Orphan nuclear receptor transcription factors as drug targets.

NR2E3, also known as the photoreceptor-specific nuclear receptor (PNR), is a transcription factor crucial for retinal photoreceptor development and maintenance. It regulates rod and cone differentiation by enhancing rhodopsin expression and repressing cone opsins, ensuring proper vision. Mutations in NR2E3 are linked to retinal degenerative diseases such as Enhanced S-cone syndrome, retinitis pigmentosa, and Goldman-Favre disease [ 236 , 237 ]. NR2E3 interacts with CRX, NR1D1, and NRL to drive rod-specific gene expression essential for retinal function [ 238–240 ]. Beyond ophthalmology, NR2E3 exhibits tumor-suppressive properties in hepatocellular carcinoma (HCC) by counteracting Wnt/β-catenin signaling through interactions with Sp1, β-catenin, and p300, thereby maintaining epigenetic stability [ 241 ]. NR2E3 also regulates the expression of the aryl hydrocarbon receptor (AHR) [ 242 ] and activates both wild-type and mutant p53 in multiple myeloma, enhancing tumor-suppressor gene expression and offering therapeutic potential in p53-mutant cancers [ 243 , 244 ]. NR2E3 functions as a tumor suppressor across various cancers. In HCC, its loss leads to Wnt/β-catenin activation, resulting in aggressive tumor growth and poor prognosis [ 241 ]. In multiple myeloma, NR2E3 promotes p53 acetylation, enhancing apoptosis and reducing cancer cell survival, making it a promising target for p53-mutant therapy [ 243 ]. In estrogen receptor-positive (ER+) breast cancer, NR2E3 regulates ESR1 (estrogen receptor α), improving clinical outcomes and tamoxifen response. It also influences stem-like cancer cell properties, suggesting a role in tumor plasticity and resistance [ 245 , 246 ]. NR2E3 also plays a protective role in liver function by maintaining chromatin accessibility and activating p53 during liver injury, preventing severe damage [ 241 ]. NR2E3 also regulates the expression of the AHR, and higher levels of both receptors are prognostic factors for increased survival of liver cancer patients [ 242 ]. Moreover, NR2E3 supports oxidative stress response by maintaining ERα expression, protecting against oxidative damage, and highlighting its role in cellular homeostasis [ 247 ]. Overall, NR2E3 is a key regulator in photoreceptor development, cancer suppression, and metabolic regulation, making it a significant therapeutic target in both vision disorders and malignancies. Biliverdin has been identified as the endogenous ligand for the PNR/NR2E3 receptor, forming a light-sensitive retinal transcription system ( Figure 16 ). Using gel-filtration chromatography-mass spectrometry, biliverdin, a pigment derived from heme metabolism, was found to bind NR2E3 [ 248 , 249 ]. Azacyclonol (AZA) ( Figure 16 ) enhances the Nr2e3/Tet2 pathway, increasing synaptic protein expression (PSD95, NMDAR1) and dendritic spine density, contributing to its antidepressant effects. While it shows promise for depression treatment, its clinical application is still under investigation [ 250 ]. The small-molecule agonist 11a promotes p53 acetylation, activating tumor-suppressing genes and inducing apoptosis in multiple myeloma (MM) cells. It exhibits synergistic effects with other therapies and promising preclinical results but 11a has not yet been approved for clinical use [ 251 ]. Figure 16. Endogenous and synthetic NR2E3 agonists exhibit different structures and exhibit cell context dependent effects [ 248–251 ]. Endogenous and synthetic NR2E3 agonists exhibit different structures and exhibit cell context dependent effects [ 248–251 ].

