The structure-based drug design (SBDD) technique was employed for studying the interactions between the already existing analogues of DAP and the enzyme’s active site. Further, SBDD was utilized for designing the new analogues, thio-DAP and oxa-DAP (one and two). The SBDD approach increased the inhibitor binding affinity and cellular potency towards the target site. However, very few investigations on the target characteristics of this amino acid in the development of antibacterial drugs is reported. Inhibitors against the enzyme dapF of the lysine biosynthetic pathway were developed. In the present work, compounds one and two were developed in accordance with the reported analogues’ structure-activity relationships and studies of the active sites of the enzymes (Figure 2) [16,28]. In addition, structural DAP analogues with carboxyl groups replaced with heterocycle moieties, such as thiazole and oxazole, as well as the introduction of a sulphur moiety at 2–3 carbon lengths from the terminal heterocycle, were designed and synthesized. These analogues underwent testing as dapF enzyme inhibitors and, consequently, as antibacterial agents. Because of their potential biological effects as antibacterial, herbicidal, antitubercular, fungicidal, anti HIV, pesticidal, antiinflammatory, antiprotozoal, antihypertensive, and schizophrenia medications, medicinal chemists are increasingly interested in designing oxazole and thiazole heterocyclic analogues [18,29]. These heterocyclic analogues mimic the important aspects of protein structures or their functions and thus act as bioactive molecules. These heterocycles contain oxygen, nitrogen, or sulphur atoms, which provide interactions with the target site like enzymes, receptors and nucleic acids via forces, such as hydrogen bonding, hydrophobic interactions, weak Vander Waals forces, and ionic bonding, as in the case of captopril binding with ACE [30]. These substances were designed and synthesized to act as reversible or irreversible inhibitors of lysine pathway enzymes [16,31]. These DAP analogues might most likely be changed into analogues of tight-binding transition states at the dapF active site [14]. Because these heterocycles act as an important facet of protein structures and provide interactions with the target site or enzymes, they exhibit excellent competitive dapF inhibitory activity [16,22]. The synthesized analogues did not exhibit any of the examined toxicity parameters in tests of toxicity risk assessments utilizing the protox-II property explorer. These molecules may therefore serve as good potential candidates for novel antibacterial compounds. 3.3. Molecular Docking Study(a)

Protein structure designing

The protein structure preparation (PDB ID: 5BNR) was performed using the protein preparation wizard within the software programme Maestro (version 11.5, implanted in Schrodinger) (made by Schrodinger.com in New York, USA). The pre-processing of proteins was conducted with the help of Prime, in which the addition of missing side chains was incorporated, and restrained minimization was also performed. The addition of hydrogen bond assignments was also performed. Finally, no problem was found in the protein structure. While the inner water molecules were preserved, the solvent water molecules near the 6-OCH3 location, as well as the waters beyond 5.0 A˚ from the ligand, were removed. The pH value was set to 7.0 ± 2.0, and Epik was used to produce the states of the ligands. The PROPKA program’s H-bond assignment was used to optimize the water molecules’ orientations, and the pH level was set to 7.0. The minimization of the energy of the added hydrogens was conducted via the OPLS3 force field. For the generation of the grid, the protein of compound three in the complex with dapF was utilized.

The LigPrep programme with default parameters was used for the preparation of the ligands. An OPLS3 force field was used for energy minimization. A ligand docking module was used to carry out the docking studies. No constraints were applied, and all the parameters were left at their default values. The glide SP mode was used for the docking of the compounds.

The ligand-target docking was performed using the “Grip-Based Docking Tool” from the wizard within Maestro in Schrodinger for analyzing the structural complex of our target protein, viz., dapF, with LL-DAP and compounds one and two, for understanding the structural basis of this protein’s target specificity. The ligand and protein interactions were revealed by choosing the conformers from the file. The purified protein was opened, and grid generation was performed with a cubic box of specific dimensions centered on the protein cavity. Then, the ligand conformers were selected along with the folder simultaneously. The output folder was selected to save the results of docking. The protein-ligand interactions were investigated for the hydrophobic and hydrophilic properties of these complexes in order to understand the binding affinity of the ligand towards the protein.

