Pharmacological covalent inverse agonists stabilize a repressive 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 ± s.e.m.). (c,d) 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 ± std. err.). (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. Grey 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.

TR-FRET profiling of non-covalent ligand binding to PPARγ 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 ± s.d.). (c,d) 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 ± s.d.).

TR-FRET assay profiling of non-covalent ligand cobinding to PPARγ LBD with a covalent inhibitor and increasing NCoR1 peptide concentrations.

(a,b) 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 ± s.d.). MRL-24 and nTZDpa Ki values are noted with vertical dotted orange lines, and a vertical grey line denotes log M = -7 as a visual cue to compare the concentration-response curves.

Corepressor peptide binding synergizes with covalent inhibitor inverse agonism to weaken non-covalent ligand cobinding.

Fitted parameters extracted from TR-FRET NCoR1 ligand cobinding assays for the (a) MRL-24 or (b) nTZDpa titrations 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 ± s.d.). MRL-24 and nTZDpa Ki values are noted with vertical dotted orange lines.

Non-covalent ligand and covalent inhibitor cobinding shifts the PPARγ 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. (b,c) 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 ± s.d.).

Non-covalent ligand cobinding occurs in the presence of 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 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. (c,d) 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 ± s.d.).