Continuous evolution of compact protein degradation tags regulated by selective molecular glues

Science, Год журнала: 2024, Номер 383(6688)

Опубликована: Март 14, 2024

Conditional protein degradation tags (degrons) are usually >100 amino acids long or triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame SD40 using prime editing degraded otherwise inert PT-179. Cryo-electron microscopy structures ligand-bound revealed mechanistic insights into basis SD40's activity specificity. Our efforts establish system provide ZF overcome shortcomings associated existing degrons.

Язык: Английский

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Degradation bottlenecks and resource competition in transiently and stably engineered mammalian cells

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Янв. 2, 2025

Abstract Degradation tags, otherwise known as degrons, are portable sequences that can be used to alter protein stability. Here, we report degron-tagged proteins compete for cellular degradation resources in engineered mammalian cells leading coupling of the rates independently expressed when constitutively targeted human degrons adopted. We show effect this competition dependent on context degrons. By considering different proteins, degron position and hosts, highlight how impact both strength resource changes, with identification orthogonal combinations. adopting inducible bacterial plant also controlled uncoupling synthetic construct from native machinery achieved. then build a genomically integrated capacity monitor tagged confirm between genomic transiently DNA constructs. This work expands characterisation including systems, providing framework optimisation heterologous expression systems advance applications fundamental applied biological research.

Язык: Английский

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Small molecule-mediated targeted protein degradation of voltage-gated sodium channels involved in pain

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 22, 2025

The voltage-gated sodium channels (VGSC) NaV1.8 and NaV1.7 (NaVs) have emerged as promising high-value targets for the development of novel, non-addictive analgesics to combat chronic pain epidemic. In recent years, many small molecule inhibitors against these been developed. successful clinical trial VX-548, a NaV1.8-selective inhibitor, has spurred much interest in expanding arsenal subtype-selective channel therapeutics. Toward that end, we sought determine whether NaVs are amenable targeted protein degradation with degraders, namely proteolysis-targeting chimeras (PROTACs) molecular glues. Here, report degron-tagged potently rapidly degraded by degraders harnessing E3 ubiquitin ligases cereblon (CRBN) Von Hippel Lindau (VHL). Using LC/MS analysis, demonstrate PROTAC-mediated proximity between CRBN results ubiquitination on 2 nd intracellular loop, pointing toward potential mechanism action demonstrating ability recognize VGSC neosubstrate. Our foundational findings an important first step realizing immense NaV-targeting degrader pain.

Язык: Английский

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Degron tagging for rapid protein degradation in mice

Disease Models & Mechanisms, Год журнала: 2024, Номер 17(4)

Опубликована: Апрель 1, 2024

Degron tagging allows proteins of interest to be rapidly degraded, in a reversible and tuneable manner, response chemical stimulus. This provides numerous opportunities for understanding disease mechanisms, modelling therapeutic interventions constructing synthetic gene networks. In recent years, many laboratories have applied degron successfully cultured mammalian cells, spurred by rapid advances the fields genome editing targeted protein degradation. this At Glance article, we focus on efforts apply mouse models, discussing distinct set challenges posed vivo environment.

Язык: Английский

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The emerging role of targeted protein degradation to treat and study cancer

The Journal of Pathology, Год журнала: 2024, Номер 263(4-5), С. 403 - 417

Опубликована: Июнь 17, 2024

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase monoclonal antibody therapies being deployed across a range solid organ haematological malignancies. However, novel approaches are required target transcription factors oncogenic fusion proteins that central biology generally eluded successful drug development. Thalidomide analogues causing protein degradation been cornerstone multiple myeloma, but lack in-depth mechanistic understanding initially limited progress field. When cereblon (CRBN) was found mediate thalidomide analogues' action CRBN's neo-targets were identified, existing development accelerated, applications outside including non-Hodgkin's lymphoma, myelodysplastic syndrome, acute leukaemias. Critically, first canonical targets described. In addition broadening application protein-degrading drugs, resistance mechanisms overcome is widening scope druggable against which ineffective. Examples degraders include molecular glues proteolysis targeting chimeras (PROTACs): heterobifunctional molecules bind interest cause proximity-induced ubiquitination proteasomal via linked E3 ligase. Twenty years since their inception, PROTACs begun progressing through clinical trials, early success oestrogen androgen breast prostate respectively. This review explores important developments treat study cancer. It also considers potential advantages challenges translational aspects developing new treatments. © 2024 Author(s). Journal Pathology published by John Wiley & Sons Ltd on behalf Pathological Society Great Britain Ireland.

