-tetrazole-based agent VT-1161 [(R)-2-(2,4-difluorophenyl)-1,1-difluoro-3-(1H-tetrazol-1-yl)-1-{5-[4-(2,2,2-trifluoroethoxy)phenyl]pyridin-2-yl}propan-2-ol], which is currently in two phase 2b antifungal clinical trials, was found to be a tight-binding ligand (apparent dissociation constant [Kd], 24 nM) and a potent inhibitor of cytochrome P450 sterol 14α-demethylase (CYP51) from the protozoan pathogen Trypanosoma cruzi. Moreover, VT-1161 revealed a high level of antiparasitic activity against amastigotes of the Tulahuen strain of T. cruzi in cellular experiments (50% effective concentration, 2.5 nM) and was active in vivo, causing >99.8% suppression of peak parasitemia in a mouse model of infection with the naturally drug-resistant Y strain of the parasite. The data strongly support the potential utility of VT-1161 in the treatment of Chagas disease. The structural characterization of T. cruzi CYP51 in complex with VT-1161 provides insights into the molecular basis for the compound's inhibitory potency and paves the way for the further rational development of this novel, tetrazole-based inhibitory chemotype both for antiprotozoan chemotherapy and for antifungal chemotherapy.

FIG 1

(A) Spectral response of T. cruzi CYP51 to the addition of the heme-coordinating ligand 1-tetrazole VT-1161, shown as the difference in the type 2 binding spectra. The P450 concentration was 0.3 μM, and the optical path length was 5 cm. (Inset) The titration curve obtained using equation 1. ΔA(423-386), the difference in absorption between A₄₂₃ and A₃₈₆. (B) The corresponding titration curves and type 2 spectral responses (insets) of the A. fumigatus (top) and C. albicans (bottom) CYP51 orthologs.

FIG 2

Inhibitory effects of VT-1161 on the enzymatic activities of sterol 14α-demethylases from the protozoan parasite T. cruzi and the opportunistic fungal pathogens C. albicans and A. fumigatus. The incubation time was 60 min. E/I, molar ratio enzyme/inhibitor. P450 concentration was 0.5 µM; the concentration of the substrate (see Materials and Methods) was 25 µM. The experiments were performed in triplicate, and the results are presented as means ± SEs.

FIG 3

Cellular effects of VT-1161 in T. cruzi Tulahuen-infected cardiomyocytes. Cardiomyocyte monolayers were exposed to green fluorescent protein-expressing trypomastigotes (10 parasites per cell) for 24 h and then treated with VT-1161 or with the corresponding volumes of DMSO. (A to C) Fluorescence microscopic observations of T. cruzi inside cardiomyocytes treated with DMSO (control) (A) or with 10 nM (B) and 25 nM (C) VT-1161 at 72 h after infection. Green, T. cruzi amastigotes; blue, cardiomyocyte nuclei; red, cardiomyocyte actin myofibrils. (D) Dose-dependent clearance of the parasite. The infection was quantified by determination of the fluorescence, indicated as relative fluorescence units (RFU), at 72 h. The experiments were performed in triplicate, and the results are presented as means ± SEs.

FIG 4

VT-1161 suppresses parasitemia in mice infected with T. cruzi. Groups of Swiss female mice (n = 6) were infected i.p. with 10⁴ blood trypomastigotes of the Y strain of T. cruzi and not treated (control) or treated with 0.5% carboxymethyl cellulose (vehicle) or 50 mg/kg of VT-1161. The treatment was started on day 5 after infection and performed by oral gavage. The bars represent means ± SEs. Three days of treatment with VT-1161 caused >99.8% suppression of parasitemia.

FIG 5

(A) Structural formula (top) and 2Fo-Fc electron density map (bottom) of VT-1161 coordinated to the T. cruzi CYP51 heme iron. Here and in Fig. 6, VT-1161 is presented as a stick model, and carbon atoms are green. The map is shown as a gray mesh and contoured at 1.3σ. The heme is depicted as a wire model, and carbon atoms are gray. (B) The 19 amino acid residues that surround VT-1161 in the T. cruzi CYP51 active site. The corresponding secondary structural elements of the enzyme are presented as semitransparent ribbons and marked. (The corresponding residues in the aligned sequences of CYP51 from C. albicans and A. fumigatus are listed in Table 2).

FIG 6

VT-1161 (shown as a stick model with carbon atoms in green) bound to T. cruzi CYP51. (A) Overall view. The protein backbone is depicted as a rainbow ribbon color succession of the secondary structure from blue (N terminus) to red (C terminus). A surface representation is shown alongside to outline the view of VT-1161 through the entrance into the enzyme substrate access channel. (B) Enlarged view of panel A as a slice through the protein surface showing the location of VT-1161 within the CYP51 active-site cavity. The orientation is about the same as that in panel A. (C) Superimposition of VT-1161 with imidazole LFD (blue), the triazole posaconazole (khaki), and pyridine UDO (yellow) in the T. cruzi CYP51 active site. The PDB accession numbers of the corresponding structures are 4CK8, 3K1O, and 3ZG2, respectively.