Exploration of precision coregulator TR-FRET identifies diverse signatures for LXR ligands relevant to discovery of nonlipogenic ABCA1 inducers
- Department of Chemistry & Biochemistry, Colleges of Science & Medicine, University of Arizona, United States
- Department of Pharmacology & Toxicology, R Ken Coit College of Pharmacy, University of Arizona, United States
- Arizona Center for Drug Discovery, University of Arizona, United States
Figures
Figure 1 with 1 supplement
Liver X receptor (LXR) activation mediates beneficial effects and unwanted lipogenesis.
LXR transcriptional complexes are retinoid X receptor (RXR) heterodimers that can be activated by a canonical mechanism of coregulator recruitment or an alternative de-repression mechanism of (A) a constitutively repressed complex by (B) corepressor displacement. (C) ABCA1 gene transcription leads to cholesterol efflux and mobilization, apolipoprotein-E (APOE) lipidation and potentially Aβ clearance, in addition to other mechanisms of potential benefit in Alzheimer’s disease and related dementia (ADRD); whereas, SREBF1 transcription initiates a lipogenic program in the liver leading to unwanted low-density lipoprotein (LDL) formation and triglyceride (TG) elevation.
Figure 1—figure supplement 1
Chemical structures and compound codes of literature liver X receptor (LXR) ligands with biphenyl sulfone ligands shown in blue.
Figure 2 with 1 supplement
Liver X receptor (LXR) isoform selectivity from coregulator TR-FRET (CRT) measurements.
Concentration-response for recruitment of steroid receptor coactivator 1 (SRC-1) to LXRα (dotted lines) and LXRβ (solid line), determined by CRT assay, showing mean and SD from triplicate experiments, normalized to T0 (100%) as a full LXR agonist (shown in turquoise for reference). GSK2033 (R) and SR9238 (S) titrations were run in the presence of 24-hydroxycholesterol 24HC (3 µM) and neither ligand stabilized the LXR:SRC1 complex alone (T, U).
Figure 2—figure supplement 1
nuclear hormone receptor (NR) panel results.
(A) Concentration-response for recruitment of coregulator D22 to RXRα (dotted lines) and RXRβ (solid line), determined by coregulator TR-FRET (CRT) assay, showing mean and SD, normalized to SR11237 as a full agonist. (B) Concentration-response for recruitment of coregulator SRC2 to farnesoid X receptor (FXR) determined by CRT assay, showing mean and SD, normalized to Obeticholic acid (OCA) as a full agonist. (C) Concentration-response for recruitment of coregulator C33 to PPARδ determined by CRT assay, showing mean and SD, normalized to GW501516 as a full agonist. (D) Concentration-response for recruitment of coregulator D22 to RARα (dotted lines) and recruitment of coregulator SRC2-2 RARβ (solid line), determined by CRT assay, showing mean and SD, normalized to TTNPB as a full agonist. (E) Concentration-response for recruitment of coregulator SRC1-4 to pregnane X receptor PXR determined by CRT assay, showing mean and SD, normalized to T0901317(T0) as a full agonist.
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Figure 2—figure supplement 1—source data 1
Summary of nuclear hormone receptor (NR) panel parameters.
- https://cdn.elifesciences.org/articles/109146/elife-109146-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Coregulator peptide sequences.
- https://cdn.elifesciences.org/articles/109146/elife-109146-fig2-figsupp1-data2-v1.pdf
Figure 3 with 1 supplement
ATP-binding cassette-A1 (ABCA1)-luc reporter in CCF cells.
Concentration-response for liver X receptor (LXR) ligands in astrocytoma cells measured after 24 hr incubation, normalized to T0 response (100%) showing mean and SD from triplicate experiments (see Figure 3—figure supplement 1 for biological replicates).
Figure 3—figure supplement 1
Concentration-response curves of the control compound, T0, in CCF ATP-binding cassette-A1 (ABCA1)-luc reporter assay and HepG2 sterol response element (SRE)-luc reporter assay (A, B) show high reproducibility across independent experiments.
Figure 4 with 1 supplement
Correlating potency in cell-free and cell-based assays.
(A–F) Correlation of potency for steroid receptor coactivator 1 (SRC1) coactivator stabilization by liver X receptor (LXR) ligands from coregulator TR-FRET (CRT) data with potency for ATP-binding cassette-A1 (ABCA1) and sterol response element (SRE) reporter assays in CCF and HepG2 cells, respectively. Pearson and Spearman correlation data are shown with significance and Deming slope with F-test, showing poor correlation of LXRα CRT data with ABCA1 activation. Solid line and 95% confidence limits are from simple linear fit with dashed line showing Deming fit. (G–H) Correlation of potency for ligand-dependent recruitment of SRC2-2, D22, and TRAP220 to LXR α and β showing correlation statistics, best-fit line, and 95% confidence intervals. (I) Hierarchical clustering analysis of cell-free and cell-based relative response at 1 µM ligand. (J–K) Correlation of potency for ligand-dependent recruitment of SRC1 to LXR α and β isoforms showing Pearson and Spearman correlations and best-fit line and 95% confidence intervals from simple linear regression.
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Figure 4—source data 1
Data used in correlation plots and heatmaps.
- https://cdn.elifesciences.org/articles/109146/elife-109146-fig4-data1-v1.docx
Figure 4—figure supplement 1
Correlating potency in cell-free and cell-based assays.
