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Science (New York, N.Y.) Aug 2023Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs...
Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a β strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation.
Topics: Nuclear Proteins; Proteasome Endopeptidase Complex; Ubiquitin; Ubiquitination; Proteolysis; Genes, Immediate-Early; HEK293 Cells; NIH 3T3 Cells; Transcription, Genetic
PubMed: 37616343
DOI: 10.1126/science.adh5021 -
Angewandte Chemie (International Ed. in... Sep 2023Proteolysis Targeting Chimeras (PROTACs) represent a promising therapeutic modality to address undruggable and resistant issues in drug discovery. However, potential...
Proteolysis Targeting Chimeras (PROTACs) represent a promising therapeutic modality to address undruggable and resistant issues in drug discovery. However, potential on-target toxicity remains clinically challenging. We developed a generalized caging strategy to synthesize a series of stimuli-responsive PROTACs (sr-PROTACs) with diverse molecular blocks bearing robust and cleavable linkers, presenting "turn on" features in manipulating protein degradation. By leveraging pathological cues, such as elevated ROS, phosphatase, H S, or hypoxia, and external triggers, such as ultraviolet light, X-Ray, or bioorthogonal reagents, we achieved site-specific activation and traceless release of original PROTACs through de-caging and subsequent self-immolative cleavage, realizing selective uptake and controlled protein degradation in vitro. An in vivo study revealed that two sr-PROTACs with phosphate- and fluorine-containing cages exhibited high solubility and long plasma exposure, which were specifically activated by tumor overexpressing phosphatase or low dosage of X-Ray irradiation in situ, leading to efficient protein degradation and potent tumor remission. With more reactive biomarkers to be screened from clinical practice, our caging library could provide a general tool to design activatable PROTACs, prodrugs, antibody-drug conjugates, and smart biomaterials for personalized treatment, tissue engineering or regenerative medicine.
Topics: Humans; Proteolysis; Neoplasms; Drug Discovery; Ubiquitin-Protein Ligases
PubMed: 37470380
DOI: 10.1002/anie.202306824 -
Nature Chemistry Feb 2024Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide,...
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 high-throughput 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 analogues to understand how functionalizing different positions of the phthalimide ring, hydrogen bonding, and 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 anaplastic lymphoma kinase oncoprotein-targeting PROTACs with enhanced potency and minimal off-target degradation.
Topics: Proteolysis; Ubiquitin-Protein Ligases; Proteins; Thalidomide
PubMed: 38110475
DOI: 10.1038/s41557-023-01379-8 -
Cell Chemical Biology Jul 2023Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention, but options to target cell surface proteins and receptors remain...
Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention, but options to target cell surface proteins and receptors remain limited. Here we introduce ROTACs, bispecific WNT- and BMP-signaling-disabled R-spondin (RSPO) chimeras, which leverage the specificity of these stem cell growth factors for ZNRF3/RNF43 E3 transmembrane ligases, to target degradation of transmembrane proteins. As a proof-of-concept, we targeted the immune checkpoint protein, programmed death ligand 1 (PD-L1), a prominent cancer therapeutic target, with a bispecific RSPO2 chimera, R2PD1. The R2PD1 chimeric protein binds to PD-L1 and at picomolar concentration induces its lysosomal degradation. In three melanoma cell lines, R2PD1 induced between 50 and 90% PD-L1 protein degradation. PD-L1 degradation was strictly dependent on ZNRF3/RNF43. Moreover, R2PD1 reactivates cytotoxic T cells and inhibits tumor cell proliferation more potently than Atezolizumab. We suggest that signaling-disabled ROTACs represent a paradigm to target cell surface proteins for degradation in a range of applications.
Topics: Receptors, G-Protein-Coupled; Proteolysis; B7-H1 Antigen; Wnt Signaling Pathway; Ubiquitin-Protein Ligases
PubMed: 37321224
DOI: 10.1016/j.chembiol.2023.05.010 -
Angewandte Chemie (International Ed. in... Sep 2023Proteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post-translational protein degradation capabilities. However, off-target...
