Summary of Targeted Protein Degradation in Clinical Trials

1. Huateng Pharma https://us.huatengsci.com Summary of Targeted Protein Degradation in Clinical Trials Targeted Protein Degradation (TPD) is a rapidly evolving field that has the potential to revolutionize the way we treat diseases. The TPD strategy, which uses small molecules to selectively degrade disease-causing proteins, exhibits multiple advantages over traditional small molecule inhibitors. The concept of TPD has been around for decades and has gained a lot of attention in recent years along with increasing research in the fields of chemical biology and drug discovery. Targeted Degradation Pathways After more than 20 years of development, more than 20 different subdivisions of TPD have been developed, and new technologies continue to be invented every year. In terms of degradation pathways, these technologies can be categorized into ubiquitin-proteasome systems and lysosomal pathway, both of which have different application scenarios. Ubiquitin-proteasome System (UPS) The ubiquitin-proteasome system (UPS) is responsible for the degradation of most intracellular proteins, such as kinases, nuclear receptors, and transcription factors, and therefore plays an essential regulatory role in critical cellular processes including cell cycle progression, proliferation, differentiation, angiogenesis and apoptosis. PROTAC and molecular glues are the two main modes of TPD technology based on the UPS. Among them, PROTAC works by recruiting E3 ubiquitin ligase and inducing target proteins to approach E3 ubiquitin ligase, leading to ubiquitination and degradation of target proteins. On the other hand, molecular glues are designed to promote or induce protein-protein interactions (PPI) between E3 ubiquitin ligase and target proteins by modifying the surface of the ubiquitin ligase, leading to ubiquitination and then degradation of the target protein. Figure 1. Proteasome pathway. Source: references [1]

2. Huateng Pharma https://us.huatengsci.com Lysosomal Systems Newly developed lysosomal systems (e.g., LYTAC), on the other hand, are an alternative strategy for degradation techniques based on the ubiquitin-proteasome system. The endosome-lysosome pathway degrades proteins extracellularly and at the cell membrane, while the autophagy-lysosome pathway degrades a broader range of intracellular targets, including aggregated proteins, lipid droplets, and damaged organelles. These technologies are still in the preclinical proof-of-concept stage with no clinical pipeline. Figure 2. Endosome-lysosome pathway. Source: references [1] Development of TPD Drugs TPD drugs have made significant breakthroughs in clinical application. Several TPD drugs are currently undergoing clinical trials for different types of cancers,

3. Huateng Pharma https://us.huatengsci.com such as breast cancer, prostate cancer, and multiple myeloma, and have shown positive therapeutic effects in early-stage studies, providing patients with more treatment options. Compared with other TPD drugs, molecular glue degradation drugs have been the first to enter the market. At present, there are three TPD drugs (molecular glues) approved and marketed worldwide, namely, thalidomide (Contergan), lenalidomide (Revlimid), and pomalidomide (Pomalyst) for the treatment of multiple myeloma, myelodysplastic syndromes, etc. Figure 3. Approved molecular glues All three are molecular glues with molecular weights below 300 Da, which work by recruiting the E3 ubiquitin ligase CRBN to degrade target proteins including the transcription factors IKZF1/3. Molecular Glues in Clinical Trials According to statistics, there are 16 molecular glue drugs in the clinical research stage (Table 1), and more than 20 active compounds have been reported in the preclinical stage, of which the highest research status is clinical phase II, and the main leading companies are Celgene (BMS), Novartis, C4 Therapeutics, and BioTheryX, etc. The majority of the new drugs developed so far have been used to degrade proteins through CRBN, a component molecule of E3, mainly for the treatment of MM, hematologic tumors and solid malignant tumors. In terms of mechanism of action, IKZF1/3 are the most popular targets, and new targets such as GSPT1, DCAF15, IKZF2, BCL-xL, SEL1L2 have also been developed.

