Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs–s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs– with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs– showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs– activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs–, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs– in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.


SUPPLEMENTARY MATERIALS Supplementary
34 49 39 43 a Determined by HPLC using the same conditions as those for compound purity analyses (see below). After 46 h, the reactions were worked up, and DIM-Ph-4-X + OMss were isolated in 36% and 32% yields, respectively.    Table 4.  Table 1 were determined by interpolation of these curves. were transfected with either siRNA targeting Nur77 (NR4A1) or control siRNA as described in the methods. Knockdown efficiency and specificity was analyzed by immunofluorescence staining with NR4A1 XP ® monoclonal antibody (Cell Signaling Technologies) as described in the methods. Immunofluorescence staining demonstrated that the NR4A1 XP ® monoclonal antibody specifically recognizes NR4A1. Immunofluorescence results were confirmed with another antibody (NR4A1 polyclonal antibody, Sigma). (B) The same tests performed in (A) were done in MCF7 cells. (C) The same tests performed in (A) were done in LNCaP-SKP2 cells. (D) NR4A1 knockdown efficiency in LNCaP-SKP2 cells was determined by immunoblotting with NR4A1 XP ® monoclonal antibody. Actin is shown for reference. (E) Nuclear and cytoplasmic fractions from LNCaP-SKP2 and MCF7 cells were analyzed by immunoblotting with the NR4A1 XP ® monoclonal antibody. Signals obtained with nuclear marker PARP and cytoplasmic tubulin are shown for reference. (F) 293T cells were transfected with either siRNA targeting Nur77 (NR4A1), myc-TR3 plasmid DNA that overexpresses TR3 (NR4A1) or both. NR4A1 XP ® monoclonal antibody (Cell Signaling Technologies) specificity was further validated through immunofluorescence staining and analysis.

Compound purity analyses
Reversed-phase HPLC analysis of target compounds in Supplementary Table 2 was performed on a Shimadzu 20A HPLC system using elution from a 4.6-mm × 150mm TITAN C 18 column (5-μm particle size), with the UV absorbance detection at 254 nm, and the mixed A/B solvent system described below at a flow rate of 1.0 mL/min:

DIM-Ph-4-CF 3 + docks to NR4A1 LBD structure 3V3Q
Although the pose in Figure S6A shows that the 4-CF 3 -Ph group and benzo portions of the indole rings of the cation are more exposed on the LBD surface, a partial π-π stacking between its phenyl ring and that of the H5 Tyr122 and two F···HO interactions between its Fs and the Tyr122 OH are observed. One indole NH could H-bond with the H5 Arg119 C = O and the other indole NH could H-bond with the H1 Pro46 C = O. The pose in Figure  S6B suggests a H-bond between one F of the cation and the helix H12 Phe261 NH and hydrophobic interactions between its phenyl ring and H4 Phe112 and Leu113, H11 Leu239 and H12 Ile260 side-chains. One indole NH of the cation could H-bond with the H5 Glu114 CO 2 H or the H4 Ser110 OH.

Docking of Csn-B to the apo-NR4A1 LBD (PDB 2QW4)
Our docked pose for Csn-B showed that its 2-octanoyl side chain could extend into the interior of the allosteric pocket to form van der Waals contacts with several pocket residues ( Figure 3E). Its 3-OH and 5-OH groups could form H-bonds with the loop L7-8 Val167 backbone C = O and the helix H7 His163 backbone C = O and N, respectively. A partial π-π stacking interaction could occur between the phenyl rings of Csn-B and the H5 Tyr122.

Supplemental synthetic methods and target compound characterizations General methods
Chemicals and solvents were obtained from commercial sources and used without purification. Unless mentioned, anhydrous and/or oxygen-sensitive reactions were carried out under argon gas. Reactions were monitored by thin-layer chromatography on silica gel (mesh size 60, F 254 ) with visualization under UV light. Unless otherwise specified, the standard workup involved washing the organic extract with water and brine and then drying it over anhydrous sodium sulfate followed by filtration and concentration under reduced pressure. Chromatography refers to flash column chromatography on silica gel (Merck 60, 230-400 mesh). Most experimental procedures were not optimized. Melting points of compounds were determined in capillary tubes using a Mel-Temp II apparatus and were not corrected. Infrared spectra of powdered or liquid samples were obtained using an FT-IR Mason satellite spectrophotometer. Unless mentioned, 1 H and 13 C NMR spectra were obtained on compounds dissolved in CDCl 3 or the specified solvent using a 300-MHz Varian Unity Inova or a 400-MHz ECS Jeol spectrometer. Proton chemical shifts are expressed in ppm (δ) relative to CHCl 3 as the internal standard. High-resolution mass spectra were determined using an AgilentTechnologies 6224A accurate mass TOF LC/MS system at Sanford-Burnham Medical Research Institute (Lake Nona, FL). A Shimadzu HPLC system was used to analyze the oxidation rate of DIM-Ph-4-CF 3 to DIM-Ph-4-CF 3 + OMs -(Supplementary Table 1) and the purity of target molecules (Supplementary Table  2). The purity of compounds used in biological assays was ≥ 95% as determined by HPLC. Compound nomenclature used in these Methods follows that currently employed by Chemical Abstracts.
The mixture was stirred for 22-52 h, diluted with Et 2 O (100 mL) and filtered through a short pad of Celite ® (Et 2 O rinse). The filtrate was concentrated at reduced pressure, and the residue was purified by chromatography to give the 4-substituted di(1H-indol-3-yl)methylbenzene (DIM-Ph-4-X).

