Molecular interactions study
Covaxin is demonstrated to produce antibodies and show a robust response against the COVID-19 virus . It is approved in thirteen countries and shows minimal to no adverse events (https://covid19.trackvaccines.org/vaccines/approved/). However, there are some concerns about tolerability, molecular interactions, and safety. These issues were addressed here using the computational biology approach. The binding and interactions activity of HSA, ACE2, S Protein, and ingredients of the Covaxin were studied. The molecular interaction study provides the binding affinity of 2-phenoxyethanol, Imidazoquinolinone, S protein, ACE2, and HSA.
The molecular interaction study provided the binding affinity of 2-phenoxyethanol, Imidazoquinolinone, S protein, ACE2, and HSA. Analysis of the binding interactions of 2-Phenoxyethanol, imidazoquinolinone with virus-cell surface protein S protein, human cell membrane receptor ACE2 and drug carrier protein HSA was carried out using the Autodock Vina 1.1.2. The binding energies of the molecular interaction study were determined as ΔGb-5.3 Kcal/mol and ΔGb-8.5 Kcal/mol when ACE2 interacts with 2-Phenoxyethanol and imidazoquinolinone, respectively (Figure 1, Figure 2). In contrast, ΔGb-5.3 Kcal/mol and ΔGb-9.1 Kcal/mol were the energies, respectively, when HSA interacts with 2-Phenoxyethanol, Imidazoquinolinone (Figure. 3, Figure 4). On the contrary, the binding affinities scored to be ΔGb-5.2 Kcal/mol and ΔGb-8.5 Kcal/mol when S protein interacts with the above molecules (Figure 5, Figure 6).
The imidazoquinolinone bound S protein through multiple bonds and interactions, including Vander Waal's (Asn317, Ser316, Thr315, Thr761, Thr302, Tyr313, Thr768, Gln314, Asn764, Thr739); Conventional hydrogen bond (Thr302) Pi-Anion (Asp-737) Pi-alkyl (Cys760, Leu303) Alkyl (Arg765) (Figure 6). Imidazoquinolinone engage with ACE2 with Vander Waal’s (Ala348, Thr347, Glu402, His401, Trp69); Conventional hydrogen bond (Tyr385); Pi-Pi T-shaped (His378); Pi-Pi stacked (Phe390, Phe40); Pi-alkyl (Arg393); Salt bridge (Asp350, Asp382) (Figure 2). Furthermore, imidazoquinolinone engages with HSA by Van der Waal's (Met123, Phe134, Glu141, Tyr138, Phe157, Gly189, His146, Leu115); Conventional hydrogen bond (Tyr161, Leu185); Pi-sigma (Ile142); Pi-alkyl (Arg186, Lys190); Alkyl (Lys137) (Figure 4).
Similarly, 2-Phenoxyethanol bind with S protein of coronavirus by Van der Waal's (Gln564, Phe565, Val576, Phe543, Leu517, Cys391, Ala522, Leu518, Pro521); Carbon hydrogen bond (Asn544) Conventional Hydrogen Bond (Asn544); Pi-Alkyl (Leu546) (Figure 5), with ACE2 by Van der Waal's (Leu91, Asn210, Lys562, Ala396, Glu564); Carbon hydrogen bond (Pro565); Unfavourable donor (Trp566); Pi-alkyl (Val212, Val209); Pi-sigma (Leu95) and HSA by Van der Waal's (Asn391, Ala449, Leu387, Val433, Phe403, Tyr411); Conventional hydrogen bond (Cys392, Ile388); Pi-sigma (Leu453); Pi-alkyl (Leu430, Leu407) (Figure 1, Figure 3).
From the above molecular interaction data, it was observed that 2-Phenoxyethanol binds with RBD (Receptor Binding Domain) of S protein which spans from 319 amino acids to 591 amino acid residues in S protein . Similarly, imidazoquinolinone strongly interacts with S protein compared to 2-Phenoxyethanol with the proximity of the RBD site. Both compound form hydrogen bonds with S protein which, play an essential role in molecular interaction . The binding affinity and position of the above two compounds suggest that these molecules may cause hindering in S Protein function, which was also reflected in the protein-protein interaction study.
Imidazoquinolinone strongly interacts with the IB domain (Span from 108-196) of HSA with a binding affinity ΔGb -9.1 Kcal/mol, which plays a vital role in drug delivery [7,8]. Similarly, 2-Phenoxyethanol binds with IIIA domain (Span from 384-497) of HSA with a binding affinity ΔGb-5.3 Kcal/mol, an effective drug binding site .
The evidence above showed that Covaxin (2-Phenoxyethanol, imidazoquinolinone) ingredients show a robust binding affinity towards ACE2 and S protein HSA (Table 1). Imidazoquinolinone showed the highest affinity with S protein, ACE2, and HSA with energies ΔGb-8.5 Kcal/mol, ΔGb -8.5 Kcal/mol, and ΔGb -9.1 Kcal/mol, respectively.
Protein-protein interaction study
There are 10 top docking models listed on the ClusPro web server with various free energies. A grouping criterion was used based on the overall RMSD . Our study analyzed 5 ClusPro docking models chosen based on S Protein probability, S Protein with 2-Phenoxyethanol, and imidazoquinolinone to engage with the anticipated binding sites ACE2 as well as the lowest binding energy during such interactions. For the S Protein-ACE2 interaction, the average binding energy for all 5 binding sites is -901.2 kJ/mol. Nevertheless, the average binding energy for S Protein-ACE2 in the presence of 2-Phenoxyethanol is -696.64 kJ/mol, and imidazoquinolinone is -589.46 kJ/mol (Figure 7, Figure 8 and Figure 9; Table 2).
