*3.3. Computational Analysis of the Preferred Site of NSGMs Binding to MMP-8*

To identify a plausible site of binding of the active sulfated benzofurans and sulfated quinazolinones, we performed genetic algorithm-based molecular docking and scoring studies. Docking was performed using the available MMP-8 crystal structure (PDB ID: 5H8X) and AutoDock 4.2

(Scripps Research). The studies revealed that the sulfate group of NSGM **26** interacts with the catalytic Zn2<sup>+</sup> and the triad of histidine residues (H197, H201, and H207, Figure 5A,B). This is similar to the previously reported interactions of MMPs with the phosphate groups [48]. Additionally, the oxygen atoms of the furan ring and the linker form a bidentate interaction with S151 (Figure 5B), and the aromatic substituent at the R<sup>4</sup> position contributes towards hydrophobic and cation-Π interactions with residues in the unprimed S<sup>2</sup> and S<sup>3</sup> pockets. In contrast, other sulfated benzofurans did not have any interaction with S151, either because of their sub-optimal chain length (NSGMs **15**–**25**) or the bulk of their substituents (NSGMs **27**–**35**). This explained the observed lack of MMP-8 inhibition with these NSGMs. Π

**Figure 5.** (**A**) Surface representation of MMP-8 (cyan) showing the potential interacting sites of sulfated benzofuran **26** (blue) and sulfated quinazolinone **38** (magenta). The S<sup>1</sup> ′ pocket and specificity loop are shown in pale yellow and orange, respectively. (**B**,**C**) Polar interactions of NSGM **26** (**B**, shown in sticks) and NSGM **38** (**C**, shown in sticks) with MMP-8 (cartoon). Interacting residues of MMP-8 are shown as lines, catalytic Zn+<sup>2</sup> ion as a sphere, and polar interactions as grey dotted lines.

In the manner of NSGM **26**, sulfated quinazolinone dimers **36**–**42** also bound near the catalytic site of MMP-8 (Figure 5A,C). For NSGM **38**, one monomeric unit of the quinazolinone was found to insert deep into the S<sup>1</sup> ′ pocket (shown as a pale-yellow surface in Figure 5A), which resulted in a strong binding of the sulfate with L193, L214, A220, and R222 (Figure 5C). The insertion of the quinazolinone ring into the S<sup>1</sup> ′ pocket positioned the triazole linker close to the catalytic Zn+<sup>2</sup> ion. However, based on these experiments, NSGM **38** did not make polar interactions with the catalytic Zn+<sup>2</sup> or the histidine triad. Instead, the nitrogen atom of the triazole ring, along with the nitrogen of the quinazolinone ring form hydrogen bonds with the backbone oxygen of P217. Additional polar, hydrophobic, and cation-Π interactions were observed with multiple residues present in the site of binding (see Table S2). The insertion of sulfated quinazolinone dimers (**36**–**42**) in the S<sup>1</sup> ′ pocket of MMP-8 and their interactions with the residues in the S<sup>1</sup> ′ specificity loop is particularly interesting (Figure 5A). Such GAG-like molecules inhibiting MMP-8 by binding in the electropositive S<sup>1</sup> ′ pocket have the potential to be selective towards MMP-8. Thus, the identification of NSGM hits in this work is likely to be of significant value for both drug discovery and chemical biology campaigns against MMP-8.

Several other aspects of structure–activity dependence were also observed. For example, when one of the molecules of quinazolinone in the dimers that inhibited MMP-8 (**38**, **40**, **41**, and **42**) was replaced with quercetin, the resulting quercetin–quinazolinone heterodimers (**43**–**46**) lost their inhibition towards MMP-8 (Figure 3). Although the core scaffold of quinazolinones and quercetins studied here are very similar, i.e., fused bicyclic ring attached to a phenyl group, the presence of three sulfate groups on the quercetins compared with one sulfate on the quinazolinones adds both steric bulk and negative charges. Based on computational modeling, this addition of steric bulk drives the heterodimeric molecules away from the S<sup>1</sup> ′ pocket (Figure S1), resulting in a loss of MMP-8 inhibition.
