12 results on '"Nicholas A. Eleuteri"'
Search Results
2. A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets
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Hillary M. Layden, Nicholas A. Eleuteri, Scott W. Hiebert, and Kristy R. Stengel
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Cell Biology ,Flow Cytometry/Mass Cytometry ,Genomics ,Molecular Biology ,CRISPR ,Protein Biochemistry ,Science (General) ,Q1-390 - Abstract
Summary: Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors.For complete details on the use and execution of this protocol, please refer to Stengel et al., 2021).
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- 2021
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3. Acute pharmacological degradation of Helios destabilizes regulatory T cells
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Katherine A. Donovan, Kenneth H. Ngo, Eric S. Wang, Eric S. Fischer, Verano Alyssa, Radosław P. Nowak, Prafulla C. Gokhale, Nathanael S. Gray, Nicholas A. Eleuteri, Patrick H. Lizotte, J Christine Yuan, and Hong Yue
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Models, Molecular ,Ubiquitin-Protein Ligases ,HeliOS ,T-Lymphocytes, Regulatory ,Article ,Cell Line ,Substrate Specificity ,Small Molecule Libraries ,Ikaros Transcription Factor ,Jurkat Cells ,Mice ,03 medical and health sciences ,Animals ,Humans ,Receptor ,Molecular Biology ,Transcription factor ,Adaptor Proteins, Signal Transducing ,030304 developmental biology ,0303 health sciences ,Molecular Structure ,biology ,Chemistry ,Cereblon ,030302 biochemistry & molecular biology ,Cell Biology ,Phenotype ,Small molecule ,Ubiquitin ligase ,Cell biology ,DNA-Binding Proteins ,Mutation ,biology.protein ,Reprogramming ,Transcription Factors - Abstract
The zinc finger transcription factor Helios is critical for maintaining the identity, anergic phenotype, and suppressive activity of regulatory T cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor Cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of regulatory T cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity., Graphical Abstract
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- 2021
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4. Exploring the target scope of KEAP1 E3 ligase-based PROTACs
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Guangyan Du, Jie Jiang, Nathaniel J. Henning, Nozhat Safaee, Eriko Koide, Radosław P. Nowak, Katherine A. Donovan, Hojong Yoon, Inchul You, Hong Yue, Nicholas A. Eleuteri, Zhixiang He, Zhengnian Li, Hubert T. Huang, Jianwei Che, Behnam Nabet, Tinghu Zhang, Eric S. Fischer, and Nathanael S. Gray
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Pharmacology ,Kelch-Like ECH-Associated Protein 1 ,NF-E2-Related Factor 2 ,Ubiquitin-Protein Ligases ,Clinical Biochemistry ,Ligands ,Biochemistry ,Mice ,Focal Adhesion Protein-Tyrosine Kinases ,Drug Discovery ,Animals ,Molecular Medicine ,Ubiquitins ,Molecular Biology - Abstract
Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.
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- 2022
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5. Structure-Guided Design of a 'Bump-and-Hole' Bromodomain-Based Degradation Tag
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Katherine A. Donovan, Radosław P. Nowak, Yuan Xiong, Nadia Kirmani, Eric S. Fischer, Nicholas A. Eleuteri, Joann Kalabathula, and J Christine Yuan
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Dose-Response Relationship, Drug ,Molecular Structure ,Chemistry ,Cereblon ,Chemical biology ,Cell Cycle Proteins ,Computational biology ,Protein degradation ,Bromodomain ,Structure-Activity Relationship ,Drug Design ,Drug Discovery ,Proteome ,Molecular Medicine ,Structure–activity relationship ,Degradation (geology) ,Humans ,Protein depletion ,Transcription Factors - Abstract
Chemical biology tools to modulate protein levels in cells are critical to decipher complex biology. Targeted protein degradation offers the potential for rapid and dose-dependent protein depletion through the use of protein fusion tags toward which protein degraders have been established. Here, we present a newly developed protein degradation tag BRD4BD1L94V along with the corresponding cereblon (CRBN)-based heterobifunctional degrader based on a "bump-and-hole" approach. The resulting compound XY-06-007 shows a half-degradation concentration (DC50, 6 h) of 10 nM against BRD4BD1L94V with no degradation of off-targets, as assessed by whole proteome mass spectrometry, and demonstrates suitable pharmacokinetics for in vivo studies. We demonstrate that BRD4BD1L94V can be combined with the dTAG approach to achieve simultaneous degrader-mediated depletion of their respective protein fusions. This orthogonal system complements currently available protein degradation tags and enables investigation into the consequences resulting from rapid degradation of previously undruggable disease codependencies.
