Amphoteric PROTAC with Enhanced Pharmacokinetic Properties Targeting ALK
Anaplastic lymphoma kinase and ALK gene fusion
Anaplastic lymphoma kinase (ALK) is a transmembrane protein tyrosine kinase belonging to the insulin receptor kinase subfamily. The forms of ALK gene mutation include overexpression, fusion gene formation with other genes and point mutation. It was first identified in anaplastic large cell lymphoma (ALCL) with a nuclear phosphoprotein (NPM)-ALK fusion form, a subtype of T-cell non-Hodgkin lymphoma that is often associated with chromosomal translocations. Since then, many cancers have been found to be associated with different forms of ALK fusion, which can produce fusion proteins with more than 20 different genes in cancer, constitutive activation of the ALK signaling pathway. These include non-small cell lung cancer (NSCLC, EML4-ALK), inflammatory myofibroblastoma (IMT, TPM3-ALK), and diffuse large B-cell lymphoma (DLBCL, CLTC-ALK). The fusion of ALK gene with echinodermal microtubule-associated protein-4 (EML4) gene is an important driver of NSCLC and a crucial pathway in the formation and progression of lung adenocarcinoma. In addition, amplification of the ALK gene and mutations of the wild-type ALK protein have been reported in various tumors.
ALK has become an attractive therapeutic target, in part because of its low levels in normal adult tissue, which is thought to reduce the chance of off-target toxicity caused by ALK inhibitors. Therefore, therapeutic strategies that inhibit ALK kinase activity are thought to produce fewer side effects. To date, three generations of ALK inhibitors have been approved by the FDA. Although these ALK inhibitors have shown good efficacy in clinical applications, resistance remains a serious challenge over time.
Targeted protein degradation technology breaks through the limitations of traditional inhibitors that must continue to occupy the active site of the protein to play the inhibitory role, and it is not easy to develop drug resistance. The protein degradation targeting chimera (PROTAC) molecule is a typical bi-functional molecule, one end is the ligand that targets the target protein, the other end is the ligand that binds the E3 ubiquitin ligase, and the middle is connected by a certain length of Linker. In this way, the target protein and E3 ubiquitin ligase can be pulled closer in vivo, so that the target protein is labeled by ubiquitin, and then degraded by the proteasome, and the target protein can be "knocked out" after translation. Compared with traditional small molecule drugs, PROTACs has the following advantages: 1) it can target proteins without obvious "pockets" and non-targeted proteins; 2) PROTACs contains two ligands, with bidirectional selection, which improves drug selectivity and reduces off-target toxicity; 3) the dosage is small, recycling, and drug resistance is not easy to occur. Therefore, it is of great significance to design PROTACs targeting ALK degradation to solve the above problems.
The ALK inhibitor ceritinib-derived PROTAC 91 effectively reduced the levels of NPM-ALK in SU-DHL-1 cells and EML4-ALK in H2228 cells. PROTAC 91 could significantly inhibit the proliferation of SU-DHL-1 cell line. In vivo studies have shown that at 12 h after administration (50mg/kg, intraperitoneal injection), 91 can reach a blood concentration of 340 nM in mice, which is much higher than its IC50 value for the SU-DHL-1 cell line. Ceritinib-based PROTAC 92 significantly accelerated the degradation of ALK in NSCLC cells H3122 and ALCL cells Karpas 299 in a UPS dependent manner. Unlike the CRBN-recruited PROTACs mentioned above, PROTAC 93 containing VHL ligand can effectively induce ALK degradation and inhibit the proliferation of SU-DHL-1 and H3122 cell lines.