Proteolysis targeting chimera (PROTAC) is an emerging pharmacological modality with innovated post-translational protein degradation capabilities. However, off-target induced unintended tissue effects and intrinsic "hook effect" hinder PROTAC biotechnology to be maturely developed. Herein, an intracellular fabricated nano proteolysis targeting chimeras (Nano-PROTACs) modality with a center-spoke degradation network for achieving efficient dose-dependent protein degradation in tumor is reported. The PROTAC precursors are triggered by higher GSH concentrations inside tumor cells, which subsequently in situ self-assemble into Nano-PROTACs through intermolecular hydrogen bond interactions. The fibrous Nano-PROTACs can form effective polynary complexes and E3 ligases degradation network with multi-binding sites, achieving dose-dependent protein degradation with "anti-hook effect". The generality and efficacy of Nano-PROTACs are validated by degrading variable protein of interest (POI) such as epidermal growth factor receptor (EGFR) and androgen receptor (AR) in a wide-range dose-dependent manner with a 95 % degradation rate and long-lasting potency up to 72 h in vitro. Significantly, Nano-PROTACs achieve in vivo dose-dependent protein degradation up to 79 % and tumor growth inhibition in A549 and LNCap xenograft mice models, respectively. Taking advantages of in situ self-assembly strategy, the Nano-PROTACs provide a generalizable platform to promote precise clinical translational application of PROTAC.
Topics: Humans; Animals; Mice; Proteolysis; Ubiquitin-Protein Ligases; Proteins; Neoplasms; Binding Sites
PubMed: 37486792
DOI: 10.1002/anie.202308049 -
Nature Reviews. Drug Discovery Feb 2024Targeted protein degradation (TPD) has emerged in the past decade as a major new drug modality to remove intracellular proteins with bispecific small molecules that... (Review)
Review
Targeted protein degradation (TPD) has emerged in the past decade as a major new drug modality to remove intracellular proteins with bispecific small molecules that recruit the protein of interest (POI) to an E3 ligase for degradation in the proteasome. Unlike classic occupancy-based drugs, intracellular TPD (iTPD) eliminates the target and works catalytically, and so can be more effective and sustained, with lower dose requirements. Recently, this approach has been expanded to the extracellular proteome, including both secreted and membrane proteins. Extracellular targeted protein degradation (eTPD) uses bispecific antibodies, conjugates or small molecules to degrade extracellular POIs by trafficking them to the lysosome for degradation. Here, we focus on recent advances in eTPD, covering degrader systems, targets, molecular designs and parameters to advance them. Now almost any protein, intracellular or extracellular, is addressable in principle with TPD.
Topics: Humans; Proteolysis; Proteasome Endopeptidase Complex; Membrane Proteins; Drug Discovery; Ubiquitin-Protein Ligases
PubMed: 38062152
DOI: 10.1038/s41573-023-00833-z -
Advanced Science (Weinheim,... Sep 2023Oxaliplatin is a widely used chemotherapy drug for patients with advanced colorectal cancer (CRC); however, frequent drug resistance limits its therapeutic efficacy in...
Oxaliplatin is a widely used chemotherapy drug for patients with advanced colorectal cancer (CRC); however, frequent drug resistance limits its therapeutic efficacy in patients. Here, this work identifies cyclin-dependent kinase 1 (CDK1) as a critical contributor to oxaliplatin resistance via in vitro and in vivo CRISPR/Cas9 screening. CDK1 is highly expressed in oxaliplatin-resistant cells and tissues due to the loss of N6-methyladenosine modification. Genetic and pharmacological blockade of CDK1 restore the susceptibility of CRC cells to oxaliplatin in vitro and in cell/patient-derived xenograft models. Mechanistically, CDK1 directly binds to and phosphorylates Acyl-CoA synthetase long-chain family 4 (ACSL4) at S447, followed by recruitment of E3 ubiquitin ligase UBR5 and polyubiquitination of ACSL4 at K388, K498, and K690, which leads to ACSL4 protein degradation. Reduced ACSL4 subsequently blocks the biosynthesis of polyunsaturated fatty acid containing lipids, thereby inhibiting lipid peroxidation and ferroptosis, a unique iron-dependent form of oxidative cell death. Moreover, treatment with a ferroptosis inhibitor nullifies the enhancement of CRC cell sensitivity to oxaliplatin by CDK1 blockade in vitro and in vivo. Collectively, the findings indicate that CDK1 confers oxaliplatin resistance to cells by suppressing ferroptosis. Therefore, administration of a CDK1 inhibitor may be an attractive strategy to treat patients with oxaliplatin-resistant CRC.