Differential scanning calorimetry
Experiments were performed at a scanning rate of 1 K/min under 3.0 atm of pressure using an N-DSC II differential scanning calorimeter (Calorimetry Sciences Corp.). Samples contained NR4A1 LBD (20 µM, 0.51 mg/ml) alone or with compound (30 or 60 µM), and 5% DMSO in PBS, pH 7.4. The reference contained 5% DMSO in PBS. Data were analyzed using the NanoAnalyse software package (TA Instruments). Melting temperature (T m ) corresponds to the maximum thermal transition temperature. Calorimetric enthalpy (ΔH) was calculated as the area under the excess heat capacity function (C p ).

Molecular modeling
Docking of the cation (DIM-Ph-4-CF 3 + ) of DIM-Ph-4-CF 3 + OMsinto the pocket corresponding to allosteric site 1 in the crystal structure of the human NR4A1 (TR3) LBD protein (PDB 2QW4) [43] employed BioMed Cache vs. 6.2 software, which is no longer supported by Fujitsu Limited, and our previously described methods [44]. Briefly, the LBP was derived by selecting all neighboring residues within a 10-Å radius to ensure that all residues were encompassed. In the docking process, the energyminimized pose of Csn-B was kept rigid to maintain the geometry used for docking by Wu and colleagues [45], whereas the carbocation was allowed to be flexible. In both cases, the side chains of pocket residues were allowed to be flexible. The docked poses for Csn-B and DIM-Ph-4-CF 3 + ( Figure 3E) were analyzed by measuring inter-atom distances after superposing the helical backbones of the docked NR4A1 LBD poses.
Docking of DIM-Ph-4-CF 3 + into allosteric sites 1 and 2 on the structure (PDB 3V3Q) [11] of the complex of the NR4A1 LBD with two molecules of the Csn-B analogue (structure in Figure S1) used the GOLD docking engine in the Discovery Studio 3.5 package (Accelrys Inc.). The LBP for each site was derived by selecting all neighboring residues within an 8-Å radius of the analogue [11]. The geometry of DIM-Ph-4-CF 3 + was optimized before docking and during docking was allowed to be flexible, whereas the side chains of pocket residues were kept rigid. Possible interactions between DIM-Ph-4-CF 3 + and the residues lining both sites were analyzed and are shown in Figure S6.

Cancer cell line characteristics
HCT-116 colorectal carcinoma cells are poorly differentiated, express a constitutively active mutant p21ras (Ki-ras) proto-oncogene [13] and a mutant β-catenin, which is not inhibited by wild-type APC [14], and over-express c-myc [13]. This cell line also expresses wild-type p53, PPARγ and NR4A1 [12]. Of the breast cancer lines, MCF-7 expresses estrogen receptors and its growth is estrogen-dependent [15,18], whereas MDA-MB-231 does not express estrogen receptors and its growth is estrogen-independent [18]. Wild-type tumor suppressor p53 and mutant nonfunctional p53 are expressed in MCF-7 and MDA-MB-231 cells, respectively [26]. NR4A1 is expressed in MCF-7 cells [1], but not in MDA-MB-231 cells [1], and PPARγ is expressed in both lines [17,18]. Of the prostate cancer cell lines, LNCaP cells are androgen-dependent and express the mutant androgen receptor (AR) (Thr877Ala), which is activated by both the androgen dihydrotestosterone (DHT) and the anti-androgen hydroxyflutamide [25]. They also express low levels of PPARγ1 [24] and after treatment with the PPARγ ligand 15d-Pg J 2 express modest levels of PPARγ2 [24]. The calcium ionophore A23187, anti-cancer drug etoposide and androgen induce the expression of NR4A1 [27]. LAPC-4 cells are androgen-dependent and express wild-type AR [23,28]. 22Rv1 cells are reported to grow independently of androgen in vivo and to express both a DHT and hydroxyflutamide-activated full-length AR(His874Tyr) mutant [25] and a truncated 80-KDa AR mutant [29]. They also express NR4A1 [24], but not PPARγ [24]. The expression of NR4A1 and PPARγ in LAPC-4 cells [23] has not been reported. The growth of PC-3 cells, which are AR null, is androgen independent [25]. They weakly express PPARγ1 [25] and after treatment with 15d-Pg J 2 robustly express PPARγ2 [25], A23187 or etoposide treatment induces their expression of NR4A1 [27]. PC-3 cells do not express p53 [26], whereas the other three lines do [26]. The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) enzyme is reported to antagonize AR transactivation and Akt-PI3K signaling, and its loss is associated with advanced prostate cancer [28]. PTEN is expressed and functional in LACP-4 [28] and 22Rv1 [28,29] cells, although in the former its expression is reported to be low [29], PTEN is absent in LNCaP and PC-3 cells [28].