The c-ros oncogene 1 (ROS1), an oncogenic driver, is known to induce non-small cell lung cancer (NSCLC) when overactivated, particularly through the formation of fusion proteins. Traditional targeted therapies focus on inhibiting ROS1 activity with ROS 1 inhibitors to manage cancer progression. However, a new strategy involving the design of protein degraders offers a more potent approach by completely degrading ROS1 fusion oncoproteins, thereby effectively blocking their kinase activity and enhancing anti-tumour potential. Utilizing PROteolysis-TArgeting Chimera (PROTAC) technology and informed by molecular docking and rational design, we report the first ROS1-specific PROTAC, SIAIS039. This degrader effectively targets multiple ROS1 fusion oncoproteins (CD74-ROS1, SDC4-ROS1 and SLC34A2-ROS1) in engineered Ba/F3 cells and HCC78 cells, demonstrating anti-tumour effects against ROS1 fusion-driven cancer cells. It suppresses cell proliferation, induces cell cycle arrest, and apoptosis, and inhibits clonogenicity. The anti-tumour efficacy of SIAIS039 surpasses two approved drugs, crizotinib and entrectinib, and matches that of the top inhibitors, including lorlatinib and taletrectinib. Mechanistic studies confirm that the degradation induced by 039 requires the participation of ROS1 ligands and E3 ubiquitin ligases, and involves the proteasome and ubiquitination. In addition, 039 exhibited excellent oral bioavailability in a mouse xenograft model, highlighting its potential for clinical application. In conclusion, our study presents a promising and novel therapeutic strategy for ROS1 fusion-positive NSCLC by targeting ROS1 fusion oncoproteins for degradation, laying the foundation for the development of further PROTAC and offering hope for patients with ROS1 fusion-positive NSCLC.

Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer\'s disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.

This review explores various methods for modulating protein stability to achieve target protein degradation, which is a crucial aspect in the study of biological processes and drug design. Thirty years have passed since the introduction of heat-inducible degron cells utilizing the N-end rule, and methods for controlling protein stability using the ubiquitin-proteasome system have moved from academia to industry. This review covers protein stability control methods, from the early days to recent advancements, and discusses the evolution of techniques in this field. This review also addresses the challenges and future directions of protein stability control techniques by tracing their development from the inception of protein stability control methods to the present day.

Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we designed and synthesized a novel series of proteolysis targeting chimeras (PROTACs) using a SHP2 allosteric inhibitor as warhead, with the goal of achieving SHP2 degradation both inside the cell and in vivo. Among these molecules, compound P9 induces efficient degradation of SHP2 (DC50 = 35.2 ± 1.5 nM) in a concentration- and time-dependent manner. Mechanistic investigation illustrates that the P9-mediated SHP2 degradation requires the recruitment of the E3 ligase and is ubiquitination- and proteasome-dependent. P9 shows improved anti-tumor activity in a number of cancer cell lines over its parent allosteric inhibitor. Importantly, administration of P9 leads to a nearly complete tumor regression in a xenograft mouse model, as a result of robust SHP2 depletion and suppression of phospho-ERK1/2 in the tumor. Hence, P9 represents the first SHP2 PROTAC molecule with excellent in vivo efficacy. It is anticipated that P9 could serve not only as a new chemical tool to interrogate SHP2 biology but also as a starting point for the development of novel therapeutics targeting SHP2.

\"Undruggable\" targets such as KRAS are particularly challenging in the development of drugs. We devised a novel chemical knockdown strategy, CANDDY (Chemical knockdown with Affinity aNd Degradation DYnamics) technology, which promotes protein degradation using small molecules (CANDDY molecules) that are conjugated to a degradation tag (CANDDY tag) modified from proteasome inhibitors. We demonstrated that CANDDY tags allowed for direct proteasomal target degradation independent of ubiquitination. We synthesized a KRAS-degrading CANDDY molecule, TUS-007, which induced degradation in KRAS mutants (G12D and G12V) and wild-type KRAS. We confirmed the tumor suppression effect of TUS-007 in subcutaneous xenograft models of human colon cells (KRAS G12V) with intraperitoneal administrations and in orthotopic xenograft models of human pancreatic cells (KRAS G12D) with oral administrations. Thus, CANDDY technology has the potential to therapeutically target previously undruggable proteins, providing a simpler and more practical drug targeting approach and avoiding the difficulties in matchmaking between the E3 enzyme and the target.