Protein-protein interactions are highly specific physical contacts created by electrostatic forces, hydrogen bonding, and hydrophobic interactions between two or more protein molecules [20,21]. An analysis of protein-protein interactions can provide important information about the molecular networks that comprise a living cell . Furthermore, the interaction of protein-protein plays a significant role in predicting the protein activity of molecules that target protein and drug ability . In the presence of 2-Phenoxyethanol, imidazoquinolinone, the binding energy of S protein, ACE2, was reduced during protein-protein interaction. In comparison to direct binding, S protein interaction with ACE2 in 2-phenoxyethanol resulted in a substantial decrease in the binding energy of 204.56 kJ/mol and 311.74 kJ/mol in the presence of Imidazoquinolinone (Figure 7, Figure 8, and Figure 9). As a result, it's possible that 2-phenoxyethanol, or imidazoquinolinone, can prevent the RBD site of S protein from attaching to the ACE2 receptor protein.
Drug likeliness analysis
We investigated the drug-likeness of the lead compounds, which are 2-phenoxyethanol and imidazoquinolinone, using the SwissADME tool. It determined the connection between the molecule's pharmacokinetics and physicochemical properties. The physicochemical properties of 2-Phenoxyethanol (C8H10O2) were determined, i.e.,138.16 g/mol molecular weight, molar refractivity of 38.90, 10 heavy atoms, 3 rotatable bonds, 2 hydrogen bond acceptors, 1 hydrogen bond donor, and topological polar surface area of 29.46 Å². Similarly, physicochemical properties for Imidazoquinolinone (C19H21N5O) were determined, i.e.,335.40 g/mol molecular weight, molar refractivity of 100.12, 25 heavy atoms, 2 rotatable bonds, 3 hydrogen bond acceptors, 1 hydrogen bond donor, and topological polar surface area of 66.81 Å². The average lipophilicity score of 2-Phenoxyethanol iLOGP, XLOGP3, WLOGP, MLOGP, and SILICOS-IT models computes to 1.35. The bioavailability Score of the molecule is 0.55. It has been noted that 2-Phenoxyethanol has very solubility in water. SwissADME uses five distinct criteria to predict drug-likeness (Lipinski, Ghose, Veber, Egan, Muegge). Lipinski, Veber, and Egan obey the drug-likeness property of 2-Phenoxyethanol, whereas Ghose and Muegge are not. Similarly, for lipophilicity score of Imidazoquinolinone iLOGP, XLOGP3, WLOGP, MLOGP, and SILICOS-IT models compute to 2.27. The bioavailability Score of the molecule is 0.55. It is observed that Imidazoquinolinoneis soluble in water. All 5 models render Imidazoquinolinonecompetent for an adequate drug molecule (Table ST1, ST2). The BOILED-Egg model assumes that 2-phenoxyethanol and imidazoquinolinone can easily pass through the blood-brain barrier and be absorbed by human gastrointestinal absorption (HIA) . Based on the model, the parameters such as lipophilicity, solubility, drug-likeness, and pharmacokinetics that imidazoquinolinone could be a potential drug candidate. The 2-phenoxyethanol's pharmacokinetics and drug-likeness properties have sparked debate about whether it should be considered a drug molecule. Swiss Target prediction, a webserver, plays a central, critical role in identifying ligand-target of known molecules [20,25,26]. It accurately predicts the targets to modulate their behavior, elucidating the molecular mechanism and predicting cross-reactivity in 2D and 3D similarity events with known ligands [27,28]. It also detects potential side effects and assists in the repurposing of molecules for new uses [25,29]. Using the SwissTarget method, 2-phenoxyethanol shows a high predictive performance level of interaction with A G and C G coupled receptors, kinases, enzymes, and nuclear receptors. The major drug-likeness targets from this prediction are A G coupled receptors and enzymes. In addition (Fig. SF4), imidazoquinolinone shows a variety of A G and B G coupled receptors, enzymes, and histone-modifying enzymes without affecting the vaccine ingredients' function (Fig. SF3).
Molecular interaction data suggested that imidazoquinolinone had a robust binding affinity compared to 2-Phenoxyethanol, corroborating the protein-protein interaction data. Imidazoquinolinone hinders maximally the S Protein ACE2 interaction as compared to 2-phenoxyethanol. Molecular interaction data also suggest that imidazoquinolinone binds to the RBD site of S Protein which may cause hindering in S Protein-ACE2 complex formation. Protein-protein interactions regulate various biological functions, including cell to cell interactions, metabolic regulation, and developmental control . This could open the door to repurposing/designing appropriate treatment to deter viral penetration using 2-Phenoxyethanol and imidazoquinolinone in vaccine covaxin. A molecule must achieve the target in optimum concentration and be usable in the bioactive form before the necessary biological events arise for it to be an effective drug. The SwissADME technology reduces the time and resources required for drug development. To be considered an oral drug candidate, development products' structural or physicochemical properties must be evaluated for drug-likeness. A molecule's drug-likeness is determined for bioavailability by qualitatively assessing the likelihood of the molecule is to be formed into an oral drug. Bioavailability Radar defines the optimal set of properties like lipophilicity, saturation, scale, polarity, size, and flexibility for the input molecule drug-likeness of 2-phenoxyethanol and Imidazoquinolinone (Fig. SF1, SF2). Most protein targets are predicted using experimentally defined vaccine adjuvants and molecular similarities, according to the study. These adjuvants are effective against virus entry but less effective against biological targets in humans.