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- 2021
6. A protocol for rapid degradation of endogenous transcription factors in mammalian cells and identification of direct regulatory targets
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Kristy R. Stengel, Hillary M. Layden, Nicholas A. Eleuteri, and Scott W. Hiebert
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Science (General) ,Transcription, Genetic ,Endogeny ,Genomics ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Q1-390 ,RNA interference ,Protein Biochemistry ,Protocol ,CRISPR ,Animals ,Flow Cytometry/Mass Cytometry ,Transcription factor ,Molecular Biology ,Cells, Cultured ,General Immunology and Microbiology ,General Neuroscience ,Transcript analysis ,Cell Biology ,Identification (biology) ,RNA Interference ,Transcription (software) ,RNA, Guide, Kinetoplastida ,Transcription Factors - Abstract
Summary Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors. For complete details on the use and execution of this protocol, please refer to Stengel et al., 2021)., Graphical abstract, Highlights • A chemical genetic approach for small-molecule-induced degradation of endogenous proteins • Genomic analysis identifies direct targets of transcriptional regulators • Powerful system for interrogating mechanisms of transcriptional control • Step-by-step protocol from sgRNA design to integration of genomic data sets, Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors.
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- 2021
7. IKAROS and MENIN coordinate therapeutically actionable leukemogenic gene expression in MLL-r acute myeloid leukemia
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Brandon J. Aubrey, Jevon A. Cutler, Wallace Bourgeois, Katherine A. Donovan, Shengqing Gu, Charlie Hatton, Sarah Perlee, Florian Perner, Homa Rahnamoun, Alexandra C. P. Theall, Jill A. Henrich, Qian Zhu, Radosław P. Nowak, Young Joon Kim, Salma Parvin, Anjali Cremer, Sarah Naomi Olsen, Nicholas A. Eleuteri, Yana Pikman, Gerard M. McGeehan, Kimberly Stegmaier, Anthony Letai, Eric S. Fischer, X. Shirley Liu, and Scott A. Armstrong
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Cancer Research ,Ikaros Transcription Factor ,Leukemia, Myeloid, Acute ,Oncology ,Gene Expression ,Humans ,Myeloid Ecotropic Viral Integration Site 1 Protein ,Chromatin ,Transcription Factors - Abstract
Acute myeloid leukemia (AML) remains difficult to treat and requires new therapeutic approaches. Potent inhibitors of the chromatin-associated protein MENIN have recently entered human clinical trials, opening new therapeutic opportunities for some genetic subtypes of this disease. Using genome-scale functional genetic screens, we identified IKAROS (encoded by IKZF1) as an essential transcription factor in KMT2A (MLL1)-rearranged (MLL-r) AML that maintains leukemogenic gene expression while also repressing pathways for tumor suppression, immune regulation and cellular differentiation. Furthermore, IKAROS displays an unexpected functional cooperativity and extensive chromatin co-occupancy with mixed lineage leukemia (MLL)1-MENIN and the regulator MEIS1 and an extensive hematopoietic transcriptional complex involving homeobox (HOX)A10, MEIS1 and IKAROS. This dependency could be therapeutically exploited by inducing IKAROS protein degradation with immunomodulatory imide drugs (IMiDs). Finally, we demonstrate that combined IKAROS degradation and MENIN inhibition effectively disrupts leukemogenic transcriptional networks, resulting in synergistic killing of leukemia cells and providing a paradigm for improved drug targeting of transcription and an opportunity for rapid clinical translation.
