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Design of PROTACs utilizing the E3 ligase GID4 for targeted protein degradation

Nature Structural & Molecular Biology (2025)Cite this article

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Abstract

Proteolysis targeting chimeras (PROTACs) hijack E3 ligases and the ubiquitin–proteasome system to achieve selective degradation of neo-substrates. Their ability to target otherwise intractable substrates has rendered them a valuable modality in drug discovery. However, only a handful of over 600 human E3 ligases have been functionalized for PROTAC applications. Here we show that the E3 ligase GID4 (glucose-induced degradation deficient complex 4) can be leveraged for targeted protein degradation using a noncovalent small molecule. We design and synthesize GID4-based PROTACs, exemplified by NEP162, which can eliminate endogenous BRD4 in a GID4- and ubiquitin–proteasome system-dependent manner. NEP162 exhibits antiproliferative activity and inhibits tumor growth in a xenograft model, hinting toward potential anticancer applications. We further present the crystal structures of GID4–PROTAC–BRD4 ternary complexes in three distinct states, unveiling plastic interactions between GID4 and BRD4. These structural insights, combined with in vitro and in vivo data, decipher the molecular basis by which the hereby developed PROTACs recruit BRD4 to GID4 for targeted degradation and expand our arsenal of PROTAC-exploitable E3 ligases.

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Fig. 1: Design and characterization of NEP108 for BRD4 degradation.
Fig. 2: Crystal structure of the GID4–NEP108–BRD4 ternary complex.
Fig. 3: Design and characterization of NEP162 for BRD4 degradation.
Fig. 4: Structural analysis of the GID4–NEP162–BRD4 ternary complex.
Fig. 5: NEP162 degrades BRD4 via GID4 and UPS and inhibits cell proliferation.
Fig. 6: NEP162 inhibits tumor growth in vivo.
Fig. 7: GID4-based PROTACs degrade additional target proteins.

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Data availability

Atomic coordinates and structure factors have been deposited in the PDB with the accession codes 8X7G (GID4–NEP108–BRD4 ternary complex) and 8X7H (GID4–NEP162–BRD4 ternary complex). The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository84 with the dataset identifier PXD053449. Source data are provided with this paper.

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Acknowledgements

We thank the staff at beamlines BL19U1 of Shanghai Synchrotron Radiation Facility for assistance in X-ray data collection. This work was supported by National Natural Science Foundation of China grant nos. 82321001 (to C.D.), 32271265 (to C.D.), 82425040 (to L.S.), 82230101 (to L.S.), 22307093 (to D.C.) and 82473144 (to X.Y.), National Youth Top-Notch Talent Support Program in China, Tianjin Municipal Science and Technology Commission grant nos. 22JCZDJC00440 (to C.D.) and 23JCQNJC00540 (to D.C.), Research Foundation of Tianjin Municipal Education Commission grant nos. 2022KJ189 (to D.C.) and 2022KJ191 (to B.Z.), Postdoctoral Fellowship Program and China Postdoctoral Science Foundation (grant nos. GZB20240530 and 2024T170654 to K.B.) and Initiative (grant no. 2024NITFID313) by the National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Yanran Li, Kaiwen Bao, Jiyue Sun, Ruixin Ge.

Authors and Affiliations

  1. Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, State Key Laboratory of Experimental Hematology, Tianjin Medical University Cancer Institute and Hospital, Tianjin Medical University, Tianjin, China

    Yanran Li, Kaiwen Bao, Jiyue Sun, Qiqing Zhang, Bing Zhang, Xiaojie Yan, Junlin Li, Lei Shi & Cheng Dong

  2. Center for Cell Structure and Function, Collaborative Innovation Center of Cell Biology in Universities of Shandong, College of Life Sciences, Shandong Normal University, Jinan, China

