Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors

Abstract

Summary Patients with non-small cell lung cancer (NSCLC) that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR where the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may impact drug activity and safety.

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

“…To assess the structural impact of the EGFR G724S mutation on the EGFR kinase we performed structural analysis based on a previously published co-crystal structure of rociletinib bound to EGFR (PDB ID: 5UWD) (Fig. 3a ) 30 . As described before the glycine-rich loop is an essential element for ligand binding and the glycine at position 724 is in direct contact with the adjacent ELREA motif that is subject to deletion mutations in affected patients.…”

“…As described before the glycine-rich loop is an essential element for ligand binding and the glycine at position 724 is in direct contact with the adjacent ELREA motif that is subject to deletion mutations in affected patients. The ELREA sequence plays a crucial role in the alignment of the regulatory helix αC that is a key element in the transition between the active and inactive kinase domain conformations 30 . It is therefore conceivable that the EGFR G724S mutation influences structure and dynamics of the binding site and thereby the affinity toward third-generation EGFR inhibitors.…”

Evaluation and Optimization of Chemically-Cleavable Linkers for Quantitative Mapping of Small Molecule-Protein Interactomes

“…Dacomitinib is a pan-erythroblastic leukemia viral oncogene homologue (ERBB) inhibitor which targets wildtype and mutant forms of EGFR that possess activating L858R or exon 19 deletion mutations in addition to T790M mutation 42,43 . For the purpose of our study, we utilized the A431 epidermoid cancer cell line as it was previously reported to be characterized by a high expression of wildtype EGFR 44 , and has been utilized in experiments profiling EGFR covalent inhibitors 45,46 .…”

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…Derivatisation of ABX‐1431 with an alkyne enrichment handle (ABX‐1431_ABP, Figure B) confirmed CNS penetrance and in vivo target engagement by ABX‐1431 in the mouse brain after a 4 h treatment with a single 25 mg kg −1 oral dose . This general approach has also been used to profile the cysteine‐targeted covalent drugs afatinib (EGFR inhibitor) and ibrutinib (BTK inhibitor), as well as the newly developed third generation EGFR inhibitors osimertinib, PF‐06747775 and rociletinib . Additionally, a dichlorotriazine‐based probe has been utilised recently to label bromodomains on lysine and tyrosine residues, and the high selectivity profile of a lysine‐targeted covalent inhibitor of the lipid kinase PI3Kδ was also determined using similar strategies …”