Identification of Actionable Targeted Protein Degradation Effector Sites through Site-Specific Ligand Incorporation-Induced Proximity (SLIP)

Abstract

Targeted protein degradation (TPD) is a rapidly emerging and potentially transformative therapeutic modality. However, the large majority of >600 known ubiquitin ligases have yet to be exploited as TPD effectors by proteolysis-targeting chimeras (PROTACs) or molecular glue degraders (MGDs). We report here a chemical-genetic platform, Site-specific Ligand Incorporation-induced Proximity (SLIP), to identify actionable ("PROTACable") sites on any potential effector protein in intact cells. SLIP uses genetic code expansion to encode copper-free "click" ligation at a specific effector site in intact cells, enabling the in situ formation of a covalent PROTAC-effector conjugate against a target protein of interest. Modification at actionable effector sites drives degradation of the targeted protein, establishing the potential of these sites for TPD. Using SLIP, we systematically screened dozens of sites across E3 ligases and E2 enzymes from diverse classes, identifying multiple novel potentially PROTACable effector sites which are competent for TPD. SLIP adds a powerful approach to the proximity-induced pharmacology (PIP) toolbox, enabling future effector ligand discovery to fully enable TPD and other emerging PIP modalities.

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SLIP enables direct prioritization of specific effector sites for targeted protein degradation (TPD). (a) Ligand binding domain (“tag”) such as Halo or FKPB12 is fused to the N- or C-terminus of an effector to enable ligand-induced proximity to a protein of interest (POI) through a conserved motif; (b) SLIP employs genetic code expansion (GCE) to incorporate an unnatural amino acid (UAA), followed by an in-cell copper-free click reaction to precisely induce proximity at an actionable site with minimal disruption to effector structure or function. SLIP enables the identification of specific PROTACable effector sites. This figure was generated by using BioRender.

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Site-specific ligand incorporation via genetic code expansion. (a) All-in-one plasmid encoding the orthogonal Pyl-RS/tRNA pair and VHL_Cys77TAG-HA for the expression of UAA-modified VHL. GFP is used to monitor transfection efficiency and analysis. (b) Structures of UAAs and tetrazine derivatives used to perform Cu-free “click” ligations in cells. (c) Immunoblot analysis of protein expression efficiency with different UAA supplements; cells were transfected and treated with DMSO or different UAAs for 30 h, and expression analyzed by anti-HA Western blot, with β-actin as loading control. (d) 2-TCOK dose-dependency study; cells were transfected and incubated with different concentrations of 2-TCOK for 30 h, and expression analyzed by anti-HA Western blot. (e) Time-dependence of VHL_Cys77TAG-HA expression with the addition of 50 μM 2-TCOK, analyzed by anti-HA Western blot. (f) In-gel fluorescence analysis of 2-TCOK substituted protein labeling in live cells; cells were transfected and incubated with or without 50 μM 2-TCOK for 30 h, then washed, pretreated with DMSO or 500 nM FT3 for 30 min before labeling with TAMRA-tetrazine (TT) at the concentration indicated for 1 h, and cell lysates analyzed by in-gel fluorescence and anti-HA Western blot.

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Site-specific FKBP12^F36Vligand incorporation induces TPD of the FKBP12^F36V-mCherry protein. (a) Schematic hypothesis for FKBP12^F36V ligand-bioconjugation-induced proximity and subsequent protein degradation. FKBP12^F36V-mCherry degradation can be assessed by fluorescent signal reduction. (b) HEK293 cells stably expressing FKBP12^F36V-mCherry were transfected with plasmid containing VHL_C77TAG in the presence of 50 μM 2-TCOK for 30 h, washed, treated with DMSO or FT3 (100 nM) for 16 h, and analyzed by flow cytometry. (c) HEK293 cells stably expressing FKBP12^F36V-mCherry were transfected with VHL_C77TAG in the presence of 50 μM 2-TCOK for 30 h, washed, treated with DMSO or FT3 (100 or 500 nM) for 16 h, and sorted by GFP expression using FACS between the top 10% (GFP-high) and the bottom 90% (GFP-low); FKBP12^F36V-mCherry levels were then analyzed by immunoblot detected by anti-mCherry antibody, which was normalized to loading control Vinculin. (d) Quantification of protein levels in (c). Values are shown as means ± SD (n = 3, *p < 0.05). (e) FKBP12^F36V-mCherry expressing cells were transfected to express VHL_C77TAG-HA in the presence of 50 μM 2-TCOK for 30 h, washed, pretreated with Cl-T or Bortezomib (BTZ) for 1 h, then with FT3 (100 nM) for 16 h, followed by cytometry analysis. Values are shown as means ± SD (n = 3, *p < 0.05 and **p < 0.01). (f) 2-TCOK modified VHL was immobilized on HA magnetic beads, followed by treatment with FT3 (100 nM) for 1 h, after which beads were incubated with cell lysate containing FKBP12^F36V-mCherry for 1 h, and proteins analyzed by immunoblot.

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SLIP enables combinatorial and differential interrogation of PROTACable sites and linker chemistry. (a) Structure of ternary complex BRD4-MZ1-VCB (VHL and adaptor proteins Elongin C and Elongin B), mutated sites highlighted in green (PDB: 5T35 ). (b) HEK293 cells were transiently transfected with wild-type or different variants of VHL in the presence of 50 μM 2-TCOK for 30 h, and VHL protein levels were accessed by immunoblot using anti-HA antibody. (c) HEK293 cells stably expressing FKBP12^F36V-mCherry were transfected with different VHL variants in the presence of 50 μM 2-TCOK for 30 h, washed, treated with DMSO (ctrl), dTAG PROTACs, or different concentrations of tetrazine probes for 16 h, as indicated; mCherry reduction was analyzed by flow cytometry as described above and normalized to the efficiency of VHL_His110. (d) Population of generated feasible low-energy complex conformations formed between ligand-modified VCB and FKBP12^F36V. The computational studies were performed using the fast Fourier transform (FFT)-based PROTAC complex modeling method.

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Broad identification of PROTACable sites and effectors using SLIP. (a) FKBP12^F36V-mCherry expressing cells were transfected with different variants in the presence of 50 μM 2-TCOK for 30 h, washed, treated with different concentrations of tetrazines for 16 h, and mCherry level analyzed via flow cytometry normalized to the maximal effect of VHL_H110TAG. (b) Structure of GSPT1/CC-885/CRBN/DDB1 complex (PDB: 5HXB ). (c) Structure of SOCS2-Elongin C/B complex (PDB: 2C9W ). (d) Structure of UBE2B (PDB: 2YB6 ). Figures were prepared using PyMOL. Effector proteins are shown in magenta, and key residues are highlighted in cyan.

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SLIP induces targeted degradation of endogenous Aurora A protein. (a) Structures of tetrazine-modified Aurora A ligand AT1 and AT2. (b) Cells were transfected with CRBN_H353TAG all-in-one plasmid (see Figure a) for 30 h in the presence of 50 μM 2-TCOK, washed, treated with 71 μM cycloheximide (CHX, which blocks additional protein synthesis) and AT1 at the concentration indicated for 4 h, separated by FACS into GFP high and low populations, and Aurora A protein levels analyzed by immunoblot following cell lysis. (c) Following the same protocol as in (b), cells were transfected with either SOCS2_C111TAG or RNF146_C185TAG, treating with AT1 or AT2 for 4 h. Nontransfected cells were used as a control.