Anticancer Agents

Abstract

In an embodiment, the disclosure relates to a method for treating a disease by inducing activity in a cell. Typically, the method comprises administering a bisindole derived compound to a subject in need thereof. In some embodiments, the method further comprises binding to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2) by the bisindole derived compound. In another embodiment, the disclosure relates to a compound for treating a disease by inducing activity in a cell. Typically, the compound comprises a bisindole derived compound. In some embodiments, the bisindole derived compound binds to at least one of NR4A1 and NR4A2.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to U.S. Provisional Application No. 62 / 880,801, filed on July 31, 2019, and the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates generally to anticancer agents, and more particularly, but not by way of limitation, to NR4A2 ligands as anticancer agents. Background Art

[0004] This section provides background information to facilitate a better understanding of the various aspects of the present disclosure. It should be understood that the statements in this section of this document should be read in this light, and not as admissions of prior art.

[0005] Nuclear receptor 4A2 (NR4A2) is an orphan nuclear receptor that is expressed in many cell types, induced under stress and overexpressed in tumors. Initially, the bisindole-derived compound 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl) and its p-bromo analog (DIM-C-pPhBr) were identified as NR4A2 antagonists, and these compounds were shown to induce vasoactive intestinal peptide (VIP) in pancreatic cancer cells. Using this assay, the present disclosure identifies other bisindole-derived compounds as inducers of VIP in pancreatic cancer cells and identifies a "second generation" NR4A2 ligand set. In addition, it has been identified that certain patient-derived glioblastoma cells express NR4A2, and that NR4A2 is pro-oncogenic and is inhibited by bisindole-derived NR4A2 ligands.

[0006] There are currently no drugs available for targeting NR4A2 in cancer or other NR4A2-dependent anti-inflammatory diseases, and the bisindole-derived compounds disclosed herein will be unique and very suitable for targeting NR4A2 in cancer. Therefore, the present disclosure describes bisindole-derived ligands and their role as NR4A2 antagonists, which exhibit broad-spectrum anticancer activity. The bisindole-derived NR4A2 ligands disclosed herein can be used to develop anticancer drugs targeting NR4A2, which is highly beneficial for glioblastoma patients who currently have a poor survival prognosis. Summary of the invention

[0007] This Summary is provided to introduce selected concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0008] In embodiments, the disclosure relates to methods for treating diseases by inducing activity in cells. Typically, the method comprises administering a bisindole derived compound to a subject in need thereof. In some embodiments, the method further comprises binding to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2) by the bisindole derived compound. In some embodiments, the activity induced in the cell is at least one of anticancer activity and anti-inflammatory activity. In some embodiments, the bisindole derived compound (CDIM) comprises two or more substituents on its benzene ring. In some embodiments, the bisindole derived compounds include but are not limited to 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl; 4-Cl), 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), 1,1-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (N-Me-4-OH), 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br), 1-bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr), 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), and 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8). Substituted analogs CDIM8-3,5-(CH3)2, CDIM8-3,5-Br2, CDIM8-3,5-Cl2, CDIM8-3-Br-5-OCH3, CDIM8-3-Cl-5-OCH3, CDIM8-3-Cl-5-Br, CDIM8-3-Cl-5-F, CDIM, 3,5-disubstituted analogs of CDIM CDIM-3,5-Br2, CDIM-2,5-Br2, CDIM-3,5-Cl2, CDIM-3,5-(CH3)2, CDIM-3-Br-5-OCF3, CDIM-3-Br-5-OCH3, CDIM-3-Cl-5-OCH3, CDIM-3-Cl-5-OCF3, CDIM-3-Cl-5-CF3 and combinations thereof.

[0009] In some embodiments, the bisindole derived compound exerts a function on the cell, including but not limited to inducing NR4A1-dependent transactivation in the cell, inducing NR4A2-dependent transactivation in the cell, inhibiting the growth of the cell, inducing apoptosis in the cell, inhibiting the survival of the cell, inhibiting the migration of the cell, and combinations thereof. In some embodiments, the cells include but are not limited to A172, U87-MG, U98G, CCF-STTG1, 1708, 15037, 14004s, 14015s, 15049, glioblastoma multiforme (GBM) cells, and combinations thereof. In some embodiments, the bisindole derived compound is at least one of a bisindole derived NR4A1 ligand and a bisindole derived NR4A2 ligand. In some embodiments, at least one of the bisindole-derived NR4A1 ligand and the bisindole-derived NR4A2 ligand functions, including but not limited to antagonizing NR4A1 in the cancer cell, targeting NR4A1 in the cancer cell, antagonizing NR4A2 in the cancer cell, targeting NR4A2 in the cancer cell, and combinations thereof. In some embodiments, the cell comprises at least one of NR4A1 and NR4A2 in the cancer cell. In some embodiments, the cancer cell corresponds to a cancer, including but not limited to brain cancer, breast cancer, kidney cancer, colon cancer, pancreatic cancer, lung cancer, and combinations thereof. In some embodiments, the disease includes but is not limited to cancer, brain cancer, breast cancer, kidney cancer, colon cancer, pancreatic cancer, lung cancer, inflammatory diseases, asthma, chronic peptic ulcer, tuberculosis, rheumatoid arthritis, periodontitis, ulcerative colitis, Crohn's disease, sinusitis, active hepatitis, and combinations thereof.

[0010] In other embodiments, the disclosure relates to methods of inducing anticancer activity in tumors. Generally, the method comprises administering a bisindole derived compound to a subject in need thereof. In some embodiments, the method further comprises binding to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2) via the bisindole derived compound. In some embodiments, the bisindole derived compound (CDIM) comprises two or more substituents on its phenyl ring. In some embodiments, the bisindole derived compounds include but are not limited to 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl; 4-Cl), 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), 1,1-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (N-Me-4-OH), 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br), 1-bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr), 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), and 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8). Substituted analogs CDIM8-3,5-(CH3)2, CDIM8-3,5-Br2, CDIM8-3,5-Cl2, CDIM8-3-Br-5-OCH3, CDIM8-3-Cl-5-OCH3, CDIM8-3-Cl-5-Br, CDIM8-3-Cl-5-F, CDIM, 3,5-disubstituted analogs of CDIM CDIM-3,5-Br2, CDIM-2,5-Br2, CDIM-3,5-Cl2, CDIM-3,5-(CH3)2, CDIM-3-Br-5-OCF3, CDIM-3-Br-5-OCH3, CDIM-3-Cl-5-OCH3, CDIM-3-Cl-5-OCF3, CDIM-3-Cl-5-CF3 and combinations thereof.

