Characterization of a continuous fluorogenic assay for calpain I. Kinetic evaluation of peptide aldehydes, halomethyl ketones and (acyloxy)methyl ketones as inhibitors of the enzyme

Z-Leu-Arg-(7-methoxynaphthyl)amide (1) is a substrate for calpain I. The specificity constant for 1 (kcat/Km = 1405 ± 40 M−1s−1) is 10x greater than for any previously reported fluorogenic substrate. Using this substrate, a sensitive, continuous fluorogenic assay was developed permitting the identification of Z-(D)Ala-Leu-Phe-(OCO-2,6-Fl2-Ph) (69) as the first selective (>100-fold versus cathepsins B and L) time-dependent inhibitor of the enzyme.