Figure 2. Inhibitory activity of compound 4b . ( A ) Inhibition of versicanase activity. ADAMTS-4 (5.5 nM) and -5 (0.4 nM) were incubated either with compound 4b or DMSO 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 ADAMTS-generated versican fragments (versikine) quantified by sandwich ELISA. The relative versicanase activity is presented and 100% activity corresponds to that in the presence of DMSO alone. ( B ) Inhibition of ADAMTS-5 aggrecanase activity. Compounds (Cpd) 4b , 5b and 6 were incubated with ADAMTS-5 (1 nM) for 2 h at 37 °C before addition of aggrecan (20 μg). Following SDS-PAGE and immunoblot, fragments cleaved at the Glu392 ↓ Ala393 bond were detected by a monoclonal neoepitope antibody recognizing the new C-terminal fragment (anti-ARGSV) and analyzed by densitometric analysis. Data are presented as mean ± SEM (n = 4). * p < 0.05 and *** p < 0.001, compared to DMSO controls; ## p < 0.01, compared to the same concentration of compound 6 (Mann–Whitney of GM6001 (Ilomastat, 0–800 nM), a known zinc-binder and broad-spectrum metalloproteinase inhibitor 22,24 , in