Small heterodimer partner (SHP, NR0B2) and dosage-sensitive sex reversal DSS-adrenal hypoplasia critical region on chromosome X, gene1 (DAX-1, NR0B1) are orphan NRs based on their domain structures, however, both receptors are unique since they lack a DNA-binding domain [ 3 , 4 ]. Thus their mechanisms of action are different from of other NRs, namely they cannot activate transcription by binding DNA. However, both receptors interact with a large number of proteins including DNA-bound transcription factors other NRs and they are important for regulating multiple genes and pathways to maintain cellular homeostasis and in pathophysiology. SHP plays an important role in the function of various hepatic metabolic pathways associated with regulation of bile acid and lipid metabolism circadian rhythms and also hepatocellular carcinogenesis [ 195 , 196 ]. DAX-1 is a transcriptional repressor and interacts with other NRs to decrease activation of genes, and it also plays a key role in steroidogenesis, early, embryonic development and testicular endocrine function. Mutations in the DAX-1 gene can cause X-linked adrenal hypoplasia congenital, characterized by adrenal insufficiency and hypogonadotropic hypogonadism [ 197 , 198 ], as well as delayed testis development and dosage-sensitive sex reversal (DSS) syndrome in XY individuals with Xp21 duplications, leading to female development. Both DAX-1 and SHP contain LXXLL motifs that facilitate interactions with other NRs and stapled LXXLL-based peptides have been developed to target the DAX-1/SHP-NR interactions [ 199 ]. Interactions of natural or synthetic ligands with DAX-1 have not been reported, however, 5-(diethylsulfamoyl)-3-hydroxynaphthalene-2-carboxylic acid (DSHN) has been discovered as a novel SHP activator ( Figure 14 ) [ 201 ]. DSHN bound SHP protein and activated a SHP-luciferase promoter construct [ 201 ]. Activation of SHP by DSHN inhibited migration and invasion of hepatic carcinoma cells by antagonizing CCl2 expression, and this compound has clinical potential for inhibiting liver cancer metastasis. The effectiveness of DSHN was recently confirmed in breast and other tumor types and correlations between SHP expression and positive prognosis for cancer patients was observed. In this study, several DSHN analogs were synthesized, and a more potent methyl ester derivative was identified as a SHP ligand. SHP also functions in the immune system to modulate the inflammasome in myeloid cells resulting in decreased immune-suppressive Treg cells and the methoxy DSHN analog enhanced this response [ 200 ]. These results are promising and suggest that SHP ligands can be effectively used as anticancer agents by targeting the receptor in the tumor and in immune cells. Figure 14. DSHN: a novel SHP ligand that modulates, cancer development and immune cell function [ 200 ]. DSHN: a novel SHP ligand that modulates, cancer development and immune cell function [ 200 ].

TLX (NR2E1) is an orphan nuclear receptor primarily expressed in the central nervous system (CNS), where it plays a crucial role in maintaining neural stem cell (NSC) self-renewal and neurogenesis [ 202 , 203 ]. It is also involved in retinogenesis and regulates the Pax-2 gene, essential for vision [ 204 , 205 ]. TLX is predominantly localized in the nucleus and functions as a transcriptional repressor [ 205 , 206 ]. Despite its importance, no natural ligands have been identified for TLX. Unlike classic nuclear receptors, TLX interacts with atrophin, BCL11A, LSD1, and HDACs to mediate transcriptional repression [ 206–209 ]. TLX is implicated in glioblastoma multiforme (GBM), a highly aggressive brain tumor. In glioblastoma stem cells (GSCs), TLX expression is elevated, and its overexpression in astrocytes induces a GSC-like phenotype. Knockdown of TLX in GSCs significantly reduces their growth and self-renewal, resulting in smaller tumors and prolonged survival in xenograft models [ 210 , 211 ]. TLX is also linked to metabolic disorders such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD). Elevated TLX expression in diabetic patients suggests a role in inflammation, while TLX ablation impairs β-cell function [ 212 , 213 ]. In