4. Discussion
Antibody mimetics is a new technique based on highly-structured scaffolds originating from natural proteins. Mutation in their key residues, especially in variable loops, can alter their affinity towards new targets [18]. In the present study, we conducted a CDR grafting technique to find new binders with a high affinity toward EGFR. In our report, we retrieved the extracellular domain 3 of EGFR from EGFR/panitumumab complex (5SX4) to be used as the target in our structure-based virtual screening method for 36 CDR-acceptors designed by loop alteration technique. Further, the docking hits were applied for molecular dynamic (MD) simulation, and the result was analyzed by different parameters such as RMSF, H-bond, and MMPBSA. Therefore, the current study was undertaken to analyze the behavior of the designed complexes using protein-receptor complex against domain III EGFR. In addition to the scFv structure originating from panitumumab, three grafting scaffolds among all designed ones were selected to be docked against EGFR DIII. Then, the complex of protein-EGFR DIII was simulated through molecular dynamic analysis to be compared in their binding site. Based on the previous research on the crystal structure of the panitumumab-EGFR complex[19], three CDR loops H2, H3 from the heavy chain, and L3 from the light chain of panitumumab with the most effective interaction against receptor selected to be grafted in new scaffolds. The docking scores and analyzing the CDR loop positions against receptor were used as the main criteria for the binder selection. Therefore, we focused on the three top hits with the best docking scores that share a similar binding site to the reference molecules. The epidermal growth factor protein binding by EGFR occurs within a cleft between two extracellular domains, DI and DIII [20]. The binding site of panitumumab partially overlaps the EGF binding site and prevents the dimerization of the EGFR ectodomain and its activation [19]. The overall binding of panitumumab to domain III is similar to cetuximab. Based on a mutational analysis, Voigt et al. described critical amino acids overlap in panitumumab and cetuximab epitopes with EGF binding site: K443 and D355 in panitumumab and D355, K443, Q408, H409, S468 in cetuximab binding site [21]. The panitumumab complex with EGFR DIII shows that upon binding, all 6 CDRs in the heavy and light chains interact without any conformational alteration. According to previous studies, CDRs L3, H2, and H3 commonly make the largest contribution to antigen binding [9]. The L3 CDR lines up adjacent to the final B-strand of domain III with two interactions of D92 and L94. The heavy chain showed most of the specific hydrogen bond interactions made by H2 and H3. Most notably, three tyrosine residues interacted with the B-strand of domain III at the same position as EGF. Y54, Y55, N58, and T59 in H2 interacted with N384, D420, K443, respectively, while in H3, D100 has a water-mediated hydrogen bond with S468. H3 makes an additional hydrogen bond between T103 and K465 of domain III [19]. It can be visualized that interaction with S468 had the highest interaction rate (Fig.5). This residue interacts with EGF, panitumumab, cetuximab, scFv, and all three designed scaffolds. The scFv structure interacted with critical residues of S468, Q408, and H409 through 5 H-bonds in CDR regions which overlap with the cetuximab binding site. Scaf1 interacted with the same epitope as cetuximab through 3 H-bonds in inserted CDR loops against S468 and Q408. Scaf2, in addition to S468, interacted with D355, which is involved in both panitumumab and cetuximab binding sites. It indicates that this scaffold mimics the function of these two antibodies. In contrast to the other two scaffolds, Scaf3 could not make any hydrogen interaction in CDR regions with critical residues in the panitumumab binding site. However, Q1 in Scaf3 interacted with S468 and made hydrophobic contact with F30 against F412 in the panitumumab binding site. It shows that Scaf3 can be used as an alternative to panitumumab but with lower binding affinity. Therefore, all three scaffolds that exhibit the interaction with the same amino acid residues (S468, D355, Q408, and H409) are subjected to a 50 ns simulation process. The MD results confirmed the stability of scFv and three selected scaffolds throughout the simulation. The RMSF was calculated for domain III of EGFR, scFv, and three designed binders. It refers to the stability of the complex as high fluctuations related to more flexible and unstable bounds. Although there are some fluctuations in Scaf2 amino acid residues, they are not at the inserted CDR loops nor involved in protein interaction. The fluctuation during all interactions was below 0.2 nm, which is totally acceptable. To validate the docking energy of the protein-receptor complex, an MMPBSA calculation was performed. The designed structures presented comparatively acceptable MM-PBSA scores compared to the scFv structure. The calculated binding free energy of these binders were -26.4, -39.89, -9.47, -13.51 KJ mol-1, for scFv, Scaf1, Scaf2, and Scaf3, respectively. Therefore, they represent excellent candidates for further investigation in vitro analysis. The only exception is the Scaf3 complex which showed a slightly lower MMPBSA value which is in accordance with its visual analysis results. By analyzing the binding energy and stability through dynamic simulation, we have shown that scFv, Scaf1, and Scaf2 may be potential binders that mimic the function of panitumumab. We tested in vitro and in vivo function of scFv structure against EGFR (unpublished data), which confirmed the results of in silico report. However, we also believe that the function of the other three designed scaffolds in vitro also should be investigated to approve their binding potential to EGFR protein.