PROTAC-mediated activation, rather than degradation, of a nuclear receptor reveals complex ligand-receptor interaction network

Abstract

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules containing a ligand for a protein of interest linked to an E3 ubiquitin ligase ligand that induce protein degradation through E3 recruitment to the target protein. Small changes in PROTAC linkers can have drastic consequences, including loss of degradation activity, but the structural mechanisms governing such changes are unclear. To study this phenomenon, we screened PROTACs of diverse targeting modalities and identified dTAG-13 as an activator of the xenobiotic-sensing pregnane X receptor (PXR), which promiscuously binds various ligands. Characterization of dTAG-13 analogs and precursors revealed interplay between the PXR-binding moiety, linker, and E3 ligand that altered PXR activity without inducing degradation. A crystal structure of PXR ligand binding domain bound to a precursor ligand showed ligand-induced binding pocket distortions and a linker-punctured tunnel to the protein exterior at a region incompatible with E3 complex formation, highlighting the effects of linker environment on PROTAC activity.

Keywords: PROTAC; cytochrome P450; drug design; metabolism; nuclear receptor; pregnane X receptor; targeted protein degradation.

Figure 1.. dTAG-13 is a partial PXR agonist.

(A) Chemical structures of PXR modulators. (B-C) HepG2 cells were transfected with PXR-expressing plasmid and a plasmid encoding firefly luciferase under the control of a PXR-responsive CYP3A4 promoter (pGL3-CYP3A4-luc), treated with compounds for 24 h, and assessed for luciferase activity. Results are expressed as the mean ± standard deviation (SD) from three replicates. In (C), cells were cotreated with the indicated compound and 5 μM rifampicin to assess antagonistic effects. (D) Time-resolved fluorescence resonance energy transfer (TR-FRET) PXR LBD binding assay measuring displacement of a fluorescent probe from PXR LBD. Results are expressed as the mean ± SD from four replicates. (E-G) HepG2 cells were transfected with VP16-PXR LBD plasmid, a coregulator plasmid (GAL4-SRC-1 or GAL4-NCoR), and a GAL4 firefly luciferase reporter plasmid. Cells were treated for 24 h and assessed for luciferase activity. Results are expressed as the mean ± SD from at least four replicates. In (G), cells were cotreated with the indicated compound and 1 μM SPA70 to evaluate disruption of NCoR-PXR LBD complex. (H) SNU-C4 cells were treated with 5 μM rifampicin, 10 μM SPA70, or 10 μM dTAG-13 for 24 h, and RNA was extracted and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to measure expression of CYP3A4, PXR, and GAPDH. Data were normalized to 18S RNA and represent fold change (FC) relative to the DMSO control for each gene. (I) SNU719 cells were treated and processed as in (H). (J) SNU719 cells were treated with 0, 0.3, 3, or 30 μM dTAG-13 in the absence or presence of 5 μM rifampicin for 24 h. CYP3A4 RNA abundance was measured as in (H-I). Results in (H-J) are expressed as the mean ± SD from three replicates.

Figure 2.. dTAG-13 does not induce PXR degradation.

(A) SNU-C4 HiBiT-PXR KI cells were treated with dTAG-13 for 24 h and assessed for HiBiT level. Results are expressed as the mean ± SD from four replicates. (B) SNU-C4 3xFLAG-PXR KI cells were treated with dTAG-13 for 2 h or 24 h, and western blots were performed with antibodies to FLAG and β-Actin. (C) HepG2 cells were transfected with 1) 20 ng pcDNA3-HiBiT-FKBP12^F36V-2xHA-PXR or pcDNA3-HiBiT-PXR and 2) 20 ng pcDNA3 (empty vector, EV) or pcDNA3-HA-CRBN. Cells were treated with dTAG-13 for 24 h and assessed for HiBiT level. Results are expressed as the mean ± SD from three replicates. (D) HepG2 cells were transfected with pcDNA3-LgBiT-CRBN and either pcDNA3-SmBiT-FKBP12^F36V-2xHA-PXR or pcDNA3-SmBiT-PXR. Cells were treated with dTAG-13 for 30 min and assessed for luminescence. RLU, relative light units. Results are expressed as the mean ± SD from four replicates. (E-H) HepG2 cells were transfected with 1) 100 ng pcDNA3-HiBiT-FKBP12^F36V-2xHA-PXR or pcDNA3-HiBiT-PXR, 2) 100 ng pcDNA3 (EV) or pcDNA3-HA-CRBN, and 3) 2 μg pGL3-CYP3A4-luc reporter. Cells were treated with compounds for 24 h and assessed for luciferase activity. Results are expressed as the mean ± SD from three replicates. In (F and H), cells were cotreated with the indicated compound and 5 μM rifampicin.