PROTACs (Proteolysis-Targeting Chimeras) have emerged as a groundbreaking class of chemical tools that facilitate the degradation of target proteins by leveraging the ubiquitin-proteasome system (UPS). However, the effective utilization of PROTACs in chemical biology studies and therapeutics encounters significant challenges when it comes to achieving cell-selective protein degradation and in vivo applications. This review article aims to shed light on recent advancements in the development of Pro-PROTACs, which exhibit controlled protein degradation capabilities in response to external stimuli or disease-related endogenous biochemical signals. The article delves into the specific chemical strategies employed to regulate the interaction between PROTACs and E3 ubiquitin ligases or target proteins. These strategies enable spatial and temporal control over the protein degradation potential of Pro-PROTACs. Furthermore, the review summarizes recent investigations regarding the delivery of PROTACs using biodegradable nanoparticles for in vivo applications and targeted protein degradation. Such delivery systems hold great promise for enabling efficient and selective protein degradation in vivo. Lastly, the article provides a perspective on the future design of multifunctional PROTACs and their intracellular delivery mechanisms, with a particular focus on achieving cell-selective protein degradation.

Targeted Protein Degradation is an emerging and rapidly developing technique for designing and treating new drugs. With the emergence of a promising class of pharmaceutical molecules, Heterobifunctional Proteolysis-targeting chimeras (PROTACs), TPD has become a powerful tool to completely tackle pathogenic proteins with traditional small molecule inhibitors. However, the conventional PROTACs have gradually exposed potential disadvantages of poor oral bioavailability and pharmacokinetic (PK) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics due to their larger molecular weight and more complex structure than the conventional small-molecule inhibitors. Therefore, 20 years after the concept of PROTAC was proposed, more and more scientists are committed to developing new TPD technology to overcome its defects. And several new technologies and means have been explored based on \"PROTAC\" to target \"undruggable proteins\". Here, we aim to comprehensively summarize and profoundly analyze the research progress of targeted protein degradation based on PROTAC targeting the degradation of \"undruggable\" targets. In order to clarify the significance of emerging and highly effective strategies based PROTACs in the treatment of various diseases especially in overcoming drug resistance in cancer, we will focus on the molecular structure, action mechanism, design concepts, development advantages and challenges of these emerging methods(e.g., aptamer-PROTAC conjugates, antibody-PROTACs and folate-PROTACs).

Targeted protein degradation (TPD) is a useful approach in dissecting protein function and therapeutics. Technologies such as RNA interference or gene knockout that are routinely used rely on protein turnover. However, RNA interference takes a long time to deplete target proteins and is not suitable for long-lived proteins, while a genetic knockout is irreversible, takes a long time to achieve and is not suitable for essential genes. TPD has the potential to overcome the limitations of RNA interference and gene editing approaches. We have established the Affinity directed PROtein Missile (AdPROM) system, which harnesses nanobodies or binders of target proteins to redirect E3 ubiquitin ligase activity to the target protein to induce TPD through the ubiquitin proteasome system. Here we provide a step-by-step protocol for using the AdPROM system for targeted proteolysis of endogenously GFP-tagged K-RAS through an anti-GFP nanobody. This protocol can be amended to target a wide range of different proteins of interest (POIs) either by replacing the anti-GFP nanobody with a nanobody recognising the POI or by endogenously tagging the POI with GFP through CRISPR/Cas9 genome editing.

The development and application of traditional drugs represented by small molecule chemical drugs and biological agents, especially inhibitors, have become the mainstream drug development. In recent years, targeted protein degradation (TPD) technology has become one of the most promising methods to remove specific disease-related proteins using cell self-destruction mechanisms. Many different TPD strategies are emerging based on the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP), including but not limited to proteolysis-targeting chimeras (PROTAC), molecular glues (MG), lysosome targeting chimeras (LYTAC), chaperone-mediated autophagy (CMA)-targeting chimeras, autophagy-targeting chimera (AUTAC), autophagosome-tethering compound (ATTEC), and autophagy-targeting chimera (AUTOTAC). The advent of targeted degradation technology can change most protein targets in human cells from undruggable to druggable, greatly expanding the therapeutic prospect of refractory diseases such as metabolic syndrome. Here, we summarize the latest progress of major TPD technologies, especially in metabolic syndrome and look forward to providing new insights for drug discovery.

Chronic myeloid leukemia (CML) is a malignant disease of the hematopoietic system with crucial pathogenic protein named BCR-ABL, which endangers the life of patients severely. As a milestone of targeted drug, Imatinib has achieved great success in the treatment of CML. Nevertheless, inevitable drug resistance of Imatinib has occurred frequently in clinical due to the several mutations in the BCR-ABL kinase. Subsequently, the second-generation of tyrosine kinase inhibitors (TKIs) against BCR-ABL was developed to address the mutants of Imatinib resistance, except T315I. To date, the third-generation of TKIs targeting T315I has been developed for improving the selectivity and safety. Notably, the first allosteric inhibitor has been in market which could overcome the mutations in ATP binding site effectively. Meanwhile, some advanced technology, such as proteolysis-targeting chimeras (PROTAC) based on different E3 ligand, are highly expected to overcome the drug resistance by selectively degrading the targeted proteins. In this review, we summarized the current research progress of inhibitors and degraders targeting BCR-ABL for the treatment of CML.