[0011] In some embodiments, the bisindole derived compound exerts a function on the tumor cells, including but not limited to inducing NR4A1-dependent transactivation in the cells, inducing NR4A2-dependent transactivation in the tumor cells, inhibiting the growth of the tumor cells, inducing apoptosis in the tumor cells, inhibiting the survival of the tumor cells, inhibiting the migration of the tumor cells, and combinations thereof. In some embodiments, the tumor includes cells, including but not limited to A172, U87-MG, U98G, CCF-STTG1, 1708, 15037, 14004s, 14015s, 15049, glioblastoma multiforme (GBM) cells, and combinations thereof. In some embodiments, the bisindole derived compound is at least one of a bisindole derived NR4A1 ligand and a bisindole derived NR4A2 ligand. In some embodiments, at least one of the bisindole-derived NR4A1 ligand and the bisindole-derived NR4A2 ligand functions, including but not limited to, antagonizing NR4A1 in the tumor cells, targeting NR4A1 in the tumor cells, antagonizing NR4A2 in the tumor cells, targeting NR4A2 in the tumor cells, and combinations thereof. In some embodiments, the cells of the tumor are cancerous. In some embodiments, the tumors include but are not limited to brain tumors, breast tumors, kidney tumors, colon tumors, pancreatic tumors, lung tumors, and combinations thereof.

[0012] In further embodiments, the present disclosure relates to methods of inducing anticancer activity in glioblastoma multiforme (GBM) cells. Typically, the method comprises administering a bisindole derived compound to a subject in need thereof. In some embodiments, the method further comprises binding to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2) via the bisindole derived compound. In some embodiments, the bisindole derived compound (CDIM) comprises two or more substituents on its phenyl ring. In some embodiments, the bisindole derived compounds include but are not limited to 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl; 4-Cl), 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), 1,1-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (N-Me-4-OH), 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br), 1-bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr), 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), and 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8). Substituted analogs CDIM8-3,5-(CH3)2, CDIM8-3,5-Br2, CDIM8-3,5-Cl2, CDIM8-3-Br-5-OCH3, CDIM8-3-Cl-5-OCH3, CDIM8-3-Cl-5-Br, CDIM8-3-Cl-5-F, CDIM, 3,5-disubstituted analogs of CDIM CDIM-3,5-Br2, CDIM-2,5-Br2, CDIM-3,5-Cl2, CDIM-3,5-(CH3)2, CDIM-3-Br-5-OCF3, CDIM-3-Br-5-OCH3, CDIM-3-Cl-5-OCH3, CDIM-3-Cl-5-OCF3, CDIM-3-Cl-5-CF3 and combinations thereof.

[0013] In some embodiments, the bisindole-derived compound functions on the GBM cells, including but not limited to inducing NR4A1-dependent transactivation in the GBM cells, inducing NR4A2-dependent transactivation in the GBM cells, inhibiting the growth of the GBM cells, inducing apoptosis in the GBM cells, inhibiting the survival of the GBM cells, inhibiting the migration of the GBM cells, and combinations thereof. In some embodiments, the GBM cells include but are not limited to A172, U87-MG, U98G, CCF-STTG1, and combinations thereof. In some embodiments, the bisindole-derived compound is at least one of a bisindole-derived NR4A1 ligand and a bisindole-derived NR4A2 ligand. In some embodiments, at least one of the bisindole-derived NR4A1 ligand and the bisindole-derived NR4A2 ligand functions, including but not limited to antagonizing NR4A1 in the GBM cells, targeting NR4A1 in the GBM cells, antagonizing NR4A2 in the GBM cells, targeting NR4A2 in the GBM cells, and combinations thereof. In some embodiments, the GBM cells are cancerous cells. In some embodiments, the cancerous cells include, but are not limited to, brain cancer cells, breast cancer cells, kidney cancer cells, colon cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof.

[0014] In another embodiment, the disclosure relates to compounds for treating diseases by inducing activity in cells. Typically, the compounds include bisindole derived compounds. In some embodiments, the bisindole derived compounds bind to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2). In some embodiments, the activity induced in the cells is at least one of anticancer activity and anti-inflammatory activity. In some embodiments, the bisindole derived compound (CDIM) comprises two or more substituents on its phenyl ring. In some embodiments, the bisindole derived compounds include but are not limited to 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl; 4-Cl), 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), 1,1-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (N-Me-4-OH), 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br), 1-bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr), 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), and 3,5-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8). Substituted analogs CDIM8-3,5-(CH3)2, CDIM8-3,5-Br2, CDIM8-3,5-Cl2, CDIM8-3-Br-5-OCH3, CDIM8-3-Cl-5-OCH3, CDIM8-3-Cl-5-Br, CDIM8-3-Cl-5-F, CDIM, 3,5-disubstituted analogs of CDIM CDIM-3,5-Br2, CDIM-2,5-Br2, CDIM-3,5-Cl2, CDIM-3,5-(CH3)2, CDIM-3-Br-5-OCF3, CDIM-3-Br-5-OCH3, CDIM-3-Cl-5-OCH3, CDIM-3-Cl-5-OCF3, CDIM-3-Cl-5-CF3 and combinations thereof.

[0015] In some embodiments, the bisindole derived compound functions, including but not limited to inducing NR4A1-dependent transactivation in cells, inducing NR4A2-dependent transactivation in cells, inhibiting cell growth, inducing apoptosis in cells, inhibiting cell survival, inhibiting cell migration, and combinations thereof. In some embodiments, the cells include but are not limited to A172, U87-MG, U98G, CCF-STTG1, 1708, 15037, 14004s, 14015s, 15049, glioblastoma multiforme (GBM) cells, and combinations thereof. In some embodiments, the cells include at least one of NRA1 and NR4A2 in the cells. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells include but are not limited to brain cancer cells, breast cancer cells, renal cancer cells, colon cancer cells, pancreatic cancer cells, lung cancer cells, and combinations thereof. In some embodiments, the bisindole derived compound is at least one of a bisindole derived NR4A1 ligand and a bisindole derived NR4A2 ligand. In some embodiments, at least one of the bisindole-derived NR4A1 ligand and the bisindole-derived NR4A2 ligand performs functions including but not limited to antagonizing NR4A1, targeting NR4A1, antagonizing NR4A2, targeting NR4A2, and combinations thereof. In some embodiments, the diseases include but are not limited to cancer, brain cancer, breast cancer, kidney cancer, colon cancer, pancreatic cancer, lung cancer, inflammatory diseases, asthma, chronic peptic ulcer, tuberculosis, rheumatoid arthritis, periodontitis, ulcerative colitis, Crohn's disease, sinusitis, active hepatitis, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] A more complete understanding of the subject matter of the present disclosure may be obtained by referring to the following detailed description when taken in conjunction with the accompanying drawings, in which:

[0017] Figures 1A-1D To illustrate the effects of bisindole analogs and quinoline derivatives on vasoactive intestinal polypeptide (VIP) gene expression;

[0018] Figures 2A-2C To describe the expression and function of nuclear receptor 4A (NR4A) 2 in glioblastoma cells;

[0019] Figures 3A-3C Describe the expression and function of NR4A3;

[0020] Figures 4A-4C Indicates the NR4A2 ligand-dependent effect on transactivation;

[0021] Figures 5A-5D illustrate the responses induced by NR4A2 antagonists;

[0022] Figures 6A-6CIt is shown that NR4A2 antagonists induce apoptosis in glioblastoma cells;

[0023] Figure 7A-7B It is shown that NR4A2 antagonists inhibit migration / invasion and glioblastoma tumor growth;

[0024] Figure 8 The binding of 2,4-dichlorophenyl analogs to NR4A1 and NR4A2 is illustrated;

[0025] Fig. 9 The binding of 3-chloro-5-methoxyphenyl analogs to NR4A1 and NR4A2 is illustrated;

[0026] Fig.10 Description of the prototypical NR4A2 ligand. DETAILED DESCRIPTION

[0027] It should be understood that the following disclosure provides many different embodiments or examples for implementing the different features of various embodiments. Specific examples of components and arrangements are described below to simplify the disclosure. Of course, these are merely examples and are not intended to be limiting. The section headings used herein are for organizational purposes and should not be construed as limiting the subject matter described.

[0028] The orphan nuclear receptor 4A (NR4A1) family contains three receptors, NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (Nor1), which show significant structural similarities in the ligand binding domain (LBD) and DNA BD, while their N-terminal (A / B) domains containing the activation function 1 (AF1) are highly divergent. The initial discovery of the NR4A receptors was associated with their rapid induction by multiple stimuli in various tissues / cells and organs. These responses play an important role in coping with exogenous and endogenous stressors and in tissue-specific expression and induction of NR4A receptors, which contributes to the specificity of the receptors. Ongoing studies have identified multiple roles for NR4A receptors in maintaining cellular homeostasis and pathophysiology, including but not limited to cancer. Preliminary studies in knockout mouse models have shown that the combined loss of NR4A1 and NR4A3 leads to the development of acute myeloid leukemia in mice, suggesting tumor suppressor-like activity of these receptors against leukemia. In contrast, there is a large body of evidence showing that NR4A1 is highly expressed in most solid tumors, and that overexpression of NR4A1 in tumors from lung, colon, and breast cancer patients is an unfavorable prognostic factor, whereas little is known about the functions of NR4A2 and NR4A3 in solid tumors. Ongoing studies of breast, kidney, colon, pancreatic, and lung cancers, as well as rhabdomyosarcoma (RMS) cells, show that NR4A1 plays an important role in these responses by regulating genes that drive cancer cell growth, survival, and migration / invasion. In addition, recent studies have shown that transforming growth factor β (TGFβ)-induced invasion of breast and lung cancer cells is also NR4A1-dependent and is due to nuclear export of the receptor that promotes proteasome-dependent degradation of SMAD7.

[0029] The role of NR4A2 in cancer and the role of synthetic NR4A2 ligands are not well defined, although most available data suggest that, similar to NR4A1, NR4A2 is also pro-oncogenic in most cancer cell lines. Furthermore, in many of these tumors, NR4A2 is a negative prognostic factor for patient survival, and the overall profile of NR4A2 and NR4A1 is similar across cancer types. Both orphan receptors also bind and inactivate p53.

[0030] NR4A2 has been extensively characterized in subcellular regions of the brain and - / -The mice do not generate midbrain dopaminergic neurons and die shortly after birth. Several laboratories have studied the role of NR4A2 in Parkinson's disease, and studies have demonstrated that the NR4A2 agonist 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane [DIM-C-pPhCl (C-DIM12)] crosses the blood-brain barrier and accumulates in the brain, and in vivo studies have shown that DIM-C-pPhCl inhibits 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons and other markers of neurodegeneration.

[0031] The expression of NR4A receptors and the potential role of ligands of these receptors in glioblastoma and other neuronal tumors have not been studied, although one study showed drug-induced NR4A1 expression in glioblastoma multiforme (GBM) cell lines. Therefore, NR4A expression was initially screened in several established GBM cell lines and five patient-derived GBM cell lines. Western blot analysis of cell lysates showed that four established cell lines expressed NR4A1, NR4A2, and NR4A3; in patient-derived cells, the expression of NR4A1 and NR4A3 was variable, while NR4A2 was highly expressed in all five cell lines. Therefore, glioblastoma cells serve as an ideal model for studying the role of NR4A2 in this tumor and the role of NR4A2 ligands (e.g., DIM-C-pPhCl). The results demonstrate that NR4A2 is proto-oncogenic in glioblastoma and that NR4A2 ligands act as antagonists and therefore represent a new class of chemotherapeutic agents for the treatment of this deadly disease.

