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

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12F36V (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) to hijack the CRL4CRBN complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate.

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A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Source data are provided with this paper. Reagents are available upon request. All NanoTACs are available upon request. Uncropped western blots are provided in the source data file. The mass spectrometry proteomics data generated in this study have been deposited in the ProteomeXchange Consortium via the PRIDE42 partner repository with the dataset identifier PXD031371.

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
For in vitro assays no statistical analysis was used to determine sample size, but each experiment was performed in triplicate or quadruplicate technical repeats and repeated a minimum of 3 times independently to verify the results. Given the differing protein expression levels between experiments, samples sizes were taken based on consistent trends across biological replicates which were clear within the replicates performed.
Data exclusions No data was excluded from the analyses.

Replication
All attempts to replicate experimental data were successful, where possible each individual experimental data point is indicated. Western blots: 3 independent replicates. Luminescence assays: 3 independent replicates with 4 technical replicate wells per experiment. Flow cytometry analysis: 3 independent replicates.
Randomization For all experiments other than those mentioned here randomisation is always employed. In vitro experimental samples to treatment groups were designated at random using numerical identifiers.

Blinding
Blinding was not necessary as all samples were analysed simultaneously using identical assay conditions and any analysis performed was not through subjective scoring methods.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Validation
HaloTag and NanoLuc antibodies were validated using cDNA encoding the HaloTag or NanoLuc with inducible expression systems and appropriate molecular weight markers to ensure that each antibody was on target. When expressed in HEK293T and MDF cells, the appropriate sized bands were only observed upon HaloTag-or NanoLuc-tagged protein induction.
From manufacturers website: HaloTag: Little to no cross-reactivity with other non-HaloTag proteins. NanoLuc: Mobility approximated the expected mobility for each (NanoLuc) fusion protein. Background bands are evident in all lanes, including HEK293 cell lysate, which lacks expression of a NanoLuc fusion protein, but the NanoLuc fusion bands are more prominent.
MLKL antibody has been previously validated using a knockout model for MLKL (reference included in manuscript) and was further validated through inducible induction in knockout cells in our manuscript.
FKBP, from manufacturers website: Detects human, mouse, and rat FKBP12 in Western blots. In Western blots, no cross-reactivity with other FKBP family members is observed.

Mycoplasma contamination
All cell lines are routinely tested for mycoplasma and the cell lines used in this study were negative for mycoplasma.

Commonly misidentified lines (See ICLAC register)
There were no commonly misidentified cell lines used in this study.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals Species: Mouse. Strain: C57BL/6J mice. Animals were selected independently of their gender for generating MDFs, as we find gender does not impact signalling responses in MDFs. Female and male mice were at least 6-weeks old at the time of experimentation. None of the mice used in our experiments had been previously used for other procedures.

Wild animals
The study did not involve wild animals.
Field-collected samples The study did not involve samples collected from the field.

Ethics oversight
All procedures for this study were approved by the Walter and Eliza Hall Institute (WEHI) Animal Ethics Committee, Australia.
Note that full information on the approval of the study protocol must also be provided in the manuscript.