Schematic diagram of chemical structure of PROTACs targeting ALK. (Zhao, H. Y., 2023)
Bugatinib, a second-generation ALK inhibitor, was approved in 2017 for the treatment of ALK-positive metastatic NSCLC. When used in combination with EGFR antibodies, bugatinib is able to overcome resistance caused by triple mutant EGFR. PROTAC 95 acts as a potent ALK depressant, effectively killing ALK in SR cells and significantly inhibiting the proliferation of SR (and H2228 cell lines). 95 can induce degradation of resistant mutant ALKG1202R in 293T cells. PROTAC 95 had an IC50 value of 146.4 nM against the 293T cell line, exceeding bugatinib. CRBN-recruited PROTAC 96 has strong cellular activity against SR cell lines with an IC50 value of 2.0 nM, slightly lower than bugatinib (IC50 = 3.3 nM). 96 can significantly induce the degradation of ALK at concentrations as low as 10 nM.
Using the second-generation ALK inhibitor alitinib as an ALK ligand, PROTAC 97 was discovered. PROTAC 97 showed nanomolar degradation of ALK in H3122 and Karpas cell lines. In vivo studies showed that Karpas 299 cell xenotransplantation model given 97 (10mg/kg/ day) could reduce tumor weight by 75.82%, more than alitinib, 20mg/kg/ day, tumor weight reduction of 63.82%.
PROTACs targeting ALK at BOC Sciences
With extensive expertise in PROTAC targets, BOC Sciences also provides drug development services for PROTAC molecules to target protein kinases.
Novel amphoteric ALK PROTAC with enhanced pharmacokinetic properties
The EML4-ALK fusion gene is considered to be one of the most important drivers of non-small cell lung cancer. Several EML4-ALK degraders based on second-generation ALK inhibitors have been developed and have demonstrated their potential for protein degradation. However, these molecules have solubility or bioavailability problems, such as B3 and AP-1. In order to overcome the problems of poor water solubility, low bioavailability and insufficient selectivity of tumor cells in PROTAC technology, ensure the degradation ability of EML4-ALK protein and maintain the hydrophobicity of small molecule degraders, Shirui Wang et al. from the Cancer Center of West China Hospital of Sichuan University developed ALK degraders with alkyl linkers, and then connected PEG branches through disulfide bonds to generate the target amphipathic PROTAC molecule B1-PEG. B1-PEG can self-assemble into micelles in water and release its active form in the high glutathione (GSH) environment of tumor cells, taking advantage of the difference in GSH levels between tumor cells and normal cells, improving drug selectivity and stability, and enhancing pharmacokinetic properties. The bioavailability of B1-PEG was up to 84.8%, which was significantly better than that of unmodified PROTAC molecule B1.
PEG modification of PROTAC. (Wang, S., 2024)
In H3122 xenografted mouse models, B1-PEG showed significant antitumor effects and was able to effectively inhibit tumor growth, demonstrating its strong potential as a potential cancer therapy. B1-PEG degrades EML4-ALK fusion protein through mechanisms dependent on the ubiquitin-proteasome system (UPS), which is considered an important driver of non-small cell lung cancer. B1-PEG not only reduced the level of ALK protein, but also inhibited its phosphorylation, leading to apoptosis of tumor cells, especially at higher concentrations, almost completely inhibited the G0/G1 phase of the cell cycle, inducing early apoptosis.
Pharmacokinetic analysis showed that B1-PEG had faster absorption rate and higher plasma peak concentration, and its absolute bioavailability was significantly improved compared with B1. In the H3122 xenograft model, B1-PEG demonstrated excellent tumor growth inhibition and performed well in terms of safety, with no significant side effects observed.
The study also evaluated the safety of B1-PEG, which showed stable survival and body weight during administration, with no significant side effects observed. Histopathological analysis also showed that B1-PEG had good safety in vivo. However, the study also pointed out that too long PEG chains may increase the molecular weight of the drug, require higher doses, and too many PEG fragments may cause immunogenicity issues, which need to be further explored in subsequent studies. This study provides a new direction for overcoming bioavailability limitations in PROTAC drug design and is expected to accelerate the clinical development process of PROTAC drugs.
References:
- Zhao, H. Y., et al. Progress of small molecules for targeted protein degradation: PROTACs and other technologies. Drug Development Research. 2023, 84(2): 337-394.
- Wang, S., et al. Novel amphiphilic PROTAC with enhanced pharmacokinetic properties for alk protein degradation. Journal of Medicinal Chemistry. 2024.