[0032] Working Example

[0033] Reference will now be made to more specific embodiments of the present disclosure and data providing support for such embodiments. However, it should be noted that the following disclosure is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0034] Cell lines, antibodies, and reagents1,1-Bis(3'-indolyl)-1-(p-chlorophenyl)methane [DIM-C-pPhCl (C-DIM12)], 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), and 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxyphenyl)methane (2-OH-4-Br) were synthesized in the laboratory. Patient-derived xenografts (PDXs) from human glioma cell lines 17008, 15037, 14104s, 14015s, and 15049 were generated from fresh tumor specimens collected from newly diagnosed patients without prior chemotherapy or radiotherapy treatment. Established human malignant glioma cell lines U87-MG, A172, T98G, and CCF-STTG1 were purchased from the American Type Culture Collection (Manassas, VA). PDX cells were maintained in a 50 / 50 mixture of Dulbecco's Modified Eagle's Medium (DMEM) / Hams F-12 supplemented with L-glutamine, 10% fetal bovine serum (FBS), 1× MEM non-essential amino acids, and 10 μg / ml gentamicin (Gibco, Dublin, Ireland). U87-MG, A172, T98G, and CCF-STTG1 were maintained in DMEM1X supplemented with 10% FBS. All cells were maintained at 37°C in the presence of 5% CO2, and the solvents used in the experiments (dimethyl sulfoxide; DMSO) were ≤0.2%. DMEM, DMEM F-12 50 / 50 mixture, FBS, formaldehyde, and trypsin were purchased from Sigma-Aldrich (St. Louis, MO). Cleaved poly (ADP-ribose) polymerase (cPARP, cat# 9541T), cleaved caspase-8 (cat# 9496T), cleaved caspase-7 (cat# 9491T), anti-rabbit Alexa Fluor 488 conjugate (cat# 4412s), and anti-mouse Alexa Fluor 488 conjugate (cat# 4408s) antibodies were obtained from Cell Signaling (Boston, MA); NR4A1 (cat# ab109180) antibody was purchased from Abcam (Cambridge, MA); NR4A2 (cat# sc-991), Ki67 (sc-23900), and NR4A3 (cat# sc-133840) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and β-actin (cat# A5316) antibody was obtained from Sigma-Aldrich (St. Louis, MO).Chemiluminescent reagents (Immobilon Western) for western blot imaging were purchased from Millipore (Billerica, MA). Apoptosis, necrosis, and healthy cell quantification kits were purchased from Biotium (Hayward, CA), invasion chambers (cat # 354480) were purchased from Corning Inc. (Corning, NY), and XTT cell viability kits were obtained from Cell Signaling (Boston, MA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA). Luciferase reagent (cat # E1483) was purchased from Promega (Madison, WI). NR4A2-specific antisense oligonucleotides 3 and 4 were purchased from AUM Biotech (Philadelphia, PA). The siRNA complexes purchased from Sigma-Aldrich and used in the study were as follows: siGL2-5': CGU ACG CGG AAU ACU UCG A (SEQ ID NO: 1), siNR4A1 (SASI_Hs02_00333289), siNR4A2 (SASI_Hs02_00341055), and siNR4A3 (SASI_Hs01_00091655).

[0035] Transactivation assay. Cells (8 × 10 4 ) / well were plated in 12-well plates. After 24 h of growth, different amounts of DNA [i.e., UAS x5 -Luc (400 ng), GAL4-Nurr1 (40 ng) and β-gal (40 ng)] were co-transfected into each well. 5-6 hours after transfection, cells were treated with plating medium containing solvent (DMSO) or the specified concentration of compound (as described above) for 24 hours. The cells were then lysed using a freeze-thaw protocol, and 30 μL of cell extracts were used for luciferase and β-gal assays. LumiCount (Packard, Meriden, CT) was used to quantify luciferase and β-gal activity. Luciferase activity values ​​were standardized for the corresponding β-gal activity values ​​and protein concentrations determined by the Lowry method.

[0036] Cell viability assay.Cells were plated in 96-well plates at a density of 10,000 / well with DMEM and DMEM F-12 50 / 50 containing 2.5% activated carbon-treated FBS. Cells were treated with DMSO (solvent control) and different concentrations of C-DIM 12, 3-CF3-4-Cl and 2-OH-4-Br and DMEM containing 2.5% activated carbon-treated FBS for 0-48 hours. After treatment, 25 μL (XTT with 1% electron coupling solution) were added to each well and incubated for 4 hours as described in the manufacturer's instructions (Cell Signaling, Boston, MA). After incubation for 4 hours at 37°C in 5% CO2, absorbance was measured at 450 nm wavelength in a 96-well plate reader.

[0037] Measurement of apoptosis (Annexin V staining). Cancer cells were collected at a rate of 1.5 × 10 5 Cells were seeded at a density of 1:100 / ml in 6-well plates and treated with vehicle (DMSO) or compound for 24 hours. Cells were then stained and analyzed by flow cytometry using a dead cell apoptosis kit and an Alexa Fluor 488 assay kit according to the manufacturer's protocol (Invitrogen, Carlsbad CA).

[0038] Scratch and invasion assays. In 6-well plates maintained at 80% confluence, scratches were created using sterile pipette tips, and cell migration toward the scratch was determined 24 hours later. BD-Matrigel invasion chambers (24-transwell, polycarbonate membrane with 8 μm pore size) were used for the modified Boyden chamber assay. The culture medium in the lower chamber contained complete culture medium for GBM, which served as a chemoattractant. PDX cells (5×10 4 Cells / insert) were plated in the upper chamber with or without various concentrations of compounds and incubated at 37°C, 5% CO2 for 24 hours; non-invading cells were removed from the upper surface of the membrane with a wet cotton swab. Invading cells were fixed on the lower surface with 10% formalin for 10 minutes and stained in hematoxylin and eosin Y solution (H&E). After washing and drying, the number of cells in five adjacent fields was counted.

[0039] Small interfering RNA interference assay. The cells (2×10 5Cells / well) were plated in 6-well plates in complete medium. After 24 hours, cells were transfected with 100 nM of each siRNA duplex using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol for 6 hours. Antisense oligonucleotides targeting NR4A2 were used directly in 6-well plates and the final concentration was 10 μM. For siRNA-mediated transfection, the culture medium was replaced with fresh culture medium containing 10% FBS, while the culture medium was not replaced for antisense oligonucleotides. Both transfection conditions were incubated for 42 hours. After incubation, cells were treated with vehicle (DMSO) or different concentrations of compounds and collected for further experiments.

[0040] Immunofluorescence. 15037, 14015s and U87-MG cells (1.0×10 5 / ml) were plated in complete medium and treated with DMSO or C-DIM 12 for 24 hours or with siCt or siNR4A2 for 48 hours. The cells were then fixed with 37% formalin, blocked, and treated with fluorescent Ki67 primary antibody for 24 hours. The cells were then washed with PBS and treated with anti-mouse IgG Fab2 Alexa Fluor 488 secondary antibody at room temperature for 2 hours. Finally, the cells were observed with a Zeiss confocal fluorescence microscope.

[0041] Western blot analysis. 17008, 15037, 14104s, 14015s, 15049, U87-MG, A172, T98G and CCF-STTG1 cells were cultured at 1.5 × 10 5 / ml density was seeded in 6-well plates and treated with various concentrations of compounds, and whole-cell proteins were extracted with RIPA lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100 (w / v), 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS), with a mixture of protease and phosphatase inhibitors. Protein concentration was measured using the Lowry method, and equal amounts of protein were separated in 10% and 15% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were incubated overnight at 4°C with the primary antibody in 5% skim milk and incubated for 2-3 hours with the secondary antibody conjugated to horseradish peroxidase (HRP). The membranes were subsequently exposed to HRP-substrate, and immunoreactive proteins were detected with chemiluminescent reagents.

