Fig 1: Inhibition of ADAM10 and 17 proteolytic activities using a kinetic enzymatic cleavage assay. (A) Kinetic cleavage of a fluorogenic TNF-α substrate peptide ES003 by recombinant ADAM10 or 17 (R&D systems, Inc) in the presence and absence of 2 μM BB-94. In the presence of BB-94, the kinetic ES003 cleavage curves by ADAM10 and 17 are indistinguishable from that of ES003-only curve without the enzyme. (B,C) Inhibition of ADAM17 (B) or ADAM10 (C) cleavage of ES003 by various compounds at 2 μM concentration. All statistics are calculated between no inhibitor and respective inhibitor data sets using multiple t-tests with p-values indicated above their data. The data are a representative of two repeated experiments. (D,E) Inhibition of ADAM10 (D) and ADAM17 (E) enzymatic activities by titrating amount of BB-94, GI254023X, prinomastat, and 14mb. The compound concentrations are indicated on the x-axis. The enzymatic activities measured at 4-h time point of the reactions are normalized against zero compound condition and plotted on the y-axis. The inhibition curves are fitted with non-linear regression with inhibition constants (Ki) for each compound listed in Table 2. (F) Enzymatic cleavage of a fluorogenic CD62L peptide by recombinant ADAM10 or 17. The CD62L cleavage curves in the presence of 2 μM BB-94 are indistinguishable with that of fluorogenic peptide CD62L-only without the enzyme. (G,H) Inhibition of ADAM10 cleavage of CD62L substrate peptide by various compounds at 2 μM concentration. (I) Inhibition of ADAM10 cleavage of CD62L by titrating amount of 14mb. P-values are ****<0.0001.