SIAIS-126

A new class of EGFR PROTACs based on pomalidomide was developed, synthesised, and tested for their cytotoxic activity against a panel of human cancer cells. Compounds 15-21 were showed to be more effective against the four tested cell lines than erlotinib. In particular, compound 16 was found to be the most potent counterpart as it was 5.55, 4.34, 5.04, and 7.18 times more active than erlotinib against MCF-7, HepG-2, HCT-116, and A549 cells, respectively. Compound 15 was revealed to be more active than doxorubicin against the four tested cell lines. Furthermore, the most potent cytotoxic compounds were studied further for their kinase inhibitory effects against EGFR^WT and EGFR^T790M using HTRF test. Compound 16 showed to be the most effective against both kinds of EGFR, with IC₅₀ values of 0.10 and 4.02 µM, respectively. Compound 16 could effectively degrade EGFR protein through ubiquitination (D_max = 96%) at 72 h in the tested cells.

Targeted therapy of treating patients with specific tyrosine kinase inhibitors (TKIs) is currently the standard care for epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer. However, the inevitably developed drug resistance in patients to EGFR TKIs is the biggest obstacle for cancer targeted therapy. About 60% of drug resistance to the 1st generation of EGFR TKIs was resulted from an acquired T790M mutation in the kinase domain of EGFR protein. Proteolysis targeting chimera (PROTAC) is a lately-developed technology to target point of interest proteins for degradation. Because EGFR-mutant lung cancers are highly dependent on EGFR proteins, designing specific PROTAC molecules to degrade EGFR proteins from cancer cells provides a very promising strategy to treat such patients and eradicate drug resistance. Currently, there is no cereblon (CRBN)-based PROTAC reported able to degrade T790M-containing EGFR resistant proteins. In this study, we synthesized two novel CRBN-based EGFR PROTACs, SIAIS125 and SIAIS126, based on EGFR inhibitor canertinib and cereblon ligand pomalidomide. These two degraders displayed potent and selective antitumor activities in EGFR TKI resistant lung cancer cells. Firstly, they could selectively degrade EGFR^L858R+T790M resistant proteins in H1975 cells at the concentration of 30-50 nM, and EGFR^Ex19del proteins in PC9 cells. But they did not degrade EGFR^{Ex19del+T790M} mutant proteins in PC9Brca1 cells or wild type EGFR in A549 lung cancer cells. They could also selectively inhibit the growth of EGFR mutant lung cancer cells but not that of normal cells or A549 cells. Secondly, the degradation of EGFR^L858R+T790M proteins was long lasting up to 72 h. Thirdly, these degraders displayed better inhibition of EGFR phosphorylation in H1975 cells and PC9Brca1 cells comparing to canertinib. Finally, these degraders could also induce significant apoptosis and cell cycles arrest in H1975 cells. Pre-incubation with canertinib, pomalidomide or ubiquitination inhibitor MLN4924 totally blocked EGFR degradation by PROTACs. Mechanistic studies showed that PROTAC could induce autophagy in lung cancer cells. PROTAC-induced EGFR degradation acted through both ubiquitin/proteosome system and ubiquitin/autophagy/lysosome system. Elevating autophagy activities enhanced EGFR degradation and cell apoptosis induced by PROTACs. Our research not only offered a novel PROTAC tool to target EGFR TKI drug resistance in lung cancer, but also firstly demonstrated that the involvement of autophagy/lysosome system in PROTAC- mediated target protein degradation.