Identification of a new class of nonpeptidic inhibitors of cruzain

Abstract

Cruzain is the major cysteine protease of Trypanosoma cruzi, which is the causative agent of Chagas disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the substrate activity screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored beta-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group p K a, with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second-order inactivation constant of 147,000 s (-1) M (-1). This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas disease.

Figure 1

Most advanced inhibitor of cruzain.

Figure 2

Alignment of cathepsin S (PDB ID: 2H7J) and cruzain (PDB ID: 1F2C) amino acid sequences with areas around the catalytic triad highlighted in yellow. Identical residues are indicated with “*” and similar residues are indicated with “.”. The figure was produced using Swiss-Pdb Viewer (http://ca.expasy.org/spdbv/).

Figure 3

(a) Crystal structure of cathepsin S (PDB ID: 2H7J) and (b) molecular replacement model of cruzain (PDB ID: 1F2C) with chloromethyl ketone inhibitor 14. The atoms are shaded according to element: protein carbons are green, inhibitor carbons are grey, nitrogens are blue, and oxygens are red. The figure was produced using PyMOL (www.pymol.org).

Figure 4

Time-dependence of (a) vinyl sulfone 38 and (b) β-chloro vinyl sulfone 43.

Figure 5

Mechanism of inhibition of cysteine proteases by (a) vinyl sulfones and (b) β-chloro vinyl sulfones.

Scheme 1

Fluorogenic substrate screening

Scheme 2

General Synthesis of Focused Library 1,2,3-Triazole Substratesa ^a Reagents: (a) CuI, i-Pr₂EtN, THF, rt; (b) R’CO₂H, triphosgene, THF, rt; (c) R’COH, NaBH(OAc)₃, AcOH, THF, rt; (d) CF₃CO₂H, CH₂Cl₂, rt.

Scheme 3

Synthesis of Vinyl Sulfone Inhibitor 38a ^a Reagents: (a) CuI, i-Pr₂EtN, THF, rt; (b) diazomethane, THF, rt; (c) DIBAL-H, Et₂O, −78 °C; (d) (OEt)₂P(O)CH₂SO₂Ph, LiBr, NEt₃, CH₃CN, −40 °C.

Scheme 4

Synthesis of β-Chloro Vinyl Sulfone Inhibitor 43a ^a Reagents: (a) diazomethane, THF, rt; (b) methylphenylsulfone, n-BuLi, THF, 0 °C to −78 °C; (c) Tf₂O, i-Pr₂EtN, THF, −20 °C to rt; (d) TBACl, THF, rt; (e) Na ascorbate, CuSO₄, 1:1 H₂O:t-BuOH, 39, rt.

Scheme 5

Synthesis of Aryl- and Acyl-oxymethyl Ketone Inhibitorsa ^a Reagents: (a) isobutyl chloroformate, N-methylmorpholine, THF, −40 °C; (b) diazomethane, THF, 0 °C; (c) HBr, THF, 0 °C; (d) KF, DMF, 0 °C; (e) Na ascorbate, CuSO₄, 1:1 H₂O:t-BuOH, rt.

Scheme 6

Synthesis of Diastereomerically Pure Aryloxymethyl Ketone Inhibitor 58a ^a Reagents: (a) NaBH₄, 95:5 THF:H₂O, 0 °C to rt; (b) Na ascorbate, CuSO₄, 1:1 H₂O:t-BuOH, rt; (c) Dess-Martin periodonane, CH₂Cl₂, rt.

Scheme 7

Configurational Lability of Aryloxymethyl Ketone Inhibitor 54