Figure 4.

Rapid dilution assay for SMAIs. (A) Dilution scheme for testing the reversibility of a SMAI. Upon 100-fold dilution, the inhibitor concentration decreases from $10-\mathrm{f}\mathrm{o}\mathrm{l}\mathrm{d}>K_i^{*\mathrm{a}\mathrm{p}\mathrm{p}}$ (91% inhibition) to $10-\mathrm{f}\mathrm{o}\mathrm{l}\mathrm{d}<K_i^{*\mathrm{a}\mathrm{p}\mathrm{p}}$ (9% inhibition). (B, C) Cruzain activity rapidly recovered from inhibition by 2 and 6–10. (D–F) Cruzain activity recovery showed a significant lag for 1, 11, and 12. All curves were fitted to eq 1 to provide $k_{\text{obs}}$ values as listed in Table S1.