[0042] Three-dimensional (3D) tumor spheroid invasion assay.The cells were suspended in complete culture medium (2 × 10 4 Spheroids were generated by inoculating 200 μl of cell suspension into the wells of a 96-well round-bottom ultra-low attachment culture plate (Costa, #7007). After incubation for 24 hours at 37°C in a 5% CO2 incubator, 100 μl / well of growth medium was removed from the spheroid plate and 100 μl / well of Matrigel (Corning, #356234) was added to the bottom of each well. The plate was transferred to an incubator for 1 hour and supplemented with 100 μl of complete medium containing 3 times the desired final concentration of the compound, and then incubated for 3-5 days and then fixed in 4% formaldehyde. Spheroid invasion was determined by measuring the cross-sectional area of ​​the center and edge of the spheroid of the invaded cells with ImageJ.

[0043] Xenograft studies. Female athymic nu / nu mice (4-6 weeks old) were purchased from Harlan Laboratories (Houston, TX). U87-MG cells (1×10 6 ), and suspended in ice-cold Matrigel (1: 1 ratio), and subcutaneously injected into either side of the flank area of ​​mice. One week after tumor cell inoculation, mice were divided into two groups of 5 animals in each group. The first group received 100 μL vehicle (corn oil), while the second group of animals received intraperitoneal injections of 30 mg / kg / day C-DIM 12 in 100 μL volume corn oil for three weeks. All mice were weighed once a week during the treatment to monitor changes in body weight. Tumor volume could not be determined during treatment because xenografted tumors were relatively deep. After three weeks of treatment, mice were killed and tumor weights were determined.

[0044] Statistical analysis. One-way ANOVA and Dunnett's test were used to determine the statistical significance between the two groups. In order to confirm the reproducibility of the data, the experiment was performed at least three times independently, and the results were expressed as mean ± standard deviation (SD). P values ​​less than 0.05 were considered statistically significant.

[0045] Structure-activity studies. In the structure-activity study, two quinoline derivatives, chloroquine (CQ) and amodiaquine (AQ), were used as "control" NR4A2 ligands, which have been previously studied in Parkinson's disease models. The effects of bisindole and quinoline derivatives on the activation of the NR4A2-regulated gene vasoactive intestinal polypeptide (VIP) were investigated. Panc1 ( Figure 1A and Figure 1B ) and Panc28 ( Figure 1C and Figure 1D ) cells. The relative expression level of VIP was determined by quantitative PCR analysis. For each treatment, the results are expressed as the mean ± SD of at least six individual determinations. The asterisk (*) indicates significant gene induction (P < 0.01) of the highest concentration treatment relative to the solvent control (DMSO). Figure 1A The bisindole compounds were shown to significantly induce VIP in Panc1 cells, with up to 330-fold induction observed using 2-OH-4-Br ( Figure 1A Although CQ and AQ induced VIP in Panc1 cells (2.3-4.1-fold), the magnitude of the response was significantly lower than that of the NR4A2-active bisindole compounds ( Figure 1B Differences in VIP induction in Panc28 cells between bisindoles and quinolines (respectively Figure 1C and Figure 1D ) were similar to those observed in Panc1 cells. The least active bisindole compound, N-Me-4-OH, was a more potent inducer of VIP in both Panc1 and Panc28 cells than CQ or AQ. Thus, a new NR4A2 ligand more potent than DIM-C-pPhCl (4-Cl) and DIM-C-pPhBr (4-Br) has been identified.

[0046] Quenching of NR4A1 tryptophan fluorescence by direct ligand binding. Tryptophan fluorescence spectra were obtained as described herein. Briefly, the histidine-tagged ligand binding domain (LBD) of NR4A1 was used for fluorescence measurements at a final concentration of 0.5 μmol / L in 1.0 mL phosphate-buffered saline (PBS, pH 7.4). The protein was incubated for 3 minutes at 25°C in a temperature-controlled (Quantum NorthwestTC125) fluorescence spectrophotometer (Varian Cary Eclipse). Fluorescence spectra were obtained using an excitation wavelength of 285 nm (excitation slit width = 5 nm) and an emission wavelength in the range of 300-420 nm (emission slit width = 5 nm). An aliquot of the ligand (0.1 μL / aliquot) (10 mmol ligand / L ethanol) was then added to a cuvette containing NR4A1 to a final concentration of 30 μmol ligand / L. After each aliquot of ligand, the NR4A1 / ligand solution was incubated at 25°C for 3 minutes and the NR4A1 tryptophan fluorescence was measured as described above. The addition of ethanol alone (to a final volume of 3.0 μL) had no effect on the NR4A1 tryptophan fluorescence (data not shown). According to the references listed above, the ligand binding affinity (Kd) to NR4A1 was determined by measuring the fluorescence intensity of NR4A1 tryptophan at an emission wavelength of 330 nm. As described in the references above, the fluorescence intensity of the ligand alone at each ligand concentration was used to correct the NR4A1 tryptophan fluorescence intensity.

[0047] Ligand binding to the NR4A1 LBD: bisANS displacement assay. bisANS (Molecular Probes, Inc / ThermoFisher) is essentially non-fluorescent in aqueous solution; however, when bound to a protein (e.g., NR4A1), bisANS fluorescence increases significantly. The binding affinity (Kd) and binding stoichiometry (Bmax) of NR4A1 / bisANS were determined essentially as described in FEBS Journal, 2014, 281, 2266-2283. For NR4A1 / bisANS, the binding affinity (Kd) was determined to be 0.84 μmol / L, and the binding stoichiometry (Bmax) was determined to be 0.80 mol bisANS / mol NR4A1. The ability of the ligand to displace bisANS was analyzed essentially as described in FEBS Journal, 2014, 281, 2266-2283. Briefly, histidine-tagged NR4A1 LBD (final concentration = 0.05 μmol / L PBS, pH 7.4) was incubated for 3 min at 25°C in the presence of 5.0 μmol bisANS / L. Fluorescence spectra of bisANS were acquired using a Varian Cary Eclipse fluorescence spectrophotometer with an excitation wavelength of 365 nm (excitation slit width = 5 nm) and an emission wavelength in the range of 400-600 nm (emission slit width = 5 nm). Ligand titration was performed as described above for direct ligand binding using a stock ligand concentration of 10 mmol ligand / L ethanol. Addition of ethanol alone had no effect on NR4A1 / bisANS fluorescence (data not shown). Ligand binding affinity (Ki) of NR4A1 was determined by measuring NR4A1 / bisANS fluorescence intensity at an emission wavelength of 500 nm according to the above reference. The ligand / bisANS fluorescence intensity at each ligand concentration was used to correct the NR4A1 / bisANS / ligand fluorescence intensity as described in the above references.