NAFLD, the loss of TLX disrupts lipid metabolism, increases cholesterol, triglycerides, and liver fat accumulation [ 214 , 215 ]. TLX is evolutionarily conserved, with its homolog, tailless ( tll ), playing a critical role in embryogenesis and optic lobe development in Drosophila [ 216 , 217 ]. In mammals, TLX regulates genes such as Gsh2, Pax2, Pten, p21, p57, S100b, Aqp4, Plce1, Wnt7a, Bmp4, and Gfap , acting as both a repressor and an activator depending on the cellular context [ 198–208 , 218–223 ]. TLX influences neurogenesis and angiogenesis, contributing to neuroblastoma progression by stabilizing hypoxia-inducible factor (HIF) and activating vascular endothelial growth factor (VEGF) [ 224 ]. In cancer, TLX plays a significant role in the progression of castration-resistant prostate cancer (CRPC) and triple-negative breast cancer. It suppresses androgen receptor transcription and promotes tumor growth. TLX also prevents oncogene-induced senescence, enabling continuous cell proliferation and survival [ 225–227 ]. TLX also contributes to neuroblastoma by activating MMP-2 and promoting tumor sphere formation, correlating with poor patient outcomes. Despite its potential as a therapeutic target, the identification of ligands for TLX has been challenging. Recent research has focused on discovering both natural and synthetic ligands that can modulate TLX activity, which is crucial for advancing therapeutic strategies for neurological disorders and brain tumors [ 228 , 229 ]. Both natural and synthetic retinoids including all-trans and 11-cis retinaldehyde are natural retinoids involved in the visual cycle and bind to and modulate TLX function. Synthetic retinoids either activate or inhibit TLX’s transcriptional repressor activity, highlighting their potential in therapeutic applications [ 228 ]. Compounds such as caffeine and istradefylline have been identified as TLX modulators. These xanthine derivatives interact with the TLX ligand-binding domain, counteracting its repressor activity and indicating a possible role for the ligands in modulating TLX function through protein–protein interactions [ 230 ]. Oleic acid has been identified as an endogenous ligand of TLX, promoting hippocampal neurogenesis. This discovery opens new avenues for therapeutic modulation of TLX to address cognitive decline associated with aging and diseases [ 231 ]. Propranolol is a TLX ligand, which promotes TLX-regulated gene expression in glioblastoma cells, resulting in inhibition of cell proliferation and migration [ 232 ]. Through computer-aided drug discovery, small molecules have been identified that inhibit transcriptional activity of TLX ( Figure 15 ). Three compounds that target TLX include famprofazone (CCRP1), 1(1-(1,5-dimethylpyrazole-3-carbonyl)-4-(diphenylmethyl) piperazine (CCRP2), and dydrogesterone (CCRP3). These ligands target the Atro-box binding site of TLX, representing a novel class of compounds for potential therapeutic applications [ 233 , 234 ]. Figure 15. Small molecule inhibitors of TLX receptor that exhibit large differences in structure [ 228 , 230 , 231 , 233 ]. Small molecule inhibitors of TLX receptor that exhibit large differences in structure [ 228 , 230 , 231 , 233 ]. VPC-33010, VPC-33017, and VPC-33035 were identified by virtual screening and luciferase reporter assays, and they inhibited TLX-mediated transcriptional activity [ 234 ]. Recently, both synthetic and natural retinoids as regulators of TLX activity were identified and they act through direct interaction with the ligand-binding domain (LBD). This study identified specific compounds, such as BMS453 and BMS493 that act as agonists, while others, including CD437, CD1530, and all-trans-retinal, functioned as inverse agonists [ 228 ]. Through molecular dynamic simulations and reporter assays, along with site-directed mutagenesis, it was confirmed that these retinoids, like CCRP compounds, effectively bind to the LBD of TLX and modulated its function [ 228 ]. A rational fragment fusion approach was used to discover TLX agonist with submicromolar efficacy and their results demonstrated that this was it a valuable tool for identifying selective TLX modulators [ 235 ].

