PROTAC targeting EGFR mutants

Targeted therapy for non-small cell lung cancer with EGFR activating mutations has made tremendous progress, with first, second and third generation EGFR-TKIs being approved and marketed in succession, with significant clinical benefits for patients.

The first generation EGFR-TKI targets the “L858R” or “exon19 del” mutation in the structure of EGFR kinase region, and the representative drugs listed are gefitinib, erlotinib and erlotinib; the researchers based on the first generation EGFR-TKI anilinoquinazolium. The second generation of EGFR tyrosine kinase covalent inhibitors were developed based on the first generation of EGFR-TKI quinazoline backbone, which contains a cysteine residue (C797) near the ligand binding site, and can capture the cysteine residue by replacing the quinazoline ring with a Michael receptor (such as acrylamide). Afatinib and Daclatinib; the third generation EGFR-TKI has been used for “L858R” combined with “T790M” and “exon19 del” combined with “T790M”. The third generation EGFR-TKIs have shown significant inhibitory activity against EGFR mutated tumor cells with “T790M”, while possessing certain selectivity (not significantly inhibiting wild type).

In order to solve the problem of drug resistance, the third generation EGFR-TKI, such as Ocitinib, will inevitably also have drug resistance phenomenon. The third generation EGFR-TKI resistance mechanism is divided into EGFR-related and EGFR non-related, of which EGFR-related resistance accounts for about 30%~45%, and C797S mutation accounts for about 20%. The tertiary C797S (Cys 797 → Ser 797 ) mutation disrupts the covalent formation between Cys797 and acrylamide warhead.

Overcoming triple mutations in EGFR containing C797S (L858R/T790M/cis C797S or Del19/T790M/cis C797S) is generally referred to as a fourth generation EGFR-TKI, and no drugs of this class are currently on the market.

The above four generations of small molecule drugs, all of which are similar drug design strategies targeting the EGFR adenosine triphosphate (ATP) binding site, have both advantages and disadvantages, and these compounds may always be difficult to avoid suffering from drug-induced EGFR mutations. There is a clear need for a new drug design strategy to develop compounds with different mechanisms of action, and in the last decade, targeted protein degradation induced by protein degradation targeted chimeras (PROTACs) has emerged as an attractive strategy for drug discovery.

PROTACs (Proteolysis Targeting Chimera) are heterogeneous bifunctional small molecule compounds that contain two different ligands in their structure, one for the E3 ubiquitin ligase and the other for binding to the target protein in the cell, which are linked by a Linker to form a “trimeric” polymer – target protein ligand-Linker-E3 ligand.

PROTAC binds to E3 ubiquitin ligases and target proteins in the cell and forms a ternary complex: target protein-PROTAC-E3 ubiquitin ligase, leading to polyubiquitination of the target protein and subsequent recognition and degradation by the 26S proteasome. After triggering target protein ubiquitination, PROTAC can dissociate from the complex and participate in the next catalytic cycle.PROTAC may increase the selectivity of protein degradation and evade resistance mechanisms such as acquired resistance mutations and target protein overexpression.

The authors provide a brief review of some of the publicly reported PROTACs targeting common mutants of EGFR.

In 2018, the Crews group reported gefitinib-based PROTAC 1 targeting EGFR del19, and afatinib-based PROTAC 2 targeting EGFR L858R/T790M. 1 treatment of EGFR del19-expressing HCC827 (non-small cell lung cancer cells) cells reached a DC50 of 11.7 nm, and 2 treatment of cells expressing the double H1975 (human lung adenocarcinoma cells) cells expressing the double mutant (L858R/T790M) EGFR, with a DC50 of 215.8 nm.

In 2020, Zhang, S.-Q’s group found that compound 3, based on a fourth-generation EGFR-TKI, was effective in inducing EGFR del19 degradation; however, 3 showed poor degradation activity against EGFR L858R/T790M.

In 2020, Jian Jin’s group described the discovery of a novel E3 ligase VHL recruiting EGFR degradation agent MS39 (compound 4) and E3 ligase CRBN recruiting EGFR degradation agent MS154 (compound 5) using protein hydrolysis targeting chimera technology. These compounds efficiently induced degradation of mutant but not wild-type EGFR in cancer cell lines in an E3 ligase-dependent manner and effectively inhibited the growth of lung cancer cells compared to the corresponding negative control.

