Identification of PKC-directed ligands from different sources
3.3 Results
3.3.4 Selected molecules bind strongly to PKC under in-silico conditions and form many interactions with the C1b domain of PKC: After the docking simulation was over, the PKC-ligand
molecular models were taken for analysis. This final PKC-ligand structure was analyzed in molecular visualization software PyMOL v0.99 (Schrodinger, 2010). Molecular docking study indicates that Danazol, Flunarizine and Cinnarizine fits well into the polar binding groove of the C1b domain of PKC- α (Figure 3.4).
Figure 3.4: Selected molecules fit well into the C1b domain. Danazol (blue), Flunarizine (orange), Cinnarizine (magenta), Chlorogenic acid (yellow), Epigallocatechin gallate (green), Gallic acid (cyan) and β-Glycyrrhetinic acid (red) fit well into C1b domain. All the molecules bind to the polar ligand binding pocket of the C1b domain of PKC-α. Docked complexes of the ligands with the C1b domain were visualized in pyMOL using the “vaccum electrostatics” option.
Similarly, docking studies have also revealed that many phytochemicals bind to the C1b domain at the same binding site as PMA albeit with minor variations in the ligand orientations about the lipophilic binding pocket (Figure 3.4).The molecular interactions between ligand and receptor help us to understand better the affinity of a particular ligand for a receptor. These molecular interactions between bound ligand molecules and PKC C1b domain residues were sketched using LigPlot+ software (Wallace et al., 1995).
All the three selected drugs Danazol, Flunarizine and Cinnarizine, were analysed for interactions with PKC C1b domain. It is seen that all the three drugs fit well & deep, as well as span the entire binding pocket of the C1b domain. They form many hydrophobic interactions as well as strong hydrogen bonding interactions which indicate strong binding characteristics. Robust hydrophobic and hydrogen bonding interactions (Figure 3.5) is expected to confer a high level of stability to ligand-PKC complexes.
The Danazol-PKC molecular model analysis indicates extensive interaction of bound Danazol with the protein residues lining the C1b domain. It forms many hydrophobic interactions as well as strong
Chapter 3|89 hydrogen bonding interactions. Danazol makes hydrophobic interaction with Pro 28, Tyr 25, Leu 41, Gly26, Tyr 39, Gln44, Ser 27 and Thr 29 (Figure 3.5). Also, it makes two hydrogen bonds with Leu 38 and Gly 40. Flunarizine forms hydrophobic interactions with the amino acid residues His 23, Thr 24, Leu 41, Gly 26, Pro 28, Gly 40, Gln 44, Ser 27 and Leu 38 of the C1b polar binding pocket (Figure 3.5). It also forms hydrogen bonding interactions with Tyr 25 and Thr 29. Similarly, Cinnarizine formed hydrophobic interactions with the amino acid residues Leu 37, Tyr 39, Leu 38, Gln 44, Gly 40, Thr 29, Tyr 25, Ser 27, Gly 26, Leu 41 and Pro 28 of the polar binding pocket (Figure 3.5). But cinnarizine doesn’t form any hydrogen bonding interaction.
Figure 3.5: Molecular interaction of PKC-α with Danazol, Flunarizine and Cinnarizine. Danazol, Flunarizine andCinnarizine form stronger hydrogen bonding interactions with PKC. The PKC-ligand molecular models were used to draw molecular interaction using LigPlot+ software.
Similarly, it was revealed that many other molecules utilize a combination of hydrogen bonding and hydrophobic interactions to position themselves most efficiently for binding to the lipophilic pocket.
This strategy ultimately results in an optimal reduction of free energy of the system which is relatively indicated by the docking scores predicted by Autodock software. Similarly, the selected phytochemicals β-Glycyrrhetinic acid, Gallic acid, Epigallocatechin gallate & Chlorogenic acid were also analysed for interactions with PKC C1b domain. All the selected phtochemicals also fit well & deep, as well as span the entire binding pocket of the C1b domain. In addition, they form many hydrophobic interactions as well as strong hydrogen bonding interactions. The interaction analysis of the molecules Chlorogenic acid, Gallic acid, Epigallocatechin gallate and β-Glycyrrhetinic acid are summarized in Figure 3.6.
Except for β-Glycyrrhetinic acid, all other phytochemicals are observed to utilize hydrogen bonding interactions in addition to hydrophobic interactions for binding to the ligand binding domain of C1b domain. Chlorogenic acid makes hydrophobic interaction with Pro 28, Gln 44, Leu 37, Ser 27, Tyr
25, Gly 26 and Thr 24 (Figure 3.6). In addition, it makes three hydrogen bonds with Thr 29, Leu 41 and Leu 38. On the other hand, Gallic acid makes hydrophobic interaction with Pro 28, Gln 44, Gly 26 and Tyr 39, whereas it makes hydrogen bonding interactions with Thr 29, Gly 40, Leu 38, Tyr 25 and Ser 27 (Figure 3.6). Epigallocatechin gallate makes hydrophobic interactions with Tyr 25, Leu 41, Gly 26, Pro 28, Ser 27, Leu 37 and Gly 40, while it makes hydrogen bonding interactions.
Figure 3.6: Molecular interaction of PKC-α with phytochemicals. Except β-Glycyrrhetinic acid, all other phytochemicals form stronger hydrogen bonding interactions with PKC. The PKC-ligand molecular models were used to draw molecular interaction using LigPlot+ software.
with Gln 44, Thr 29 and Leu 38. However, β-Glycyrrhetinic acid only makes hydrophobic interactions with Tyr 25, Leu 41, Leu 38, Gly 26, Ser 27, Gln 44, Leu 37, Tyr 39, Pro 28 and Gly 40 (Figure 3.6).
To further understand the strong binding affinity of these ligands to the C1b domain of PKC, all the selected PKC-directed molecules were sent to Dr. Debasis Manna’s laboratory, Department of Chemistry, IIT Guwahati for in-vitro ligand binding assay. The dissociation constant (KD) of each ligand
FLUNARIZINE
Chapter 3|91 was determined and given to us by them. The dissociation constant (KD) of Flunarizine, Cinnarizine, Danazol, Chlorogenic acid, β-Glycyrrhetinic acid, Gallic acid and Epigallocatechin gallate towards PKC is given in Table 3.6.
Table 3.6: Dissociation constants (KD) of PKC-directed molecules S No. Molecule Dissociation constant, KD (μM)
1. Danazol 5.64±1.27
2. Flunarizine 4.51±0.51
3. Cinnarizine 10.75±1.87
4. Chlorogenic acid 28.84±3.95
5. β-Glycyrrhetinic acid 10.14 ± 1.13
6. Gallic acid 0.91 ± 0.11
7. Epigallocatechin gallate 0.88 ± 0.14
The dissociation constant (KD) of in-vitro binding of PKC directed molecules towards the C1b domain of PKC. Each of the KD values was calculated by a fellow student in Dr.
Debasis Manna’s laboratory, Department of Chemistry, IIT Guwahati.
Thus, all the top hit molecules make extensive interactions with most of the amino acid residues lying around the periphery of the ligand binding pocket of the C1 domain. The binding of all the ligands to the C1b domain under in-silico and in-vitro conditions confirms that Danazol, Flunarizine, Cinnarizine, Chlorogenic acid, β-Glycyrrhetinic acid, Gallic acid and Epigallocatechin gallate are strong ligands of PKC.