We appreciate the comments of the reviewer and the following are the answers in response to the question for the kind consideration of the reviewer- Tyrosinase is a key enzyme in melanogenesis, which is essential for pigmentation. Dysfunction of tyrosinase may cause skin cancer. It is well-known that tyrosinase of Bacillus Megaterium bacteria is an attractive target for the development of antimicrobials or antibiotic adjuvants for the treatment of hyperpigmentation because of its similarity (33.5%) to the human enzyme [97-103]. That’s why, to investigate and compare the antimicrobial activity of the synthesized compounds with in vitro data, docking analysis of L1 and L2 against tyrosinase from Bacillus Megaterium was performed. Also, molecular docking with other proteins of B. Megaterium was conducted, but 46ju provided better binding affinity with the synthesized compounds. For this reason, we aim to study the structure of tyrosinase from the bacteria. Preliminary antimicrobial studies were carried out to validate the docking protocol. At present, we are doing the fluorescence activities of these ligands and L2 showed excellent fluorescent emission at λmax 520 nm. So, we hope that we will carry out ALA scan (Alanine scanning) based MD experiments and 100ns Molecular dynamics (MD) simulation guided protein-ligand stability in the future for our next study since within this shortest time, we are unable to do these experiments. The active binding site of tyrosinase was predicted by CASTp. We have included this in the manuscript as follows: To investigate and compare the antimicrobial activity of the synthesized compounds, docking analysis of L1 and L2 against tyrosinase from Bacillus Megaterium were performed. It is well-known that tyrosinase of Bacillus Megaterium bacteria is an attractive target for the development of antimicrobials or antibiotic adjuvants for the treatment of hyperpigmentation because of its similarity (33.5%) to the human enzyme [97-100]. The binding energies for L1 and L2 with Bacillus Megaterium were −7.7 and −8.8 kcal mol−1, respectively, which were calculated by AutoDock Vina. The interactions of the 4j6u with compounds L1 and L2 are shown in the Fig. Results of docking studies revealed that L1 and L2 formed bonds to the active site of tyrosinase and showed strong interactions with Tyr267A, Ala44A, and ALA44B of tyrosinase enzyme (PDB ID: 4j6u), which also supports the literature [101-103]. Thus, computational results are in good agreement with in vitro antibacterial behaviour of our compounds for novel antibacterial drug design.
4. 3. 2. Third one their binding study: 1. Why did the authors consider Tyrosinase from Bacillus megaterium N205A mutant pdb id 4j6u? As they did not report any enzyme specific inhibition related experiments. Docking is a preliminary experiment. How did the author validate the docking protocol? The authors are suggested using some decoy ligands and calculating the enrichment value in order to justify the docking protocol followed by ALA scan (Alanine scanning) based MD experiments. In addition, authors are suggested to report at list 100ns Molecular dynamics (MD) simulation guided protein-ligand stability report followed by either MM-GB or PB-SA based thermo data analysis. The pdb id 4j6u does not contain any bound ligand. How did the author select the binding site? If a few sets of amino acids were considered to define the binding site, then on what basis they select them? The authors did not report any Tyrosinase enzyme inhibition assay, they are requested to justify Tyrosinase inhibition as the probable mechanism of action for these sets of ligands by providing suitable literature references.