Ghaedi, H.

Mahboudi, F.

Omidinia, E.

Omrani, M. D.

Tabasinezhad, M.

Talebkhan, Y.

Wenzel, Wolfgang

English

KITopen

the study was limited to investigate missense mutations found in the EGFR ectodomain (n = 22) and S492R variant.7.2 Modeling and docking analysis resultDocking of the wild-type EGFR and 23 EGFR mutant models with four anti-EGFR mAbs including Cetuximab, Panitumumab, Matuzumab, and Necitumumab was performed by ClusPro server. We found the lowest energy of binding for EGFRWT-Cetuximab, -Panitumumab, -Matuzumab, and -Necitumumab complex was as  1158.4, 900.7, 800.5, and 920.8 kJ/mol, respectively. Further, we showed that several mutations in the case of each antibody unfavorably changed the docking energy of interactions (Figure 3). Data showed out of the 23 mutants, EGFRS492R revealed most positive docking energy in complex with Cetuximab (Figure 3A). The complex of Panitumumab with EGFRR377S identified to be thermodynamically most unfavorable complex as compared to an interaction of this antibody with wild-type EGFR and also other EGFR mutants (Figure 3B). In the case of Matuzumab, data showed that its complex with EGFRG465R mutant has a more positive docking energyTABLE 1 The list of missense mutations within theEGFRectodomainNo. Position (aa) Mutation No. Position (aa) Mutation1 363 p.T363I 13 427 p.R427L2 364 p.S364F 14 430 p.T430I3 375 p.A375T 15 437 p.S437Y4 377 p.R377S 16 441 p.V441I5 384 p.T384S 17 447 p.S447Y6 387 p.L387 M 18 451 p.R451F7 387 p.L387 V 19 459 p.G459A8 390 p.Q390K 20 465 p.G465R9 397 p.T397S 21 473 p.N473D10 400 p.E400K 22 475 p.I475V11 411 p.P411R 23 492 p.S492Ra12 427 p.R427CAbbreviation: EGFR, epidermal growth factor receptor.aThis mutation is not resided within the EGFR ectodomain, but still islocated in the EGFR binding site.Complex of Cetuximab with WT/mutants EGFRLowest Energy (KJ/mol)S492RV441IR427LR377SS447YS437YR427Cl387VR451FA375TT384SN473DT430IP390KI475VL387MS364FT363IWTT397SE400KG459AP411RG465R-1300-1250-1200-1150-1100-1050-1000Complex of Panitumumab with WT/mutants EGFRLowest Energy (KJ/mol)R377SP411RG465RN473DS447YV441IS437YR427LG459AR451FL387MR427CA375TL387VT430IT384SQ390KWTT397SE400KT363IS364FI475VS492R-950-900-850-800Complex of Necitumumab with WT/mutants EGFRLowest Energy (KJ/mol)V441IR377SG465RR427LS447YG459AR427CP411RA375TR451FQ390KT384SS364FE400KN473DL387VI475VT430IS437YWTT397ST363IL387MS492R-1050-1000-950-900-850-800Complex of Matuzumab with WT/mutants EGFRLowest Energy (KJ/mol)G465RR427CL387MT384SA375TS492RT430IE400KI475VS364FR451FT363IR427LT397SWTV441IR377SL387VQ390KP411RS447YG459AN473DS437Y-900-850-800-750-700-650(A) (B)(C) (D)F IGURE 3 Docking energy of native and mutants EGFR with anti-EGFR mAbs. A-D represent docking energy of EGFRs in complex withCetuximab, Panitumumab, Matuzumab, and Necitumumab, respectively. EGFR, epidermal growth factor receptor [Color figure can be viewed atwileyonlinelibrary.com]TABLE 4 The result of G MMPBSA calculation for free energies of Matuzumab-EGFRs complexesEGFR type Van der Waals (kJ/mol) Electrostatic (kJ/mol) Polar solvation (kJ/mol) SASA energy (kJ/mol) Binding energy (kJ/mol)EGFRWT 147 903.01 ± 5406.20 277.12 ± 56.44 754.87 ± 91.76 41.89 ± 7.46 148 338.87 ± 5409.66EGFRL387M 147 351.55 ± 4662.00 345.99 ± 60.25 826.79 ± 101.08 36.08 ± 7.17 147 796.26 ± 4650.98EGFRG465R 147 282.82 ± 4181.21 306.16 ± 97.19 729.44 ± 151.56 33.98 ± 7.40 147 672.12 ± 4187.71EGFRR427C 147 069.20 ± 4618.36 283.04 ± 72.92 712.02 ± 119.41 41.58 ± 7.54 147 456.59 ± 4633.51EGFRA375T 146 886.549 ± 4428.74 391.37 ± 65.28 557.04 ± 128.38 39.48 ± 7.06 147 012.74 ± 4340.41EGFRT384S 146 605.138 ± 4687.64 160.715 ± 78.35 508.592 ± 117.39 32.872 ± 7.89 146 920.143 ± 4265.16Abbreviations: EGFR, epidermal growth factor receptor; SASA, surface accessible surface area.TABLE 5 The result of G MMPBSA calculation for free energies of Necitumumab-EGFRs complexesEGFR type Van der Waals (kJ/mol) Electrostatic (kJ/mol) Polar solvation (kJ/mol) SASA energy (kJ/mol) Binding energy (kJ/mol)EGFRV441I 147 184.22 ± 5039.10 313.63 ± 98.76 523.76 ± 119.32 72.43 ± 6.74 147 322.65 ± 5234.19EGFRR377S 146 953.26 ± 4829.70 415.63 ± 74.14 729.11 ± 146.86 84.48 ± 6.32 147 192.45 ± 4779.86EGFRWT 146 719.48 ± 4391.80 536.11 ± 69.78 1056.56 ± 151.4 94.73 ± 6.81 147 145.39 ± 4682.43EGFRR427L 146 867.11 ± 4580.35 134.28 ± 54.83 912.94 ± 124.90 42.33 ± 6.97 146 949.41 ± 4938.74EGFRS447Y 146 220.14 ± 4611.30 319.85 ± 67.43 773.19 ± 117.39 90.88 ± 6.14 146 583.89 ± 4847.10EGFRG465R 145 941.68 ± 4362.76 299.34 ± 56.46 757.61 ± 134.74 90.45 ± 6.48 146 309.47 ± 4687.36Abbreviations: EGFR, epidermal growth factor receptor; SASA, surface accessible surface area.Complex of Cetuximab with WT/mutants EGFRFree Energy of Binding (KJ /mol)V441I S492R R377S WT S447Y R427L146000146500147000147500148000148500Complex of Pantitumab with WT/mutants EGFRFree Energy of Binding (KJ /mol)R377S S447Y G465R N473D WT P411R146000146500147000147500148000148500Complex of Matazumab with WT/mutants EGFRFree Energy of Binding (KJ /mol)Free Energy of Binding ( KJ /mol)WT L387M G465R R427C A375T T384S146000146500147000147500148000148500Complex of Necitumumab with WT/mutants EGFRV441I R377S WT R427L S447Y G465R146000146500147000147500(A) (B)(C) (D)F IGURE 4 The free energy of binding of the mAbs in complex with different EGFR mutant models. A-D represent the free energy of bindingof EGFRs in complex with Cetuximab, Panitumumab, Matuzumab, and Necitumumab, respectively. EGFR, epidermal growth factor receptor[Color figure can be viewed at wileyonlinelibrary.com]complex with EGFRWT were measured (Figure 5). All of the com-plexes reached equilibrium after 10 ns of the simulation phase. This indicated that the trajectories of the MD simulations for all of the complexes next to equilibrium are reliable for further assessments. The RMSD of EGFRWT, EGFRS492R, and EGFRV441I in complex with Cetuximab is under 0.3 nm until 30 ns with a slight increase in the RMSD value of the EGFRS492R and EGFRV441I. However, after  30 ns  a noticeable deviation in the backbone RMSD value of these mutant complex structures is evident. The RMSD value of the mutants con-tinued to increase more than 0.4, although the RMSD value of EGFRWT was drop down to 0.2 nm until 50 ns (Figure 5A). Our find-ings suggested that mutation of Ser492 to Arg and mutation of Val441 to IIe could potentially destabilize EGFR interaction with Cetuximab.As Figure 5B illustrates, RMSD values of EGFRWT, EGFRR377S, and EGFRS447Y in complex with Panitumumab are about 0.3 nm until 30 ns. One can see that for EGFRWT-Panitumumab complex, the max-imum RMSD value reached a level of about 0.3 nm, while for the EGFRR377S- and EGFRS447Y-Panitumumab complexes, RMSD values exceeded to 0.3 nm, and in the case of EGFRR377S-Panitumumab, the value reached a level of 0.5 nm until 50 ns. Our result evidenced that S447Y and R377S mutations in the EGFR could change the interac-tion of epitopes with Panitumumab and consequently reduced the stability of the complexes.The RMSD value of EGFRWT- and EGFRV441I-Necitumumab was about 0.3 nm until 40 ns (Figure 5C). After this time point, RMSD value of EGFRV441I-Necitumumab started to increase and finally reached 0.4 nm at 50 ns. Such findings suggest a destabilizing effect of V441I substitution on the EGFR interaction with Necitumumab.7.5 Interaction surface analysisIn order to obtain deeper insights into the effects of the mutations with unfavorable consequences (decreasing both mAb-EGFR complexes sta-bility and binding affinity) on the mAbs-EGFR complex, we performed a close inspection on their interactions at the binding pockets of the mAb-EGFR complexes.We found that Ser492 residue of EGFRWT is involved in hydro-phobic interactions with Trp94 on the light chain of Cetuximab (Figure 6A,B). Substitution of Ser492 with Arg on the ectodomain of EGFR (Figure 6D) significantly changed the binding pose of EGFRS492R from hydrophilic negatively charged (Figure 6A) to a  