Expanding the horizons of targeted protein degradation:A non-small molecule perspective

Targeted protein degradation(TPD)represented by proteolysis targeting chimeras(PROTACs)marks a significant stride in drug discovery.A plethora of innovative technologies inspired by PROTAC have not only revolutionized the landscape of TPD but have the potential to unlock functionalities beyond degradation.Non-small-molecule-based approaches play an irreplaceable role in this field.A wide variety of agents spanning a broad chemical spectrum,including peptides,nucleic acids,antibodies,and even vaccines,which not only prove instrumental in overcoming the constraints of conventional small molecule entities but also provided rapidly renewing paradigms.Herein we summarize the burgeoning non-small molecule technological platforms inspired by PROTACs,including three major trajectories,to provide insights for the design strategies based on novel paradigms.

Machine Learning Modeling of Protein-intrinsic Features Predicts Tractability of Targeted Protein Degradation

Targeted protein degradation(TPD)has rapidly emerged as a therapeutic modality to eliminate previously undruggable proteins by repurposing the cell’s endogenous protein degradation machinery.However,the susceptibility of proteins for targeting by TPD approaches,termed“degradability”,is largely unknown.Here,we developed a machine learning model,model-free analysis of protein degradability(MAPD),to predict degradability from features intrinsic to protein targets.MAPD shows accurate performance in predicting kinases that are degradable by TPD compounds[with an area under the precision–recall curve(AUPRC)of 0.759 and an area under the receiver operating characteristic curve(AUROC)of 0.775]and is likely generalizable to independent non-kinase proteins.We found five features with statistical significance to achieve optimal prediction,with ubiquitination potential being the most predictive.By structural modeling,we found that E2-accessible ubiquitination sites,but not lysine residues in general,are particularly associated with kinase degradability.Finally,we extended MAPD predictions to the entire proteome to find964 disease-causing proteins(including proteins encoded by 278 cancer genes)that may be tractable to TPD drug development.

Hydrophobic tag tethering degrader as a promising paradigm of protein degradation:Past,present and future perspectives

Small molecule inhibitors have dominated the pharmaceutical landscape for a long time as the primary therapeutic paradigm targeting pathogenic proteins.However,their efficacy heavily relies on the amino acid composition and spatial constitution of proteins,rendering them susceptible to drug resistance and failing to target undruggable proteins.In recent years,the advent of targeted protein degradation(TPD)technology has captured substantial attention from both industry and academia.Employing an event-driven mode,TPD offers a novel approach to eliminate pathogenic proteins by promoting their degrada-tion,thus circumventing the limitations associated with traditional small molecule inhibitors.Hydropho-bic tag tethering degrader(HyTTD)technology represents one such TPD approach that is currently in the burgeoning stage.HyTTDs employ endogenous protein degradation systems to induce the degrada-tion of target proteins through the proteasome pathway,which displays significant potential for medical value.In this review,we provide a comprehensive overview of the development history and the reported mechanism of action of HyTTDs.Additionally,we delve into the physiological roles,structure-activity re-lationships,and medical implications of HyTTDs targeting various disease-associated proteins.Moreover,we propose insights into the challenges that necessitate resolution for the successful development of HyTTDs,with the ultimate goal of initiating a new age of clinical treatment leveraging the immense po-tential of HyTTDs.

Targeted Degradation of DNA/RNA Binding Proteins via Covalent Hydrophobic Tagging

Targeted protein degradation(TPD)holds great promise for biological inquiry and therapeutic development.However,it still remains elusive to destruct DNA/RNA binding proteins(DBPs/RBPs)previously deemed undruggable.Herein,we report ligandassisted covalent hydrophobic tagging(LACHT)as a modular strategy for TPD of these difficult-totarget proteins.Guided by a noncovalent protein ligand,LACHT leverages a reactive N-acyl-N-alkyl sulfonamide group to covalently label the protein target with a hydrophobic adamantane,which further engages the cellular quality control mechanism to induce proteolytic degradation.Using a smallmolecule ligand,we demonstrated that LACHT allowed TPD of a DBP,bromodomain-containing protein 4,in human leukemia cells with high efficiency.Mechanistic studies revealed that LACHT-mediated TPD dependent on ligand-directed irreversible tagging and the covalently labeled proteins underwent polyubiquitination before removal through both the proteasome and the lysosome.Furthermore,when an RNA ligand was employed,we showed that LACHT also enabled TPD of an RBP,Lin28a,leading to upregulation of its downstream let-7 miRNA.This study thus provides a generalizable platform to expand the TPD toolbox for biomedical applications.

