Molecular docking virtual screening, one of the methods applied in structure-based drug design, was used to screen fifty sets of EGFR inhibitors in order to identify hit compound that could be used to design new EGFR inhibitors by investigating their binding interactions in the active site of EGFR tyrosine kinase enzyme (3IKA) (Supplementary Table 2). The binding affinities of this docking study presented in Supplementary Table 2 in kcal/mol range from − 7.1 kcal/mol to − 9.3 kcal/mol. Also, all possible interactions (hydrogen bond, hydrophobic, and electrostatic interactions) between the ligands and the target protein were shown in the same table. The results of the best top 10 compounds under investigation with higher binding affinities/lower docking scores is presented in Table 1 out of which the best three are discussed.
The best three hit compounds identified were compound 6 with the highest binding affinity of − 9.3 kcal/mol, followed by compounds 5 and 8, each with the binding affinity of − 9.1 kcal/mol, respectively. The best hit compound 6 bound to EGFR tyrosine kinase receptor via four different types of interactions including conventional hydrogen, carbon-hydrogen, electrostatic, and hydrophobic bond interactions, respectively. Nitrogen one (N1) of the quinazoline ring of compound 6 binds to EGFR tyrosine kinase receptor via conventional hydrogen bond interaction with GLN791 amino acid residue with a bond distance of 2.87813 Å. PRO794 and GLY796 amino acid residues formed carbon-hydrogen bonds with the active site of the receptor each with a bond distance of 3.44185 Å and 3.37399 Å, respectively. EGFR tyrosine kinase receptor interacted also via three hydrophobic interactions with PHE723, LEU718, and VAL726 amino acid residues with different parts of the compound, respectively. Furthermore, electrostatic interaction was observed between compound 6 and the active site of the receptor with LYS745 and ASP855 amino acid residues, respectively.
Compound 5 was found to bind with the active site of EGFR tyrosine kinase receptor via two hydrogen bonds (conventional and carbon-hydrogen), four hydrophobic, and two electrostatic interactions. The conventional hydrogen bonds were between the hydrogen attached to one of the nitrogen of the quinazoline ring, oxygen of the acrylamide moiety, and fluorine on the benzyl ring of compound 5 with ASP855 (2.8768 Å), LYS745 (2.83195 Å), and CYS797 (2.80635 Å) amino acid residue of the receptor, respectively. The carbon-hydrogen bond was between the carbon and oxygen attached to the tetrahydrofuran-3-yl moiety and also carbon of the flourobenzyl ring with ASN842, PRO794, and GLY796 amino acid residues of the receptor each with a bond distance of 3.65564 Å, 3.22827 Å, and 3.2556 Å, respectively. A different part of the ligand was also observed to bind with the binding pocket of the receptor via two electrostatic and four hydrophobic bonds with ASP855 (2), LEU718, VAL726 (2), and PHE723 amino acids.
Compound 8 among the hit compounds formed two conventional hydrogen bonds with GLN791 and ASP855 amino acid residues, with a bond distance of 2.92147 Å and 2.69743 Å, respectively. Carbon-hydrogen bonds were also observed between the compound and the active site of the receptor with PRO794 (3.3526 Å) and GLY796 (3.30683 Å) residues, respectively. The compound was further observed to bind with the active site of the receptor protein via hydrophobic and electrostatic interactions with LYS745, LEU718, and VAL726 amino acid residues, respectively.
The common amino acid residues to these hit compounds were GLN791, LYS745, PRO794, GLY796, LEU718, and VAL726, respectively. These amino acid residues might be responsible for their higher binding affinities. Furthermore, Fig. 3a, b shows the 2D structures of compounds 6 and 8 using discovery studio visualizer.
Drug-likeness and ADME property prediction of the studied compounds
The drug-likeness and ADME properties of all the compounds under investigation were predicted using SWISSADME online web tool. The drug-likeness and ADME properties of all the compounds are presented in Supplementary Tables 3 and 4 following the notable Lipinski’s rule of five. It states that the permeation of an orally administered compound is more likely to be better if the molecule satisfies the following conditions: (i) hydrogen bond donors ≤ 5 (OH and NH groups), (ii) hydrogen bond acceptors ≤ 10 (N and O atoms), (iii) molecular weight < 500, (iv) calculated WlogP < 5 (v), and TPSA ≤ 140, respectively. Any compound that violates more than two of these conditions may have bioavailability-related problems. The results of the best top 10 compounds are presented in Tables 2 and 3.
All the compounds satisfied the Lipinski’s rule of five without violating more than one of the conditions stated except compound 16 which has two violations. Thus, predicting their good permeability properties, easy transportation, absorption, and diffusion. The number of hydrogen bond acceptors and donors for all the compounds under investigation was less than 5 and 10, respectively, per the notable RO5. From all these predicted parameters, it can be predicted that all the compounds under investigation including the three hit compounds can be orally bioavailable and also orally active as they obeyed the notable RO5.
