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 test).