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Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells

Nature Protocols (2024)Cite this article

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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.

Key points

  • This protocol describes the application of NanoLuciferase- and HaloTag-based screening technologies to measure the whole cascade of targeted protein degradation from binary complexes to live-cell target degradation kinetics.

  • Compared with conventional endpoint assays such as western blots or quantitative proteomics, live-cell assays take dynamic cellular processes into account such as the complete degradation machinery, intracellular competitors or posttranslational modifications during time-resolved measurements.

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Fig. 1: Assay strategies based on bioluminescence and BRET.
Fig. 2: Schematic representation of the cloning strategy used for the generation of the NLuc and HaloTag fusion constructs.
Fig. 3: Schematic representation of the different steps for the generation of the endogenously HiBiT-tagged CRISPR cell line.
Fig. 4: Proposed plate layout for dose–response assays and schematic principle of the NanoBRET technology.
Fig. 5: Principle of the HiBiT-CETSA.
Fig. 6: Schematic overview of the ternary complex assay setup and principle.
Fig. 7: Schematic overview of the HiBiT-based protein degradation measurement.

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Data availability

Any additional information required to reanalyze the data reported in this manuscript is available upon request from the lead contact. Plasmids have been made available on Addgene.

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Acknowledgements

M.P.S., K.S., S.M. and S.K. are grateful for support by the Structural Genomics Consortium, a registered charity (no. 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute, EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant 875510), Janssen, Pfizer and Takeda and by the German Cancer Research Center DKTK and the Frankfurt Cancer Institute. M.P.S. is funded by the Deutsche Forschungsgemeinschaft (German Research Foundation), CRC1430 (project ID 424228829). M.P.S. is thankful for the help of B.-T. Berger during assay establishment.

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Authors and Affiliations

  1. Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany

    Martin P. Schwalm, Krishna Saxena, Susanne Müller & Stefan Knapp

  2. Structural Genomics Consortium (SGC), Buchmann Institute for Life Sciences, Frankfurt am Main, Germany

    Martin P. Schwalm, Krishna Saxena, Susanne Müller & Stefan Knapp

  3. German Cancer Consortium (DKTK)/German Cancer Research Center (DKFZ), DTKT Site Frankfurt-Mainz, Heidelberg, Germany

    Martin P. Schwalm & Stefan Knapp

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  1. Martin P. Schwalm
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Contributions

M.P.S. conceived the study and designed experiments. The manuscript and figures were prepared by M.P.S. and edited by K.S., S.M. and S.K.

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Correspondence to Martin P. Schwalm or Stefan Knapp.

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Key references using this protocol

Schwalm, M. P. et al. Front. Cell Dev. Biol. 10, 886537 (2022): https://doi.org/10.3389/fcell.2022.886537

Schwalm, M. P. et al. Cell Chem. Biol. 30, 753–765.e8 (2023): https://doi.org/10.1016/j.chembiol.2023.06.002

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Schwalm, M.P., Saxena, K., Müller, S. et al. Luciferase- and HaloTag-based reporter assays to measure small-molecule-induced degradation pathway in living cells. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-00979-z

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