high positive surface potential region (Figure 6C). Since there is a difference in charge between the Ser  and Arg  amino acid, the  S492R mutation introduces a positive charge at this position. Con-sequently, this can cause repulsion between the mutant residue and neighboring residues at the binding pocket.The mutation of Val441 to Ile (Figure 7B,D) could increase positive surface potential at the binding pose of EGFRV441I (Figure 7A,C). These alterations on the surface of EGFR would lead to a weaker nonpolar and dispersion forces with Tyr102 on the heavy chain of Cetuximab.Substitution of Arg377 with Ser on the EGFR extended a nega-tively charged hydrophilic surface on the region (Figure 8A,C). TheTime (ns)RMSD (nm)0 10       20     30     40     500.00.10.20.30.40.5EGFRW T / CetuximabEGFRS492R / CetuximabEGFRV441I / CetuximabTime (ns)RMSD (nm)0   10     20      30    40   500.00.10.20.30.40.5EGFRW T/ NecitumumabEGFRV441I / NecitumumabTime (ns)RMSD (nm)0    10    20     30     40    500.00.10.20.30.40.5EGFRR377S / PanitumumabEGFRW T / PanitumumabEGFRS447Y / Panitumumab(A)(B)(C)F IGURE  5  Root mean square deviation of anti-EGFR mAbs complexes with mutant EGFRs along the MD simulation. A–C represent RMSD plot of Cetuximab-EGFRs, Panitumumab–EGFRs, and Necitumumab-EGFRs, respectively. EGFR, epidermal growth factor receptor; MD, molecular dynamics; RMSD, root mean square deviation7.4 Stability of mAb-EGFR complexesTo gain further insights into the structural effect of the mutationswhich significantly decreased binding affinity, the stability of EGFR-anti-EGFR complexes were predicted by RMSD analysis.The RMSD values of the Cetuximab-EGFRS492R, Cetuximab-EGFRV441I, Panitumumab-EGFRR377S, Panitumumab-EGFRS447Y, andNecitumumab-EGFRV441I backbone atoms relative to these mAbs inF IGURE 6 Conformation change ofEGFR binding poses for Cetuximab as aresult of S492R mutation occurrence.A and C are a representation of theinteraction surface for EGFRWT- andEGFRS492R-Cetuximab, respectively.B represents amino acid interactions atbinding sites of EGFRWT in complex withCetuximab. D represents amino acidinteractions at binding sites of EGFRS492Rin complex with Cetuximab which theinteraction of EGFRWT-Cetuximab woulddisturb by the S492R mutation. EGFR,epidermal growth factor receptor [Colorfigure can be viewed atwileyonlinelibrary.com]F IGURE 7 Conformation change ofthe EGFR binding site for Cetuximab dueto V441I mutation. A and C are therepresentation of the interaction surfacefor EGFRWT- and EGFRV441I-Cetuximab,respectively. B represents amino acidinteractions at the binding sites ofEGFRWT in complex with Cetuximab.D represents amino acid interactions atbinding sites of EGFRV441I in complexwith Cetuximab which the interaction ofEGFRWT-Cetuximab would disturb by theV441I mutation. EGFR, epidermal growthfactor receptor [Color figure can beviewed at wileyonlinelibrary.com]modification at the binding interaction of EGFRR377S, contributed todisturb interaction of Arg377 of EGFRR377S with Ser30 on the lightchain of Panitumumab (Figure 8B,D) and decreased the electrostaticinteractions. The difference in the charge between wild-type andmutant residue will disturb the ionic interaction made by the original,wild-type residue.As a consequence of Ser447 mutation to Tyr on EGFR, a phenolgroup is introduced at this position (Figure 9B,D). This partially changesthe electrostatic surface potential of the antigen binding site(Figure 9A,C) and it would decrease strong electrostatic interactionsof EGFR with Panitumumab. Also the wild-type residue, Ser447forms a hydrogen bond with Asp at position 416 of EGFR. The sizeF IGURE 8 Conformation change ofthe EGFR binding site for Panitumumabbecause of R377S mutation. A and C arethe representation of interaction surfacefor EGFRWT- and EGFRR377S-Panitumumab, respectively. B representsamino acid interaction at the binding sitesof EGFRWT in complex withPanitumumab. D represents amino acidinteractions at binding sites of