A Mechanistic Study of a Potent and Selective Epidermal Growth Factor Receptor Inhibitor against the L858R/T790M Resistance Mutation

Abstract

Covalent targeting is a promising strategy for increasing the potency and selectivity of potential drug candidates. This therapeutic approach was recently reported for the epidermal growth factor receptor (EGFR), wherein a covalent binder, 20g [N-(3-{7-[2-methoxy-4-(4-methylpiperazin-1-yl)­phenylamino]-3,4-dihydro-3-isopropyl-2,4-dioxopyrimido­[4,5-d]­pyrimidin-1­(2H)-yl}­phenyl)­acrylamide], demonstrated significant selectivity and inhibitory activity toward the EGFR L858R/T790M double mutant (EGFRDM) relative to the EGFR wild-type form (EGFRWT). The enhanced therapeutic potency of 20g against EGFRDM is 263 times greater than that against EGFRWT, which necessitates a rational explanation for the underlying selective and inhibitory mechanisms. In this work, we investigate the differential binding modes of 20g with EGFRWT and EGFRDM using molecular dynamics simulations coupled with free energy calculations and further identify key residues involved in the selective targeting, binding, and inhibitory mechanisms mediated by 20g. We find that systematic orientational and conformational changes in the α-loop, p-loop, active loop, and αC-helix are responsible for the disparate binding mechanisms and inhibitory prowess of 20g with respect to EGFRWT and EGFRDM. The calculated binding free energies show good correlation with the experimental biological activity. The total binding free energy difference between EGFRWT-20g and EGFRDM-20g is −11.47 kcal/mol, implying that 20g binds more strongly to EGFRDM. This enhanced binding affinity of 20g for EGFRDM is a result of a large increase in the van der Waals and electrostatic interactions with three critical residues (Met790, Gln791, and Met793) that are chiefly responsible for the high-affinity interactions mediated by 20g with EGFRDM relative to EGFRWT.

Impact of HEC72702 chirality on the selective inhibition of hepatitis B virus capsid dimer: A dynamics–structure–energetics perspective

“…Moreover, estimations of high RoG can be predictive of a systematic loss in structural compactness while a marked reduction in RoG could correlate with a structurally compact protein. (Akher, Farrokhzadeh, Olotu, et al., 2019; Akher, Farrokhzadeh, Ravenscroft, et al., 2019; Farrokhzadeh, Akher, Olotu, et al., 2019; Lobanov, Bogatyreva, & Galzitskaya, 2008) Using the FE‐RoG, we observed minimal differences in the compactness of the entire protein structure (bound and unbound), with mean values of 23.72 Å, 23.43 Å, and 23.64 Å for the unbound, ( R,R ) ‐7‐ bound, and ( R,S ) ‐7‐ bound systems, respectively (Figure 6a). On the other hand, the active site regions of HBVCd exhibited more compactness in the presence of ( R,R ) ‐7 while active site RoG values were relatively higher for the ( R,S ) ‐7 and unbound systems.…”

“…This helps identify crucial binding site residues and their interaction patterns which would facilitate further structure‐based drug design efforts. Herein, the MM/GBSA method was used to estimate the differential binding affinities of ( R,R ) ‐ and ( R,S ) ‐7 diastereomers toward HBVC dimer (HBVCd) (Akher, Farrokhzadeh, Olotu, Agoni, & Soliman, 2019b; Akher, Farrokhzadeh, Ravenscroft, & Kuttel, 2019c; Akher, Farrokhzadeh, & Soliman, 2019a; Farrokhzadeh, Akher, Olotu, Soliman, & Van Heerden, 2019b; Farrokhzadeh, Akher, & Soliman, 2019a; Genheden & Ryde, 2012). This enabled the determination of the binding energy profiles that included other components such as electrostatic and van der Waals energies.…”

Deciphering the Mechanism of Binding Selectivity of Chlorofluoroacetamide-Based Covalent Inhibitors toward L858R/T790M Resistance Mutation

“…Epidermal growth factor receptor (EGFR), a member of the ERBB receptor tyrosine kinase family, plays an indispensable role in cellular signaling related to cell growth, proliferation, differentiation, migration, adhesion, and survival. − Dysregulation and overactivity of EGFR have been implicated in various human cancers, including lung, breast, gastric, head, and neck cancers. − Activating mutations in the EGFR kinase domain, primarily the L858R point mutation and exon 19 deletions, give rise to non-small-cell lung cancer. , Patients carrying one of these activating mutations benefit enormously from treatment with the first-generation reversible and 4-amino-quinazoline-based EGFR tyrosine kinase inhibitors (TKIs) gefitinib ( 1 , Figure ) and erlotinib ( 2 , Figure ) that elicit excellent clinical response rates of 50–80% as compared to traditional chemotherapy. − However, patients eventually develop an additional specific point mutation (T790M) at the gatekeeper position of EGFR that limits the therapeutic efficacy of the first-generation EGFR TKIs. ,− Replacement of a threonine residue with a larger methionine provokes steric repulsion in the drug-binding site, resulting in an altered conformation of the catalytic domain that accounts for the dramatic reduction in inhibitory activity. ,, In addition, as compared to the L858R point mutation that causes a substantial loss of ATP affinity to EGFR, the T790M mutants restore the ATP affinity to almost the same level as in wild-type EGFR (EGFR WT ), resulting in reduced cellular potency for ATP-competitive inhibitors in the presence of cellular ATP concentrations. , To overcome the resistance caused by T790M mutation, second-generation irreversible EGFR TKIs dacomitinib ( 3 , Figure ), afatinib ( 4 , Figure ), canertinib, and CI-1033 ( 5 , Figure ) were developed. These have an electrophilic warhead that covalently targets a unique cysteine (Cys797) located at the lip of the ATP binding site in EGFR. , Although these covalent inhibitors have improved clinical performance relative to their reversible counterparts, their efficacy in patients is restricted due to dose-limiting toxic side effects that are correlated with inhibition of EGFR WT . ,…”

Molecular Mechanism Exploration of Potent Fluorinated PI3K Inhibitors with a Triazine Scaffold: Unveiling the Unusual Synergistic Effect of Pyridine-to-Pyrimidine Ring Interconversion and CF3 Defluorination

“…Molecular dynamics (MD) simulation combined with the molecular mechanics/generalized Born surface area (MM/GBSA) free energy calculation method is as an alternative to docking-based scoring functions . This method is successfully used as a powerful computational method to provide valuable insights into the detailed binding process and compare the binding mode of inhibitors with different activities. − However, a major bottleneck of the MD simulation method for large systems is the huge computational cost associated with adequate conformational sampling . Apart from this, the MD simulation method is affected by the accuracy of underlying force fields that can be a concern as well .…”