Antitrypanosomal lead discovery: identification of a ligand-efficient inhibitor of Trypanosoma cruzi CYP51 and parasite growth

Abstract

Chagas disease is caused by the intracellular protozoan parasite Trypanosomal cruzi , and current drugs are lacking in terms of desired safety and efficacy profiles. Following on a recently reported high-throughput screening campaign, we have explored initial structure-activity relationships around a class of imidazole-based compounds. This profiling has uncovered compounds 4c (NEU321) and 4j (NEU704), which are potent against in vitro cultures of T. cruzi and are greater than 160-fold selective over host cells. We report in vitro drug metabolism and properties profiling of 4c and show that this chemotype inhibits the T. cruzi CYP51 enzyme, an observation confirmed by X-ray crystallographic analysis. We compare the binding orientation of 4c to that of other, previously reported inhibitors. We show that 4c displays a significantly better ligand efficiency and a shorter synthetic route over previously disclosed CYP51 inhibitors, and should therefore be considered a promising lead compound for further optimization.

Figure 1

Anti-T. cruzi compounds previously discovered via HTS approaches, leading to the design principle 4.

Figure 2

Reversibility of compound 4c. T. cruzi growth inhibition assays were performed adding the compounds 4c or benznidazole at the time of infection and washing them off at the indicated times. EC₅₀ was calculated for each condition after 96 h of incubation. Left panel shows T. cruzi Tulahuen and right panel T. cruzi Y strains.

Figure 3

NIH-3T3 fibroblasts were incubated with T. cruzi trypomastigotes for 2 h before washing of extracellular T. cruzi and addition of drugs. Cells were incubated for 3 days, stained with an anti-T. cruzi antibody and DAPI to visualize DNA. Control infection (A) or infection in the presence of benznidazole (B) and 4c (C) at 9.5 and 0.4 µM, respectively (5 times the EC₅₀ concentration). These are representative images from a total of 50 fields observed in each condition.

Figure 4

Other azole-based inhibitors of T. cruzi CYP51.

Figure 5

Spectroscopic assay data for 4c binding to T. cruzi CYP51.

Figure 6

X-ray structure of T. cruzi CYP51 complexed to 4c (PDB code 4H6O). Electron density maps and stereoview of the active site are available in the Supporting Information. (a) Overall view from the distal side of the CYP51 molecule. The polypeptide chain is depicted as a ribbon diagram, the secondary structural elements forming the substrate access channel entrance are colored in purple. The C atoms of the heme and 4c are displayed as orange and blue stick models, respectively. The semitransparent surface of 4c (probe radius 1.4 Å) is colored by interpolated charge. (b) Enlarged view of 4c in the T. cruzi CYP51 active site. The 13 residues located within 4.5 Å from the inhibitor are depicted by lines and labeled. (c) Superimposed co-crystal structures of T. cruzi CYP51 with 4c and VNF (PDB code 3KSW). VNF and the heme from the 3KSW structure are colored in salmon. The distance between the heme ring propionate and OH of Y116 (3.7 Å, dashed line) is 1 Å longer than in the ligand-free or posaconazole-bound CYP51 structures (2.7 Å). (d) Superimposed co-crystal structures of T. cruzi CYP51 with 4c and posaconazole (PDB code 3K1O). Posaconazole, heme, and Y116 from the 3K1O structure are colored in green. VNF and compound 4c both adopt the orientation in the CYP51 active site, which is opposite to the orientation of posaconazole.

Scheme 1

a Preparation of analogs 4a–e. ^aReagents and conditions: (a) N-bromosuccinimde, CH₂Cl₂; (b) imidazole, DMF, MW 180 °C, 10 min; (c) H₂N-Ar, Pd₂(dba)₃, DPEPhos, DMF, MW 170 °C, 20 min; (d) CuI, HO-Ar, Cs₂CO₃, Me₂NCH₂CO₂H; (e) 4-chlorobenzyl bromide or 1-(2-bromoethyl)-4-chlorobenzene, K₂CO₃, DMF, 60 °C, 1h; (f) LiAlH₄, THF; (g) N-bromosuccinimde, PPh₃; (h) imidazole, DMF, 100 °C, 1 h.

Scheme 2

a Synthesis of various N-linked imidazole replacements 15. ^aReagents and conditions: (a) 4-chloro-3,5-dimethylphenol, Cs₂CO₃, CuI, 2-(dimethylamino)acetic acid, 1,4-dioxane, 180°C, 12 h; (b) TMSCl, 1,1,3,3-tetramethyldisiloxane, LiBr, CH₃CN, 80 °C, overnight; (c) heterocycle, amine or indole, K₂CO₃, DMF, 50 °C, 3 h.

Scheme 3

a Synthesis of various C-linked imidazole replacements 20 and 21. ^aReagents and conditions: (a) 4-chloro-3,5-dimethylphenol,Cs₂CO₃,CuI, 2-(dimethylamino)acetic acid, 1,4-dioxane, 180 °C, 9 h; (b) THF, LiOH, H₂O, rt, 2h; (c) H₂NNHAc, HOBT, EDC, DMF, rt, 5 h; (d) POCl₃, 110 °C, 1h; or Lawesson’s reagent, 1,4-dioxane, 100°C, 2h.