Cited by 45Protein Degradation and InhibitorsAdvanced Fluorescence Microscopy TechniquesChemical Synthesis and Analysis

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

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Payne, N. Connor, Maksoud, Semer, Tannous, Bakhos A., Mazitschek, Ralph·2022·Cell Chemical Biology

Published version of a preprint

Abstract

We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1 h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

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Crossref · 10.1016/j.chembiol.2022.05.003

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  • Payne et al. applied TR-FRET to profile PROTAC-induced degradation of BRD4.

    “Recently, Payne et al. applied time-resolved fluorescence resonance energy (TR-FRET) to profile PROTAC-induced degradation of BRD4.165”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [165]

  • By combining a fluorescent JQ1-based tracer with a primary BRD4 antibody and a labeled nano-secondary antibody, the authors monitored BRD4 levels.

    “By combining a fluorescent JQ1-based tracer with a primary BRD4 antibody and a labeled nano-secondary antibody, the authors were able to monitor BRD4 levels.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [165]

  • The assay was miniaturized to a 96-well plate format, enabling scalable profiling of molecules in unmodified biological systems.

    “The assay was miniaturized to a 96-well plate format, enabling the scalable profiling of molecules in unmodified biological systems.”

    From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [165]

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2 of the 36 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. Yue H, Nowak RP, Overwijn D, Payne NC, Fischinger S, Atyeo C, Baden LR, Nilles EJ, Karlson EW, Yu XG, (2020). Rapid ‘mix and read’ assay for scalable detection of SARS-CoV-2 antibodies in patient plasma. medRxiv.

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Authors
Payne, N. C., Maksoud, S., Tannous, B. A., & Mazitschek, R.
Affiliations
  • Harvard University (US)
  • Broad Institute (US)
Corresponding author
Mazitschek, Ralph
Venue
Cell Chemical Biology
Publisher
Elsevier BV
Published
2022-08
Volume
29
Issue
8
Pages
1333-1340.e5
ISSN
  • 2451-9448 (ISSN-L)
  • 2451-9456
OpenAlex
W4281661759
Cited by
44
References
36
Citations by year
2026: 7 · 2025: 12 · 2024: 12 · 2023: 8 · 2022: 5
FWCI
3.10
Type
journal article
Language
en
Open access
Locations
  • Cell chemical biology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • NIH (1R01NS064983)
  • National Science Foundation (DGE1745303, 1830941)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
Keywords
PROTACs, heterobifunctional degraders, BRD4, celastrol, protein quantification, target engagement, nanobodies, E3 ubiquitin ligase, targeted protein degradation, p-quinone methides, high-throughput screening, bromodomain proteins
MeSH terms
Bromodomain Containing Proteins, Humans, Nuclear Proteins, Transcription Factors, Cell Cycle Proteins, Ubiquitin-Protein Ligases, Pentacyclic Triterpenes, Proteolysis

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