Figure 7 –

Case studies. a) Noncovalent first-generation EGFR inhibitor gefitinib was fashioned into covalent molecules (e.g. PF-168393) by adding an electrophilic acrylamide warhead to target cysteines in the ATP binding pocket (C771, C797). Further optimization yielded second-generation inhibitor afatinib, which covalently targets C797 to achieve superior potency and overcome resistance mutations that enhance ATP binding affinity. Third-generation EGFR inhibitors (Osimertinib, rociletinib, PF-06747775) are selective for mutants over wild-type EGFR, and each possess distinct off-target profiles despite being developed to target the same set of mutants. b) Fragment 6H05 was identified as a hit in a disulfide tethering primary screen against KRAS G12C. Linker removal and additional optimization yielded lead compound 12. Chemoproteomic experiments were used to profile off-target landscapes of analogs, resulting in clinical candidates such as AMG-510 and MRTX849. c) Toxicity mechanisms of BIA-10-2474 were investigated using multiple chemoproteomic approaches. van Esbroeck et al. used fluorophosphonate-biotin, a serine hydrolase ABP, to compete against the parent inhibitor, metabolite BIA-10-2639, and negative control compound PF04457845 and identified serine hydrolase off-targets involved in neuronal lipid metabolism, revealing a potential toxicity mechanism. Huang et al. appended alkyne moieties onto BIA-10-2474 and metabolites for IBPs, performed a competitive pulldown, and identified proteome-wide off-targets including ALDH2, which has been reported to protect the brain from reactive oxygen species.