The testicular orphan nuclear receptor 2 (TR2), also referred to as nuclear receptor superfamily 2 group C member 1 (NR2C1) or TR2–11, was initially identified in 1989 from cDNA libraries of human testis [ 180 ]. Testicular orphan nuclear receptor 4 (TR4), also called NR2C2 or TAK, a related homologs was later cloned from the human and rat testes, hypothalamus, and prostate libraries in 1994 [ 180 , 181 ]. TR2 and TR4 were named due to their abundance in testes and skeletal muscle [ 182 ]. TR4 is also expressed ubiquitously in the central nervous system and significant peripheral organs including the liver, spleen, adrenal gland, and thyroid gland at a similar level [ 180 , 182–185 ]. TR2 and TR4 play crucial roles in regulating the physiological functions of fertility, growth, and metabolism [ 180 , 184 ]. This section will focus on the current understanding of drug/ligand discovery developments that differentially modulate TR2/4 function and can be used for various tissue/cell context-dependent treatments. Like other NRs, TR2 and TR4 are transcriptional factors, and they can be induced, inhibited, or repressed. The C-terminal, LBD binds natural or synthetic ligands, which then induce conformational changes, thus affecting their transcriptional activity [ 182 ]. A few natural ligands that function as TR2/4 ligands/activators include polyunsaturated fatty acids (PUFAs), PUFA metabolites, genistein, vitamin A, and retinoids. These compounds directly bind the LBD of TR2/4 and induce conformational changes in the receptors [ 186 ]. Genistein is one of the most potent activators of TR4, and other potential natural ligands promote TR4-dependent transcriptional activity at a modest level [ 182 ]. Thiazolidinediones (TZDs) are a class of drugs with two FDA – approved options, in which pioglitazone (Actos) and rosiglitazone (Avandia), are used to treat type 2 diabetes and bind PPARγ. TZDs are also TR4 ligands/activators, however, TR4 and PPARγ act in an inverse fashion. Activation of PPARγ enhances insulin sensitivity, whereas TR4 activation results in loss of insulin sensitivity [ 187–189 ]. This opposing function of TR4 and PPARγ presents a challenge in drug development, and suggests that drugs activating TR4 might counteract the beneficial effects of a drug that activates PPARγ. Metformin, a widely used antidiabetic drug that induces AMPK-mediated phosphorylation decreased TR4 transactivation [ 187 ] ( Figure 13 ), and is accompanied by altered SCD1 gene expression resulting in changes to lipid oxidation and lipogenesis pathways [ 187 ]. TR4 deficient mice are protected from insulin resistance and obesity-induced inflammation [ 191 ]. The potential of modulating TR4 activity as a platform for drug discoveries for metabolic syndromes is a promising avenue for future research. Figure 13. Clinically approved drugs, metformin, Binimetinib (MEK162) and Nolotinib that bind TR2 and TR4 and can be used for treatment of metabolic diseases, myeloid leukemia, prostate cancer [ 182 , 187 , 190 ]. Clinically approved drugs, metformin, Binimetinib (MEK162) and Nolotinib that bind TR2 and TR4 and can be used for treatment of metabolic diseases, myeloid leukemia, prostate cancer [ 182 , 187 , 190 ]. Nilotinib is an FDA-approved- second-generation tyrosine kinase inhibitor (TKI) developed for treating Philadelphia chromosome-positive chronic myeloid leukemia ( Figure 13 ). Nilotinib binds the LBD of TR2/4 and inhibits the TR2/4-dependent transactivation [ 182 ]. Tanabe and colleagues demonstrated that a TR2/TR4 heterodimer forms part of the direct repeat erythroid-definitive (DRED) complex, which suppresses fetal globin transcription in definitive erythroid cells [ 192 ]. TR2 and TR4 recruit epigenetic transcriptional corepressors in differentiated adult erythroid cells to the embryonic β-type globin promoters [ 193 ]. This suggests that targeting TR2/4 could be a potential strategy for developing drugs to treat erythrocyte-related diseases. Binimetinib (MEK162) is an FDA-approved medication for treating cancers with NRAS or BRAF mutations ( Figure 13 ). MEK162 inhibits TR4 protein expression and blocks the recruitment of TR4 binding to its consensus promoter site [ 190 ]. Repression of TR4 contributes to the effect of MEK162 by disrupting the MAPK/ERK pathway that inhibits tumor cell proliferation and decreases the tumor-derived circulating hormone ACTH [ 190 ]. In prostate cancer, TR4 is highly expressed in patients after brachytherapy [ 194 ] and higher expression of TR4 correlates with a high Gleason-scored lesions [ 189 ]. Functional analyses suggest that TR4 facilitates prostate cancer cell invasion and metastasis by transactivating the CCL2/CCR2 signaling pathway [ 189 ]. Therefore, ligands such as MEK162 that inhibit TR4 are promising pharmaceutical targets for treating prostate cancer.

