Design, synthesis, and biological evaluation of Osimertinib-Cy7 (OSA-Cy7) conjugate as potential theranostic agent targeting activating EGFR mutations

Abstract

Accurately predicting the therapeutic response of non-small cell lung cancer (NSCLC) patients to tyrosine kinase inhibitors (TKIs) is of significant clinical importance. The use of TKIs in clinical is primarily guided by the detection of EGFR gene mutations. However, the current EGFR mutation assays face challenges such as inconsistent correlation with therapeutic outcomes, inconvenient sample availability and limited sensitivity. To address these, we have designed and synthesized a novel theranostic agent, OSA-Cy7, by conjugating the third-generation EGFR-TKI osimertinib with the near-infrared (NIR) fluorophore Cy7. This conjugate aims to enable fluorescence-based detection of mutant EGFR and targeted therapy of NSCLC. Our studies demonstrated that OSA-Cy7 selectively accumulates in EGFR-mutant NSCLC cell lines, such as PC9 (exon 19 deletion) and H1975 (L858R/T790M), exhibiting enhanced fluorescence signals, while showing minimal uptake in wild-type EGFR A549 cells. Western blot analysis confirmed that OSA-Cy7 effectively inhibits EGFR phosphorylation in mutant cell lines, with negligible effects on wild-type EGFR phosphorylation. Furthermore, OSA-Cy7 treatment resulted in significant suppression on cell proliferation and colony formation in EGFR-mutant cells, indicating its potent anticancer activity. These findings suggest that OSA-Cy7 holds promise as a theranostic agent for the selective imaging and treatment of EGFR-mutant NSCLC, potentially improving patient stratification and therapeutic monitoring.

Keywords: EGFR mutation dectection; Fluorescence-based assay; NSCLC; Osimertinib; TKI response evaluation; Theranostic agent.

Fig. 1

Structure and optical property of OSA-Cy7. (a) Synthetic route and chemical structure of OSA-Cy7; (b): UV-Vis absorbance spectrum of OSA-Cy7; (c) Excitation and fluorescence spectrum of OSA-Cy7

Fig. 2

Selective targeting of active mutant EGFR by OSA-Cy7 in various NSCLC cell lines. (a) Confocal fluorescence imaging (20× magnification) of NSCLC cells treated with OSA-Cy7: PC9 (EGFR 19del mutant), H1975 (L858R/T790M mutant), and A549 (wild-type EGFR). The red fluorescence represents the Cy7 signal, nuclei are counter stained with Hoechst 33,342 (blue channel). (b) Quantitative analysis of mean fluorescence intensity (MFI) from images in (a), demonstrating differential uptake of OSA-Cy7 among cell lines (n = 3). (c) Flow cytometry analysis assessing the selectivity of OSA-Cy7 binding in PC9, H1975, and A549 cell lines (the dotted line represents the MFI of A549 cells). (d) Quantification of MFI from (c) after normalization to the corresponding MFI of untreated control cells, demonstrating the preferential accumulation of OSA-Cy7 in EGFR-mutant cells. All statistical P values were calculated by the two-tailed student’s t test (n = 3, ***P < 0.001, data represent the mean ± SD)

Fig. 3

Effects of OSA-Cy7 on cell proliferation and colony formation in three NSCLC cell lines. (a) Cytotoxicity of OSA-Cy7 and osimertinib was evaluated in A549 (wild-type EGFR), H1975 (L858R/T790M mutant), and PC9 (19del mutant) cells after 48 h of treatment (n = 3, student’s t-test, *P < 0.05, ***P < 0.001, ns = not significant). (b) Representative image of colony formation, with colonies stained using crystal violet

Fig. 4

Effect of OSA-Cy7 on EGFR phosphorylation in representative mutant EGFR lines (PC-9 and H1975) and wild-type EGFR cell line (A549). EGFR phosphorylation (p) and typical downstream AKT signaling pathway were significantly inhibited by OSA-Cy7 in PC-9 and H1975 cells, while exhibiting minimal activity against EGFR phosphorylation in A549 cells. The specific blot for phosphorylated and total EGFR, AKT derives from parallel gels run with identical lysate aliquots, representative bands were selected based on band clarity