Topics: Humans; CDC2 Protein Kinase; Colorectal Neoplasms; Ferroptosis; Oxaliplatin; Proteolysis
PubMed: 37428466
DOI: 10.1002/advs.202301088 -
European Journal of Medicinal Chemistry Dec 2023Targeted protein degradation (TPD) is one of the key strategies of current targeted cancer therapy, and it can eliminate some of the root causes of cancer, and... (Review)
Review
Targeted protein degradation (TPD) is one of the key strategies of current targeted cancer therapy, and it can eliminate some of the root causes of cancer, and effectively avoid drug resistance caused by traditional drugs. Proteolysis targeting chimera (PROTAC) is a hot branch of the TPD strategy, and it has been shown to induce the degradation of target proteins by activating the inherent ubiquitin-proteasome system (UPS) in tumor cells. PROTACs have been developed for more than two decades, and some of them have been clinically evaluated. Although most of the proteins degraded by PROTACs are intracellular, degradation of some typical membrane proteins has also been reported, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), programmed death ligand 1 (PD-L1), and G-protein-coupled receptor (GPCR). In addition, some other effective membrane protein-degrading strategies have also emerged, such as antibody-based PROTAC (AbTAC), lysosome targeting chimera (LYTAC), molecular glue, and nanoparticle-based PROTAC (Nano-PROTAC). Herein, we discussed the advantages, disadvantages and potential applications of several important membrane protein degradation techniques. These techniques that we have summarized are insightful in paving the way for future development of more general strategies for membrane protein degradation.
Topics: Humans; Proteolysis; Membrane Proteins; Proteolysis Targeting Chimera; Lysosomes; Neoplasms; Ubiquitin-Protein Ligases
PubMed: 37924709
DOI: 10.1016/j.ejmech.2023.115911 -
Clinical Pharmacology and Therapeutics Sep 2023Targeted protein degradation (TPD) has emerged as a potentially transformational therapeutic modality with considerable promise. Molecular glue degraders remodel the... (Review)
Review
Targeted protein degradation (TPD) has emerged as a potentially transformational therapeutic modality with considerable promise. Molecular glue degraders remodel the surface of E3 ligases inducing interactions with neosubstrates resulting in their polyubiquitination and proteasomal degradation. Molecular glues are clinically precedented and have demonstrated the ability to degrade proteins-of-interest (POIs) previously deemed undruggable due to the absence of a traditional small molecule binding pocket. Heterobifunctional proteolysis targeting chimeras (PROTACs) possess ligands for an E3 complex and the POIs, which are chemically linked together, and similarly hijack the ubiquitin machinery to deplete the target. There has been a recent surge in the number of degraders entering clinical trials, particularly directed toward cancer. Nearly all utilize CRL4 as the E3 ligase, and a relatively limited diversity of POIs are currently targeted. In this review, we provide an overview of the degraders in clinical trials and provide a perspective on the lessons learned from their development and emerging human data that will be broadly useful to those working in the TPD field.
Topics: Humans; Proteins; Ubiquitin-Protein Ligases; Proteolysis; Neoplasms
PubMed: 37399310
DOI: 10.1002/cpt.2985 -
Trends in Pharmacological Sciences Nov 2023Targeted protein degradation (TPD) is an emerging modality for research and therapeutics. Most TPD approaches harness cellular ubiquitin-dependent proteolytic pathways.... (Review)
Review
Targeted protein degradation (TPD) is an emerging modality for research and therapeutics. Most TPD approaches harness cellular ubiquitin-dependent proteolytic pathways. Proteolysis-targeting chimeras (PROTACs) and molecular glue (MG) degraders (MGDs) represent the most advanced TPD approaches, with some already used in clinical settings. Despite these advances, TPD still faces many challenges, pertaining to both the development of effective, selective, and tissue-penetrant degraders and understanding their mode of action. In this review, we focus on progress made in addressing these challenges. In particular, we discuss the utility and application of recent proteomic approaches as indispensable tools to enable insights into degrader development, including target engagement, degradation selectivity, efficacy, safety, and mode of action.
Topics: Humans; Proteolysis; Proteomics; Proteolysis Targeting Chimera; Ubiquitin-Protein Ligases
PubMed: 37778939
DOI: 10.1016/j.tips.2023.08.007