We chose compounds one and two for molecular docking and compared them with a natural substrate, LL-DAP. The docking studies suggested that compounds one and two interact more ardently with dapF than LL-DAP. The anticipated binding free energies (kcal/mol) were used to determine the molecular docking scores [40,41]. Each of our compounds’ docked positions was evaluated, and the pose with the lowest binding free energy was selected. Figure 3 shows the hydrogen bond interactions between compound one and the specific amino acid residues in the target protein, viz., dapF. The strongest ligand-protein affinity is shown by the best dock score, which has the lowest binding free energy. Compounds one and two had anticipated binding free energies of −9.823 and −10.098 kcal/mol with dapF, respectively, while LL-DAP, the natural substrate, had a binding free energy of −9.426 kcal/mol. This indicates that our synthesized molecules have a greater dock score and a more stable conformation with dapF than LL-DAP, thereby concluding their greater target-ligand affinity compared with LL-DAP.The docking studies of compound one revealed good binding interactions with dapF compared to LL-DAP, which had a binding energy of −9.823 kcal/mol with dapF. Compound one formed eight hydrogen bonds with active site residues Asn74, Asn159, Asn194, Glu212, Arg213, and Thr223 at bond distances of 2.45, 0.81, 3.24, 1.82, 2.07, and 2.73 A˚, whereas LL-DAP formed twelve hydrogen bonds, as shown in Figure 3a. As shown in Table 4, the active site residues ASN74 and ARG213 formed two hydrogen bonds, one with the carbonyl oxygen and the other with the amine of the L-terminal of compound one, with short distances of 0.98, 3.24, 1.82, and 3.47 A˚, respectively. Figure 3a depicts a hydrophobic interaction between compound one and dapF that involves the active site residues Phe17, Tyr72, Ala80, Met82, Cys83, Pro160, Val215, and Cys221. The hydrophobic interaction of compound one with the active site residues (Cys83 and Cys221) of dapF is associated with the nitrogen of the thiazole ring of compound one at the site of epimerization on the enzyme’s catalytic site. The active site residues, Cys83 and Cys221, are the main catalytic residues involved in the epimerization of LL-DAP to meso-DAP, as shown in Figure 3a. The negatively charged residue of dapF Glu212 is involved in the hydrogen bonding and ionic interaction with NH3+ of the L-terminal of compound one. As shown in Figure 3, the polar active site residues of dapF ASN15, ASN74, ASN85, ASN159, ASN194, and THR223 are associated with an electronegative inductive attraction with the atoms nitrogen, sulphur, the two oxygens of thiazole, and the NH3+ group of compound one’s L-terminal. One of the positively charged active site residues of dapF viz., Arg213, is involved in the ionic interaction with the negatively charged carbonyl oxygen of the L-terminal of compound one, which is actually a salt bridge. The glide H-bond value was found to be −1.532, which indicates the formation of a stable hydrogen bonding network between dapF and compound one as compared to the dapF complex with LL-DAP. All the hydrophobic and hydrogen bonding interactions were observed in both terminals of compound one, viz., the epimerization terminal and L-terminal (the substrate recognition terminal).The binding free energy value obtained for compound two with dapF was −10.098 kcal/mol, which is slightly better compared to compound one (−9.823 kcal/mol) and LL-DAP (−9.426 kcal/mol). Thus, these values are lower than those shown by both compound one and LL-DAP. These docking studies suggest that compound two has a greater affinity and potency. Compound two establishes H-bonds with the amino acid residues Asn74, Asn159, Asn194, Arg213, Glu212, and Thr223 of dapF with the cumulative bond distances of 2.42, 0.78, 0.83, 2.26, 1.05, 2.83, 2.09, and 1.88 A˚, as presented in Figure 3b and Table 4. The active site residues of dapF, viz, Asn74 and Arg213, formed two hydrogen bonds, one with the carbonyl oxygen and another with the amine in the L-terminal of compound two, with the bond distances of 0.83, 2.26, 1.05, and 2.83 A˚, respectively, as provided in Table 4. Compounds one and two interact hydrophobically with dapF via the active site residues Phe17, Tyr72, Ala80, Met82, Cys83, Pro160, Val215, and Cys221 (Figure 3b). The hydrophobic interaction with the active site residues of dapF, viz., Cys83 and Cys221, is also associated with the nitrogen of the oxazole ring of compound two (similar to the case of compound one) at the site of epimerization on the