Peroxisome proliferation-activated receptor δ agonist GW0742 interacts weakly with multiple nuclear receptors, including the vitamin D receptor

Abstract

A high-throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes for modulating gene regulation mediated by VDR. Peroxisome proliferator-activated receptor (PPAR) δ agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations of > 12.1 μM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor. Surprisingly, GW0742 behaved as a PPAR agonist and antagonist, activating transcription at lower concentrations and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742 increased the fluorescence intensity and level of fluorescence polarization at an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a dose-dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced level of expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3, and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

Figure 1

IC₅₀ values of 1938 hit compounds using VDR-LBD and SRC2–3 labeled with: A Alexa Fluor 647; B Texas-Red in correlation with Alexa Fluor 647; C Fluorescein in correlation with Alexa Fluor 647.

Figure 2

Inhibition for the VDR-SRC2–3 Alexa Fluor 647 interaction by small molecules (50 µM) in the presence of 1 mM or 100 mM of 2-mercaptoethanol (ME).

Figure 3

VDR–GW0742 interactions. A chemical structure of GW0742; B FP VDR ligand competition assay; C Fluorescence interaction between GW0742 and rhodamine-labeled VDR ligand.

Figure 4

Summary of nuclear receptor-coactivator inhibition in the presence of GW0742 using FP. A Inhibition of the VDR–SRC2–3 interaction; B Inhibition of the TRβ–SRC2–2 interaction; C Inhibition of the PPARγ–DRIP2 interaction; D Inhibition of the AR–SRC2–3 interaction. The conditions for different NRs were as follows: VDR-LBD (0.8 µM), Alexa Fluor 647 labeled SRC2–3 (7 nM), and LG190178 (2 µM); TRβ-LBD (1 µM), Alexa Fluor 647 labeled SRC2– 2 (7 nM), and triiodothyronine (10 nM); PPARγ-LBD (5 µM), Alexa Fluor 647 labeled DRIP2 (7 nM), and rosiglitazone (5 µM); AR-LBD (5 µM), Alexa Fluor 647 labeled SRC2–3 (7 nM), and dihydrotestosterone (10 nM) were incubated with GW0742 for 2h.

Figure 5

Agonistic and antagonist effect of GW0742 with respect to PPARγ. A FP assay using PPARγ-LBD and Alexa Fluor 647-labeled DRIP2; B One-hybrid transcription assay employing PPARγ.

Figure 6

Inhibition of the VDR–SRC2 interaction by GW0742 analyzed by a Western pull-down assay.

Figure 7

Gene regulation by GW0742 (20 µM, or 50 µM for H) in LNCaP cells after 18 hours (or as indicated) in the presence and absence of NR ligands. A and B DHT (10 nM); C Rosiglitazone (5 µM); D Triiodothyronine (10 nM); E-H 1,25(OH)₂D₃ (10 nM). Standard errors of mean were calculated from four biological independent experiments performed in triplicates. Stars represent P<0.05 (*), P<0.01 (**), P<0.001 (***) (Student’s t-test).

Figure 8

Inhibition of differentiation of HL-60 cells induced by 1,25(OH)₂D₃ (50 nM) in the presence of GW0742.