Shifting the PPARγ conformational ensemble toward a transcriptionally repressive state improves covalent inhibitor efficacy
- Undergraduate Program in Biochemistry and Chemical Biology, Vanderbilt University, United States
- Department of Biochemistry, Vanderbilt University, United States
- Department of Integrative Structural and Computational Biology, Scripps Research and The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, United States
- School of Basic Medical Sciences, Guangzhou Laboratory, Guangzhou Medical University, China
- Department of Molecular Medicine, Scripps Research and The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, United States
- Center for Structural Biology, Vanderbilt University, United States
- Vanderbilt Institute of Chemical Biology, Vanderbilt University, United States
Figures
Figure 1
A possible allosteric mechanism by which a covalent ligand inhibits binding of other non-covalent ligands.
Crystal structures of T0070907-bound PPARγ ligand-binding domain (LBD) cobound with (a) non-covalent agonist MRL-24 (PDB 8ZFS) and (b) NCoR1 corepressor peptide (PDB 6ONI). Helix 12 (h12) can adopt a solvent-exposed active conformation—or a solvent-occluded repressive conformation within the orthosteric ligand-binding pocket that would physically clash and block binding of an orthosteric ligand.
Figure 2
Pharmacological covalent inverse agonists stabilize a repressive ligand-binding domain (LBD) conformation.
(a) Chemical structures of four covalent ligands with different pharmacological properties including transcriptionally neutral antagonism (GW9662) or transcriptionally repressive partial inverse agonism (T0070907) and a full inverse agonism (SR33065 and SR36708). (b) Cell-based luciferase reporter assay in HEK293T cells transfected with an expression plasmid encoding full-length PPARγ and a 3xPPRE-luciferase reporter plasmid (n = 4 technical replicates; mean ± SEM). One-way ANOVA using Dunnett’s multiple comparisons test was used to compare DMSO (control) to ligand-treated conditions; ****p ≤ 0.0001. Fluorescence polarization (c) FITC-labeled NCoR1 corepressor and (d) FITC-labeled TRAP220/MED1 coactivator peptide binding assays (n = 3 technical replicates) with fitted affinities shown in the legend (mean ± [95% CI]). (e) Location of Gly399 in the crystal structure of PPARγ LBD bound to NCoR1 peptide and inverse agonist T0070907 (PDB 6ONI). (f) One-dimensional (1D) traces extracted from two-dimensional (2D) [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ LBD in the absence or presence of the indicated covalent ligands. The gray dotted line denotes the Gly399 backbone amide chemical shift in the apo/ligand-free form; black lines denote the active and repressive chemical shift values when bound to T0070907.
Figure 3
Time-resolved fluorescence resonance energy transfer (TR-FRET) profiling of non-covalent ligand binding to PPARγ ligand-binding domain (LBD).
(a) Chemical structures of non-covalent PPARγ partial agonists MRL-24 and nTZDpa and the full agonist rosiglitazone. (b) TR-FRET ligand displacement assay (n = 3 technical replicates) with fitted Ki values shown in the legend (mean ± SD). TR-FRET coregulator interaction assays where non-covalent agonists were titrated with in the presence of (c) 400 nM FITC-labeled NCoR1 corepressor or (d) 400 nM FITC-labeled TRAP220/MED1 coactivator peptide (n = 3 technical replicates; mean ± [95% CI]; n.d. = not determined).
Figure 4
Time-resolved fluorescence resonance energy transfer (TR-FRET) assay profiling of non-covalent ligand cobinding to PPARγ ligand-binding domain (LBD) with a covalent inhibitor and increasing NCoR1 peptide concentrations.
TR-FRET NCoR1 corepressor peptide interaction assays where (a) MRL-24 or (b) nTZDpa were titrated with increasing concentrations of FITC-labeled NCoR1 corepressor peptide to saturate the peptide-bound forms of PPARγ LBD bound to the covalent ligands profiled in Figure 1 (n = 3 technical replicates; mean ± SD). MRL-24 and nTZDpa Ki values are noted with vertical dotted orange lines, and a vertical gray line denotes log M = −7 as a visual cue to compare the concentration–response curves.
Figure 5
Corepressor peptide binding synergizes with covalent inhibitor inverse agonism to weaken non-covalent ligand cobinding.
Fitted parameters extracted from time-resolved fluorescence resonance energy transfer (TR-FRET) NCoR1 ligand cobinding assays (Figure 5—source data 1) with (a) MRL-24 or (b) nTZDpa including potency (IC50) and cooperativity (hill slope) values (n = 2 biological replicates derived from a fit of TR-FRET data with n = 3 technical replicates; mean ± SD). MRL-24 and nTZDpa Ki values are noted with vertical dotted orange lines.
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Figure 5—source data 1
Data underlying the plots in Figure 5.
- https://cdn.elifesciences.org/articles/106697/elife-106697-fig5-data1-v1.xlsx
Figure 6 with 4 supplements
Non-covalent ligand and covalent inhibitor cobinding shifts the PPARγ ligand-binding domain (LBD) conformational ensemble to an active state.