catalytic site of this enzyme. The negatively charged residue of dapF, viz., Glu212, is involved in the hydrogen bonding and ionic interaction with the NH3+ L-terminal of both compounds (one and two). As shown in Figure 3a,b, some polar active site residues of dapF, namely Asn15, Asn74, Asn85, Asn159, Asn194, and Thr223, are associated with an electronegative inductive attraction with the atoms nitrogen, the oxygen of oxazole, and two oxygens and the NH3+ group of the L-terminal of both compounds one and two. Arg213 is involved in the ionic interaction with the carbonyl oxygen at the L-terminal of both compounds one and two (a salt bridge). The glide H-bond value was found to be −1.757 kcal/mol, which indicates the formation of a stable hydrogen bonding network between dapF and compound two as compared to the dapF complex with LL-DAP. All the hydrophobic and hydrogen bonding interactions were observed in both terminals of compound two, viz., the epimerization terminal and L-terminal (the substrate recognition terminal). The active site residues of dapF, viz., Cys83 and Cys221, are the main catalytic residues involved in epimerization, as represented in Figure 3b and Figure 5. The side chains of Cys83 and Cys221 were found to play an important role in the acid-base catalysis performed by this enzyme, dapF. DapF catalyzes epimerization by employing a “two-base” mechanism. The stereoinversion involves two active site cysteine residues acting in concert as a base (thiolate) and an acid (thiol). During the design of compounds one and two, we replaced the central carbon atom of LL-DAP with a sulphur atom, which involves neither H-bonding nor hydrophobic interactions. This means that the isosteric replacement of the central carbon with sulphur, nitrogen, oxygen and other atoms did not affect the binding properties of the molecule at the active site.The binding free energy of the natural substrate LL-DAP was found to be −9.426 kcal/mol with dapF, which is more than both compounds one (−9.823 kcal/mol) and two (−10.098 kcal/mol). The energy value of the conformers shows that LL-DAP has a higher docking score and less affinity than compounds one and two. LL-DAP was found to establish 12 H-bonds with the active site residues of dapF, viz., Asn15, Asn74, Gly84, Asn85, Glu212, Arg213, Asn159, Asn194, Gly222, and Thr223 with the bond distances of 1.34, 1.42, 4.22, 1.13, 4.53, 2.14, 1.21, 2.75, 2.23, 1.41, 3.14, and 3.31 A˚, respectively, while compounds one and two formed 8 H-bonds each as shown in Figure 3a–c. The active site residues of dapF, viz., Glu 212 and Arg213, formed two hydrogen bonds each, one with the carbonyl oxygen and another with the amine of the L-terminal of LL-DAP, with the bond distances of 1.13, 4.53, 1.42, and 4.22 A˚, respectively, as provided in Table 4. DapF H-bonding residues Asn74, Asn159, Asn194, Glu212, Arg213, and Thr223, are common in hydrogen bonding scenarios with compounds one, two, and LL-DAP, though the bond lengths differ slightly. LL-DAP shows hydrophobic interactions with dapF that involve its active site residues, viz., Phe17, Tyr72, Ala80, Met82, Cys83, Val215, Val215, and Cys221 (Figure 3c). The amino acid residues of dapF, viz., Cys83 and Cys221, were found to be crucial in the epimerization and binding. These Cys83 and Cys221 residues govern the epimerization of LL-DAP. At a neutral pH, these amino acid residues exist as rapidly equilibrating thiolate-thiol pairs in the presence of the substrate. Both these residues viz., Cys83 and Cys221 of dapF appear in complex formation with compounds one, two, and LL-DAP. The negatively charged residue of dapF, viz., Glu212, is involved in the hydrogen bonding and ionic interactions with the NH3+ of the L-terminal of compounds one, two, and LL-DAP. Some polar active site residues of dapF, viz., Asn15, Asn74, Asn85, Asn159, Asn194, Thr218, Ser220, and Thr 223, are associated with an electronegative inductive attraction with two carboxylic groups and the NH3+ group of both the L-terminals of LL-DAP, as shown in Figure 3c. One of the positively charged active site residues of dapF, viz., Arg213, is involved in the ionic interactions with the carbonyl oxygen of the L-terminal of compounds one, two, and LL-DAP (a salt bridge). The glide H-bond value was found to be −1.455 kcal/mol, which indicates the formation of a stable hydrogen bonding network between dapF and LL-DAP. All the hydrophobic and hydrogen bonding interactions were observed in both the terminals of compound one, viz., the epimerization terminal and L-terminal (the substrate recognition terminal).