Molecular glue triggers degradation of PHGDH by enhancing the interaction between DDB1 and PHGDH

Cancer stem cells(CSCs)play a pivotal role in tumor initiation,proliferation,metastasis,drug resistance,and recurrence.Consequently,targeting CSCs has emerged as a promising avenue for cancer therapy.Recently,3-phosphoglycerate dehydrogenase(PHGDH)has been identified as being intricately associated with the regulation of numerous cancer stem cells.Yet,reports detailing the functional regulators of PHGDH that can mitigate the stemness across cancer types are limited.In this study,the novel“molecular glue”LXH-3-71 was identified,and it robustly induced degradation of PHGDH,thereby modulating the stemness of colorectal cancer cells(CRCs)both in vitro and in vivo.Remarkably,LXH-3-71 was observed to form a dynamic chimera,between PHGDH and the DDB1-CRL E3 ligase.These insights not only elucidate the anti-CSCs mechanism of the lead compound but also suggest that degradation of PHGDH may be a more viable therapeutic strategy than the development of PHGDH inhibitors.Additionally,compound LXH-3-71 was leveraged as a novel ligand for the DDB1-CRL E3 ligase,facilitating the development of new PROTAC molecules targeting EGFR and CDK4 degradation.

Artificial Nucleic Acid Tractor-Directed Simultaneous Depletion of Oncogenic Membrane Proteins Without Hijacking Proteolysis-Specific Actuator

Targeted protein degradation(TPD)is an emerging tool for degrading proteins of interest,which affords an attractive modality for cancer therapy.However,the present TPD technologies must engage a proteolysis-specific actuator to initiate degradation of targeted proteins in the proteasome or lysosome.Herein,we report an artificial tractor that can induce endocytosis-mediated protein depletion without hijacking a proteolysis-specific actuator.In this design,bispecific aptamer chimeras(BSACs)are established,which can bridge human epidermal growth factor receptor 2(ErbB-2),an important biomarker in a common important biomarker in cancer,with membrane proteins of interest.Taking advantage of the property of aptamer-induced endocytosis and digestion of ErbB-2,another membrane protein is translocated into the lysosome in a hitchhike-like manner,resulting in lysosomal proteolysis along with ErbB-2.This strategy frees the TPD from the fundamental limitation of proteolysis-specific actuator and allows simultaneous regulation of the quantity and function of two oncogenic receptors in a cell-type-specific manner,expanding the application scope of TPD-based therapeutics.

靶向难成药蛋白——蛋白质降解剂的力量

Undruggable targets typically refer to a class of therapeutic targets that are difficult to target through conventional methods or have not yet been targeted,but are of great clinical significance.According to statistics,over 80%of disease-related pathogenic proteins cannot be targeted by current conventional treatment methods.In recent years,with the advancement of basic research and new technologies,the development of various new technologies and mechanisms has brought new perspectives to overcome challenging drug targets.Among them,targeted protein degradation technology is a breakthrough drug development strategy for challenging drug targets.This technology can specifically identify target proteins and directly degrade pathogenic target proteins by utilizing the inherent protein degradation pathways within cells.This new form of drug development includes various types such as proteolysis targeting chimera(PROTAC),molecular glue,lysosome-targeting Chimaera(LYTAC),autophagosometethering compound(ATTEC),autophagy-targeting chimera(AUTAC),autophagy-targeting chimera(AUTOTAC),degrader-antibody conjugate(DAC).This article systematically summarizes the application of targeted protein degradation technology in the development of degraders for challenging drug targets.Finally,the article looks forward to the future development direction and application prospects of targeted protein degradation technology.

Current strategies for improving limitations of proteolysis targeting chimeras

Proteolysis targeting chimeras(PROTACs)are bifunctional degrader molecules via hijacking the ubiquitinproteasome system(UPS)to specifically eliminate targeted proteins.PROTACs have gained momentum as a new modality of attractive technologies in the drug discovery landscape,since it allows to degrade disease-related proteins effectively.Although some PROTACs drugs reached the clinical research,they are still facing some bottlenecks and challenges that should not be neglected,such as poor oral bioavailability and potential toxic side effects.To overcome these limitations,herein,we provide an overview of recent strategies for improving the durability of PROTACs by enhancing cell permeability and reducing toxic side effects.Meanwhile,the impact of these strategies on improving oral bioavailability as well as their advantages and drawbacks will also be discussed.This review will give a useful reference toolbox for PROTACs design and further promote its clinical application.