Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

NanoTACs: NanoLuc-Targeting Protein Degraders that Parallel Existing Technology

ABSTRACT: NanoLuc-targeting PROTACs (NanoTACs) hijack the CRBN and VHL cullin-RING ligase complexes to trigger proteasomal degradation of substrates tagged with the 19 kDa luminescent NanoLuc tag. In order to validate the system and confirm selective degradation of the target protein and assess any potential off-target degradation, here DDA-PASEF label-free quantification was performed to monitor target-induced degradation following addition of NC4.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Simon Cobbold

LAB HEAD: Simon Cobbold

PROVIDER: PXD031371 | Pride | 2022-05-20

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms.

Grohmann Christoph C Magtoto Charlene M CM Walker Joel R JR Chua Ngee Kiat NK Gabrielyan Anna A Hall Mary M Cobbold Simon A SA Mieruszynski Stephen S Brzozowski Martin M Simpson Daniel S DS Dong Hao H Dorizzi Bridget B Jacobsen Annette V AV Morrish Emma E Silke Natasha N Murphy James M JM Heath Joan K JK Testa Andrea A Maniaci Chiara C Ciulli Alessio A Lessene Guillaume G Silke John J Feltham Rebecca R

Nature communications 20220419 1

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12<sup>F36V</sup> (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) ...[more]

PMID: 35440107

Similar Datasets

Repurposing E3 ubiquitin ligases as cell surface protein degraders using Proteolysis Targeting Antibodies

Project description:The majority of current therapeutics targeting plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. Typical mammalian proteins, however, consist of multiple domains executing discrete but coordinated activities, and saturating inhibition of one functional domain often incompletely suppresses the totality of the protein’s function. Recent work on targeted protein degradation technologies including Proteolysis Targeting Chimeras (PROTACs) has highlighted clinically important distinctions between target inhibition and target degradation. However, the generation of heterobifunctional compounds requiring linkage of two small molecules, each with high affinity for their targets, is highly complex, particularly with respect to achieving oral bioavailability. Here we describe the development of Proteolysis Targeting Antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target ubiquitination and subsequent degradation. PROTAB-mediated degradation drives deeper pathway inhibition than inhibitory antibodies and is functional in vivo. The scope of this technology is also demonstrated through the identification of additional cell surface E3 ubiquitin ligases that can function as “on demand” degraders of various cell surface proteins. The generality of this approach enables tissue-selective degradation, as suggested by the Wnt-responsive ligases RNF43 and ZNRF3. Furthermore, through engineering of various optimized antibody formats, we offer insights on the ground rules governing optimal target degradation. Taken together, this work describes a strategy for the rapid development of potent, bioavailable and tissue selective degradation of cell surface proteins.

2022-07-21 | GSE208372 | GEO

TMT analysis of MOLM-13 cells treated with series 3 CK1a degraders

Project description:Targeted Protein Degradation (TPD) encompasses two approaches that generate small molecules with similar proteasome-dependent mechanisms of action: Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glues (MGs). Our goal is to develop novel MG approaches that exploit the E3 ubiquitin ligase Cereblon (CRBN) to degrade key oncogenic drivers in the leading causes of childhood cancer death: acute myeloid leukemia (AML). This project will identify new vulnerabilities and compounds that degrade hitherto undruggable transcription factors in high-risk acute leukemia (AL) and medulloblastoma (MB). Based on our preliminary data, we hypothesize that these compounds target AML cells via CK1a degradation. Our aims are to understand the mechanism by which compounds SJ001041330, SJ001043149, and SJ001043301 target AML cells using TMT proteomic approach and confirm CK1a degradation by these compounds leading to AML cell death.

2023-12-01 | PXD045230 | Pride

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia

Project description:We evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against Janus kinases. Solving the structure of FDA-approved type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 JH1 tyrosine kinase domain enabled the rational design and optimization of Cereblon (CRBN)-directed JAK PROTACs utilizing multiple derivatives of JAK inhibitors, linkers and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation by proteomic approaches, and activity tested in CRLF2-rearranged cell line and xenograft models of ALL.

| EGAS00001005180 | EGA

Proteolysis Targeting Chimeras With Reduced Off-targets

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

Repurposing E3 ubiquitin ligases as cell surface protein degraders using Proteolysis Targeting Antibodies

Project description:The majority of current therapeutics targeting plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. Typical mammalian proteins, however, consist of multiple domains executing discrete but coordinated activities, and saturating inhibition of one functional domain often incompletely suppresses the totality of the protein's function. Recent work on targeted protein degradation technologies including Proteolysis Targeting Chimeras (PROTACs) has highlighted clinically important distinctions between target inhibition and target degradation. However, the generation of heterobifunctional compounds requiring linkage of two small molecules, each with high affinity for their targets, is highly complex, particularly with respect to achieving oral bioavailability. Here we describe the development of Proteolysis Targeting Antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target ubiquitination and subsequent degradation. PROTAB-mediated degradation drives deeper pathway inhibition than inhibitory antibodies and is functional in vivo. The scope of this technology is also demonstrated through the identification of additional cell surface E3 ubiquitin ligases that can function as on demand degraders of various cell surface proteins. The generality of this approach enables tissue-selective degradation, as suggested by the Wnt-responsive ligases RNF43 and ZNRF3. Furthermore, through engineering of various optimized antibody formats, we offer insights on the ground rules governing optimal target degradation. Taken together, this work describes a strategy for the rapid development of potent, bioavailable and tissue selective degradation of cell surface proteins.

