In vitro and in vivo studies of the trypanocidal properties of WRR-483 against Trypanosoma cruzi

Abstract

Background: Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas' disease.

Methodology/principal findings: WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 A crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported.

Conclusions: We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas' disease.

Figure 1. Structure of vinyl sulfone inhibitors, K11777 (1) and WRR-483 (2).

The P₁ –P₃ subsites of K11777 are labeled.

Figure 2. Synthesis of WRR-483 (2):

(a) (i) benzyl alcohol (BnOH), 4-dimethylaminopyridine, 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-methylmorpholine, CH₂Cl₂; (ii) piperidine, CH₂Cl₂, 90% (two steps); (b) (i) triphosgene, sodium bicarbonate (NaHCO₃), CH₂Cl₂; (ii) N-methylpiperazine, CH₂Cl₂, 93% (two steps); (c) H₂, 5% palladium on carbon (Pd/C), MeOH, 92% (d) (i) trifluoroacetic acid (TFA), CH₂Cl₂; (ii) 6, N-hydroxybenzotriazole (HOBT), 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-methylmorpholine, dimethyl formamide (DMF), CH₂Cl₂, 84%; (g) 3:1 TFA:CH₂Cl₂, 86%.

Figure 3. Structure of WRR-483 bound to cruzain

. (A) Crystallographic unit of the WRR-483-cruzain complex. (B) View of the active site of cruzain. The catalytic Cys25 and residues involved in binding to the inhibitor are also shown. Figures prepared with PyMol.

Figure 4. Comparison of the binding modes of WRR-483 and Z-RA-FMK.

Superimposition of the cruzain-WRR-483 structure (blue) on cruzain-Z-RA-FMK (yellow). Bound inhibitors are colored as their respective cruzain model. The glutamate residue in the S₂ pocket which binds to the guanidine moiety of the inhibitor (Glu205 and 208 for Z-RA-FMK and WRR-483, respectively) are highlighted. Figure prepared with PyMol.