*2.3. Pharmacophore Study*

The prospect of repositioning ampicillin for KC treatment may seem beneficial especially for complicated cases suffering from secondary bacterial infections. However, its unattended use for prolonged periods of time increases the risk of bacterial resistance. For this purpose, we extended our study on the MMP-9 inhibitors to propose pharmacophoric features with a high potential of a MMP-9 inhibitory goal for future use.

The co-crystallized ligand of MMP-9 (PDB ID: 1GKC) was used to generate the pharmacophore model using the Discovery studio software. In this test, the protocol of receptorligand pharmacophore generation was applied. In this protocol, the software identifies the essential features of **NFH** (co-crystalized ligand) during its interaction with the receptor. The library was then screened, and fit value data were calculated (Supplementary Table S1). Ampicillin, aztreonam, cephalexin, and lincomycin achieved the highest fit value, even comparable to **NFH,** as shown in Table 4.


**Table 4.** Fit value of the top four compounds and the co-crystallized ligand of MMP-9 (PDB ID: 1GKC).

The formed pharmacophore model consisted of six features: two H-bond donors (HBD), two H-bond acceptors (HBA), and two hydrophobic centers (H). The features were distributed in a pyramidal shape with each feature occupying a 1.6 A<sup>0</sup> radius (Figure 17). In accordance with the data acquired so far, the overlay of each compound on the pharmacophore (Figure 18) demonstrates the ability of the compounds to span across the entire pharmacophoric features in a similar fashion to the co-crystallized inhibitor **NFH**.

**Figure 17.** (**A**) The generated 3D pharmacophore geometry with six features: two hydrogen bond donors (pink color) and two hydrogen bond acceptors (green), and two hydrophobic centers (blue). (**B**) The 3D-pharmacophore with vector direction of each feature.

**Figure 18.** Mapping of the tested compounds on the generated pharmacophore: (**A**) **NFH** on the generated pharmacophore (fit value = 4.74), (**B**) ampicillin (fit value = 3.76), (**C**) aztreonam (fit value = 4.13), (**D**) cephalexin (fit value = 4.06), and (**E**) lincomycin (Fit value = 3.76).