(a) 2D [1H,15N]-TROSY-HSQC data zoomed into the Gly399 backbone amide peaks of 15N-labeled PPARγ LBD bound individually or cobound to the indicated non-covalent and covalent ligands. The active (act) and repressive (rep) peaks when bound to covalent ligand alone are labeled; black arrows denote the shift in the peaks between the covalent ligand bound alone vs. cobound with the non-covalent ligand. Time-resolved fluorescence resonance energy transfer (TR-FRET) TRAP220/MED1 coactivator peptide interaction assays where (b) MRL-24 or (c) nTZDpa were titrated into PPARγ LBD pretreated with the indicated covalent inhibitors (n = 3 technical replicates; mean ± [95% CI]; n.d. = not determined).
Figure 6—figure supplement 1
2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ ligand-binding domain (LBD) bound individually or cobound to MRL-24 (top), nTZDpa (bottom), and GW9662 as indicated.
Figure 6—figure supplement 2
2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ ligand-binding domain (LBD) bound individually or cobound to MRL-24 (top), nTZDpa (bottom), and T0070907 as indicated.
Figure 6—figure supplement 3
2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ ligand-binding domain (LBD) bound individually or cobound to MRL-24 (top), nTZDpa (bottom), and SR33065 as indicated.
Figure 6—figure supplement 4
2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ ligand-binding domain (LBD) bound individually or cobound to MRL-24 (top), nTZDpa (bottom), and SR36708 as indicated.
Figure 7 with 1 supplement
Non-covalent ligand cobinding occurs in the presence of the covalent inhibitor SR16832.
(a) Chemical structure of the dual-site covalent inhibitor SR16832. (b) 2D [1H,15N]-TROSY-HSQC data zoomed into the Gly399 backbone amide peaks of 15N-labeled PPARγ ligand-binding domain (LBD) bound individually or cobound to MRL-24, nTZDpa, and SR16832 as indicated. Black arrows denote the shift in the peaks, or LBD conformation, between the covalent ligand bound alone vs. cobound with the non-covalent ligand. Time-resolved fluorescence resonance energy transfer (TR-FRET) coregulator interaction assays using (c) FITC-labeled NCoR1 corepressor or (d) FITC-labeled TRAP220/MED1 coactivator peptides where MRL-24 (orange) or nTZDpa (purple) were titrated with two concentrations of FITC-NCoR1 corepressor peptide to saturate the peptide-bound forms of PPARγ LBD bound to SR16832 (n = 3 technical replicates; mean ± SD).
Figure 7—figure supplement 1
2D [1H,15N]-TROSY-HSQC NMR data of 15N-labeled PPARγ ligand-binding domain (LBD) bound individually or cobound to MRL-24 (top), nTZDpa (bottom), and SR16832 as indicated.
Figure 8
Model for improved covalent inhibitor efficacy.
Covalent ligands that when bound to PPARγ ligand-binding domain (LBD) increase the occupancy of helix 12 (h12) within the orthosteric pocket show improved efficacy of inhibiting other ligands from binding to the orthosteric pocket. Our data suggest that this is an allosteric mechanism in the sense that the covalent inhibitor does not physically clash with the non-covalent ligand itself; instead, increased occupancy of h12 within the orthosteric pocket resulting from the bound covalent inhibitor results in a clash or competition with a non-covalent orthosteric ligand.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | PPARG | UniProt | PPARG_HUMAN | Residues 203–477, isoform 1 numbering |
| Strain, strain background (Escherichia coli) | BL21(DE3) | New England Bioscience | #C2527H | Electrocompetent cells used for bacterial expression of proteins |
| Cell line (Homo sapiens) | HEK293T | ATCC | #CRL-3216; RRID:CVCL_0063 | Used for luciferase reporter assays |
| Antibody | LanthaScreen Elite Tb-anti-His Antibody | Thermo Fisher Scientific | #PV5895; RRID:AB_3720338 | TR-FRET antibody |
| Recombinant DNA reagent | Human PPARγ LBD - pET46 (plasmid) | Hughes et al., 2012 | Used to express human PPARγ ligand-binding domain (LBD) protein | |
| Peptide, recombinant protein | Human NCoR1 ID2 motif | LifeTein | FITC-labeled peptide for TR-FRET | |
| Peptide, recombinant protein | Human TRAP220/MED1 ID2 motif | LifeTein | FITC-labeled peptide for TR-FRET | |
| Chemical compound, drug | T0070907 | Cayman Chemical | #10026 | Covalent PPARγ inverse agonist |
| Chemical compound, drug | GW9662 | Cayman Chemical | #70785 | Covalent PPARγ antagonist |
| Chemical compound, drug | SR33065 | MacTavish et al., 2025 | Covalent PPARγ inverse agonist | |
| Chemical compound, drug | SR33068 | MacTavish et al., 2025 | Covalent PPARγ inverse agonist | |
| Chemical compound, drug | MRL-24 | Abbexa | #abx282275 | PPARγ partial agonist |
| Chemical compound, drug | nTZDpa | Tocris | #2150 | PPARγ partial agonist |
| Chemical compound, drug | Rosiglitazone | Cayman Chemical | #71740 | PPARγ agonist |
| Software, algorithm | Prism | GraphPad | Version 10 | Used to plot assay data |
| Software, algorithm | PyMOL | Schrödinger | Version 3 | Used to generate structural plots |
| Other | X-tremegene 9 | Roche | Transfection reagent | |
| Other | Britelite Plus | PerkinElmer | Luciferase reagent |
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Shifting the PPARγ conformational ensemble toward a transcriptionally repressive state improves covalent inhibitor efficacy
eLife 14:RP106697.
https://doi.org/10.7554/eLife.106697.3
