Molecular Dynamics Analysis of Binding of Kinase Inhibitors to WT EGFR and the T790M Mutant

Abstract

Epidermal growth factor receptor (EGFR) inhibitors interrupt EGFR-dependent cellular signaling pathways that lead to accelerated tumor growth and proliferation. Mutation of a threonine in the inhibitor binding pocket, known as the "gatekeeper", to methionine (T790M) confers acquired resistance to several EGFR-selective inhibitors. We studied interactions between EGFR inhibitors and the gatekeeper residues of the target protein. Thermodynamic integration (TI) with Amber14 indicates that the binding energies of gefitinib and AEE788 to the active state of the T790M mutant EGFR is 3 kcal/mol higher than to the wild type (WT), whereas ATP binding energy to the mutant is similar to the WT. Using metadynamics MD simulations with NAMD v2.9, the conformational equilibrium between the inactive resting state and the catalytically competent activate state was determined for the WT EGFR. When combined with the results obtained by Sutto and Gervasio, our simulations showed that the T790M point mutation lowers the free energy of the active state by 5 kcal/mol relative to the inactive state of the enzyme. Relative to the WT, the T790M mutant binds gefitinib more strongly. The T790M mutation is nevertheless resistant due to its increased binding of ATP. By contrast, the binding of AEE788 to the active state causes a conformational change in the αC-helix adjacent to the inhibitor binding pocket, that results in a 2 kcal/mol energy penalty. The energy penalty explains why the binding of AEE788 to the T790M mutant is unfavorable relative to binding to WT EGFR. These results establish the role of the gatekeeper mutation on inhibitor selectivity. Additional molecular dynamics (MD) simulations, TI, and metadynamics MD simulations reveal the origins of the changes in binding energy of WT and mutants.

Dynamic, structural and thermodynamic basis of insulin-like growth factor 1 kinase allostery mediated by activation loop phosphorylation

“… a This value was estimated as the average of the Src, 25 EGFR, 76 and CDK5  29 simulation results. b This value was determined based on the experimental results that >90% of IRK is in the inactive conformation in its basally active kinase form. 81 …”

“…Our analysis shows that this occurs by destabilization of the inactive conformation by 12.7 kcal mol –1 , whereas the active conformation contributes only 0.5 kcal mol –1 to the conformational change. In the absence of the phosphorylation, the inactive conformation is favored by ∼4.0 kcal mol –1 (based on the Src, 25 EGFR 76 and CDK5 (ref. 29) results).…”

Fast and flexible gpu accelerated binding free energy calculations within the amber molecular dynamics package

“…Previous work by several groups has shown the CPU implementation of TI to be already capable of predicting experimental free energies . Thus for the purposes of validating our GPU implementation we consider values that agree within statistical error to the CPU implementation to indicate success.…”

Drug resistance analysis of gefitinib-targeted therapy in non-small cell lung cancer

“…Secondary mutation theory considered that EGFR gene exon 20 occurred to the secondary mutation in the gefitinib treatment process, resulting in threonine in EGFR790 locus being substituted by methionine (7). Threonine was located outside the tyrosine kinase contacting core reaction region and the hydrogen bond with high affinity formed with the adjacent gefitinib anilino, to ensure the antitumor effect.…”