For a drug to be orally active, it is expected to have high gastrointestinal absorption and all the compounds under investigation including the three hit compounds exhibited high gastrointestinal absorption except compounds 2, 16, 21, 34, and 39 with low gastrointestinal absorption. None of them was seen to permeant through the blood-brain barrier indicating lower toxicity. The most vital factor indicating good absorption was recognized to be the bioavailability score (which give the amount of drug present in the plasma). All the compounds under investigation including the three hit compounds were found to have high bioavailability scores of 0.55 except compound 16 which has a lower bioavailability score of 0.17. P-gp substrate served as a defender to the central nervous system (CNS) from xenobiotics. Also, the P-gp substrate of all the compounds under investigation including the 3 hit compounds was predicted. Some were found to be a substrate to P-gp while some were not. Moreover, synthetic accessibility scores refer to how easy a compound can be synthesized in a laboratory and scales of easy to hard range between 0 and 10. Synthetic accessibility score of all the compounds under investigation including the three (3) hit compounds was less than 5 showing that they can be easily synthesized in the laboratory. The compounds under investigation including the three hit compounds were predicted to have good pharmacokinetic profile and drug-likeness except compound 16 [24, 25].
Based on the virtual screening carried out using molecular docking and pharmacokinetic studies on quinazoline derivatives, compound 6 with the highest binding affinity of − 9.3 kcal/mol, good pharmacokinetic profile, and drug-likeness property was identified as the best hit compound in the analogs. Compound 6 being the best hit was used as the template for designing new compounds. Sixteen new compounds (Table 4) were designed by carrying out structural modification on the meta position of the flourobenzyl ring of compound 6 (the template). Studying the designed compounds, the addition of phenyl-amino rings and halo substituted phenyl-amino rings on the meta position of the flourobenzyl ring attached to “oxy-phenyl amino ring” moiety of the template significantly increase the binding affinities of the designed compounds.
Molecular docking investigation of the newly designed compounds
The newly developed compounds were also docked at the active site of EGFR tyrosine kinase receptor (PDB code: 3IKA). Table 5 shows the docking results of all the newly designed compounds in the active site of the target protein (EGFR tyrosine kinase receptor). The binding affinities of these newly designed compounds range from − 9.5 kcal/mol to − 10.2 kcal/mol. When compared with the template and the control afatinib, the docked-designed compounds were seen to have better binding affinities than the template (f − 9.3 kcal/mol) and the control (− 7.9 kcal/mol).
Designed compound SED10 was found to be the best among all designed compounds with a binding affinity of − 10.2 kcal/mol. It interacted with the binding pocket of EGFR tyrosine kinase receptor via three conventional hydrogen bonds, two carbon-hydrogen bonds, four halogen bonds, three electrostatic bonds, and nine hydrophobic bonds with the following amino acid residues: ARG841, GLN791, GLY796, LYS745 (2), ASP855, LEU844, LEU718 (2), PRO877 (2), LYS875, VAL726, ALA743, and LEU858, respectively (Table 5).
The second-best among the designed compounds was SAD14. It bounded to the binding pocket of its target via three carbon-hydrogen bonds, four halogen bonds, three electrostatic bonds, and nine hydrophobic bonds with the following amino acids GLN791, GLY796, ASP837 (2), LYS745 (3), ASP855, LEU844, PHE723, PRO877, LEU718 (2), ALA743, VAL726, and LEU858, respectively.
The following amino acid residues GLN791, GLY796, LYS745, ASP855, LEU844, LEU718, PRO877, VAL726, ALA743, and LEU858 were common to the best two designed compounds. This may be the reason they have higher binding affinities. Furthermore, Afatinib, the positive control, was used to validate the docking procedure in this study, then compared with the designed compounds. The designed compounds were observed to have better binding affinities than Afatinib with a binding affinity of − 7.9 kcal/mol. The 2D-structures of designed compound SED10 and SED14 in complex with the receptor were presented in Fig. 4a, b.
Drug-likeness and ADME prediction of the newly designed compounds
The drug-likeness of the newly designed compounds was also predicted following Lipinski’s rule of five (Table 6). None of the designed compounds was found to violate more than two of the permissible limit set by the Lipinski’s rule of five filters for small molecules. Based on that, their permeability across the cell membrane, easy transportation, absorption, and diffusion was predicted [24, 26].
ADME properties of these newly designed compounds were also predicted (Table 7). All were observed to have low gastrointestinal absorption. But none was observed to permeate through the brain indicating lower toxicity. All designed compounds have higher bioavailability score of 0.55 except compounds 9, 13, and 14 with lower bioavailability scores of 0.17. Interestingly, all the designed compounds have a good synthetic accessibility score of < 5 except molecule 9 with the synthetic accessibility score > 5 (5.16) which indicates that these designed compounds can be easily synthesized in the laboratory [25, 27].