Uncovering the Molecular Basis for the Better Gefitinib Sensitivity of EGFR with Complex Mutations over Single Rare Mutation: Insights from Molecular Simulations

Abstract

Epidermal growth factor receptor (EGFR) is an intensively focused target for anti-tumor compounds used in non-small cell lung cancer (NSCLC) therapy. Compared to the classical activating mutations, there are still many uncommon EGFR mutations associated with poorer responses to EGFR inhibitors. A detailed understanding of the molecular basis for multiple EGFR mutants exhibiting diverse responses to inhibitors is of critical importance for related drug development. Herein, we explored the molecular determinants contributing to the distinct responses of EGFR with a single rare mutation (G719S) or combined mutations (G719S/L858R and G719S/l861Q) to Gefitinib (IRE). Our results indicated that interactions, formed within the tetrad of residues S768 (in the αC-helix), D770 (in the αC-β4 loop), Y827 (in the αE-helix), and R831 (in the catalytic loop), play an important role in the stability of αC-helix and the maintenance of K745-E762 salt bridge in the absence of IRE, which are weakened in the EGFR^G719S system and enhanced in the EGFR^G719S/L858R system upon IRE binding. Besides, the introduced hydrogen bonds by the co-occurring mutation partner also contribute to the stability of αC-helix. The work done for inhibitor dissociation suggests that IRE exhibits a stronger binding affinity to EGFR^G719S/L858R mutant. Together, these findings provide a deeper understanding of minor mutations, which is essential for drug development targeting EGFR with less common mutations.

Keywords: EGFR complex mutations; EGFR rare mutants; Gefitinib (IRE); molecular dynamics simulation; steered molecular dynamics simulation.

Figure 1

The structure of EGFR is complex with IRE in the absence (A) and presence (B) of ATP. The αC-helix is marked in light blue. The residues E762 and K745 forming a salt bridge are shown as orange and magenta sticks, respectively. The small molecules IRE and ATP are shown as green and yellow sticks, respectively. The IRE binding site is highlighted by a black circle in (A), and the five regions consisting of the catalytic cleft are highlighted by circles with different colors in (B).

Figure 2

The stability of the αC-helix and KE salt bridge during the simulations for APO systems. (A) The RMSD values of the αC-helix backbone atoms in the last 200 ns MD trajectories using the initial structure as the reference. (B–D) The probability distribution profile of the distance between atoms K745/NZ and E762/(OE1, OE2) during the last 200 ns is based on all three replicates.

Figure 3

The covariance matrix maps of EGFR in the APO forms: (A) G719S-APO, (B) G719S/L858R-APO, and (C) G719S/L861Q-APO. The positively correlated regions existing in all APO systems are demonstrated by a black square box.

Figure 4

Conformational dynamics of EGFR in the APO and IRE-bound forms. The first two principal components (PCs) are shown in free energy surfaces. All APO forms are colored in black (A–C). Complex systems of G719S-IRE (A), G719S/L858R-IRE (B), and G719S/L861Q-IRE (C) are colored in green, blue, and magenta, respectively.

Figure 5

The stability of the αC-helix and KE salt bridge upon IRE binding during the simulations. The RMSD of the αC-helix backbone atoms in the absence and presence of IRE: (A) G719S mutants; (B) G719S/L858R mutants; (C) G719S/L861Q mutants. The probability distribution profiles of the distance between atoms K745/NZ and E762/(OE1, OE2) in the absence and presence of IRE: (D) G719S mutants with the APO form colored in black and with the complex form colored in green; (E) G719S/L858R mutants with the APO form colored in black and with the complex form colored in blue; (F) G719S/L861Q mutants with the APO form colored in black and with the complex form colored in magenta.

Figure 6

Hydrogen bond occupancy differs between the APO systems and the complex systems. Some residues have a hydrogen bond donor or acceptor of more than one, thus the hydrogen bond occupancy is above 1. A low panel of the red dotted line indicates the occupancy of hydrogen bonds (Y827-D770, R831-S768, R831-D770, and R831-Y827) in the APO systems, and the top panel indicates the occupancy of these hydrogen bonds in the complex systems.

Figure 7

The probability distribution profiles of the R_g values of the binding site in the six systems considering three replicates together: (A) G719S mutants; (B) G719S/L858R mutants; (C) G719S/L861Q mutants. The probability distribution profiles of the R_g values of hydrophobic residues in the six systems considering three replicates together: (D) G719S mutants; (E) G719S/L858R mutants; (F) G719S/L861Q mutants.

Figure 8

The probability distribution profiles of (A) the distance between atoms K745/NZ and IRE/F, (B) the distance between atoms E762/(OE1, OE2) and IRE/F, and (C) the solvent-accessible surface area (SASA) for the binding region of IRE benzene.

Figure 9

The average work profile for IRE dissociation with error bars of each complex system during the SMD simulations. The bars represent the standard deviation of the external works.