Estrogen receptor-related receptors (ERRs) are a family of 3 orphan receptors ERRα (NR3B1), ERRβ (NR3B2) and ERRγ (NR3B3) that have a high degree of sequence homology to the ER and also to each other. Their LBDs and hinge regions are approximately 55–66% identical with their NTDs containing only 25% sequence identical. ERRα is expressed ubiquitously, ERRγ is more highly expressed in vascular and metabolically active tissues, ERRβ expression is primarily detected in liver, eye and inner ear and all 3 receptors are critical for maintaining homeostasis in multiple tissues [ 277 , 278 ]. The existence and function of endogenous ERR ligands is not well understood however there have been many studies on the development of ligands for ERRs and their possible therapeutic applications for treating cardiovascular diseases, diabetes, cancers and osteoporosis. Steroidogenic factor-1 (SF-1, also called AD4BP and NR5A1) is an orphan nuclear receptor that plays an important role in the regulation of various physiological processes as well as the transcription of an array of different pathways and genes including those involved in endocrine development, steroid hormone biosynthesis and metabolism and expression of cytochrome P450 steroid hydroxylases, and StAR [ 279 , 280 ]. Although both ERs and ERRs have structural similarities, the ERs are ligand-activated transcription factors. ERRs do not bind an “endogenous” ligand but are constitutively active as monomers or heterodimers in combination with PGC-1 as a cofactor. Nevertheless in binding assays the stilbene analogs 4-hydroxytamoxifen, diethylstilbestrol (DES) and tamoxifen bind ERRγ but exhibit minimal binding to ERRα and ERRβ and the high-affinity ligand 4-hydroxytamoxifen inactivates ERRγ [ 281 ]. Another report also showed binding and functional interactions of DES with all 3 ERRs [ 282 ] and the differential interactions with ERRγ involve subtle structural features of the LBD [ 282–284 ]. More recent studies show that cholesterol is an ERRα agonist and induces several ERRα-dependent metabolic gene targets and also induces ERRα expression [ 285 ]. In contrast an estradienone-like steroid identified in human pregnancy urine acted as an inverse agonists of ERRα and ERRγ and this compound inhibited growth of breast cancer cells [ 286 ]. This suggests that some steroidal metabolites may be endogenous ligands for the ERR subfamily of receptor. In addition several flavonoids also bind ERR [ 228 , 287 ] ( Figure 18 ). The endogenous oxysterols such as 25, 26 or 27-hydroxycholesterol (OHC) are known inhibitors of cholesterol biosynthesis [ 289 ] and selectively enhance SF1 mediated transcriptional activity. This activation is dependent upon the SF-1 AF2 domain and are specific for SF-1. Ligand-dependent regulation of SF-1 by oxysterols has a significant effect on the steroidogenesis in vivo [ 289 ]. Figure 18. Flavonoid compounds that bind ERR [ 287 , 288 ]. Flavonoid compounds that bind ERR [ 287 , 288 ]. Structurally diverse compounds have been identified and investigated as ligands for ERRα and these compounds exhibit tissue-specific agonist and antagonist activities [ 3 ]. For example, the pyrido[1,2-α]pyrimidin-4-ones were screened for ERRα agonist activity and one of these (#5) ( Figure 19 ) was primarily an ERRα agonist with only moderate ERRγ activity and this compound induced genes such as MCAD and PDK4 in 293 FT cells that are involved in oxidative metabolism [ 294 ]. A subsequent study of N-aceyl hydrazones identified compound #24 which exhibited ERRα/β/γ pan agonist activity [ 290 ] and a boronic acid compound, (3-(5-(2-fluorophenyl)carbamoyl)thiophen-2-yl)phenyl) boronic acid was also characterized as an ERRα/β/γ pan agonist [ 291 ]. OR-449 is a potent and orally bioavailable small molecule antagonist of SF-1 and clinically used for treatment of adult and pediatric forms of adrenocortical