Hydration effects on the efficacy of the Epidermal growth factor receptor kinase inhibitor afatinib

Abstract

Small molecules targeting the EGFR tyrosine kinase domain have been used with some success at treating patients with non-small cell lung cancer driven by activating mutations in the kinase domain. The initial class of inhibitors displaced ATP noncovalently but were rendered ineffective due to the development of resistance mutations in the kinase domain. These were overcome by the development of covalent inhibitors such as afatinib which also bind in the ATP pocket. However pooled analysis of two recent clinical trials LUX-3 and LUX-6 demonstrated an unprecedented overall survival benefit of afatinib over chemotherapy for the EGFR 19del, but not the EGFR L858R. In the current study we use modelling and simulations to show that structural constraints in EGFR 19del deletion result in significantly attenuated flexibilities in the binding pocket resulting in strong hydrogen and halogen bonds with afatinib in the EGFR 19del; these constraints are modulated by buried water and result in the differential affinities of afatinib for the different mutants. SNP analysis of residues surrounding the buried water points to the likelihood of further differential effects of afatinib and provides a compelling case for investigating the effects of the SNPs towards further stratification of patients for ensuring the most effective use of afatinib.

Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors

“…Our data indicate that EGFR G724S mutations seem to primarily affect the reversible first step of third-generation inhibitor binding within the ATP-binding pocket before irreversible attachment to Cys797 can occur. We conclude that the observed fluctuation of the glycine-rich loop plays a role in this phenotype, similar to other systems where anti-correlations between flexibility and ligand-binding affinity have been observed 49 , 50 . However, although the kinetics of third-generation EGFR inhibitor binding are perturbed dramatically, second-generation EGFR inhibitors are potent enough to establish such a reversible binding despite the EGF R G724S mutation.…”

Conformational landscape of the epidermal growth factor receptor kinase reveals a mutant specific allosteric pocket

“…Increased flexibility and destabilization of the αC-helix have also been reported in earlier computational studies. 14 – 20 This clearly points to the structural requirement of a helical motif in this region (the short helix) to stabilize the kinase in its inactive state. Apart from the increased flexibility observed for the A-loop, the αC-helix and the P-loop (Fig.…”

“…The T790M mutation is thought to stabilize the active form of the kinase by stabilizing the hydrophobic spine, 12 , 13 while the L858R mutation is known to destabilize the inactive form and stabilize the kinase in an intermediate form. 14 – 20 We therefore subjected our models of the T790M, L858R and L858R/T790M mutants in their inactive states to multiple μs MD simulations. The simulations very quickly show that the L858R substitution, either as a single point mutation or in combination with T790M, is not tolerated as modelled based on the wild type conformation, i.e.…”

“…This mutation is also known to destabilize the inactive state in favour of the active state. 20 We model the EGFR 19del mutant by removing 5 amino acids ( 746 ELREA 750 ) from the β3-αC-helix loop that connects the αC-helix and β-strand (β3), from the EGFR WT in its inactive state. During the simulations of this mutant in its apo state, the volume of the allosteric pocket is much smaller as compared to that of the EGFR L858R / T790M and also smaller than that of the EGFR WT ( Fig.…”

Altered conformational landscape and dimerization dependency underpins the activation of EGFR by α C– β 4 loop insertion mutations

“…MD studies on other recurring mutations such as R776H in the αC-β4 loop have identified novel autoinhibitory interactions associated with kinase activation (25) and protonation-dependent changes in mutant EGFR functions (30). In addition, MD-based free-energy methods (26,31), molecular mechanics calculations (32), and molecular modeling studies (33) have provided new insights into mutation-induced drug resistance mechanisms and the conformational transitions connecting active and inactive states (34)(35)(36).…”