Bioresponsive lysosome-targeting chimeras for targeted protein degradation
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ABSTRACT:
PROVIDER: PRJNA1256450 | ENA |
REPOSITORIES: ENA
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Project description:Lysosome-mediated degradation of extracellular proteins represents an emerging therapeutic paradigm that exploits cellular waste-disposal machinery to eliminate pathogenic targets. Despite its promise, achieving selective degradation of disease-associated proteins remains constrained by the scarcity of discovering highly effective disease-specific lysosome-targeting receptors. To address this limitation, we engineered NeuroTAC, a lysosome-targeting chimera (LYTAC) that bridges a sortilin-binding ligand-neurotensin (NT) to a disease-specific antibody targeting proteins overexpressed in tumors and inflammatory disorders. NeuroTAC demonstrated robust degradation efficacy against both membrane-bound and extracellular proteins in experimental models. Leveraging the dysregulated activity of matrix metalloproteinases (MMPs), a characteristic feature of tumor and inflammatory microenvironments, we further developed BioresTAC as an innovative bioresponsive LYTAC variant. This advanced construct integrates an MMP-cleavable linker and terminal RGD peptides, enabling MMP-triggered activation and spatial precision in MMP-enriched pathological niches. Systematic validation demonstrated that NeuroTAC and BioresTAC mediate broad-spectrum protein degradation with microenvironmental selectivity, enhancing therapeutic efficacy in cancer and psoriasis models. These findings advance the translational potential of lysosome-engaging biologics, offering a dual strategy for precision degradation through receptor recruitment and microenvironmental sensing.
2025-05-01 | GSE295826 | GEO
Project description:Targeted protein degradation is a powerful therapeutic strategy that offers benefits over canonical target inhibition. Lysosome targeting chimeras (LYTACs) harness lysosome trafficking receptors such as the cation-independent mannose-6-phosphate receptor (CI-M6PR) to direct secreted and membrane proteins to lysosomes. Their development as therapeutics would benefit from mechanistic insights into the factors that govern their activity. We conducted a genome-wide CRISPR screen to identify modulators of LYTAC-mediated membrane protein degradation. Disrupting retromer genes improved LYTAC-induced degradation by reducing the recycling of LYTAC-CI-M6PR complexes from endosomes to the plasma membrane. We identified neddylated cullin 3 as a predictive marker for LYTAC efficacy. Quantitative proteomics revealed that a significant fraction of cell-surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Accordingly, disruption of M6P biosynthesis enhanced the internalization of LYTAC-target complexes. Our findings inform new design strategies for LYTACs with enhanced degradation activity and elucidate the biology of CI-M6PR with implications for enzyme replacement therapies.
2024-01-26 | PXD037899 | Pride
Project description:Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6-phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking.
| S-EPMC10766146 | biostudies-literature
Project description:PROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-Ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.
2022-06-26 | GSE206612 | GEO
Project description:Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.
| S-EPMC8309050 | biostudies-literature
Project description:Targeted protein degradation has generated excitement in chemical biology and drug discovery throughout academia and industry. By hijacking the machinery responsible for protein degradation via the ubiquitin proteasome system (UPS), various cellular targets have been selectively degraded. However, since the tools used, often termed PROteolysis TArgeting Chimeras (PROTACs), hijack the intracellular quality control machinery, this technology can only access targets within the cell. Extracellular targets such as growth factors, cytokines, and chemokines bind to cell surface receptors, often initiating aberrant signaling in multiple diseases such as cancer and inflammation. However, efforts to develop small molecule inhibitors for these extracellular target proteins have been challenging. Herein, we developed a proof-of-concept approach to evaluate if extracellular proteins can be internalized and degraded via the receptor-mediated endolysosomal pathway. Using a heterodimeric molecule, termed "ENDosome TArgeting Chimera" (ENDTAC), internalization and degradation of an extracellular recombinant eGFP-HT7 fusion protein was achieved by hijacking the decoy GPCR receptor, CXCR7. This proof-of-concept study suggests that using ENDTACs to co-opt the endosomal-lysosomal degradation pathway, in contrast to PROTACs using the UPS, may provide an avenue for degrading extracellular targets such as cytokines. Overall, the technology described herein provides a novel expansion to the field of targeted protein degradation.
| S-EPMC6598169 | biostudies-literature
Project description:Proteolysis Targeting Chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation. Here we developed a highthroughput platform that interrogates off-target degradation and found that reported pomalidomide-based PROTACs induce degradation of several ZF proteins. We generated a library of pomalidomide analogs to understand how functionalising different positions of the phthalimide ring, hydrogen bonding, steric and hydrophobic effects impact ZF protein degradation. Modifications of appropriate size on the C5 position reduced off-target ZF degradation, which we validated through target engagement and proteomics studies. By applying these design principles, we developed ALK oncoprotein-targeting PROTACs with enhanced potency and minimal offtarget degradation.
2023-12-20 | PXD046264 | Pride
Project description:Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.
| S-EPMC10125259 | biostudies-literature
Project description:Global proteomics and metabolic data for CD36-mediated endocytosis of proteolysis-targeting chimeras are deposited here.
2025-04-10 | PXD062797 | JPOST Repository
Project description:Global proteomics and metabolic data for CD36-mediated endocytosis of proteolysis-targeting chimeras has been deposited here.
2025-04-09 | PXD062747 | JPOST Repository