Fig 1.
Structures of nitrile-based cysteine protease inhibitors.
Fig 2.
Routes used for the synthesis of the dipeptidyl nitrile cruzain inhibitors.
Fig 3.
Structural variations at P1, P2 and P3 for dipeptidyl nitrile cruzain inhibitors.
Table 1.
Inhibition of cruzain by dipeptidyl nitriles.
Table 2.
Data collection and refinement statistics.
Fig 4.
Non-additivity in dipeptidyl nitrile SAR.
Values of pKi shown in parentheses and ΔpKi values are associated with the arrows. Differences between ΔpKi values on opposite sides of the ‘square’ reveal mutually reinforcing effects of replacement of the P3 phenyl with 1-methyl, 3-t-butylpyrazol-5-yl and introducing a 3-chloro substituent on the P2 phenyl.
Fig 5.
X-ray crystal structure of compound 5 covalently bound to cruzain (chain A), solved to a resolution of 3.13 Å and deposited at PDB with ID code 4QH6.
The inhibitor is colored green and the final 2Fo-Fc electron density map (contoured at 1σ level) is shown in cyan. The catalytic Cys25 and the main residues involved in binding to the inhibitor are also shown. Black dashed lines represent hydrogen bonds. Figure prepared with CCP4mg software [52].
Fig 6.
Superposed binding modes of compound 5 and a structural analog bound to cathepsin L (PDB code 3HHA) onto a molecular surface representation of cruzain taken from the crystal structure of 5.
Carbon atoms colored green for compound 5 in complex with cruzain and ice blue for cathepsin L analog complex. Oxygen, nitrogen, sulfur colored red, blue and yellow respectively. The binding subsites and the main residues involved at the binding are labeled. Figure prepared with CCP4mg software [52].
Fig 7.
(a) Michaelis-Menten, (b) Lineweaver-Burk and (c) reversibility assay plots compound 5.
The effect of increasing concentrations of compound 5 over the rate of reaction shown in (a) and the convergence of double-reciprocal plots over the y-axis characterizes compound 5 as a competitive inhibitor relative to the substrate Z-FR-AMC. Additionally, the recovery of enzyme activity after abrupt dilution of a solution containing cruzain and compound 5 after incubation reveals that this compound is acting by a reversible type of inhibition.
Fig 8.
In vitro screening of nitrile derivatives (250 μM) for cytotoxicity using Balb-c 3T3 cell line.
One way-ANOVA analysis for each day using benznidazole (Bzn) as control: p < 0.0001 (****), <0.001 (***), < 0.01 (**), < 0.05 (*). MTT is the control for cell growth without the addition of compounds.
Table 3.
Trypanocidal activity of dipeptidyl nitriles against Tulahuen and CL-Brener strains of T. cruzi.