carcinoma (ACC) as well as other cancers that express high levels of SF-1. The U.S. Food and Drug Administration (FDA) has granted Rare Pediatric Disease Designation (RPDD) for OR-449 for the treatment of pediatric ACC. The compound 4-[heptyloxy]phenol (AC-45594) represses SF-1 dependent transcription in transient transfection experiments in H295R cells [ 292 ]. Two analogous compounds of the isoquinolinone class, SID7969543 and SID7970631 drugs inhibited SF-1 induced proliferation in adrenocortical carcinoma cells [ 293 ]. Thus both ERR and SF-1 have been extensively used as drug targets for treating both cancer and non-cancer related diseases. Figure 19. Synthetic ligands that bind and activate ERRα and SF-1; ligand structures for both receptors are highly variable [ 290–293 ]. Synthetic ligands that bind and activate ERRα and SF-1; ligand structures for both receptors are highly variable [ 290–293 ].

The members of the orphan nuclear receptor NR2F family are also referred to as chicken ovalbumin upstream promoter transcription factors (COUP-TFs). Among them, COUP-TFI (NR2F1) and COUP-TFII (NR2F2) share 96% and 98% sequence identity, respectively, in their ligand and DNA binding domains. However, the third member, EAR2 (NR2F6), stands out as the most divergent member of the NR2F subfamily [ 151–153 ]. These NR2F receptors are expressed during early vertebrate development and play crucial roles in a variety of biological processes, such as development, differentiation, metabolism, and immune regulation. Located in the nucleus, NR2F regulates gene expression either by directly binding to AGGTCA motifs in the promoters of target genes or by interacting with co-factors, chromatin remodeling complexes, and other signaling pathways. Despite sharing some functions, the NR2F receptors exhibit non-redundant roles due to their distinct expression patterns across different tissues [ 151 ]. COUP-TFI/II regulates crucial processes such as cell differentiation, tissue development, angiogenesis, and metabolism [ 71 ]. COUP-TFI is a key transcriptional regulator involved in cortical development, cell specification, and maturation [ 154 ]. Disruptions in COUP-TFI-regulated neurodevelopmental processes can lead to conditions such as epilepsy, cognitive impairments, and visual deficits [ 71 , 151 ]. The roles of COUP-TF members in cancer progression can be either positive or negative, depending on the specific cell type and biological context [ 154 ]. In breast cancer cells, increased COUP-TFI expression promotes cell proliferation and migration [ 155 ], while in head and neck squamous cell carcinoma and prostate cancer, it induces cell cycle arrest by binding to the SOX9, RARβ, and p27 gene promoters [ 156 ]. In contrast, some cancer types exhibit lower COUP-TFI expression compared to normal tissues [ 157 , 158 ]. COUP-TFII plays a crucial role in regulating cell differentiation and angiogenesis [ 151 , 159 ]. It inhibits the Notch pathway by binding to FoxC1, NR-1, and Hey2, and modulates the expression of several genes, such as angiopoietin-1 and VEGF/VEGFR2, which are involved in angiogenic pathways [ 159–161 ]. COUP-TFII is also a key regulator and inhibitor of SMAD4-dependent transcription, necessary to overcome the TGF-β-dependent growth barrier during the malignant progression of PTEN-null prostate tumorigenesis [ 162 ]. Overexpression of COUP-TFII activates focal adhesion kinase, matrix metalloprotease 2, and urokinase receptor (uPAR), which enhance the invasiveness of lung cancer cells [ 163 ]. EAR2 (NRF6), the most divergent member of the NR2F subfamily, acts as a negative regulator of gene transcription and plays a crucial role in the central nervous system. It represses transcription of the human luteinizing hormone receptor (LHR) gene and the renin gene in mice, and negatively regulates the thyroid hormone nuclear receptor. Several