Figure 3.. dTAG-13 analogs and components have distinct functional properties.

(A) Chemical structures of dTAG-13-related compounds. (B-C) HepG2 cells were transfected with PXR-expressing plasmid and pGL3-CYP3A4-luc reporter, treated with compounds for 24 h, and assessed for luciferase activity. Results are expressed as the mean ± SD from at least four replicates. In (C), cells were cotreated with the indicated compound and 5 μM rifampicin. (D) HepG2 cells were transfected with 1) 20 ng pcDNA3-HiBiT-FKBP12^F36V-2xHA-PXR or pcDNA3-HiBiT-PXR, 2) 20 ng pcDNA3-HA-CRBN, and 3) 20 ng pcDNA3-HA-VHL. Cells were treated with compounds for 24 h and assessed for HiBiT level. Results are expressed as the mean ± SD from three replicates. (E-G) HepG2 cells were transfected with VP16-PXR LBD plasmid, a coregulator plasmid (GAL4-SRC-1 or GAL4-NCoR), and a GAL4 firefly luciferase reporter plasmid. Cells were treated for 24 h and assessed for luciferase activity. Results are expressed as the mean ± SD from eight replicates. In (G), cells were cotreated with the indicated compound and 1 μM SPA70 to evaluate disruption of NCoR-PXR LBD complex.

Figure 4.. AP1867 disrupts PXR LBD secondary structure elements.

(A) The crystal structure of AP1867-bound PXR LBD was solved to 2.60 Å resolution. AP1867 is shown as spheres. The activation function-2 (AF-2) surface is the coactivator binding site. (B) The 2Fo–Fc map is contoured in mesh at 1.0 σ and carved around AP1867 at 1.8 Å. (C) The PROTAC linker precursor (the carboxylic acid) of AP1867 is positioned over α2. (D) All PXR LBD chains (n = 81) of all deposited structures (n = 59) are overlaid. AP1867 (violet) displaces α2 compared to all other structures. (E) The all-atom root mean square deviation (RMSD) was calculated for the α2 region of all PXR LBD chains (n = 79) of all deposited structures (n = 57) compared to apo PXR LBD (n = 2 PXR LBD chains from 2 structures, PDB IDs 1ILG and 7AX8), showing that AP1867-bound α2 is substantially displaced compared to all other structures. (F) The structures of apo (PDB ID 7AX8), T0-BP-bound (PDB ID 8FPE), and AP1867-bound PXR LBD are overlaid, and the α2 region is shown.

Figure 5.. AP1867 deposes key ligand binding pocket residues.

(A) Residues within 5 Å of AP1867 are shown as gray sticks with semi-transparent surface (n = 28), and selected residues are indicated (n = 11). AP1867 is shown as violet sticks. (B) The all-atom RMSD was calculated for the ligand binding pocket of all PXR LBD chains (n = 79) of all deposited structures (n = 57) compared to apo PXR LBD (n = 2 PXR LBD chains from 2 structures, PDB IDs 1ILG and 7AX8), showing that AP1867 displaces numerous residues. Here, the ligand binding pocket was defined as residues within 5 Å of AP1867 only. (C-K) Positions of selected residues are shown for AP1867-bound (violet) and all other PXR LBD chains (white). Distance measurements and green lines in (I-J) indicate hydrogen bonds between the residue and AP1867.

Figure 6.. dTAG PROTAC binding mode may be incompatible with PXR-E3 complex formation.

(A) Cartoon and surface views are shown for AP1867-bound PXR LBD. The circles indicate chain breaks where residues were not observed in the electron density (178–195, 230–234, and 311–313). (B) Cartoon and surface views are shown for AP1867-bound FKBP12^F36V (PDB ID 1BL4).