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- 2021
8. Mapping the Degradable Kinome Provides a Resource for Expedited Degrader Development
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Inchul You, Brian J. Groendyke, Mingxing Teng, Michael P. Agius, Jinhua Wang, Irene M. Ghobrial, SeongShick Ryu, Yunju Nam, Sara J. Buhrlage, Michelle W. Ma, Xiaoxi Liu, Taebo Sim, Yanke Liang, Dennis Dobrovolsky, Wanyi Hu, Mingfeng Hao, Eric S. Fischer, John M. Hatcher, Katherine A. Donovan, Nathanael S. Gray, Kun Shi, Hong Yue, Li Tan, Jianwei Che, Jonathan W. Bushman, Hanna Cho, Debabrata Bhunia, Sandip Sengupta, Fleur M. Ferguson, Nicholas A. Eleuteri, Zhengnian Li, Theresa D. Manz, Injae Shin, and Baishan Jiang
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Adult ,Male ,Proteomics ,Proteasome Endopeptidase Complex ,Proteome ,kinase ,Ubiquitin-Protein Ligases ,Computational biology ,Protein degradation ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,PROTAC ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,Resource (project management) ,Ubiquitin ,ubiquitin ,Humans ,Kinome ,RNA, Messenger ,IMiD ,Databases, Protein ,E3 ligase ,030304 developmental biology ,0303 health sciences ,biology ,targeted degradation ,Middle Aged ,Small molecule ,Ubiquitin ligase ,Drug development ,Proteasome ,degrader ,Proteolysis ,biology.protein ,Female ,Protein Kinases ,ubiquitin proteasome system ,030217 neurology & neurosurgery - Abstract
Targeted protein degradation (TPD) refers to the use of small molecules to induce ubiquitin-dependent degradation of proteins. TPD is of interest in drug development, as it can address previously inaccessible targets. However, degrader discovery and optimization remains an inefficient process due to a lack of understanding of the relative importance of the key molecular events required to induce target degradation. Here, we use chemo-proteomics to annotate the degradable kinome. Our expansive dataset provides chemical leads for ∼200 kinases and demonstrates that the current practice of starting from the highest potency binder is an ineffective method for discovering active compounds. We develop multitargeted degraders to answer fundamental questions about the ubiquitin proteasome system, uncovering that kinase degradation is p97 dependent. This work will not only fuel kinase degrader discovery, but also provides a blueprint for evaluating targeted degradation across entire gene families to accelerate understanding of TPD beyond the kinome.
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- 2020
9. Chemo-proteomics exploration of HDAC degradability by small molecule degraders
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Nicholas A. Eleuteri, Yuan Xiong, Eric S. Fischer, Anthony Razov, Hong Yue, Radosław P. Nowak, Katherine A. Donovan, Nadia Kirmani, and Noah M. Krupnick
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Proteomics ,Clinical Biochemistry ,Protein degradation ,Biochemistry ,Article ,Histone Deacetylases ,Cell Line ,Small Molecule Libraries ,Structure-Activity Relationship ,Ubiquitin ,Drug Discovery ,Humans ,Epigenetics ,Molecular Biology ,Pharmacology ,biology ,Small molecule ,Chromatin ,Ubiquitin ligase ,Cell biology ,Histone Deacetylase Inhibitors ,Isoenzymes ,Von Hippel-Lindau Tumor Suppressor Protein ,Proteolysis ,biology.protein ,Molecular Medicine ,Target protein - Abstract
Targeted protein degradation refers to the use of small molecules that recruit a ubiquitin ligase to a target protein for ubiquitination and subsequent proteasome-dependent degradation. While degraders have been developed for many targets, key questions regarding degrader development and the consequences of acute pharmacological degradation remain, specifically for targets that exist in obligate multi-protein complexes. Here, we synthesize a pan-histone deacetylase (HDAC) degrader library for the chemo-proteomic exploration of acute degradation of a key class of chromatin-modifying enzymes. Using chemo-proteomics, we not only map the degradability of the zinc-dependent HDAC family identifying leads for targeting HDACs 1-8 and 10 but also explore important aspects of degrading epigenetic enzymes. We discover cell line-driven target specificity and that HDAC degradation often results in collateral loss of HDAC-containing repressive complexes. These findings potentially offer a new mechanism toward controlling chromatin structure, and our resource will facilitate accelerated degrader design and development for HDACs.
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- 2021
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10. Discovery of an AKT Degrader with Prolonged Inhibition of Downstream Signaling
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Emily C. Erickson, Inchul You, Nicholas A. Eleuteri, Eric S. Fischer, Nathanael S. Gray, Alex Toker, and Katherine A. Donovan
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Clinical Biochemistry ,Molecular Conformation ,Akt inhibitor ,01 natural sciences ,Biochemistry ,Piperazines ,Article ,03 medical and health sciences ,0302 clinical medicine ,Drug Discovery ,medicine ,Humans ,Protein kinase A ,Molecular Biology ,Protein Kinase Inhibitors ,Protein kinase B ,PI3K/AKT/mTOR pathway ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Oncogene ,biology ,010405 organic chemistry ,Chemistry ,Cereblon ,Cancer ,medicine.disease ,Small molecule ,0104 chemical sciences ,3. Good health ,Ubiquitin ligase ,AKT signaling cascade ,Pyrimidines ,030220 oncology & carcinogenesis ,Proteolysis ,biology.protein ,Cancer research ,Molecular Medicine ,Proto-Oncogene Proteins c-akt ,Signal Transduction - Abstract
SUMMARYThe PI3K/AKT signaling cascade is one of the most commonly dysregulated pathways in cancer, with over half of tumors exhibiting aberrant AKT activation. Although potent small molecule AKT inhibitors have entered clinical trials, robust and durable therapeutic responses have not been observed. As an alternative strategy to target AKT, we report the development of INY-03-041, a pan-AKT degrader consisting of the ATP-competitive AKT inhibitor GDC-0068 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon (CRBN). INY-03-041 induced potent degradation of all three AKT isoforms and displayed enhanced anti-proliferative effects relative to GDC-0068. Notably, INY-03-041 promoted sustained AKT degradation and inhibition of downstream signaling effects for up to 96 hours, even after compound washout. Our findings indicate that AKT degradation may confer prolonged pharmacological effects compared to inhibition, and highlight the potential advantages of AKT-targeted degradation.
