Application of PROTAC in the Treatment of NSCLC
Proteolysis targeting chimeras (PROTACs) disrupt the traditional concept of small molecule inhibitors by utilizing the ubiquitin-proteasome pathway to degrade targets. In the realm of non-small cell lung cancer (NSCLC), common mutated targets such as Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), and anaplastic lymphoma kinase (ALK) have been effectively degraded in preclinical studies using PROTAC technology. With its event-driven unique advantages, PROTAC drugs are poised to overcome acquired resistance issues associated with small molecule inhibitors and exhibit promising therapeutic potential for challenging drug targets, potentially becoming a new strategy for NSCLC treatment.
In 2001, the concept of PROTACs was introduced by Crews and Deshaies. PROTACs are bifunctional molecules consisting of a target protein ligand and an E3 ubiquitin ligase ligand connected by a linker. Upon entering the cell, the ligand targeting the desired protein binds specifically to it, while the other end recruits an E3 ligase, forming a ternary complex of target protein-PROTAC-E3 ligase. The E3 ligase mediates ubiquitination of the target protein, leading to polyubiquitination and subsequent selective degradation by the proteasome upon dissociation of the ternary complex.
While small molecule kinase inhibitors primarily exert their effects by occupying active binding sites, often leading to resistance issues, PROTACs function by inducing degradation of target proteins, offering potential advantages in overcoming resistance and targeting traditionally deemed undruggable targets. Furthermore, PROTACs possess catalytic properties, allowing them to repeatedly bind and degrade target proteins after ubiquitination, potentially reducing dosing frequency. Currently, PROTACs are in rapid development and are emerging as a new strategy for cancer treatment.
Research Progress of PROTACs in NSCLC Treatment
KRAS plays a vital role in cell survival and cycle progression and is one of the most frequently mutated genes in NSCLC. The occurrence rate of KRAS mutations in lung adenocarcinoma is approximately 30% and 5% in lung squamous cell carcinoma. Common KRAS mutations in lung cancer include G12C, G12V, G12A, G12R, G12D, and G12S. KRAS mutations lead to the generation of mutant RAS proteins, activating downstream signaling pathways, promoting tumor cell growth, proliferation, and survival. RAS proteins lack traditional druggable pockets and have a higher affinity for GTP than ATP, posing challenges in developing KRAS inhibitors, which have long been considered undruggable targets.
Developing protein degradation agents targeting KRAS can serve as a strategy to modulate mutated KRAS. In 2020, the Crews team reported the first PROTAC molecule capable of degrading KRAS G12C and conducted in-depth studies in lung cancer cells. This compound effectively degraded KRAS G12C, with DC50 values ranging from 0.25 μmol/L to 0.76 μmol/L in five different KRAS G12C mutation cell lines. The experimental results demonstrate that PROTAC molecules can degrade mutant KRAS effectively.
EGFR mutations are present in 10%-15% of NSCLC patients. EGFR-tyrosine kinase inhibitors (EGFR-TKIs) have become a hot topic in drug development, with three generations of EGFR-TKIs currently available. While first-generation EGFR-TKIs like gefitinib and erlotinib show efficacy in EGFR 19del and EGFR L858R mutant patients, resistance often develops. Second-generation EGFR-TKIs like afatinib and dacomitinib, despite enhanced inhibition, struggle to overcome the T790M mutation. Third-generation EGFR-TKIs like osimertinib have shown promise in overcoming T790M resistance and treating NSCLC brain metastases. However, resistance can still occur, necessitating the development of effective strategies against mutations such as T790M/C797S. Several research teams are utilizing PROTAC technology to target EGFR degradation, aiming to overcome EGFR resistance mutations.
In 2018, the Crews team demonstrated the feasibility of using PROTAC technology for targeted degradation of EGFR kinase. While PROTAC molecules generally have large molecular weights, issues regarding oral bioavailability and in vivo efficacy are significant concerns in PROTAC drug research. In 2019, the Jin team identified EGFR-PROTAC molecules with improved bioavailability, maintaining high blood concentrations even after 8 hours post-injection at a dose of 50 mg/kg in mouse pharmacokinetic experiments. In 2020, the Gray team utilized fourth-generation EGFR inhibitors as ligands in PROTAC drug research targeting EGFR degradation. One molecule effectively inhibited proliferation in cells carrying triple mutations EGFR L858R/T790M/C797S and EGFR L858R/T790M/L718Q, with half-maximal inhibitory concentrations (IC50) of 0.041 μmol/L and 0.028 μmol/L, respectively. Combined with osimertinib, this PROTAC molecule showed enhanced inhibition of cell proliferation. The catalytic nature of PROTAC molecules, allowing repeated binding and action after target protein ubiquitination, holds promise for reducing dosing frequency compared to small molecule inhibitors.
Approximately 4% of NSCLC cases involve ALK fusion genes, with echinoderm microtubule-associated protein-like 4 (EML4)-ALK being the primary fusion gene. EML4-ALK fusion proteins have been shown to induce lung cancer in vivo. Three generations of ALK inhibitors are available, with crizotinib being the first FDA-approved ALK inhibitor. Third-generation ALK inhibitor lorlatinib has become the last resort for overcoming ALK resistance mutations in the clinic. Research teams are currently utilizing PROTAC technology to develop degradation agents targeting ALK, offering a potential new solution for ALK resistance mutations.
In 2018, the Gray team reported the first PROTAC molecule capable of targeted degradation of ALK. Experimental results showed that this PROTAC molecule had a half-degradation concentration (DC50) of only 10 nmol/L for ALK in H3122 cell lines. Further studies revealed that this molecule not only degraded ALK protein but also showed some degradation capability towards other target proteins such as PTK2, FER, RPS6KA1, and Aurora A. Selecting appropriate ALK ligands and improving selectivity towards ALK protein and the degradation efficacy against resistance mutations are crucial directions for future ALK-PROTAC research.