    Ruixin Ge, Jinzhi Zang, Min Liu & Jun Zhou

  3. Tianjin Institute of Immunology, Department of Immunology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

    Fengying Shi & Wenyi Mi

  4. Department of Medicinal Chemistry, Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin, China

    Meiling Zhang & Dongxing Chen

  5. Department of Ophthalmology, Ministry of Education International Joint Laboratory of Ocular Diseases, Tianjin Key Laboratory of Ocular Trauma, Tianjin Institute of Eye Health and Eye Diseases, China-UK ‘Belt and Road’ Ophthalmology Joint Laboratory, Tianjin Medical University General Hospital, Tianjin, China

    Songbo Xie

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Contributions

C.D., L.S., D.C. and S.X. conceptualized the project and designed experiments. Y.L. performed protein expression, purification and crystallization with help from Q.Z. J.S. and M.Z. performed the synthesis and chemical characterization of all compounds. K.B. carried out the cellular assays. R.G. and J. Zang performed the in vivo assays. X.Y. and J.L. determined the crystal structures. C.D., L.S., D.C., S.X., M.L., J. Zhou, W.M., F.S. and B.Z. analyzed the data. C.D. wrote the paper with critical inputs from all authors.

Corresponding authors

Correspondence to Songbo Xie, Dongxing Chen, Lei Shi or Cheng Dong.

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Competing interests

C.D., L.S., D.C., J.S. and Y.L. declare the filing of a patent application covering the PROTACs described in this paper. The other authors declare no competing interests.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Design of GID4-based PROTACs for BRD4 degradation.

a, Chemical structures of initially designed GID4-based degraders 1-4. b, RT-qPCR analysis of GID4 mRNA expression level in different cell lines. Data presented are the mean ± S.D. of three independent experiments. c, Immunoblotting analysis of BRD4 degradation in U2OS cells treated with the indicated compounds (2 µM) for 18 h. Representative images, n = 3. d, Pull-down assay using His-GID4 to pull down BRD4BD1 with addition of varying concentrations of NEP108. Representative images, n = 3.

Source data

Extended Data Fig. 2 Structural comparison of the GID4-NEP108-BRD4 ternary complex with the GID4-PFI-7 and BRD4-JQ1 binary complexes.

a, Superimposition of PFI-7 bound to GID4 (PDB code: 7SLZ), JQ1 bound to BRD4 (PDB code: 3MXF) and NEP108 bound to GID4 and BRD4. b, Superimposition of JQ1 and NEP108 bound to BRD4 (gray surface). c, Superimposition of PFI-7 and NEP108 bound to GID4 (gray surface).

Extended Data Fig. 3 Structural comparison of the GID4-, CRBN- and VHL-recruiting BRD4.

a, Superimposition of GID4-NEP108-BRD4 and CRBN-dBET23-BRD4 (PDB code: 6BN7) ternary complexes against BRD4. For clarity, only one BRD4 model is shown. b, Superimposition of GID4-NEP108-BRD4 and VHL-MZ1-BRD4 (PDB code: 5T35) ternary complexes against BRD4. The ZA loop, BC loop and αC helix involved in the binding are indicated.

Extended Data Fig. 4 Structure-based design of GID4-based degraders.

a, Electrostatic potential surface of NEP108-binding pocket in GID4 and BRD4 (red, negative; blue, positive). b, Chemical structure of the GID4-based NEP179. c, Immunoblotting analysis of BRD4 degradation in U2OS cells treated with the 2 µM NEP179 and NEP162 compounds for 18 h. Representative images, n = 3. d, Immunoblotting analysis of BRD4 proteins in U2OS cells treated with 2 µM NEP162N or NEP162 for 18 h. Representative images, n = 3. e, Concentration-dependent degradation of BRD4 proteins by NEP162 in U2OS cells for 18 h. f, DC50 value of NEP162 for BRD4 in U2OS cells for 18 h. Data are derived from three biologically independent experiments. g,h,i, Immunoblotting analysis of BRD4 degradation in H3122 cells (g), H520 cells (h) and HEC-1-A cells (i) by NEP162 for 18 h. Representative images, n = 3. j, Immunoblotting analysis of BRD4 degradation in SW480 cells treated with 2 µM NEP162 or MZ1 for 18 h. Representative images, n = 3.