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- 2019
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11. Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15
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Hojong Yoon, Zhengnian Li, Radosław P. Nowak, Nicholas A. Eleuteri, Tyler B. Faust, Eric S. Fischer, Quan Cai, Katherine A. Donovan, Nathanael S. Gray, and Tinghu Zhang
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Indoles ,Ubiquitin-Protein Ligases ,Xenopus ,Amino Acid Motifs ,Protein domain ,RNA-binding protein ,Plasma protein binding ,Spodoptera ,Article ,Protein Structure, Secondary ,03 medical and health sciences ,Splicing factor ,0302 clinical medicine ,Low affinity ,Protein structure ,Protein Domains ,Protein Interaction Mapping ,Fluorescence Resonance Energy Transfer ,Animals ,Humans ,Receptor ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,Sulfonamides ,0303 health sciences ,DNA ligase ,biology ,Chemistry ,Cryoelectron Microscopy ,030302 biochemistry & molecular biology ,Intracellular Signaling Peptides and Proteins ,RNA-Binding Proteins ,Cell Biology ,biology.organism_classification ,Kinetics ,RNA Recognition Motif Proteins ,Förster resonance energy transfer ,030220 oncology & carcinogenesis ,Complementarity (molecular biology) ,Benzamides ,Proteolysis ,Investigational Drugs ,Biophysics ,Cullin-RING ligase ,Protein Binding - Abstract
The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1–DCAF15–DDA1 core ligase complex bound to RBM39 and E7820 at a resolution of 4.4 A, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a new fold stabilized by DDA1, and that extensive protein–protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrate how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high-affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders. Cryo-EM and crystal structural analysis of DDB1–DCAF15–DDA1 in complex with E7820 and RBM39 reveal that aryl-sulfonamides reshape the surface of the cullin RING ligase substrate receptor DCAF15 to bind and degrade the splicing factor RBM39.
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- 2019
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12. Development and Characterization of a Wee1 Kinase Degrader
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Nathanael S. Gray, Benika J. Pinch, Zhengnian Li, Caitlin E. Mills, Calla M. Olson, Radosław P. Nowak, Nicholas A. Eleuteri, Katherine A. Donovan, Mirra Chung, David A. Scott, Eric S. Fischer, Zainab M. Doctor, and Peter K. Sorger
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Pharmacology ,Cyclin-dependent kinase 1 ,biology ,010405 organic chemistry ,DNA damage ,Cereblon ,Clinical Biochemistry ,Cell cycle ,01 natural sciences ,Biochemistry ,PLK1 ,Article ,0104 chemical sciences ,Olaparib ,Wee1 ,chemistry.chemical_compound ,chemistry ,Drug Discovery ,Cancer cell ,biology.protein ,Cancer research ,Molecular Medicine ,Molecular Biology - Abstract
The G1/S cell cycle checkpoint is frequently dysregulated in cancer, leaving cancer cells reliant on a functional G2/M checkpoint to prevent excessive DNA damage. Wee1 regulates the G2/M checkpoint by phosphorylating CDK1 at Tyr15 to prevent mitotic entry. Previous drug development efforts targeting Wee1 resulted in the clinical-grade inhibitor, AZD1775. However, AZD1775 is burdened by dose-limiting adverse events, and has off-target PLK1 activity. In an attempt to overcome these limitations, we developed Wee1 degraders by conjugating AZD1775 to the cereblon (CRBN)-binding ligand, pomalidomide. The resulting lead compound, ZNL-02-096, degrades Wee1 while sparing PLK1, induces G2/M accumulation at 10-fold lower doses than AZD1775, and synergizes with Olaparib in ovarian cancer cells. We demonstrate that ZNL-02-096 has CRBN-dependent pharmacology that is distinct from AZD1775, which justifies further evaluation of selective Wee1 degraders.
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- 2020
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