Язык: Английский

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CRBN‐PROTACs in Cancer Therapy: From Mechanistic Insights to Clinical Applications

Chemical Biology & Drug Design, Год журнала: 2024, Номер 104(5)

Опубликована: Ноя. 1, 2024

Cereblon (CRBN), a member of the E3 ubiquitin ligase complex, has gained significant attention as therapeutic target in cancer. CRBN regulates degradation various proteins cancer progression, including transcription factors and signaling molecules. PROTACs (proteolysis-targeting chimeras) are novel approach that uses cell's system to remove disease-causing selectively. CRBN-dependent work by tagging harmful for destruction through ubiquitin-proteasome system. This strategy offers several advantages over traditional protein inhibition methods, potential overcome drug resistance. Recent progress developing CRBN-based shown promising preclinical results both hematologic malignancies solid tumors. Additionally, have enhanced our understanding CRBN's role cancer, potentially serving biomarkers patient stratification predicting responses. In this review, we delineate mechanisms action (CRBN-PROTACs), summarize recent advances clinical applications, provide perspective on future development.

Язык: Английский

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Mining the CRBN Target Space Redefines Rules for Molecular Glue-induced Neosubstrate Recognition

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Окт. 9, 2024

Abstract The CRL4 CRBN ubiquitin ligase is leveraged by molecular glue degraders, small molecules that reprogram specificity to induce degradation of clinically relevant neosubstrate proteins. Known neosubstrates share a generalizable β-hairpin G-loop recognition motif, yet systematic exploration the target landscape still pending. Through computational mining human proteome using structure-based approaches, we predict over 1,400 CRBN-compatible proteins across diverse classes, identify novel mechanisms through structurally differentiated helical motifs and surface mimicry, validate 22 representative with clinical implications. This work broadens space, redefines rules for establishes platform elimination challenging drug targets repurposing next-generation degraders.

Язык: Английский

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Differential Protein Degradation with a Super-Degron Tag and Thalidomide Analogs: EGFP is Degraded in Wild-Type Mouse Cell, While PD-1 Requires CRBN Humanization, Providing New Insights into T Cell Regulation

Опубликована: Янв. 1, 2025

It was previously considered that protein knockdown using a combination of zinc finger degron tag and thalidomide analog is impossible in mouse cells, however, EGFP expression as an indicator, we found possible super-degron (SD) with iberdomide or mezigdomide cells. Despite the efficient degradation SD-tagged wild-type PD-1 could not be degraded but human Jurkat cells human-type CEREBLON (CRBNI391V). In mice CRBNI391V, endogenous tagged SD efficiently suppressed T both cultured whole body. addition, comparison treatment anti-PD-1 antibody suggested transient downregulation might prevent cell exhaustion compared to treatment. brain also reduced by pomalidomide crosses brain-blood barrier. These results suggest analogs are effective for vitro vivo knockdown, although some conditional settings required.

Язык: Английский

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Systematic evolution of functional oligonucleotides for targeted protein degradation

Chem, Год журнала: 2025, Номер unknown, С. 102408 - 102408

Опубликована: Фев. 1, 2025

Язык: Английский

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Identification of actionable targeted protein degradation effector sites through Site-specific Ligand Incorporation-induced Proximity (SLIP)

Опубликована: Фев. 4, 2025

ABSTRACT Targeted protein degradation (TPD) is a rapidly emerging and potentially transformative therapeutic modality. However, the large majority of >600 known ubiquitin ligases have yet to be exploited as TPD effectors by proteolysis-targeting chimeras (PROTACs) or molecular glue degraders (MGDs). We report here chemical–genetic platform, Site-specific Ligand Incorporation-induced Proximity (SLIP), identify actionable (“PROTACable”) sites on any potential effector in intact cells. SLIP uses genetic code expansion (GCE) encode copper-free “click” ligation at specific site cells, enabling situ formation covalent PROTAC-effector conjugate against target interest (POI). Modification drives targeted protein, establishing these for TPD. Using SLIP, we systematically screened dozens across E3 E2 enzymes from diverse classes, identifying multiple novel PROTACable which are competent adds powerful approach proximity-induced pharmacology (PIP) toolbox, future ligand discovery fully enable TPD, other PIP modalities.

Язык: Английский

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