Pro-PEG3-BA is a PROTAC degrader targeting EML4-ALK and EGFR, effectively degrading EML4-ALK and EGFR mutants (L858R/T790M) with DC50 values of 0.42 μM and 13.50 μM, respectively. In vitro, it inhibits the proliferation of non-small cell lung cancer cells, inducing cell cycle arrest and apoptosis. Pro-PEG3-BA demonstrates favorable safety characteristics and can reduce EML4-ALK protein expression by modulating the ubiquitin-proteasome system in vivo, making it suitable for research on non-small cell lung cancer.

Pro-PEG3-BA binds to purified ALK protein in vitro with a Kd of 387 nM. Within HEK293T cells, it interacts with the GID4 protein, exhibiting a Kd of 6.05 μM. In H3122 (EML4-ALK) cells, Pro-PEG3-BA specifically degrades the EML4-ALK fusion protein without affecting wild-type or mutant ALK at concentrations of 0-20 μM for 48 hours. The compound inhibits cell growth with an IC50 of 0.16 μM in H3122 cells (EML4-ALK) over 48 hours and an IC50 of 8.8 μM in H1975 cells (EGFR-L858R/T790M) over 72 hours, while demonstrating low toxicity to normal cells (HEK293T cells). At 10 μM for 24 hours, Pro-PEG3-BA significantly reduces the abundance of ALK protein and downregulates CD2AP, MRPS23, RNF2, RAB18, and TRMT10C, indicating high selectivity in targeting ALK degradation; meanwhile, it upregulates POLR2F, WASHC2C, NCOR2, ZNF622, ALDH6A1, PRRC1, GPD1L, EXOC7, WACPAF1 in H1975 (EGFR L858R/T790M) cells. The compound, at concentrations of 10-20 μM for 0-24 hours, induces a time-dependent marked reduction of EML4-ALK or EGFR mutant proteins in H3122 (EML4-ALK) (10 μM) and H1975 (EGFR-L858R/T790M) (20 μM) cells. Pro-PEG3-BA reduces EML4-ALK and EGFR mutant levels in a proteasome-dependent manner in H3122 (EML4-ALK) and H1975 (EGFR-L858R/T790M) cells at 0.5-20 μM for 6 or 12 hours. It also induces cell cycle arrest and apoptosis in H1975 (EGFR-L858R/T790M) and H3122 (EML4-ALK) cells at 5-10 μM for 48-72 hours.

Administered at 10 mg/kg via intraperitoneal injection every other day for a total of 8 doses, Pro-PEG3-BA significantly reduces EML4-ALK protein expression in a mouse xenograft model induced with H3122 cells. It also exhibits a favorable safety profile.