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A High-Throughput Method to Prioritize PROTAC Intracellular Target Engagement and Cell Permeability Using NanoBRET

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Targeted Protein Degradation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2365))

Abstract

Target engagement and cell permeation are important parameters that may limit the efficacy of proteolysis-targeting chimeras (PROTACs). Here, we present an approach that facilitates both the quantitation of PROTAC binding affinity for an E3 ligase of interest, as well as the assessment of relative intracellular availability. We present a panel of E3 ligase target engagement assays based upon the NanoBRET Target Engagement platform. Querying E3 ligase engagement under live-cell and permeabilized-cell conditions allow calculation of an availability index that can be used to rank order the intracellular availability of PROTACs. Here we present examples where the cellular availability of PROTACs and their monovalent precursors are prioritized using NanoBRET assays for CRBN or VHL E3 ligases.

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Authors and Affiliations

  1. Promega Corporation, Fitchburg, WI, USA

    James D. Vasta, Cesear R. Corona & Matthew B. Robers

Authors
  1. James D. Vasta
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  2. Cesear R. Corona
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  3. Matthew B. Robers
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Corresponding author

Correspondence to James D. Vasta .

Editor information

Editors and Affiliations

  1. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Angela M. Cacace

  2. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    Christopher M. Hickey

  3. Platform Biology, Arvinas, Inc., New Haven, CT, USA

    MiklĂ³s BĂ©kĂ©s

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Vasta, J.D., Corona, C.R., Robers, M.B. (2021). A High-Throughput Method to Prioritize PROTAC Intracellular Target Engagement and Cell Permeability Using NanoBRET. In: Cacace, A.M., Hickey, C.M., Békés, M. (eds) Targeted Protein Degradation. Methods in Molecular Biology, vol 2365. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1665-9_14

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  • DOI: https://doi.org/10.1007/978-1-0716-1665-9_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1664-2

  • Online ISBN: 978-1-0716-1665-9

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