Chemical Probes Derived from Third-Generation EGFR-TKIs as Potential Companion Diagnostic Tools for Targeted Therapy.

Recent studies demonstrated that the third-generation EGFR-TKI (i.e., AZD9291) showed benefits not only for patients with EGFR acquired resistance mutations but also for those with EGFR sensitizing mutations. In particular, AZD9291 showed efficacy superior to that of the first- and second-generation EGFR-TKIs, leading to the extension of its approval for the first-line treatment of EGFR mutant non-small-cell lung cancer (NSCLC). Despite genetic testing being used to detect mutations for identifying patients who may benefit from EGFR-TKIs, it lacks information on the functional status of EGFR kinase, a key determinant of treatment efficacy. Here, we reported a series of chemical probes (3-6) specifically targeting the active site of the EGFR kinase domain by conjugating either an alkyne ligation handle or a fluorescent tag to the third-generation EGFR-TKIs (e.g., AZD9291, PF-06747775). Both in vitro assays and cellular studies demonstrated that PF-06747775-yne (4) showed better selectivity toward EGFR kinase than AZD9291-yne (3). Interestingly, we found that the attachment of a fluorescent tag (i.e., Cy5) to AZD9291 altered the size and chemical properties of the parent compound and enhanced its selectivity. We further showed that PF-06747775-yne (4) was capable of visualizing the active form of EGFR mutants not only in xenograft mouse models but also in clinical specimens from lung cancer patients. These results demonstrate that PF-06747775-based probes hold promise as companion diagnostic tools to guide the development of effective, personalized therapeutic strategies.