Identification of a New RXRα Antagonist Targeting the Coregulator-Binding Site

Retinoid X receptor-alpha (RXRα) is implicated in the regulation of many biological processes and also represents a unique intracellular target for pharmacologic interventions. Efforts on discovery of small molecules targeting RXRα have been primarily focused on the molecules that bind to its classical ligand-binding pocket (LBP). Here, we report the identification and characterization of a new RXRα transcriptional antagonist by using structure-based virtual screening. The new antagonist binds with submicromolar affinity to RXRα (Kd = 4.88 × 10–7 M) and selectively inhibits RXRα transactivation. The compound does not bind to the LBP but to a hydrophobic groove on the surface of RXRα. The new compound also effectively suppresses AKT activation and promotes apoptosis of cancer cells in a RXRα-dependent manner by inhibiting tRXRα interaction with the p85α subunit of PI3K. Thus, the compound represents a new RXRα modulator that regulates the nongenomic actions of RXRα by surface binding.

with exponential growth were used throughout the experiments. Transient transfection was performed using TurboFect according to the instructions of the manufacturer in regular growth medium.

Virtual screening
To undertake the virtual screening for the identification of compounds that bind to the protein-protein interaction groove on the surface of RXRα, we used the structure of RXRα LBD in complex with CD3254 and a coactivator peptide (PDB code, 3FUG). A chemical library of 200,000 compounds that is commercially available from Specs (www.specs.net) was downloaded to use as the initial screening library. A Pipeline Pilot protocol was used to filter out compounds that failed the Lipinski rules and that are potentially reactive and contain undesired groups. This exercise left us with about 102,000 compounds for docking-based VS. Schrodinger's Ligprep program was used to prepare the compounds for docking, including 3D structure generation, tautomer and protonation enumeration. Docking was performed using the Glide docking package from Schrodinger. 14 compounds were selected for purchase and biological testing after visual evaluation of the first 300 compounds with the best docking score.

Native-PAGE for the separation of RXR homodimer and tetramer
The native-PAGE was carried out by the same method as SDS-PAGE with 5% stacking gel and 8% resolving gel. But SDS was removed from both gel buffer and running buffer. MTT assays Confluent cells cultured in 96-well dishes were treated with various concentrations of compounds for 48 hr. The cells were then incubated with 2 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) for 4 hr at 37℃. MTT solution was then aspirated, and formazan in cells was instantly dissolved by addition of 150 ml of DMSO each well. Absorbance was measured at 570 nm.

SPR measurements
The binding kinetics between RXRα-LBD and 23 was analyzed at 25 o C on a BIAcore T200 machine with CM5 chips (GE Healthcare). PBSP was used for all measurements. For SPR measurements, RXRα-LBD were purified. We used the blank channel as negative control. About 1,0000 response units of RXRα-LBD were immobilized on the chip. When the data collection was finished in each cycle, the sensor surface was regenerated with Glycine-HCl 2.5. A serial of concentrations ranging from 0.5 to 10 µM were designed for the experiment. Sensograms were fit globally with BIAcore T200 analysis using 1:1 Langumuir binding mode. siRNA and transfections siRNAs against RXRα (SASI-HS01-00097639) and control siRNA (SIC001) used were from Sigma. 5 µL aliquot of 20 µM siRNA/well was transfected into cells in six-well plates using DharmaFECT 1 transfection reagent, according to the manufacturer's recommendations. Western blotting Cell lysates were prepared by lysing cells with lysis buffer containing 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, pH 7.4 with a cocktail of proteinase inhibitors on ice for 30 min. Equal amounts of the lysates were electrophoresed on an SDS-PAGE gel (8 or 12%) and transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% nonfat milk in TBST [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] for 1 hr, incubated with various primary antibodies overnight at 4°C and incubated with secondary antibodies for 1 hr at room temperature. Immunoreactive products were detected by chemiluminescence with an enhanced chemiluminescence system.

Co-immunoprecipitation (Co-IP) assays
For Co-IP assay, cells grown in 10-cm dishes were transfected with various plasmids for 24 hr. After transfection, Cell lysates were incubated with the appropriate antibody for 2 hr, and subsequently incubated with protein A-Sepharose beads for 2 hr. The protein-antibody complexes recovered on beads were subjected to western blotting using appropriate antibodies after separation by SDSpolyacrylamide gel electrophoresis.

Statistical analysis
Data were expressed as mean±SD. Each assay was repeated in triplicate in three independent experiments. Statistical significance of differences between groups was analyzed by using Student's t test or ANOVA analysis. p<0.05 was considered significant.

Supplemental Figures
Supplemental Figure 1A. Chemical structures of the 14 selected compounds. Compound 7 is highlighted. Figure 1B. Identification of RXRα-selective antagonist 7. MCF-7 cells transfected with the reporter plasmid pG5-Luc and pBind-RXRα-LBD were treated with 9-cis-RA (10 -7 M) alone or together with a compound (10 μM for compounds from Specs and 1 μM for BI1003 as "B") for 18 hr. Cells were lysed and reporter activities measured by using the Dual-Luciferase Reporter Assay System. Transfection efficiency was normalized to Renilla luciferase activity. Data for 5 (Compounds 1, 2, 3, 5 and 8) of the 14 compounds are not available due to solubility issues. Figure 2. Activation of nuclear receptors by compounds 7 and 23. MCF-7 cells cotransfected with pG5-Luc and pBind-ERα-LBD, pBind-GRα-LBD, pBind-RARα-LBD, pBind-RARγ-LBD, pBind-PPARγ-LBD, pBind-LXRα-LBD, pBind-RXRα-LBD and pBind-Nur77 were treated respectively with their specific agonists (as indicated), in the presence or absence of compounds for 18 hr. Reporter activities were measured as described above. Twenty-four hours after transfection, the medium was replaced by medium containing a respective ligand and/or a testing compound. After 18 hours, cells were washed, lysed and assayed by using the Dual-Luciferase Reporter Assay System. Transfection efficiency was normalized to Renilla luciferase activity.
Supplemental Figure 5. Mutation of Val298 impairs the antagonist effect of 23. HEK293T cells cotransfected with the reporter plasmid pG5-Luc and pBind-RXRα-LBD or pBind-RXRα-LBD-V298S were treated with 9-cis-RA (10 -7 M) alone or together with 23 for 18 hr. Cells were lysed and reporter activities measured by using the Dual-Luciferase Reporter Assay System. Transfection efficiency was normalized to Renilla luciferase activity.
Supplemental Figure 6. Expression of RXRα and mutant proteins in HEK-293T cells. HEK293T cells grown in 10-cm dishes were transfected with various plasmids for 24 hr. Lysates prepared were analyzed by Western blotting for RXRα and mutant expression.

WB: RXRα
Supplemental Figure 7. Inhibition of AKT activation by 23 in PC-3 prostate cancer cells. PC-3 cell line were pre-treated with 23 for 24 hr before exposed to TNFα (20 ng/ml) for additional 30 min. Lysates prepared were analyzed by Western blotting for AKT activation.
Supplemental Figure 8. Growth inhibitory effect of 23 can be significantly enhanced by tRXRα. Confluent MCF-7 (WT and tRXRα stable) cells cultured in 96-well dishes were treated with various concentrations of compounds for 48 hr. The cells were then incubated with 2 mg/ml 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 hr at 37℃. MTT solution was then aspirated, and formazan in cells was instantly dissolved by addition of 150 ml of DMSO each well. Absorbance was measured at 570 nm.