EGFRR377Sin complex with Panitumumab which theinteraction of EGFRWT-Panitumumabwould disturb by the R377S mutation.EGFR, epidermal growth factor receptor[Color figure can be viewed atwileyonlinelibrary.com]F IGURE 9 Conformation change ofthe EGFR binding site for Panitumumabdue to S447Y mutation. A and C are therepresentation of the interaction surfacefor EGFRWT- and EGFRS447Y-Panitumumab, respectively. B representsamino acid interaction at the binding sitesof EGFRWT in complex withPanitumumab. D represents amino acidinteractions at binding sites of EGFRS447Yin complex with Panitumumab which theinteraction of EGFRWT-Panitumumabwould disturb by the S447Y mutation.EGFR, epidermal growth factor receptor[Color figure can be viewed atwileyonlinelibrary.com](Figure 3). An increment of the docking energy is thermodynamicallyunfavorable in terms of complex stability, therefore in case of eachmAbs, we passed the top five complexes with the most positiveweighted score to a 50 ns MD simulation.In the field of computational biophysics, MD is regarded as oneof the most widely applied simulation techniques that could calculatebinding affinities. In MD, the motions of the atoms that composeproteins are calculated using a simplified model based on Newtonianmechanics. To date, several methodologies for calculating bindingaffinities from MD simulations have been successfully developed.The molecular mechanics Poisson-Boltzmann surface area (MMPBSA)has become one of the most widely used method for estimating bindingfree energies. Indeed, MMPBSA popularity is because of its claims toprovide a compromise between accuracy and speed.25,26 In the study,we calculated binding affinities of EGFRWT and mutant EGFRs in com-plex with mAbs using MMPBSA algorithm.Our findings suggest that several mutant EGFRs decreased affinityto bind with anti-EGFR mAbs. As a proof-of-principal, two previouslydescribed resistance-mediating point mutations S492R, G465R, havebeen identified by our approach as mutations abrogated antigen-antibody interaction. Herein, we considered such mutations as resis-tance conferring.We found that the EGFRS492R mutant loosed its affinity to bind toCetuximab in comparison to EGFRWT (Δbinding affinity = +975.04 kJ/mol).Also EGFRS492R -Cetuximab showed overall greater RMSD scores ascompared to EGFRWT -Cetuximab, resulting in a backbone RMSD of0.32 and 0.22 nm, respectively, at the end of the simulation. Due to adifference in charge between the Ser and Arg amino acid, S492R muta-tion would introduce positive potential in the interaction surface ofEGFRS492R and therefore it may cause repulsion between residues criticalfor binding to Cetuximab. Overall these findings evidenced that EGFRharboring S492R mutation significantly loss it affinity to Cetuximab. Sothis mutation may confer resistance to Cetuximab. In contrast, we foundthat EGFRS492R showed normal affinity as EGFRWT does to other anti-EGFR mAbs particularly Panitumumab. Until recently, no clear therapeu-tic differences have been acknowledged between Cetuximab andPanitumumab. In 2012, Montagut et al.6 attempted to know the problemof acquired resistance to Cetuximab that virtually develops in all patientsreceived this mAb.27 They realized that prolonged in vitro exposure of aCetuximab-sensitive human colorectal cancer cell line to Cetuximab,induced resistance to this mAb. The Cetuximab-resistant cells har-bored S492R mutation in the EGFR binding domain that abrogatesCetuximab binding to EGFR. Interestingly, these S492R EGFRCetuximab-resistant cells remained to be sensitive to Panitumumab.It implied that this mAb must bind to a different EGFR epitope fromthe one bound by Cetuximab. In agreement with these findings, ourresults showed that S492R mutation did not change Panitumumabaffinity to EGFR, dramatically.The mutation G465R is another example of secondary pointmutation that confers resistance to anti-EGFR therapies primarilyPanitumumab.7 In our analysis, we identified that this