(A–D) Correlation of potency for ligand-dependent recruitment of SRC2-2, D22, PGC1α, and TRAP220 to liver X receptor (LXR) α and β showing correlation statistics, best-fit line and 95% confidence intervals. Hierarchical clustering analysis of cell-free and cell-based relative response at 1 µM ligand (E) including all agonist ligands or (F,G) excluding AZ876. (I–L) Silhouette plots for dendrograms E, F, G, respectively. (M) Heatmap of hierarchical clustering for all ligands excluding AZ876. Raw data was analyzed in Prism 10.
Figure 5 with 1 supplement
Lipogenic response of HepG2 cells to treatment with liver X receptor (LXR) ligands.
(A–D) Concentration-response curves for LXR ligands normalized to maximal response for T0 (EC50=15 nM) showing a range of potency (AZ876 EC50=4.2 nM to CL2-57 2.8 µM) and maximal efficacy (BE-1218 80% to GSK3987 150%). See Figure 3—figure supplement 1 for biological replicates. (E–F) Three biphenyl sulfones gave anomalous activation of sterol response element (SRE) at higher concentrations (Figure 5—figure supplement 1): XL041 gave a partial agonist response at submicromolar concentrations (EC50=5.7 nM Emax = 22%), while SR9238 and GSK2033 had a neutral or antagonist response. (G–I) The transcriptional response to the five biphenyl sulfones was studied by RT-PCR under the same conditions as for SRE-luc measurements replicating the lipogenic induction observed in the reporter assays; both GSK2033 and SR9238 reduced response below baseline. Both N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) and 24-hydroxycholesterol (24HC) were cytotoxic at higher concentrations. Data show mean and standard deviation for triplicate measurements. See Figure 5—figure supplement 1 for full concentration-response curves.
Figure 5—figure supplement 1
Response of HepG2 cells to treatment with liver X receptor (LXR) ligands.
(A–B) Lipogenic response of HepG2 cells to treatment with liver X receptor (LXR) ligands showing full concentration-response curves for biphenyl compounds shown in Figure 5E–F. (C) Cell viability in HepG2 cells under identical conditions to those used to obtain sterol response element (SRE)-luc data.
Figure 6 with 1 supplement
Coactivator recruitment to liver X receptor (LXR) induced by LXR ligands.
Coregulator TR-FRET (CRT) data was normalized to T0 maximal response (100%) for all four CoA studied: LXRα (dashed lines) LXRβ (solid lines). For clarity, only TRAP220 and D22 data are shown. As noted in the text, most ligands were β-selective or nonselective, with the exception of AZ876 that selectively stabilizes CoA binding to LXRα. GSK2033 and SR9238 were studied in the absence of agonist. Data shown mean and SD from at least triplicate measurements.
Figure 6—figure supplement 1
Full concentration-response profiles of all tested compounds across coactivators.
Shown here for completeness; LXRα (dashed lines) and LXRβ (solid lines).
Figure 7 with 1 supplement
Corepressor binding to liver X receptor (LXR) isoforms: ligand dependence.
Concentration-response from coregulator TR-FRET (CRT) measurements of: NCOR2 binding to LXRα (dashed lines) (A); NCOR2 binding to LXRβ (solid lines) (B); SMRT2 binding to LXRα (dashed lines) (C); SMRT2 binding to LXRβ (solid lines) (D). Data shown mean and SD from at least triplicate measurements.
Figure 7—figure supplement 1
Full concentration-response profiles of all tested compounds across corepressors NCor2 and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) for both LXRα/β isoforms.
Figure 8
Ligand-induced conformational change to liver X receptor (LXR) causing corepressor (NCOR2) replacement by coactivator (steroid receptor coactivator 1, SRC1) measured by precision CRT (pCRT).
Concentration-response from CRT for SRC1 binding relative to apo-LXR (black dashed line) and NCOR2 binding relative to apo-LXR (magenta dashed line). Concentration-response from pCRT for SRC1 binding relative to apo-LXR (black solid line) and NCOR2 binding relative to apo-LXR (magenta solid line) with titration of ligand into solution of LXR, NCOR2, and SRC1. Data show mean of triplicate measurements.
Figure 9
Coregulator TR-FRET (CRT) signatures and correlation with stabilization and structure of liver X receptor (LXR):coregulator complexes.
(Table 1) Categorization of LXR ligands as agonists and antagonists conforming to CRT and precision CRT (pCRT) signatures. (A) Heat map of responses (ligand concentration at 1 µM) in cell-free and cell-based assays to LXR ligands together with theoretical response to agonist and antagonist ligand signatures. (B) Relative energy perturbations of apo and coregulator-bound LXR caused by each class of agonist and antagonist identified and classified in Table 1. (C) Co-crystal structure of LXRα:SRC1-2 co-crystal with GW3965 (PDB 3IPQ) showing key helices. (D) Superposition of LXRα:SRC1-2:GW3965 co-crystal with structure of LXRα:SMRT2 simulated using AlphaFold, showing the significant structural perturbation of H11 and H12 on ligand binding (red shades) compared to the ligand-free LXRα (blue shades).
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Figure 9—source data 1
Relative % activity at 1 μM used in heat map analysis.
- https://cdn.elifesciences.org/articles/109146/elife-109146-fig9-data1-v1.docx
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Exploration of precision coregulator TR-FRET identifies diverse signatures for LXR ligands relevant to discovery of nonlipogenic ABCA1 inducers
eLife 14:RP109146.
https://doi.org/10.7554/eLife.109146.3