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Extended Data Fig. 5 Insights into the NEP162-mediated formation of the ternary complex.

a, Pull-down assay using His-GID4 to pull down BRD4BD1 with addition of varying concentrations of NEP162. Representative images, n = 3. b,c, Fo-Fc omit map (green mesh) of the NEP162 (b; J-state and c; L-state) generated before ligand modelling countered at the 1.0 σ level. d, BLI measurements of the interaction of immobilized mutant GID4 (Y168A) and a premix of NEP162-BRD4. Data presented are the mean ± S.D. of three independent experiments. e, BLI measurements of the interaction of immobilized double mutant GID4 (E167A and Y168A) and a premix of NEP162-BRD4. Data presented are the mean ± S.D. of four independent experiments. f, BLI analysis of the binary interaction between GID4 (E167A and Y168A) and NEP162. g, BLI measurements of the interaction of immobilized double mutant GID4 (E167A and Y168A) and a premix of NEP108-BRD4. Data presented are the mean ± S.D. of three independent experiments.

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Extended Data Fig. 6 Structural comparison of GID4-based complexes.

a, Overlay of the GID4 structures derived from ternary complexes mediated by NEP108, NEP162 (J-state) and NEP162 (L-state). b, Binding modes of GID4 bound to NEP108, NEP162 (J-state) and NEP162 (L-state). Structures are overlaid in reference to GID4. c, Structural alignment of GID4 in complex with PHRV peptide (PDB code: 6CCU), PSRV peptide (PDB code: 6CD8) and PGLW peptide (PDB code: 6CDC). For clarity, only one GID4 molecule is shown in a surface representation. d, Superimposition of BRD4 structures derived from ternary complexes mediated by NEP108, NEP162 (J-state) and NEP162 (L-state).

Extended Data Fig. 7 Degradation mechanism by NEP162.

a, Analysis of BRD4 protein ubiquitination in U2OS cells pretreated with 10 µM MG132 for 12 h, followed by treatment with DMSO, 2 µM NEP108 or NEP162 for 18 h. b, Immunoblotting analysis of the degradation of natural substrate HMGCS1 in SW480 cells upon Torin1 treatment (200 nM, 16 h) in the presence of cycloheximide (50 ng/L, 16 h). c, Cell viability was conducted in U2OS cells treated with DMSO or increasing concentrations of NEP162 for the indicated times. Data are shown as mean ± S.D. (n = 3 biological replicates); two-sided t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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Extended Data Fig. 8 Comparison of the activity of NEP162 and MZ1 in vivo.

a, Schematic of the administration of tumor-bearing mice. b,c, Images (b) and volumes (c) of tumors isolated from xenograft mice after intraperitoneal injection of DMSO, NEP162N (10 mg/kg), NEP162 (10 mg/kg) or MZ1 (10 mg/kg). Data are shown as mean ± S.D. (n = 5 biological replicates); two-sided t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. d, Tumor weights of mice at the endpoint. Data are shown as mean ± S.D. (n = 5 biological replicates); two-sided t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. e, Changes in body weight of mice during the treatments. Data are shown as mean ± S.D. (n = 5 biological replicates). f, Immunofluorescence imaging of TUNEL, and immunohistochemistry imaging of Ki67 and BRD4 in U2OS tumor sections from various groups. g,h,i, Quantitative analysis of tumor sections stained with TUNEL (g), Ki67 (h) and BRD4 (i). Data are shown as mean ± S.D. (n = 5 biological replicates); two-sided t-test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Panel a created with BioRender.com.

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Li, Y., Bao, K., Sun, J. et al. Design of PROTACs utilizing the E3 ligase GID4 for targeted protein degradation. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01537-1

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