Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach

Receptor
tyrosine kinases represent one of the prime targets in
cancer therapy, as the dysregulation of these elementary transducers
of extracellular signals, like the epidermal growth factor receptor
(EGFR), contributes to the onset of cancer, such as non-small cell
lung cancer (NSCLC). Strong efforts were directed to the development
of irreversible inhibitors and led to compound CO-1686, which takes
advantage of increased residence time at EGFR by alkylating Cys797
and thereby preventing toxic effects. Here, we present a structure-based
approach, rationalized by subsequent computational analysis of conformational
ligand ensembles in solution, to design novel and irreversible EGFR
inhibitors based on a screening hit that was identified in a phenotype
screen of 80 NSCLC cell lines against approximately 1500 compounds.
Using protein X-ray crystallography, we deciphered the binding mode
in engineered cSrc (T338M/S345C), a validated model system for EGFR-T790M,
which constituted the basis for further rational design approaches.
Chemical synthesis led to further compound collections that revealed
increased biochemical potency and, in part, selectivity toward mutated
(L858R and L858R/T790M) vs nonmutated EGFR. Further cell-based and
kinetic studies were performed to substantiate our initial findings.
Utilizing proteolytic digestion and nano-LC-MS/MS analysis, we confirmed
the alkylation of Cys797.