[0048] Expression of NR4A receptors in glioblastoma cell lines (A172, U87-MG, U98G, and CCF-STTG1) and patient-derived cells (1708, 15037, 14004s, 14015s, and 15049) was determined by Western blot analysis of whole cell lysates. NR4A2 was expressed in all cell lines, while NR4A3 was expressed in most cell lines, while NR4A1 was detected in established cell lines, but only in two patient-derived cell lines. Therefore, the NR4A2 expression of patient-derived cell lines in the absence of NR4A1 is somewhat unique. In the present disclosure, the role of NR4A2 in U87-MG, 15037, and 14015s cells was studied to determine the effect of NR4A2 knockdown using antisense oligonucleotides (#3 and #4) on cell proliferation, survival, and invasion. NR4A2 knockdown was performed as follows: glioblastoma cells were transfected with oligonucleotides targeting NR4A2 (siNR4A2-#3 and #4) or a nonspecific control (NC), and whole cell lysates were analyzed by Western blot, and the effects on cell proliferation ( Figure 2A ), cell invasion ( Figure 2B ) and Annexin V staining ( Figure 2C ). Cells were transfected with nonspecific control (NC) or oligonucleotides targeting NR4A2 (#3 and #4), and apoptotic markers were determined by Western blotting of whole cell lysates. Results ( Figures 2A-2C ) are mean ± SD of at least three determinations per treatment group, and significant (P < 0.05) effects [compared to control (NC)] are indicated (*). No caspase 8 cleavage was observed in 15037 cells. Antisense oligonucleotides effectively reduced NR4A2 expression in 15037, 14015s and U87-MG cells, and this was accompanied by decreased cell proliferation ( Figure 2A ) and decreased cell invasion as measured using the Boyden chamber ( Figure 2B In addition, NR4A2 expression decreased the induction of apoptotic markers, including induction of Annexin V staining ( Figure 2C ) and cleavage of caspase 8, 7, and PARP in 15037, 14015s, and U87-MG cells (Note: cleaved caspase 8 was not detected in 15037 cells).

[0049] Since 15037, 14015s, and U87-MG cells express NR4A3, the effect of NR4A3 knockdown by RNA interference (RNAi) on the phenotypic characteristics of the cell lines was also investigated. Cells were transfected with NC or oligonucleotides targeting NR4A3, and the effects on NR4A3 expression (determined by Western blotting of whole cell lysates), cell proliferation ( Figure 3A ), cell invasion ( Figure 3B ) and Annexin V staining ( Figure 3C ). Cells were transfected with siNR4A2 or siNR4A3, and Ki67 staining was determined as described herein. Results ( Figures 3A-3C ) are expressed as mean ± SD, each treatment group was determined at least three times, and significant (P < 0.05) differences from the control / untreated group are indicated (*). Figure 3A ), invasion( Figure 3B ) or apoptosis ( Figure 3C ) had minimal effect, and staining for the Ki67 proliferation marker demonstrated that loss of NR4A3 had minimal effect on Ki67 staining. These results clearly demonstrated for the first time that NR4A2 is a proto-oncogenic factor in glioblastoma cells, while NR4A3 has minimal effect on their growth, survival, and invasion.

[0050] Previous studies have identified a series of bisindole-derived compounds (C-DIM) that induce NR4A2-dependent transactivation, and 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl,4-Cl) has been used as a prototype NR4A2 ligand (compound 1). Fig.10 Compound 1 described in shows a prototype NR4A2 ligand. Cells were treated with NR4A2 ligand. Figure 4A )、NBRE-Luc / NR4A2 expression plasmid (40 ng) ( Figure 4B ) and NurRE-Luc / NR4A2 expression plasmid (40 ng) ( Figure 4C ) transfected cells, treated with bisindole derived ligands, and luciferase activity was determined as described herein. Results are mean ± SD of three replicates per treatment group, and significant (P < 0.05) effects (compared to DMSO controls) are indicated (*).

[0051] Compound 1

[0052]

[0053] Ongoing screens in pancreatic cancer cells have identified three additional NR4A2 ligands, including 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), 1,1-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (N-Me-4-OH), and 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br). These compounds induced NR4A2-dependent transactivation in Panc1 cells; however, all of these compounds reduced transactivation (luciferase activity) in 14015s and 15037 glioblastoma cells transfected with a GAL4-NR4A2 chimera and a reporter plasmid containing a GAL4 response element (UAS5-luc). Figure 4A ). Ligand-dependent regulation of NR4A2-regulated gene expression was also investigated using the reported gene construct containing the NGF1-B response element-luciferase construct (NBRE3-luc) ( Figure 4B ) and Nur response element (NuRE3-luc) ( Figure 4C ), which bind NR4A2 as monomers and dimers, respectively. In these assays, the three ligands also reduced NR4A2-dependent transactivation, indicating that they act as NR4A2 inverse agonists / antagonists in glioblastoma cells.

[0054] DIM-C-pPhCl ( Figure 5A )、3-CF3-4-Cl ( Figure 5B ) and 2-OH-4-Br ( Figure 5C ) were used to treat 15037, 14015s and U87-MG cells to inhibit cell proliferation. Figure 5A )、1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane(3-CF3-4-Cl) ( Figure 5B ) and 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxyphenyl)methane (2-OH-4-Br) ( Figure 5C) cells were treated with 4-Cl and the effect on glioblastoma cell proliferation was determined as described herein. Athymic nude mice bearing U87-MG cells as xenografts were treated with 4-Cl (30 mg / kg / day) and the effect of control (corn oil) and 4-Cl treated mice on tumor weight and expression of apoptotic markers in tumors was determined by Western blot analysis of tumor lysates. The expression levels of various proteins in control versus 4-Cl-treated mice were determined (normalized to β-actin). Glioblastoma cells were treated with different NR4A2 antagonists and Ki-67 staining was determined as described herein. Results ( Figures 5A-5C ) are expressed as mean ± SD of at least three replicates per treatment group, and significant (P < 0.05) differences from untreated controls are indicated (*).