studies have highlighted EAR2’s significant role in carcinogenesis and tumor progression [ 151 ]. In breast cancer, EAR2 is highly expressed in both ER+ and ER− tumors compared to normal breast tissue. Cells with lower EAR2 expression show increased sensitivity to anticancer drugs targeting microtubules. EAR2 is also overexpressed in various cancers, including lymphomas, colorectal tumors, and cervical and ovarian cancers [ 164–167 ]. Knockdown of EAR2 inhibits expression of X-linked inhibitor of apoptosis protein (XIAP) and promotes apoptosis in colon cancer cells. In epithelial ovarian cancer, overexpression of EAR2 contributes to chemoresistance by activating the Notch3 signaling pathway. EAR2 may serve as a potential biomarker to identify patients who are likely to respond to treatment with gamma-secretase inhibitors that block Notch signaling [ 165 ]. Additionally, EAR2 functions as a transcriptional repressor of cytokines in mouse macrophages, while in human macrophages, it acts as an activator of chemokines. Unlike other nuclear receptors, NR2F subfamily receptors lack a classical endogenous ligand that modulates their activity. Instead, COUP-TFI/II is primarily regulated indirectly through interactions with other nuclear receptors, such as retinoic acid receptors and thyroid hormone receptors. Retinoic acids have been identified as low-affinity ligands for COUP-TFII [ 168 ]. Furthermore, COUP-TFI/II activation by retinoic acid increases the expression of Na(+)/H(+) exchanger (NHE) [ 169 ], and COUP-TFI modulates the expression of CYP26A1 and HoxA1 via retinoic acid signaling, which is crucial for embryonic development [ 170 ]. In breast cancer cells, COUP-TFII interacts with nucleolin to coactivate COUP-TFII-mediated retinoic acid receptor [ 171 ]. Additionally, the transcriptional activity of ERα in breast cancer cells is regulated by the interaction between COUP-TFs and the estrogen response element (ERE) [ 172 ], while the COUP-TFII-AF interaction governs androgen-dependent activation of prostate-specific antigen in prostate cancer cells [ 173 ]. The reciprocal interaction between COUP-TFII and the glucocorticoid receptor is involved in gluconeogenesis and lipoprotein metabolism by regulating expression of phosphoenolpyruvate carboxykinase (PEPCK) and CYP3A [ 174 ]. Moreover, COUP-TFs enhance transcription of the NGFI-A gene by recruiting the coactivator SRC-1 through its interaction with Spl [ 175 ]. Several compounds target COUP-TFII, but there are currently no approved drugs specifically aimed at the COUP-TF family. C26 functions as an agonist enhancing the expression of NR2F1 and its downstream target genes that regulate dormancy ( Figure 12 ). In C26-treated tumors, SOX9, RARβ, and p27 are all significantly upregulated [ 176 ]. CIA1 (also known as NR2F6 modulator-2) is a synthetic compound that acts as a potent and selective inhibitor of COUP-TFII (NR2F2). It binds directly to the COUP-TFII ligand-binding domain, disrupting its interaction with transcription regulators such as FOXA1 [ 177 ]. BMH-9 (also known as Compound Z5) is a modulator of NR2F6 and acts as an activator of the p53 signaling pathway. It inhibits the proliferation of human cancer cells and demonstrates antitumor efficacy in NOD-SCID mouse models [ 178 ]. Pyridaben has demonstrated potential in targeting COUP-TFII, although its primary role as an acaricide and insecticide is through inhibiting mitochondrial electron transport in pests. This effect may also influence processes such as cell differentiation, development, and cancer progression, where COUP-TFII plays a key role [ 179 ]. There are no known FDA approved drugs for NR2F sub-family members. Figure 12. Ligands that bind COUP-TF include several structurally-diverse heterocyclic compounds [ 176–179 ]. Ligands that bind COUP-TF include several structurally-diverse heterocyclic compounds [ 176–179 ].

