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@ARTICLE{Schwalm:289592,
      author       = {M. P. Schwalm$^*$ and K. Saxena and S. Müller and S.
                      Knapp$^*$},
      title        = {{L}uciferase- and {H}alo{T}ag-based reporter assays to
                      measure small-molecule-induced degradation pathway in living
                      cells.},
      journal      = {Nature protocols},
      volume       = {19},
      number       = {8},
      issn         = {1754-2189},
      address      = {Basingstoke},
      publisher    = {Nature Publishing Group},
      reportid     = {DKFZ-2024-00825},
      pages        = {2317-2357},
      year         = {2024},
      note         = {2024 Aug;19(8):2317-2357},
      abstract     = {The rational development of small-molecule degraders (e.g.,
                      proteolysis targeting chimeras) remains a challenge as the
                      rate-limiting steps that determine degrader efficiency are
                      largely unknown. Standard methods in the field of targeted
                      protein degradation mostly rely on classical, low-throughput
                      endpoint assays such as western blots or quantitative
                      proteomics. Here we applied NanoLuciferase- and
                      HaloTag-based screening technologies to determine the
                      kinetics and stability of small-molecule-induced ternary
                      complex formation between a protein of interest and a
                      selected E3 ligase. A collection of live-cell assays were
                      designed to probe the most critical steps of the degradation
                      process while minimizing the number of required expression
                      constructs, making the proposed assay pipeline flexible and
                      adaptable to the requirements of the users. This approach
                      evaluates the underlying mechanism of selective target
                      degraders and reveals the exact characteristics of the
                      developed degrader molecules in living cells. The protocol
                      allows scientists trained in basic cell culture and
                      molecular biology to carry out small-molecule
                      proximity-inducer screening via tracking of the ternary
                      complex formation within 2 weeks of establishment, while
                      degrader screening using the HiBiT system requires a
                      CRISPR-Cas9 engineered cell line whose generation can take
                      up to 3 months. After cell-line generation, degrader
                      screening and validation can be carried out in
                      high-throughput manner within days.},
      subtyp        = {Review Article},
      cin          = {FM01},
      ddc          = {610},
      cid          = {I:(DE-He78)FM01-20160331},
      pnm          = {899 - ohne Topic (POF4-899)},
      pid          = {G:(DE-HGF)POF4-899},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:38637703},
      doi          = {10.1038/s41596-024-00979-z},
      url          = {https://inrepo02.dkfz.de/record/289592},
}