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.

Proteasomal and lysosomal degradation for specific and durable suppression of immunotherapeutic targets

Cancer immunotherapy harness the body's immune system to eliminate cancer,by using a broad panel of soluble and membrane proteins as therapeutic targets.Immunosuppression signaling mediated by ligand-receptor interaction may be blocked by monoclonal antibodies,but because of repopulation of the membranevia intracellular organelles,targets must be eliminated in whole cells.Targeted protein degradation,as exemplified in proteolysis targeting chimera(PROTAC)studies,is a promising strategy for selective inhibition of target proteins.The recently reported use of lysosomal targeting molecules to eliminate immune checkpoint proteins has paved the way for targeted degradation of membrane proteins as crucial anti-cancer targets.Further studies on these molecules'modes of action,target-binding"warheads",lysosomal sorting signals,and linker design should facilitate their rational design.Modifications and derivatives may improve their cell-penetrating ability and thein vivo stability of these pro-drugs.These studies suggest the promise of alternative strategies for cancer immunotherapy,with the aim of achieving more potent and durable suppression of tumor growth.Here,the successes and limitations of antibody inhibitorsin cancer immunotherapy,as well as research progress on PROTAC-and lysosomal-dependent degradation of target proteins,are reviewed.

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.

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.

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.

Proteolysis targeting chimera technology:a novel strategy for treating diseases of the central nervous system

Neurological diseases such as stroke,Alzheimer’s disease,Parkinson’s disease,and Huntington’s disease are among the intractable diseases for which appropriate drugs and treatments are lacking.Proteolysis targeting chimera(PROTAC)technology is a novel strategy to solve this problem.PROTAC technology uses the ubiquitin-protease system to eliminate mutated,denatured,and harmful proteins in cells.It can be reused,and utilizes the protein destruction mechanism of the cells,thus making up for the deficiencies of traditional protein degradation methods.It can effectively target and degrade proteins,including proteins that are difficult to identify and bind.Therefore,it has extremely important implications for drug development and the treatment of neurological diseases.At present,the targeted degradation of mutant BTK,mHTT,Tau,EGFR,and other proteins using PROTAC technology is gaining attention.It is expected that corresponding treatment of nervous system diseases can be achieved.This review first focuses on the recent developments in PROTAC technology in terms of protein degradation,drug production,and treatment of central nervous system diseases,and then discusses its limitations.This review will provide a brief overview of the recent application of PROTAC technology in the treatment of central nervous system diseases.

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.

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.

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.

The E3 ligase XBAT35 mediates thermoresponsive hypocotyl growth by targeting ELF3 for degradation in Arabidopsis

Plants are capable of coordination of their growth and development with ambient temperatures.EARLY FLOWERING3(ELF3), an essential component of the plant circadian clock, is also involved in ambient temperature sensing, as well as in inhibiting the expression and protein activity of the thermoresponsive regulator phytochrome interacting factor4(PIF4). The ELF3 activity is subjected to attenuation in response to warm temperature;however,how the protein level of ELF3 is regulated at warm temperature remains less understood. Here, we report that the E3 ligase XB3 ORTHOLOG 5 IN ARABIDOPSIS THALIANA, XBAT35, mediates ELF3 degradation. XBAT35 interacts with ELF3 and ubiquitinates ELF3. Loss-of-function mutation of XBAT35 increases the protein level of ELF3 and confers a short-hypocotyl phenotype under warm temperature conditions. Thus, our findings establish that XBAT35 mediates ELF3 degradation to lift the inhibition of ELF3 on PIF4 for promoting thermoresponsive hypocotyl growth in plants.

Autophagic Clearance of Lipid Droplets Alters Metabolic Phenotypes in a Genetic Obesity–Diabetes Mouse Model

Lipid droplets(LDs)are intracellular organelles that store neutral lipids,and their aberrant accumulation is associated with many diseases including metabolic disorders such as obesity and diabetes.Meanwhile,the potential pathological contribu-tions of LDs in these diseases are unclear,likely due to a lack of chemical biology tools to clear LDs.We recently developed LD-clearance small molecule compounds,Lipid Droplets·AuTophagy TEthering Compounds(LD·ATTECs),that are able to induce autophagic clearance of LDs in cells and in the liver of db/db(C57BL/6J Leprdb/Leprdb)mouse model,which is a widely used genetic model for obesity–diabetes.Meanwhile,the potential effects on the metabolic phenotype remain to be elucidated.Here,using the metabolic cage assay and the blood glucose assay,we performed phenotypic characteriza-tion of the effects of the autophagic degradation of LDs by LD·ATTECs in the db/db mouse model.The study reveals that LD·ATTECs increased the oxygen uptake of mice and the release of carbon dioxide,enhanced the heat production of animals,partially enhanced the exercise during the dark phase,decreased the blood glucose level and improved insulin sensitivity.Collectively,the study characterized the metabolic phenotypes induced by LD·ATTECs in an obesity–diabetes mouse model,revealing novel functional impacts of autophagic clearance of LDs and providing insights into LD biology and obesity–dia-betes pathogenesis from the phenotypic perspective.

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.

自噬小体绑定化合物(ATTEC)靶向降解线粒体及其潜在治疗效果

功能异常的线粒体的累积会诱发多种神经退化相关疾病,如帕金森氏病(PD)和唐氏综合征(DS).借助小分子化合物促进线粒体的降解被认为可以缓解这类疾病的进程.然而,目前尚缺乏这样一类高效、安全的靶向降解线粒体的小分子化合物.本研究借助一种名为自噬小体绑定化合物(ATTEC)的新型靶向降解技术,创造性地合成一种能同时结合自噬关键分子LC3B以及线粒体外膜蛋白TSPO的小分子化合物—mT1,并发现该化合物能够有效地降解细胞中的线粒体.借助分子生物学及细胞生物学手段,作者证明mT1能够促进LC3B和TSPO结合,从而将线粒体靶向自噬小体,并进一步与溶酶体融合而降解.此外,在PD的细胞模型和DS的类器官模型中证明mT1能够通过促进线粒体的清除而改善相应的病理学表型.该研究将ATTEC技术的应用范畴拓展到亚细胞器的层面,并为以PD和DS为代表的一类线粒体异常引起的疾病提供了一种潜在的新治疗策略.