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. 2023 Apr 21;18(4):897-904.
doi: 10.1021/acschembio.3c00040. Epub 2023 Mar 20.

Targeted Protein Degradation through E2 Recruitment

Nafsika Forte  1   2   3 , Dustin Dovala  2   4 , Matthew J Hesse  2   4 , Jeffrey M McKenna  2   5 , John A Tallarico  2   5 , Markus Schirle  2   5 , Daniel K Nomura  1   2   3   6
Affiliations

Targeted Protein Degradation through E2 Recruitment

Nafsika Forte et al. ACS Chem Biol. .

Abstract

Targeted protein degradation (TPD) with proteolysis targeting chimeras (PROTACs), heterobifunctional compounds consisting of protein targeting ligands linked to recruiters of E3 ubiquitin ligases, has arisen as a powerful therapeutic modality to induce the proximity of target proteins with E3 ligases to ubiquitinate and degrade specific proteins in cells. Thus far, PROTACs have primarily exploited the recruitment of E3 ubiquitin ligases or their substrate adapter proteins but have not exploited the recruitment of more core components of the ubiquitin-proteasome system (UPS). In this study, we used covalent chemoproteomic approaches to discover a covalent recruiter against the E2 ubiquitin conjugating enzyme UBE2D─EN67─that targets an allosteric cysteine, C111, without affecting the enzymatic activity of the protein. We demonstrated that this UBE2D recruiter could be used in heterobifunctional degraders to degrade neo-substrate targets in a UBE2D-dependent manner, including BRD4 and the androgen receptor. Overall, our data highlight the potential for the recruitment of core components of the UPS machinery, such as E2 ubiquitin conjugating enzymes, for TPD, and underscore the utility of covalent chemoproteomic strategies for identifying novel recruiters for additional components of the UPS.

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Conflict of interest statement

The authors declare the following competing financial interest(s): JAT, JMK, DD, MJH, MS are employees of Novartis Institutes for BioMedical Research. This study was funded by the Novartis Institutes for BioMedical Research and the Novartis-Berkeley Translational Chemical Biology Institute. DKN is a co-founder, shareholder, and scientific advisory board member for Frontier Medicines and Vicinitas Therapeutics. DKN is a member of the board of directors for Vicinitas Therapeutics. DKN is also on the scientific advisory board of The Mark Foundation for Cancer Research, MD Anderson Cancer Center, Photys Therapeutics, Apertor Pharmaceuticals, Oerth Bio, and Chordia Therapeutics. DKN is also an Investment Advisory Board member for Droia Ventures and a16z.

Figures

Figure 1
Figure 1
Discovering a covalent recruiter for E2 ubiquitin conjugating enzyme UBE2D. (A) Gel-based ABPP screen of cysteine-reactive covalent ligands against human pure UBE2D2 C85S protein showing EN67 as the top hit. (B) Structure of EN67 with the cysteine-reactive acrylamide warhead highlighted in red. Gel-based ABPP showing competition of EN67 against IA-rhodamine binding to UBE2D2 C85S pure protein and silver staining of protein showing equal protein loading. (C) TP53 ubiquitination activity by E1 UBE1, E2 UBE2D2, E3 MDM2, FLAG-ubiquitin, and ATP showing that EN67 does not inhibit TP53 ubiquitination activity. (D) Mapping of EN67 site of modification on human pure UBE2D2 C111 by LC-MS/MS. Experiments in (B), (C), and (D) are representative of n = 3 biological replicates/group.
Figure 2
Figure 2
Validating target engagement and selectivity of EN67 in cells. (A) Synthetic route for making the alkyne-functionalized probe of EN67. (B) Gel-based ABPP of alkyne-functionalized EN67 probes against pure recombinant human UBE2D2 C85S protein and corresponding silver staining. (C) NF363C engagement and enrichment of UBE2D2 in HEK293T cells. Cells were treated with dimethyl sulfoxide (DMSO) or NF36C (50 μM) for 24 h. Probe-modified proteins were subsequently appended with an azide-functionalized biotin handle by CuAAC, and proteins were avidin-enriched and eluted for detection of UBE2D2 and an unrelated negative control protein GAPDH by Western blotting. Both input and pulldown of UBE2D2 and GAPDH are shown. (D) isoDTB-ABPP cysteine chemoproteomic profiling of EN67 in HEK293T cells. HEK293T cells were treated with DMSO vehicle or EN67 (50 μM) for 4 h. Lysates were labeled with IA-alkyne (200 μM) for 1 h, and isotopic desthiobiotin tags were appended by CuAAC and taken through the isoDTB-ABPP procedure. Shown are ratios of control/EN67-treated probe-modified peptide ratios and adjusted p-values from n = 3 biological replicates/group. Data are shown in Table S2. Data in panels (B) and (C) are representative of n = 3 biological replicates/group.
Figure 3
Figure 3
UBE2D-based BRD4 degrader. (A) Synthetic route and structure for NF90 linking UBE2D recruiter EN67 to BRD4 inhibitor JQ1. (B) Gel-based ABPP analysis of NF90 against IA-rhodamine labeling of pure human UBE2D2 C85S protein and corresponding silver staining. (C) NF90 effects on BRD4 levels in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were treated with DMSO vehicle or NF90 for 24 h, and BRD4 and loading control GAPDH levels were assessed by Western blotting. (D) BRD4 levels from (C) quantified. (E) Attenuation of BRD4 degradation by a proteasome inhibitor bortezomib (BTZ) or a NEDDylation inhibitor (MLN4924). MDA-MB-231 cells were pre-treated with DMSO vehicle, BTZ (1 μM), or MLN4924 (1 μM) for 1 h prior to treatment of cells with DMSO vehicle or NF90 (10 μM) for 24 h. BRD4 and loading control GAPDH levels were assessed by Western blotting. (F) NF90 effects on BRD4 degradation upon UBE2D knockdown. HEK293T cells with siControl or siRNA knockdown of UBE2D1, UBE2D2, UBE2D3, and UBE2D4 treated with DMSO vehicle or NF90 (10 μM) for 24 h. BRD4 short isoform and UBE2D1-4 and loading control GAPDH were assessed by Western blotting. (G) BRD4 levels quantified from panel (F). Gels and blots shown in panels (B), (C), (E), and (F) are representative of n = 3 biological replicates/group. Bar graphs shown in panels (D) and (G) show average ± standard error of the mean (sem) with individual replicate values. Statistical significance compared to vehicle-treated control expressed as *p < 0.05 and compared to NF90-treated siControl cells as #p < 0.05.
Figure 4
Figure 4
UBE2D-based AR degrader. (A) Structure of AR-targeting ligand control NF505 and UBE2D-based AR degrader NF500C linking the AR-targeting ligand to EN67. (B) AR degradation by NF500C. LNCaP prostate cancer cells were treated with DMSO vehicle or NF500C for 24 h and AR and loading control GAPDH levels were assessed by Western blotting. (C) AR degradation attenuated by NEDDylation inhibitor MLN4924. LNCaP cells were pre-treated with DMSO vehicle or NEDDylation inhibitor MLN4924 (1 μM) for 1 h prior to treating cells with DMSO vehicle or NF500C (10 μM) for 24 h. AR and loading control GAPDH levels were assessed by Western blotting. (D) TMT-based quantitative proteomic profiling of protein level changes conferred by NF500C treatment in LNCaP cells. LNCaP cells were treated with DMSO vehicle or NF500C (10 μM) for 24 h. Data are from n = 3 biological replicates/group. Gels and blots in panels (B) and (C) are representative of n = 3 biological replicates/group.
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