mutationcaused a decrease in Panitumumab binding affinity to the EGFR(Table 3). In 2015, Braig et al.7 reported that acquired resistance toPanitumumab was associated with the emergence of G465R mutationin the EGFR ectodomain. Moreover, they identified that this mutationmay also mediate resistance to Cetuximab in preclinical samples.However, we found no significant adverse consequences for thismutation on Cetuximab interaction with EGFR.In the case of Matuzumab, we found no mutation that substantiallydeteriorates binding affinity of this mAb to EGFR. It has previously beenshown that the epitope for Matuzumab is distinct from the EGFR ligandbinding region and also from the Cetuximab epitope.28 Matuzumabattaches mostly to a buried loop (amino acids 454-464) that precedesthe most C-terminal strand of the EGFR ectodomain.28 One explanationfor that why our analysis failed to find a mutation or mutations thatadversely affect Matuzumab binding to EGFR, maybe the unusual bind-ing fashion of this mAb to EGFR. Conclusively, the residues affected bythe mutations presented in Table 1, are supposed to be different fromhot spot residues for Matuzumab binding to EGFR.We identified that the EGFRV441I mutant has the lowest affinity tobind to both Cetuximab and Necitumumab in comparison withEGFRWT. This could be partially explained by the fact that these twomAbs have been shown to interact with EGFR via the same EGFR epi-topes.28 Accordingly, it is conceivable that V441I could lead to cross-resistance to both Cetuximab and Necitumumab.It is plausible that a number of limitations could have influenced theresults obtained. In the presented study we assessed the mutations thatwere available in COSMIC database v85. Newer versions provide amore comprehensive list of mutations waited to be analyzed. Oneshould bear in mind that molecular docking and MD simulation havelimitations as any other technique, so the relevant findings should beinterpreted with care. In antibody-antigen docking the important chal-lenge is the less favorable desolvation free energies and more planarinterfaces of interacting components in comparison with other protein-protein complexes. Also concerns about rigid-body docking methodimplemented into the ClusPro server worth to be mentioned here.29Theoretically, flexible docking method considers all possible structuralmodifications and has more realistic prediction than rigid-body docking.However, evaluation of all possible conformational modifications isexcessively time-consuming and computation demanding in research.Nevertheless, the majority of the ClusPro developers effort has beendirected toward solving presenting flexibility or softness in the rigid-body search and developing a scoring function that distinguishesefficiently between the correct docking solution and the lots of falsepositives that the search brings up this method considers the moleculesas rigid objects.13,30 In the case of MD simulation, it has some inherentlimitations, particularly regarding the force field accuracy. Even with thesignificant improvement in recent years, still, a deviation from experi-mental data is evident in respect to MD force filed.This study was aimed at identifying mutations that contribute toacquire resistance against anti-EGFR mAbs (Cetuximab, Panitumumab,Matuzumab, and Necitumumab). To this aim, we used the COSMICdatabase data on the somatic mutations within the EGFR ectodomainand performed molecular docking and MD simulation to find candidatemutations. The docking poses of the five top complexes of mutantEGFRs with relevant mAb, were further validated by the MD simulation

The effects of somatic mutations on EGFR interaction with anti-EGFR monoclonal antibodies: Implication for acquired resistance

https://publikationen.bibliothek.kit.edu/1000096680/149957136

The effects of somatic mutations on EGFR interaction with anti-EGFR monoclonal antibodies: Implication for acquired resistance

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