4. Huateng Pharma https://us.huatengsci.com Table 1. Molecular Glues in Clinical Trials PROTACs in Clinical Trials The development of PROTAC has also made a major breakthrough, which is the most prominent TPD technology pathway at present. In 2015, Winter et al. synthesized the first in vivo compatible PROTAC, taking the first step of PROTAC drug development. In 2019, as the world's first PROTAC small molecule drug targeting androgen receptor (AR), ARV-110 received fast-track approval from the FDA for clinical trials, marking a key step in the durability of PROTAC technology and becoming a milestone event in the field. According to incomplete statistics, more than 20 PROTACs have been approved for clinical trials worldwide, and more than 180 drugs are in the

5. Huateng Pharma https://us.huatengsci.com preclinical stage, covering many mature drug targets such as AR, estrogen receptors (ER), Bruton's tyrosine kinase (BTK) and so on. Table 2. PROTACs in clinical development Future Direction for Targeted Protein Degradation Overall, TPDs have a promising future and are expected to lead to more effective treatments for a variety of diseases. Although TPD still faces a number of challenges to overcome, the rapid momentum in the field signals that TPD will continue to be a focal point of drug discovery in the coming years. In recent years, one of the trends in TPD has been the deep integration with other subjects, such as synthetic biology, protein engineering and nucleic acid regulation, which has led to more diverse forms of TPD molecules. For example, in order to specifically degrade target proteins, researchers are designing "antibody-PROTAC" conjugates, a novel structure similar to an antibody-drug conjugate (ADC). In addition, bispecific antibody degraders have been shown to efficiently drive the cell membrane-bound E3 ubiquitin ligases RNF and ZNRF3, leading

6. Huateng Pharma https://us.huatengsci.com to the degradation of transmembrane proteins such as PD-L1, EGFR and IGF1R. Another trend is the development of protein-based biodegraders, which are centered on an intracellular chimeric protein consisting of a high-affinity target-binding domain fused to an engineered E3 ubiquitin ligase adapter. Upon delivery of mRNA encoding the biodegradable agent protein to the cell, it can target important cancer proteins such as proliferating cell nuclear antigen (PCNA). On the other hand, in an effort to broaden the scope of the degradable genome, oligonucleotides have begun to be used to target RNA- and DNA-binding proteins that lack a well-defined structure of available ligand-binding pockets. In addition to the approach by inducing protein ubiquitination, we foresee that the new proximity-induced regulatory strategies will also direct other post-transcriptional modifications (PTMs), such as deubiquitination, dephosphorylation, phosphorylation, and acetylation, which in turn will lead to a variety of functions such as stabilization, inactivation, activation, and localization of the target proteins. These innovative studies are further advancing the field of TPD and bringing more opportunities for translational applications. By combining traditional drug discovery methods with emerging technologies, researchers are continually expanding the boundaries of the applications of TPD strategies. These advances not only highlight the importance of interdisciplinary collaboration, but also the close integration between scientific research, technological innovation and drug development. PROTAC technology is currently undergoing rapid development, and it can solve the problem of the non-druggability of many target proteins. As a reliable PEG supplier, Huateng Pharma provides multi-functionalized PEG derivatives as PROTAC linkers to empower customers’ PROTAC research. References: [1] Wang, H., Zhou, R., Xu, F. et al. Beyond canonical PROTAC: biological targeted protein degradation (bioTPD). Biomater Res 27, 72 (2023). https://doi.org/10.1186/s40824-023-00385-8 [2] Teng, Mingxing, and Nathanael S Gray. “The rise of degrader drugs.” Cell chemical biology, S2451-9456(23)00198-8. 6 Jul. 2023, doi:10.1016/j.chembiol.2023.06.020 [3] Chirnomas, Deborah et al. “Protein degraders enter the clinic - a new approach to cancer therapy.” Nature reviews. Clinical oncology vol. 20,4 (2023): 265-278. doi:10.1038/s41571-023-00736-3

7. Huateng Pharma https://us.huatengsci.com [4] Maneiro, Marı A et al. “Antibody-PROTAC Conjugates Enable HER2-Dependent Targeted Protein Degradation of BRD4.” ACS chemical biology vol. 15,6 (2020): 1306-1312. doi:10.1021/acschembio.0c00285 Related Articles: Antibody + PROTAC: The Emergence of Degrader-Antibody Conjugates PROTAC Technology： An Effective Targeted Protein Degrader Application of PROTAC In The New Field of Attenuated Vaccines PROTAC Molecule For Acute Lymphoblastic Leukemia (ALL) Development And Application of "Click Chemistry" In ADC And PROTAC Covalent PROTACs, An Emerging Protein Degradation Technology