[0055] It was observed that 4-Cl (30 mg / kg / day) significantly reduced tumor weight in athymic nude mice bearing U87-MG tumor cells as xenografts ( Figure 5D ), and this was accompanied by a significant upregulation of cleaved caspase 8 but not cleaved caspase 7 and cPARP in tumors from 4-Cl treated mice compared to vehicle controls. Treatment with 4-Cl for 24 hours reduced Ki67 (a proliferation marker) in 15037, 14015s, and U87-MG cells. Thus, NR4A2 ligands and NR4A2 knockdown ( Figures 2A-2C ) were growth inhibitory, indicating that C-DIM is an NR4A2 antagonist, and this is consistent with their antagonist activity in the transactivation assay ( Figures 4A-4C ). Use 4-Cl ( Fig. 6A )、3-CF3-4-Cl ( Figure 6B ) and 2-OH-4-Br( Figure 6C ) treatment of glioblastoma cells induced annexin V staining and cleavage of caspase 7, 8 and PARP. Fig. 6A )、3-CF3-4-Cl ( Figure 6B ) and 2-OH-4-Br ( Figure 6C ) were treated with glioblastoma cells and the effect on induction of Annexin V was determined as described herein. Glioblastoma cells were treated with different concentrations of NR4A2 antagonists and whole cell lysates were analyzed for apoptotic markers by Western blot. Results ( Figures 6A-6C ) are expressed as mean ± SD of at least three determinations per treatment group, and significant (P < 0.05) responses compared to untreated controls are indicated (*). These results are consistent with those observed after knockdown of NR4A2 ( Figure 2C )quite.

[0056] The potency of the various ligands in terms of growth inhibition and apoptosis induction was ligand-, cell type-, and response-dependent, with the most pronounced difference in the fold induction of Annexin V in 15037 (high) cells versus 14015s (low) cells, and this was due in part to the relatively higher expression of Annexin V in untreated 14015s cells.

[0057] Cells were treated with 12.5 μM (3-CF3-4-Cl and 2-OH-4-Br) or 20 μM (C-DIM 12) and the effects on glioblastoma cell invasion or migration were determined in the Boyden chamber and scratch assays, respectively, as described herein. Inhibition of cell migration by NR4A2 ligands was also determined in 15037 cells in a tumor spheroid invasion assay as described herein (note: 14015s and U87-MG cells did not show invasion in this assay). NR4A1 antagonists also inhibited invasion of 15037 and 14015s cells in the Boyden chamber assay, with the latter cell line appearing to be more sensitive and less sensitive than 3-CF3-4-Cl or 2-OH-4-Br ( Fig. 7A ), 4-Cl-mediated inhibition of cell invasion required higher concentrations. Similar results were observed in scratch assays of 15037 and 14015s cells, where 20 μM DIM-C-pPhCl exhibited minimal inhibition of migration, and lower concentrations (12.5 μM) of 2-OH-4-Br and 3-CF3-4-Cl inhibited migration, with the latter compound being the most potent inhibitor. It was also observed that, using 15037 cells, knockdown of NR4A2 or treatment with 10 μM 4-Cl inhibited tumor spheroid invasion (Figure 5—figure supplement 1A), compared to cells transfected with DMSO (solvent control) or with a control oligonucleotide (siCt). Figure 7B These results demonstrate that NR4A2 is a growth-promoting, survival, and pro-invasive gene in glioblastoma and that C-DIM / NR4A2 ligand acts as an NR4A2 antagonist and represents a new chemotherapeutic approach for the treatment of this disease.

[0058] An estimated 23,880 new cases of brain and nervous system cancer will be diagnosed, and 16,380 deaths will occur from these diseases. GBM is the most frequently diagnosed malignant brain tumor, and the global incidence of the disease varies from 0.59-3.69 / 100,000. Diagnosis of GBM in adults is fatal, as patient survival ranges from 12-15 months, and patient survival ranges from 3-5% 3 years after diagnosis. Primary de novo GBM accounts for approximately 90% of all cases and occurs in elderly patients, while secondary GBM is primarily diagnosed in young patients. GBM is a complex disease involving multiple genetic changes including mutations in several genes, resulting in a highly aggressive disease that is difficult to treat. The current standard of care for newly diagnosed glioblastoma patients includes surgery, adjuvant radiation therapy, and the drug temozolomide (TMZ; alkylating agent), and the success of these treatment options is limited. The most troublesome biological feature of high-grade glioma cells is their tendency and ability to invade normal surrounding brain tissue, thereby evading the surgeon's knife and radiation delivered to the margins of surgical resection. This pool of infiltrating tumor cells forms a subpopulation of glioma stem cells that become the main source of tumor recurrence / progression, and they are generally resistant to chemoradiation and are often the cause of final patient death. The orphan nuclear receptor NR4A2 plays an important role in neuronal function, and previous studies have shown that 4-Cl and some related C-DIM compounds cross the blood-brain barrier and inhibit NR4A2-dependent inflammatory responses in a mouse Parkinson's disease model. Preliminary findings in established and patient-derived glioblastoma cell lines demonstrate the expression of NR4A1, NR4A2, and NR4A3 in these cells and that patient-derived cells predominantly express NR4A2 / NR4A3 with relatively low levels of NR4A1. The differential expression of these orphan receptors in patient-derived cells provides an opportunity to study the function of NR4A2 and the potential of targeting this receptor as a new approach to treat GBM patients.

[0059] The gene knockdown approach was initially used to determine the function of NR4A2 in patient-derived 14015s, 15037, and U87-MG glioblastoma cells. The results indicated that loss of NR4A2 resulted in inhibition of growth, induction of apoptosis, and inhibition of invasion. The effects of NR4A2 knockdown were opposite to those obtained after NR4A3 knockdown, with minimal effects on cell growth, survival, and migration. Thus, NR4A2 clearly exhibits proto-oncogenic activity in GBM, and these results are consistent with previous reports on the function of NR4A2 in other cancer cell lines and the proto-oncogenic activity of NR4A2.

[0060] Previous studies have characterized 4-Cl as an NR4A2 ligand that is effective as an anti-inflammatory drug for the treatment of several NR4A2-regulated pathways in models of Parkinson's disease. In transactivation studies in pancreatic cancer cells, 4-Cl activated NR4A2-dependent transactivation, whereas 4-Cl and two additional C-DIM analogs inhibited NR4A2-dependent transactivation in GBM cells ( Figures 4A-4C ). Thus, 4-Cl and related compounds are selective receptor modulators that exhibit cell type-specific agonist and antagonist activity with respect to NR4A2-dependent transactivation, and this has been previously observed for C-DIM binding to NR4A1.

[0061] 4-Cl and related compounds not only inhibited NR4A2-dependent transactivation, but also inhibited NR4A2-dependent cell growth, survival, and migration. In addition, similar responses were observed in athymic nude mice using U87-MG cells in a xenograft model, where 4-Cl inhibited tumor growth and induced tumor apoptosis.

