*2.3. Direct Inhibition Studies*

NSGMs were screened for the inhibition of MMP-8 using a fluorogenic substrate assay, as described earlier [20]. Briefly, 100 µg/mL pro-MMP-8 was activated by incubating with 1mM p-aminomercuric acetate at 37 ◦C for 1 hr. Activated MMP-8 (10 nM, final) was then incubated with NSGMs (100 µM, final) in Tris-buffered saline containing 10 mM CaCl2,1 µM ZnCl2, pH 7.5 for 10 min at 37 ◦C. Residual MMP-8 activity was measured by adding the fluorogenic substrate (DNP-Pro-Leu-Ala- Tyr-Trp-Ala-Arg, 20 µM, final) and monitoring initial linear rate of increase in fluorescence. IC50s were calculated using the following equation, *Y* = *Y*<sup>0</sup> + *YM*−*Y*<sup>0</sup> <sup>1</sup>+10(*log*[*I*]0−*logIC*50)×*HS* . Y is the ratio of residual enzyme activity in the presence of NSGM to that in its absence, Y<sup>0</sup> and Y<sup>M</sup> are the minimal and maximal values of Y, respectively, obtained following regression, and HS is the Hill slope of inhibition.

Screening of sulfated benzofurans and quinazolinones (100 µM, final) against MMP-9 was performed using a commercially available assay kit (Abcam catalog #ab139448) following manufacturer's protocol. All experiments were performed at least twice in duplicates.
