Identification of PKC-directed ligands from different sources
3.4 Discussion
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.
substantial diversity in the preference of the ligand-C1b domain complex for various biological membranes and even for different lipid micro-domains in the same membrane. This differential translocation potential of diverse ligands to different micro-domains of a variety of biological membranes in the cell directly results in different biological responses of PKC signalling.
Phytochemicals have been designed by evolution to target important cellular signalling networks that result in modulation of key physiological processes (Cojocneanu Petric et al., 2015; Surh, 2003).
Phytochemicals also serve as lead molecules for the design and synthesis of advanced derivatives targeted against important human ailments (Cojocneanu Petric et al., 2015). However, isolation of a new phytochemical from natural sources is time-consuming. Moreover, it is tough to isolate every phytochemical in the purest form from the crude plant extract preparations. Chemical synthesis of phytochemicals as well as their analogues in the laboratory is costly and also a time-consuming affair. It is indeed in such situations where virtual docking techniques come handy (Geromichalos, 2007). Virtual docking approaches significantly enhance the number of molecules screened as well as eliminate the need for in-vitro analysis of each and every pre-considered natural or synthetic molecule (Meng et al., 2011).In the current study, molecular docking was performed by Autodock 4.1 software in order to screen two broad categories of ligands, heterocyclic compounds and phytochemicals. Top-hit ligands against the C1b domain of PKC were identified amongst both heterocyclic compounds and phytochemicals. An agonist competition assay enabled the selection of only those ligands that bind in a similar manner to a previously well-known agonist. We have also obtained a series of novel chemically synthesized molecules known as alkyl cinnamates from our collaborator lab. Design, synthesis, molecular docking analyses and in-vitro ligand binding analyses of these alkyl cinnamates by Mamidi et. al. showed that some of these compounds strongly interact with the C1b subdomain (Mamidi et al., 2012). KD values of in-vitro PKC binding ranged from 3 μM to 21 μM (Mamidi et al., 2012). As it has already been confirmed by in-silico and in-vitro studies that alkyl cinnamates interact with PKC by our colleagues, we haven’t repeated any such experiments again. Hence, in the current chapter experiments related to alkyl cinnamates are not presented. Any subsequent experiments with alkyl cinnamates are described in detail in later chapters.
The following figure (Figure 3.7) illustrates the structure of all of the alkyl cinnamates. Curcumin is the parent molecule of alkyl cinnamates which is designated as ‘DM 2-CRMN’.
Chapter 3|93 Figure 3.7: Chemical structure of different alkyl cinnamates with their respective compound codes. DM 2- CRMN is curcumin.
Agonist binding to a receptor is dictated by the strength of the interactions (inter-molecular forces) between the ligand and receptor. Hydrogen bonding and hydrophobic interactions are considered to be more significant interactive forces operating between ligands and receptors apart from other interactions such as ionic, salt bridges and van der Waals forces (Patil et al., 2010). This is due to the fact that generally the hydrogen bonding and hydrophobic interactions are numerous between ligand and receptor; as such, they outweigh the contribution of sparingly present other kinds of interactive inter-molecular forces.
Virtual docking softwares rely mainly on calculations from the hydrogen bonding and hydrophobic interactions between ligand and receptor (Meng et al., 2011; Patil et al., 2010). In the current study, in- silico screening experiments complemented with in-vitro ligand binding studies actively suggest that certain compounds exist in all the three categories of molecules screened that can serve as excellent ligands for PKC. Thus, PKC ligands prevail in all possible classes of molecules existing throughout the world. Finding them is only a matter of effort and time.
A few conclusions can be inevitably drawn from the experiments described in this chapter.
1. Detailed interaction analysis revealed that the top hit ligands are bound to the receptor by both hydrophobic and hydrogen bonding interactions.
2. Visualization of ligand-receptor complexes also highlighted that ligands can be structurally different but bind to the same binding site with very similar binding energy. These structurally different ligands utilize different portions of themselves to bind to the same site of the C1 domain. Having identified the strongest ligands on the basis of their binding affinity to PKC, it was inevitably intriguing to explore the effects on cancer-cell cellular physiology on the treatment of these molecules.