Figure 3. Compound 4b binds to the ADAMTS-5 Mp/Dis domains in the presence of GM6001. ( A , B ) Compound 4b inhibits versicanase activity of both full-length (FL) ADAMTS-5 and its variant containing only the Mp/Dis domains (ADAMTS-5 MD). Compound 4b (100 μM) or DMSO was incubated either with ADAMTS-5 FL (0.2 nM, A ) or ADAMTS-5 MD (26 nM, B ) for 2 h at 37 °C before addition of V1-5GAG (50 nM). At each time point, reactions were stopped by addition of EDTA and versikine fragments quantified by sandwich ELISA as reported in the Methods section. In the DMSO control, complete proteolysis was achieved after 7200 s. ( C , D ) Inhibition of ADAMTS-5 peptidolytic activity by the active-site inhibitor GM6001 in the presence of compound 4b . ADAMTS-5 MDTCS was incubated with active-site inhibitor GM6001 (0–800 nM) either in the presence of DMSO or compound 4b (10 μM) for 2 h at 37 °C before addition of QF peptide. The synergistic effect exerted by compound 4b is more evident at low concentrations of GM6001 (panel D ). The relative peptidolytic activity is presented and 100% activity corresponds to that in the presence of DMSO alone. Data are presented as mean ± SEM (n =