The LuxI homologs in most of the gram-negative bacteria generate the signal molecules, AHL. Usually, these signals were detected by the LuxR homologs present in them. Whereas in Pseudomonas aeruginosa the LuxI homolog is not been found, which makes the organism not to generate the signals of their own. Hence these bacteria cannot sense the signals from the same species. Instead, it responds to the signals produced by the other pathogenic bacteria. However, it encodes a LuxR homolog, LasR which can sense the signal molecules produced by the mixed community genera [12, 13]. Thus LasR a transcriptional regulator was considered as a potential drug target.
The 3D structure of the target protein LasR from Pseudomonas aeruginosa was not available in any of the structural database, it was developed by using homology modeling method. The most homologous sequence in the Protein Data Bank was searched by using the BLASTP program. The BLASTP results showed that the Pseudomonas aeruginosa transcriptional regulator LasR is homologous with the structure CviR, LuxR- type transcriptional factor from Chromobacterium violaceum (PDB ID: 3QP5) over 40%. As all these sequences belong to the same family, the structure of 3QP5 was considered as a template structure for comparative modeling. The model was generated by using Swiss model webserver.
The multiple sequence alignment (Fig. 2) of LuxR family proteins LasR from Pseudomonas aeruginosa, CviR from Chromobacterium violaceum, and LasR from Escherichia coli, Pseudomonas aeruginosa, and Enterobacter aerogens showed that amino acids are conserved in LuxR family proteins. These alignments enlighten that LasR from Pseudomonas aeruginosa is almost conserved. Hence, the structure of LasR from Pseudomonas aeruginosa was considered for further docking studies.
Docking studies
A total of 19 compounds were found as the principle compounds of the Euphorbia hirta. The 3D structures of these compounds were retrieved as SD files from the Pubchem database and were docked with the amino acids in the binding site of CviR from Chromobacterium violaceum and LasR from Pseudomonas aeruginosa by using FlexX. Out of these 19 compounds, 17 compounds formed docking complex with all both CviR and LasR and its binding energies were analyzed by LeadIT (Table 1 and 2). Considering the binding energy score, the 3 best-docked compounds for each protein CviR and LasR were selected (Figs. 3 and 4) and their docking interaction with the active site residues were analyzed by using the pose view of LeadIT.
The binding interactions in the docking studies of Chromobacterium violaceum CviR and Pseudomonas aeruginosa LasR with the 3 best-docked compounds of the Phyllanthus emblica exposed the similar binding of AHL residues, that are responsible for Quorum sensing activity. This result indicates that in Chromobacterium violaceum CviR, it is found that Tryptophan (Trp84) and Aspartic acid (Asp86 & Asp97) plays a crucial role in exhibiting stronger interactions with ligands and these interactions were further supported by means of hydrophobic interactions by the contribution of Tyrosine (Try88). Similarly in Pseudomonas aeruginosa LasR, it is observed that Tryptophan (Trp67) and Aspartic acid (Asp80) are responsible for the bonded interactions with the ligands, and the non-bonded interaction, hydrophobic is facilitated by Tyrosine (Tyr 71 and Tyr 63).
The compounds CID_641785 (Cardamonin), CID_444539 (Cinnamic acid), and CID_5280442 (acacetin) exhibited the best docking scoring of -12.1467 kJ/mol, -11.5130 kJ/mol, and − 9.7346 kJ/mol respectively within the active site of CviR transcriptional regulator from Chromobacterium violaceum. It is observed that natural ligand 3-oxo-C6-HSL exhibited the docking score of -8.3776kJ/mol. Thus among the docked compounds, it is revealed that the compound of all the three compounds CID_641785, CID_444539, and CID_5280442 is having the highest docking score when compared to that of the natural ligand. Thus these compounds can be used to inhibit the quorum sensing mechanism in Chromobacterium violaceum.
The compounds CID_641785 (Cardamonin), CID_5481240 (Retusin), and CID_10212 (Imperatorin) exhibited the best docking scoring of -14.8740 kJ/mol, -13.5553 kJ/mol, and − 13.2575 kJ/mol respectively within the active site of LasR transcriptional regulator from Pseudomonas aeruginosa. It is observed that natural ligand 3-oxo-octanoic acid exhibited a docking score of -8.3989 kJ/mol. Thus among the docked compounds, it is revealed that the compound CID_641785 is having the highest docking score when compared to that of the natural ligand. Thus this compound can be used to inhibit the quorum sensing mechanism in Pseudomonas aeruginosa.
The overall docking results of principle compounds with CviR and LasR proteins disclose the importance of the interacting amino acids Tryptophan, Aspartic acid, and Tyrosine (Y71). The docking studies revealed the necessary crucial hydrogen bond interactions with the critical amino acids and that of the compound Cardamonin (CID_641785) from Euphorbia hirta, with the highest binding score and might have a better inhibition activity against the quorum-sensing regulation of Pseudomonas aeruginosa.