2022-05-25 | MSV000089542 | MassIVE

Transcriptome analysis PIM kinase inhibition vs degradation

Project description:PIM kinases have important pro-tumorigenic roles and mediate several oncogenic traits, including cell proliferation, survival, and chemotherapeutic resistance. As a result, multiple PIM inhibitors have been pursued as investigational new drugs in cancer; however, response to PIM inhibitors in solid tumors has fallen short of expectations. We found that inhibition of PIM kinase activity stabilizes protein levels of all three PIM isoforms (PIM1/2/3), and this can promote resistance to PIM inhibitors and chemotherapy. To overcome this effect, we designed PIM proteolysis targeting chimeras (PROTACs) to target PIM for degradation. PIM PROTACs effectively downmodulated PIM levels through the ubiquitin-proteasome pathway. Importantly, degradation of PIM kinases was more potent than inhibition of catalytic activity in inducing apoptosis in prostate cancer cell line models. In conclusion, we provide evidence of the advantages of degrading PIM kinases versus inhibiting their catalytic activity to target the oncogenic functions of PIM kinases.

2023-12-04 | GSE237256 | GEO

Enhancement of ubiquitination-dependent mitophagy by unconventionally-acting PROTACs

Project description:Enhancing mitophagy, a naturally-occurring cellular process for elimination of damaged mitochondria, holds great promise for the intervention of many human diseases. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that induce ubiquitination and subsequent proteasome-mediated degradation of a target protein through simultaneously binding to the target protein and an E3 ubiquitin ligase. However, the narrow cavity of the proteasome prevents the degradation of mitochondria. Here we show that the E3 ubiquitin ligase MAP3K1, when recruited to the outer mitochondria membrane (OMM) protein TSPO by our PROTAC-designed molecules (termed “mitophagy-enhancing chimeras”, or MECs), induced extensive K63 ubiquitination of TSPO and other OMM proteins, reminiscent of the PINK1-activated Parkin, without triggering proteasome-mediated degradation of TSPO. Aided by NBR1 and Nur77, this increased K63 ubiquitination of OMM proteins triggered mitophagy exclusively for damaged mitochondria, leading to improved mitochondria function and diminished cellular ROS. With the capability to enhance mitophagy at low nanomolar concentrations, MECs effectively inhibited NLRP3 inflammasome activation, abrogated acetaminophen-induced acute liver injury and mitigated high-fat diet-induced obesity in mice. Our work provided a proof-of-concept for developing unconventionally-acting PROTACs to achieve degradation of damaged mitochondria and possibly other organelles.

2024-02-08 | GSE255354 | GEO

The translation termination factor GSPT1 is a phenotypically relevant off-target of heterobifunctional phthalimide degraders

Project description:Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase cereblon (CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of the respective targeting ligand as a unique feature of small-molecule degraders.

2018-01-20 | GSE108763 | GEO

Competitive Profiling of Ligandable Cysteines in Staphylococcus aureus with an Organogold Compound

Project description:With the idea of exploiting metal-templated reactions to achieve selective modification of cysteines in proteins for antibacterial applications, an organometallic cyclometalated Au(III) compound was explored in a competitive chemoproteomic approach based on the isoDTB-ABPP (isotopically labelled desthiobiotin azide-activity-based protein profiling) technology in S. aureus cell extracts. In this way, more than 100 ligandable cysteines where identified, of which 10 were close to functional sites of proteins encoded by essential genes indicating potential for antibiotic development. Interestingly, more than 50% of the identified ligandable sites were not engaged by organic alpha-chloroacetamides in a previous study, indicating that organometallic compounds expand the ligandable space in bacteria. A selected interaction identified by isoDTB-ABPP was validated using an enzyme activity assay, and intact protein mass spectrometry showed that cysteine arylation of an unprecedented target occurs with the studied compound. The obtained results constitute the proof-of-concept that this family of organogold compounds has potential for therapeutic protein targeting via selective, covalent modification of cysteine residues in bacteria. Looking more broadly, our study demonstrates that the targets of cyclometalated gold compounds can be studied proteome-wide with competitive residue-specific chemoproteomics enabling the expansion of the known ligandable proteome to sites that can be addressed with this compound class.

2022-04-19 | PXD031708 | Pride

Modeling CRL4A ligase complex to predict target protein ubiquitination induced by CRBN-recruiting PROTACs

Project description:PROteolysis TArgeting Chimeras (PROTACs) are bifunctional small molecules that can simultaneously recruit target protein and E3 ligase to form a ternary complex (target protein / PROTAC / E3 ligase), leading to target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by CRBN-based PROTACs,

2022-05-19 | PXD030213 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data