Nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for Chagas disease chemotherapy

Abstract

A century after discovering that the Trypanosoma cruzi parasite is the etiological agent of Chagas disease, treatment is still plagued by limited efficacy, toxicity, and the emergence of drug resistance. The development of inhibitors of the major T. cruzi cysteine protease, cruzain, has been demonstrated to be a promising drug discovery avenue for this neglected disease. Here we establish that a nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitor substantially ameliorates symptoms of acute Chagas disease in a mouse model with no apparent toxicity. A high-resolution crystal structure confirmed the mode of inhibition and revealed key binding interactions of this novel inhibitor class. Subsequent structure-guided optimization then resulted in inhibitor analogues with improvements in potency despite minimal or no additions in molecular weight. Evaluation of the analogues in cell culture showed enhanced activity. These results suggest that nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors have the potential to fulfill the urgent need for improved Chagas disease chemotherapy.

Figure 1

Structures of potent irreversible cruzain inhibitors: dipeptidyl vinyl sulfone 1 and 1,2,3-triazole-based tetrafluorophenoxymethyl ketone 2.

Figure 2

Crystal structure of the cruzain•2 complex (PDB ID 3IUT) elucidates the binding mode of 2 in the cruzain substrate-binding site. Cruzain residues are colored pale cyan and the inhibitor is colored grey. The unbiased mFo-DFc electron density for the inhibitor is shown in violet, contoured at the 3σ level. Black dashed lines indicate hydrogen bond interactions between inhibitor 2 and amino acid residues in cruzain’s catalytic pocket. The C(5) of the 1,2,3-triazole ring is labeled.

Figure 3

The proposed mechanism of inhibition for tetrafluorophenoxymethyl ketone inhibitor 2.

Figure 4

The putative position of a second inhibitor 2 molecule, bound at low occupancy in a solvent channel. Cruzain is colored pale cyan and the inhibitor grey. The unbiased mFo-DFc electron density for the inhibitor is shown in violet, contoured at the 3σ level.

Figure 5

Structural comparison of nonpeptidic inhibitors 2 and 8. (a) Chemical structures and second-order inactivation constants of tetrafluorophenoxymethyl ketone inhibitor 2 and vinyl sulfone inhibitor 8. (b) A superimposition of cruzain•2 (PDB ID 3IUT) and cruzain•8 (PDB ID 3HD3) crystal structures. The cruzain•2 complex is colored pale cyan, while the cruzain•8 complex is colored yellow. Black dashed lines indicate hydrogen bond interactions between the inhibitors and amino acid residues in cruzain’s catalytic pocket.

Scheme 1. Synthesis of 1,4-disubstituted-1,2,3-triazole cruzain inhibitor analogs.^a

^a Reagents: (a) isobutyl chloroformate, N-methylmorpholine, THF, −40 °C; (b) diazomethane, THF, 0 °C; (c) HBr, THF, 0 °C; (d) 2,3,5,6-tetrafluorophenol, KF, DMF, 0 °C; (e) 4Å molecular sieves, toluene, rt; (f) NaBH₄, MeOH, 0 °C; (g) Sodium ascorbate, CuSO₄, 1:1 H₂O:t-BuOH, rt.