In 2020, Zhang, S.-Q group reported that P3 (compound 6) containing purine structure recruiting VHL significantly induced degradation of both EGFR del19 and EGFRL858R/T790M under P3 treatment with DC50 values of 0.51 nM and 126.2 nM, respectively, and compound P3 was able to significantly inhibit EGFR pathway signaling. In addition, compound P3 significantly induced apoptosis, blocked cell cycle and inhibited cell colony formation.

In 2020, XingxianZhang et al. reported novel EGFR degraders of PROTACs based on oxitinib and lenalidomide. All compounds were evaluated in vitro for antiproliferative effects and showed significant inhibition against PC9 cells, HCC827 cells and H1975 cells. Protein blotting assays analyzed the effect of EGFR degradation and showed that 16c (compound 7) could effectively degrade EGFR protein by ubiquitination and reached a maximum degradation rate in PC9 cells (Dmax = 68%). These findings suggest that EGFR Del19 in PC9 cells can be effectively targeted for degradation by the designed PROTACs.

In 2020, Ke Ding et al. reported a series of PROTAC-based selective EGFR L858R/T790M mutation degraders. One of the most potent compounds, 14o (compound 8), effectively and selectively degraded EGFR L858R/T790M with a DC50 value of 5.9 nM, while having no significant effect on the wild-type protein. Further mechanistic studies suggest that the degradation is mediated by the ubiquitin proteasome pathway. Compound 14o could be used as an initial lead molecule for the development of new EGFR L858R/T790M degradation-based therapies.

In 2020, Grey’s group reported PROTAC DDC-01-163 (compound 9) based on a variant EGFR-TKI. Importantly, 9 was effective against EGFR L858R/T790M, EGFR L858R/T790M/C797S and EGFR L858R/T790M/L718Q resistant mutants.DDC-01-163 showed potent anti-proliferative effects in Ba/F3-EGFR L858R/T790M/C797S mutant cells, with Its IC50 value was 0.041 μM.

In 2021, Biao Jiang et al. reported the synthesis of two novel CRBN-based EGFR PROTACs, SIAIS125 (compound 10) and SIAIS126 (compound 11), based on the EGFR inhibitor canertinib and the CRBN ligand pomalidomide. These two degradates showed potent and selective antitumor activity in EGFR TKI-resistant lung cancer cells. First, they selectively degraded EGFRL858R+T790M resistance protein in H1975 cells and EGFR del19 mutant protein in PC9 cells at concentrations ranging from 30 to 50 nM. Mechanistic studies suggest that PROTAC can induce autophagy in lung cancer cells.PROTAC-induced EGFR degradation acts through the ubiquitin/proteasome system and the ubiquitin/autophagy/lysosome system.

In 2022, Jian Li, Fang Xu,Tianfeng Xu et al. reported novel EGFR PROTACs targeting the Del19/T790M/C797S mutation.The researchers described the design and synthesis of a series of EGFR protein hydrolysis-targeting chimeras (PROTACs) that can be used in the expression of the EGFR Del19/T790M/C797S mutation. T790M/C797S in Ba/F3 cells expressing EGFR Del19/T790M/C797S to rapidly and efficiently induce EGFR degradation mutants. A representative 6h (compound 12) induced EGFR degradation in a time- and dose-dependent manner with a DC50 of 8 nM. It also showed good anti-proliferative activity against Ba/F3-EGFR Del19/T790M/C797S cells (IC50=0.02 μM). 6h could be developed as a lead compound for the treatment of EGFR C797S mutants with drug-resistant non-small cell lung cancer patients.

In 2022, Zhang, S.-Q’s group reported the discovery of covalent PROTAC CP17 (compound 13), a novel EGFR ligand with a purine structure, as an efficient degradation agent against EGFR L858R/T790M and EGFR del19 with very low DC50 values, in addition, CP17 exhibited excellent H1975 and HCC827 cell lines cellular activity with high selectivity, and mechanistic studies suggested the involvement of lysosomes in the degradation process. Importantly, the covalent binding strategy proved to be an effective approach for the design of PROTACs targeting EGFR L858R/T790M, laying a practical foundation for further development of effective and efficient PROTACs targeting EGFR.

In conclusion: Nowadays, conventional small-molecule EGFR inhibitors of various generations (three generations have been marketed) are not only facing the dilemma of serious product homogenization, but also the problem of drug resistance due to mutation of target proteins, and the emerging protein hydrolysis targeting chimeric (PROTAC) technology provides a powerful tool to remove drug resistance barriers in the path of NSCLC treatment.
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