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Targeting Drug Resistance in EGFR with Covalent Inhibitors: A Structure-Based Design Approach
Julian Engel1, André Richters1, Matthäus Getlik2
1Department of Chemistry and Chemical Biology, TU Dortmund University , Otto-Hahn-Straße 6, D-44227 Dortmund, Germany.
Abstract:
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.
Insights
Researchers designed novel irreversible inhibitors targeting epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). These inhibitors covalently bind to Cys797, offering a promising therapeutic strategy for NSCLC treatment.
Area of Science:
- Oncology
- Pharmacology
- Structural Biology
Background:
- Receptor tyrosine kinases, like epidermal growth factor receptor (EGFR), are crucial in cancer development, particularly non-small cell lung cancer (NSCLC).
- Dysregulated EGFR signaling drives NSCLC pathogenesis, making it a prime target for cancer therapies.
- Irreversible inhibitors that increase target residence time, such as by alkylating Cys797, are being developed to mitigate toxic effects.
Purpose of the Study:
- To design novel, irreversible EGFR inhibitors using a structure-based approach combined with computational analysis.
- To identify and optimize compounds targeting EGFR mutations (L858R and L858R/T790M) relevant to NSCLC.
Main Methods:
- Phenotypic screening of approximately 1500 compounds against 80 NSCLC cell lines to identify initial hits.
- Protein X-ray crystallography using an engineered cSrc model (T338M/S345C) to determine the binding mode of EGFR inhibitors.
- Chemical synthesis of novel inhibitor analogs, followed by biochemical, cell-based, and kinetic assays.
- Proteolytic digestion and nano-LC-MS/MS analysis to confirm Cys797 alkylation.
Main Results:
- A structure-based approach, informed by computational analysis, led to the design of novel irreversible EGFR inhibitors.
- Crystallographic studies elucidated the binding mode in a validated model system for EGFR-T790M.
- Synthesized compounds demonstrated increased biochemical potency and, in some cases, selectivity for mutated EGFR (L858R, L858R/T790M) over wild-type EGFR.
- Confirmation of Cys797 alkylation in EGFR was achieved through mass spectrometry-based proteomics.
Conclusions:
- The study successfully designed and validated novel irreversible EGFR inhibitors with potential for NSCLC therapy.
- The structure-based design strategy, coupled with computational and experimental validation, proved effective in optimizing inhibitor properties.
- Confirmation of Cys797 alkylation validates the mechanism of action for these irreversible inhibitors in targeting mutant EGFR in NSCLC.
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