[0062] These results confirm the proto-oncogenic activity of NR4A2 and show that NR4A2 ligands (such as C-DIM) used as antagonists represent a new approach to treating GBM. It is easy to envision studies focused on studying and identifying genes / pathways regulated by NR4A2 in GBM, as well as developing more effective NR4A2 antagonists for future clinical applications. In addition, and as previously detailed, C-DIM12 (4-chloro analog) targets NR4A2 by interacting with the coactivator binding site rather than the ligand binding domain of NR4A2. Therefore, a ligand binding assay for NR4A1 and NR4A2 was developed that measures the ligand-induced quenching of the ligand tryptophan fluorescence to further characterize the activity of NR4A1 and NR4A2. The tryptophan residue is located in the ligand binding domain (LBD) of both receptors, and the LBD of the receptor is used for binding assays. Table 1 below summarizes the binding K values ​​of C-DIM12 and other NR4A2-active compounds. D values, and they do not quench LBD-Trp, and this confirms previous modeling studies showing that C-DIM12 binds NR4A2 outside of its LBD. Similar results were obtained for the 3-Cl and 2-Cl-phenyl isomers of C-DIM12 (Table 1). However, the introduction of a second chlorine substituent on the phenyl ring resulted in several dichloro-substituted analogs binding (tryptophan quenching) to both NR4A2 and NR4A1 ( Figure 8). These unexpected results suggest the possibility that CDIMs containing two or more substituents on the phenyl ring bind to both NR4A2 and NR4A1. This hypothesis was tested using two series of compounds; a 3,5-disubstituted analog of the prototype NR4A1 ligand, C-DIM8 [1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane] (Table 2, shown below); and a set of novel 3,5-disubstituted phenyl CDIM analogs (Table 3, shown below) ( Fig. 9 ). The results showed that all of these compounds with two or more phenyl substituents bind to NR4A2 and NR4A1.

[0063] Table 1 : Binding of 4-substituted (phenyl ring) and dichloro-substituted CDIM to NR4A1 and NR4A2

[0064]

[0065] Table 2 : Binding of 3,5-disubstituted analogs of CDIM8 (1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane) to NR4A1 and NR4A2

[0066]

[0067] Table 3 : Binding of 3,5-disubstituted analogs of CDIM to NR4A1 and NR4A2

[0068]

[0069] These data highlight the promising results of CDIMs with two or more substituents on the benzene ring as ligands for NR4A2 and NR4A1. Therefore, due to the ability of bisindole-derived compounds to bind to NR4A1 and NR4A2, bisindole-derived compounds (e.g., bisindole-derived compounds with two or more substituents on the benzene ring) can be used to induce anticancer or anti-inflammatory activity in cells, as well as to treat other diseases, such as, but not limited to, cancer, brain cancer, breast cancer, kidney cancer, colon cancer, pancreatic cancer, lung cancer, inflammatory diseases, asthma, chronic peptic ulcers, tuberculosis, rheumatoid arthritis, periodontitis, ulcerative colitis, Crohn's disease, sinusitis, active hepatitis, and combinations thereof.

[0070] While various embodiments of the present disclosure have been illustrated in the drawings and described in the foregoing detailed description, it will be understood that the disclosure is not limited to the embodiments disclosed herein but is capable of many rearrangements, modifications, and substitutions without departing from the spirit of the disclosure set forth herein.

[0071] The term "substantially" is defined as most but not necessarily all of the specified content, as understood by one of ordinary skill in the art. In any disclosed embodiment, the terms "substantially", "approximately", "generally" and "about" may be replaced with "within the specified [percentage]", where percentages include 0.1%, 1%, 5% and 10%.

[0072] The features of several embodiments are summarized above so that those skilled in the art can better understand the various aspects of the present disclosure. Those skilled in the art should understand that they can easily use the present disclosure as a basis to design or modify other processes and structures for carrying out the same purpose of the embodiments introduced herein and / or realizing the same advantages of the embodiments introduced herein. Those skilled in the art should also recognize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and they can make various changes, substitutions and modifications without departing from the spirit and scope of the present disclosure. The scope of the present invention should only be determined by the language of the appended claims. The term "comprising" in the claims is intended to mean "at least including" so that the elements cited in the claims are listed as open groups. The terms "one", "an" and other singular terms are intended to include their plural forms unless explicitly excluded. Sequence Listing <110> Texas A&M University <120> Anticancer Agents <130> 13260-P197WO <150> US 62 / 880,801 <151> 2019-07-31 <160> 1 <170> PatentIn Version 3.5 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <220> <223> siGL2-5' <400> 1 cguacgcgga auacuucga 19

Claims

1. Use of a bisindole derived compound in the preparation of a medicament for treating a disease by inducing activity in a cell, wherein a bisindole derived compound is administered to a subject in need thereof, wherein the bisindole derived compound (CDIM) comprises two or more substituents on its phenyl ring, and wherein the disease is a glioma, wherein the bisindole derived compound is selected from 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl; 4-Cl), 1,1-bis(3′-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF 3- 4-Cl), 1,1-bis(3′-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br), and combinations thereof.

2. The use of claim 1, wherein the bisindole derivative compound binds to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2).

3. The use of claim 1, wherein the activity induced in the cell is at least one of an anti-cancer activity and an anti-inflammatory activity.

4. The use of claim 1, wherein the bisindole-derived compound exerts a function on the cell selected from the group consisting of: inducing NR4A1-dependent transactivation in the cell, inducing NR4A2-dependent transactivation in the cell, inhibiting the growth of the cell, inducing apoptosis in the cell, inhibiting the survival of the cell, inhibiting the migration of the cell, and a combination thereof.

5. The use of claim 1, wherein the cells are selected from A172, U87-MG, U98G, CCF-STTG1, 1708, 15037, 14004s, 14015s, 15049, glioblastoma multiforme (GBM) cells, and combinations thereof.

6. The use according to claim 1, wherein the bisindole-derived compound is at least one of a bisindole-derived NR4A1 ligand and a bisindole-derived NR4A2 ligand.

7. The use of claim 6, wherein at least one of the bisindole-derived NR4A1 ligand and the bisindole-derived NR4A2 ligand exerts a function selected from the group consisting of: antagonizing NR4A1 in the cell, targeting NR4A1 in the cell, antagonizing NR4A2 in the cell, targeting NR4A2 in the cell, and combinations thereof.

8. The use of claim 1, wherein the cell comprises at least one of NR4A1 and NR4A2 in a cancer cell.

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