Mechanistic and Structural Understanding of Uncompetitive Inhibitors of Caspase-6
Figure 4
Compound 3 inhibition of caspase-6 is dependent on the substrate’s amino acid sequence and the P1’ character of the substrate.
(A) Concentration-response analysis of compound 3 against caspase-6 cleavage of divalent R110-containing substrates with VEID (black), DEVD (red), IETD (blue) or WEHD (green) amino acid tetrapeptides. Each assay was performed using substrate concentrations within 3-fold of the Kmapparent. (B) Concentration-response analysis of compound 3 against caspase-6 cleavage of monovalent VEID-based substrates with R110 (black) or AMC (blue) fluorophores conjugated to the C-terminal aspartate residue. (C) The indicated concentration of compound 3 or VEID-CHO was incubated with caspase-6 and GST-Lamin A prior to detection of cleaved Lamin A by western blotting. Only VEID-CHO was capable of inhibiting caspase-6 cleavage of recombinant Lamin A. Concentration response curves were generated in duplicate and represent 1 of at least 3 experiments with similar results. Each curve is normalized to zero and 100% based on no enzyme or DMSO, respectively. Western blot data represents 1 of at least 2 experiments.