2.1.1. Docking of the Target Compounds into MMP-2 Catalytic Domain

All the selected compounds showed favorable binding, demonstrating ∆G (binding free energies) values in negative Kcal.mol−<sup>1</sup> , as shown in Table 1. The most promising candidates were found to be lincomycin, atenolol, and ciprofloxacin, which were able to accommodate into the MMP-2 active site with the highest binding energy score (∆G = −29.06, −28.20, and −27.87 kcal/mol, respectively) and bind the active site essential residues via several hydrogen bonding, electrostatic, and hydrophobic interactions. An analysis of the binding modes of the co-crystalized ligand (**I52**) and our top hits was then performed for a comparative study of how well our compounds conform to the intended design.

The 2D and 3D interactions of **I52** (Figure 5) revealed that the pentylbenzamide moiety formed three hydrophobic interactions with Leu137, Phe148, and Leu150, besides two hydrogen bonds with Ile141 and Thr143. Moreover, the sulfamoylphenyl moiety formed three hydrogen bonds with Leu 82, Leu83, and Ala84 and three hydrophobic interactions with Leu83 and His120, and the zinc ion. The morpholine ring interacted with His130 via a hydrogen bond and the isopropyl moiety showed a hydrophobic interaction with His85. Finally, the ZBG (hydroxamate) interacted with the zinc group as well as Glu121 via a hydrophobic interaction.


**Table 1.** The calculated ∆G (binding free energies) of the tested drugs against MMP-2 (PDB ID 1HOV).

**Figure 5.** The 2D and 3D interactions of the co-crystallized ligand (**I52**) with amino acid residues of the catalytic domain of MMP-2 (PDB ID: 1HOV).

As described in Figure 6**,** the docking pose of Lincomycin into the MMP-2 active site showed that the *2H*-pyran arm was engaged in six hydrophobic interactions with Leu116, His120, Leu137, and Tyr142, in addition to two hydrogen bonds with Ala139 and Ile141. The sugar moiety formed three hydrogen bonds with Leu83, Ala84, and Glu121. Finally, the methyl thio group formed two hydrophobic bonds with His130. Although lincomycin showed the highest binding score, it lacked the ability to interact with the zinc of the CAT. As a result, it was excluded from subsequent studies.

**Figure 6.** The 2D and 3D interactions of lincomycin with amino acid residues in the catalytic domain of MMP-2 (PDB ID: 1HOV) (hydrogen bonds = green dashed lines, electrostatic interactions = orange dashed lines, pi-pi interactions = deep pink dashed lines, and pi-alkyl interactions = light pink dashed lines).

Although atenolol interacted with the active site with a relatively lower binding, it did also bind successfully to zinc through a hydrophobic pi-cation interaction (Figure 7). Additionally, it formed five hydrogen bonds with Leu83, Ala84, Glu121, Thr142, and Thr143 through its acetamide and hydroxy moieties. Three hydrophobic interactions were observed with His120, Leu137, and Arg149.

**Figure 7.** The 2D and 3D interactions of atenolol with amino acid residues in the catalytic domain of MMP-2 (PDB ID: 1HOV).

Ciprofloxacin (a fluoroquinolone drug) occupied the MMP-2 active site via the formation of six hydrogen bonds and eight hydrophobic interactions. The fluoroquinolone nucleus formed three hydrogen bonds with Leu83 and Ala84, as well as six hydrophobic interactions with zinc, Leu83, and His120. The piperazine arm was incorporated in three hydrogen bonds with Leu137, Ala139, and Thr143, as presented in Figure 8.

**Figure 8.** The 2D and 3D interactions of ciprofloxacin with amino acid residues in the catalytic domain of MMP-2 (PDB ID: 1HOV).

2.1.2. Docking of the Target Compounds into MMP-9 Catalytic Domain

The tested compounds revealed to be able to bind into MMP-9 and showed negative ∆G (Kcal.mol−<sup>1</sup> ) scores, as shown in Table 2. It was found that ampicillin, aztreonam, and ganciclovir, the most promising candidates, achieved the highest energy score and accommodated into the MMP-9 active site (∆G = −30.81, −29.97, and −28.89 kcal/mol, respectively).

By examining the binding interactions of the co-crystalized ligand (**NFH**) to the active site of MMP-9, it showed six hydrogen bonds with Gly186, Leu188, Ala189, Tyr393, and Tyr423, in addition to two hydrophobic interactions with Leu188. The hydroxamate group interacted as expected with the zinc of the CAT, as presented in Figure 9.


**Table 2.** The calculated ∆G (binding free energies) of the tested drugs against the catalytic domain of MMP-9 (PDB ID: 1GKC).

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**Figure 9.** The 2D and 3D interactions of the co-crystallized ligand (**NFH**) with amino acid residues of the catalytic domain of MMP-9 (PDB ID: 1GKC).

This high binding score of ampicillin is attributed to the formation of six hydrogen bonds, five hydrophobic interactions, and zinc binding as well. The 3,3-dimethyl moiety of ampicillin was involved in three hydrophobic bonds with Leu187, His411, and Pro421, while the carboxylic acid and 7-oxo groups formed hydrogen bonds with Leu187, Leu188, and Tyr423. The phenyl acetamide moiety interacted with the active site by three hydrogen bonds with Leu397 and Val398, as well as two hydrophobic interactions with Leu418 and Arg424. Finally, the sulfur atom of the thiazolidine ring was able to interact with the zinc ion of the CAT through a metal–acceptor bond (Figure 10).

**Figure 10.** The 2D and 3D interactions of ampicillin with amino acid residues of the catalytic domain of MMP-9 (PDB ID: 1GKC).

On the other hand, aztreonam was unable to bind to the zinc ion of the CAT. An investigation of the top docking pose of aztreonam showed that it interacted with the MMP-9 active site by forming five hydrogen bond interactions (Leu188, Tyr420, Met422, and Tyr423) and nine hydrophobic interactions (Val398, His401, His405, His411, Met422, and Tyr423) (Figure 11).

**Figure 11.** The 2D and 3D interactions of Aztreonam with amino acid residues of the catalytic domain of MMP-9 (PDB ID: 1GKC).

Finally, the binding of ganciclovir was through four hydrogen bonds with Leu188, Glu402, and Pro421. Four hydrophobic interactions were also detected (Val398, His401, and Tyr423). It was also able to bind with zinc ion through a metal–acceptor interaction (Figure 12).

Because atenolol and ampicillin were the most promising compounds that achieved high docking scores and similar binding modes to co-crystallized ligands with the ability to interact with zinc ions in both enzymes, they were both promoted for further analysis through molecular dynamics.
