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 (GCE) to encode copper-free "click" ligation at a specific effector site in intact cells, enabling in situ formation of a covalent PROTAC-effector conjugate against a target protein of interest (POI). 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.

Keywords: Genetic code expansion; induced proximity; targeted protein degradation.

Figure 1.. SLIP enables direct prioritization of specific effector sites for targeted protein degradation (TPD).

(a) 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 using BioRender.

Figure 2.. 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 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 UAA for 30 hours, 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 hours, and expression analyzed by anti-HA Western blot. (e) Time-dependence of VHL_Cys77TAG-HA expression with addition of 50 µM 2-TCOK, analyzed by anti-HA Western blot. (f) In-gel fluorescence analysis of 2-TCOK substituted protein labelling in live cells; cells were transfected and incubated with or without 50 µM 2-TCOK for 30 hours, then washed, pretreated with DMSO or 500 nM FT3 for 30 min before labeling with TAMRA-tetrazine (TT) at the concentration indicated for 1 hour, and cell lysates analyzed by in-gel fluorescence and anti-HA Western blot.

Figure 3.. Site-specific FKBP12^F36V ligand incorporation induces TPD of 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 hours, washed, treated with DMSO or FT3 (100 nM) for 16 hours, 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 hours, washed, treated with DMSO or FT3 (100 or 500 nM) for 16 hours, 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) FKBP12^F36V-mCherry expressing cells were transfected to express VHL_C77TAG-HA in the presence of 50 μM 2-TCOK for 30 hours, washed, pre-treated with Cl-T or Bortezomib (BTZ) for one hour, then with FT3 (100 nM) for 16 hours, followed by cytometry analysis. Values are shown as means ± SD (n=3, *p<0.05 and **p<0.01).

Figure 4.. 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: 5T3537). (b) HEK293 cells were transiently transfected with wild-type or different variants of VHL in the presence of 50 µM 2-TCOK for 30 hours, VHL protein levels was 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 hours, washed, treated with DMSO (ctrl), dTAG PROTACs, or different concentrations of tetrazine probes for 16 hours, as indicated; mCherry reduction was analyzed by flow cytometry as described above. (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.

Figure 5.. 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 hours, washed, treated with different concentrations of tetrazines for 16 hours, and mCherry level analyzed via flow cytometry normalized to the maximal effect of VHL_C77TAG. (b) Structure of GSPT1/CC-885/CRBN/DDB1 complex (PDB: 5HXB⁵⁰). (c) Structure of SOCS2-Elongin C/B complex (PDB: 2C9W⁵¹). (d) Structure of UBE2B (PDB: 2YB6⁵²). Figures were prepared using PyMOL. Effector proteins are shown in magenta, and key residues are highlighted in cyan.

Figure 6.

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_H325TAG all-in-one plasmid (see Figure 2a) for 30 hours 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 hours, 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 hours.