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The dTAG system for immediate and target-specific protein degradation


Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

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Fig. 1: Heterobifunctional dTAG molecules engage and dimerize FKBP12F36V and CRBN in biochemical assays.
Fig. 2: dTAG-7 and dTAG-13 selectively degrade FKBP12F36V in a CRBN-dependent manner in cells.
Fig. 3: Selective pharmacological degradation of endogenously tagged BRD4.
Fig. 4: Rapid degradation of nuclear and cytoplasmic FKBP12F36V fusion chimeras.
Fig. 5: KRASG12V degradation rapidly reverses the deregulated proteomic and transcriptional signaling program of transformed cells.
Fig. 6: Evaluation of rapid and reversible degradation in vivo.


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We thank N. Kwiatkowski for critical reading of the manuscript, W. Kaelin for sharing referenced cell lines and the dual luciferase plasmids (Dana-Farber Cancer Institute, pLL3.7-EF1a-IRES-Gateway-nluc-2xHA-IRES2-fluc-hCL1-P2A-Puro), R. Kunz and the Thermo Fisher Scientific Center for Multiplexed Proteomics at the Harvard Medical School for the quantitative proteomics and phosphoproteomics assessment, and S. Nabet, A. Aguirre, W. Hahn, and members of the Bradner and Gray laboratories for helpful discussions. This work was supported by an American Cancer Society Postdoctoral Fellowship PF-17-010-01-CDD (B.N.), the Claudia Adams Barr Program in Innovative Basic Cancer Research (D.L.B.), Damon Runyon Cancer Research Foundation DRG-2196-14 (D.L.B.), and generous philanthropic gifts from the Hale Center for Pancreatic Cancer Research and the Katherine L. and Steven C. Pinard Research Fund.

Author information




B.N., J.M.R., and D.L.B. conceived and led the study under the supervision of N.S.G. and J.E.B. D.L.B. and S.D. designed and performed molecule synthesis. J.M.R. constructed the lentiviral and knock-in vector systems. B.N. and J.M.R. designed and performed BRD4 knock-in and target panel studies. B.N. designed and performed KRAS studies. J.M.R. and J.P. designed and performed AlphaScreen assays and IKZF1 dual luciferase assays. S.V. and G.E.W. constructed FKBP12 dual luciferase vectors and B.N., J.M.R., and S.V. performed experiments using these systems. B.N. designed and performed RNA-sequencing experiments and B.N., M.A.E., and M.A.L. performed bioinformatics analyses. B.N., A.Y., A.S., and K.-K.W. designed and performed mouse studies. A.L.L. and T.G.S. assisted in cellular experiments. J.A.P. provided technical advice and data interpretation. J.Q. contributed reagents and technical advice. B.N. and J.E.B. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Nathanael S. Gray or James E. Bradner.

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Competing interests

The authors claim the following competing financial interests: International Patent Application Nos. PCT/US2016/039048, PCT/US2016/046087, PCT/US2016/046088, PCT/US2016/046089, each filed in the name of Dana-Farber Cancer Institute, Inc. D.L.B., J.P., and A.S. are now employees of Novartis. G.E.W. is a consultant for C4 Therapeutics. N.S.G. is a Scientific Founder and member of the Scientific Advisory Board of Syros Pharmaceuticals, C4 Therapeutics, and Petra Pharmaceuticals and is the inventor on IP licensed to these entities. J.E.B. is a Scientific Founder of Syros Pharmaceuticals, SHAPE Pharmaceuticals, Acetylon Pharmaceuticals, Tensha Therapeutics (now Roche), and C4 Therapeutics and is the inventor on IP licensed to these entities. J.E.B. is now an executive and shareholder in Novartis AG.

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Supplementary information

Supplementary Text and Figures

Supplementary Table 1–6, Supplementary Figures 1–15

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Supplementary Note

Synthetic procedures and characterization of dFKBP-1, dTAG-7, dTAG-13, dTAG-48, dTAG-51, bio-SLF, bio-Thal

Supplementary Dataset 1

Entire list of normalized and scaled quantitative mass spectrometry-based proteomics data. Triplicate values from biologically independent samples of normalized percent relative abundance of quantified proteins are presented for NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13 for one hour, and 1 µM dTAG-13 for four hours.

Supplementary Dataset 2

Entire list of normalized and scaled quantitative mass spectrometry-based phosphoserine/threonine data. Triplicate values from biologically independent samples of normalized percent relative abundance of quantified phosphosites are presented for NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13 for one hour, and 1 µM dTAG-13 for four hours.

Supplementary Dataset 3

Entire list of normalized and scaled quantitative mass spectrometry-based phosphotyrosine data. Triplicate values from biologically independent samples of normalized percent relative abundance of quantified phosphosites are presented for NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13 for one hour, and 1 µM dTAG-13 for four hours.

Supplementary Dataset 4

ERCC spike-in normalized FPKM values of top 200 upregulated and 200 downregulated expressed transcripts upon comparison of mock transduced (control) NIH/3T3 cells or NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO. Triplicate FPKM values from biologically independent samples are presented for control NIH/3T3 cells treated with DMSO and NIH/3T3 cells expressing FKBP12F36V-KRASG12V treated with DMSO, 1 µM dTAG-13, or 10 nM trametinib. Dataset accompanies heatmap in Fig. 5c.

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Nabet, B., Roberts, J.M., Buckley, D.L. et al. The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol 14, 431–441 (2018).

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