*2.2. Molecular Dynamics and Molecular Mechanics–Generalized Born Surface Area (MM-GBSA) Calculations*

The potential of atenolol and ampicillin to bind to MMP-2 and MMP-9, respectively, was further investigated through molecular dynamics. This allows the extensive analysis

of the binding modes under realistic physiological conditions. Both protein files with the corresponding drugs were processed by the Schrodinger Maestro suite and simulated for 50 ns. The RMSD of the protein residues (Figure 13) in both complexes showed uniform values around 3.7 and 1.75 A<sup>0</sup> deviation for MMP-2 and MMP-9, respectively. Furthermore, both atenolol and ampicillin exhibited stable conformations with an RMSD (Supplementary Figures S1 and S2) of around 1.2 and 1.8 A<sup>0</sup> , respectively.

**Figure 13.** Root mean square deviation (RMSD) of *C*-alpha of MMP-2 (**A**) and MMP-9 (**B**) complexes with atenolol and ampicillin through 50 ns simulations.

Additionally, the flexibility of the conformers was assessed through the calculation of the root mean square fluctuations (RMSF) of the residues of the proteins and ligand atoms across the simulation time. Consistent with the calculated RMSD, the protein residues showed a low degree of fluctuations, especially with the ones in contact with the ligands as shown in Figure 14. The RMSF of the protein residues showed around 3.5 A<sup>0</sup> fluctuations of the residues exposed to drugs, while the fluctuations were lower in the case of both ligands, especially ampicillin which showed around 1A<sup>0</sup> only (Supplementary Figures S3 and S4). These uniform values obtained points to the relative stability of both proteins and drugs conformations for the entire simulation duration.

**Figure 14.** Root mean square fluctuation (RMSF) of *C*-alpha of MMP-2 (**A**) and MMP-9 (**B**) complexes with atenolol and ampicillin through 50 ns simulations. (Ligand contacts are marked green.).

For a better understanding of the binding modes, a further analysis of the interactions throughout the whole 50 ns simulation time was performed (Figure 15). For MMP-9, ampicillin successfully formed metal coordination with the zinc ion constantly through its amide carbonyl group. On the other hand, the interaction of atenolol with the zinc ion of MMP-2 was not observed; however, it interacted extensively with the binding site residues as well compensating this inability as shown in Figure 16.

**Figure 15.** *Cont*.

**Figure 15.** The 2D ligand-protein contact summary of atenolol-MMP-2 (**A**) and ampicillin-MMP-9 (**B**) complexes through 50 ns simulations.

One of the most commonly used methods for calculating the binding free energy is molecular mechanics–generalized born surface area (MM-GBSA). The lower a ligandprotein complex's projected binding free energy is, the more stable the complex is expected to be, and the higher the ligand's activity and potency (Table 3). Both complexes showed stable binding throughout the dynamic simulation.


**Table 3.** The MM-GBSA binding free energies (Kcal.mol−<sup>1</sup> ) of MMP-2/atenolol and MMP-9/ ampicillin complexes.