Hepatocyte nuclear factor 4 (HNF4), including its isoforms HNF4α (NR2A1) and HNF4γ (NR2A2), are crucial nuclear receptors involved in embryonic development, metabolism, and cellular differentiation [ 252–254 ]. Highly expressed in tissues such as the liver, pancreas, kidney, and intestine, HNF4α is essential for maintaining hepatic and pancreatic β-cell functions by regulating genes related to glucose, cholesterol, and fatty acid metabolism [ 255–258 ]. Loss of functional HNF4 results in embryonic lethality in mice, demonstrating its critical role in early development [ 259 ]. Mutations in HNF4α are associated with metabolic disorders, including Maturity Onset Diabetes of the Young 1 (MODY1) and hemophilia, due to alterations in its target genes [ 260 ]. Structurally, HNF4α and HNF4γ possess highly conserved DNA-binding and ligand-binding domains, allowing them to act as homodimers or heterodimers to regulate gene transcription [ 261 , 262 ]. HNF4α activity is further modulated through post-translational modifications, such as phosphorylation by ERK1/2, which impacts its chromatin binding and transcriptional activity [ 263 ]. While HNF4γ shares functional redundancy with HNF4α in certain tissues, particularly in the intestine and kidney, HNF4α plays a more dominant role in hepatic and metabolic regulations [ 264 , 265 ]. HNF4α functions as a tumor suppressor in liver cancer by inhibiting epithelial-to-mesenchymal transition (EMT) and maintaining hepatic differentiation, while it also regulates Wnt/β-catenin and STAT3 signaling pathways in colorectal and cervical cancers [ 266–268 ]. Its dysregulation is linked to increased cancer cell proliferation, invasion, and metastasis, making it a potential therapeutic target [ 269 ]. Conversely, HNF4γ exhibits oncogenic properties in colorectal and pancreatic cancers, where it promotes tumor growth via the PI3K/AKT pathway and interacts with FOXA1 to enhance tumor survival [ 270 ]. MicroRNA-766-3p negatively regulates HNF4γ, suggesting a potential therapeutic approach for cancer treatment [ 271 ]. Beyond its role in cancer, HNF4α is a master regulator of metabolic processes, governing lipid metabolism, insulin secretion, and bile acid synthesis, with implications in diseases such as nonalcoholic fatty liver disease (NAFLD) and obesity [ 270 , 272 ]. Both HNF4α and HNF4γ function as transcriptional regulators by binding DNA, recruiting co-regulators, and modulating metabolic and oncogenic pathways, positioning them as critical targets for drug development and therapeutic intervention [ 273 ]. HNF4α is modulated by both endogenous and synthetic ligands. Endogenous ligands such as linoleic acid (LA) and fatty acids bind to the ligand-binding domain of HNF4α, playing a critical role in regulating metabolic pathways and influencing its transcriptional activity [ 272 ]. Synthetic ligands, including fragment-like activators and inverse agonists, have been identified to modulate HNF4α activity at low micromolar concentrations, highlighting the potential for drug development targeting this receptor [ 274 ] ( Figure 17 ). Additionally, natural compounds such as chelidonine, an isoquinoline alkaloid derived from Chelidonium majus , act as inverse agonists, downregulating genes linked to gluconeogenesis and drug metabolism in liver cancer cells [ 275 ]. Furthermore, oxidized polyunsaturated fatty acids (oxo-PUFAs) form covalent bonds with HNF4α, suggesting the potential for the development of covalently linked drugs targeting HNF4α [ 276 ]. These are examples of the potential therapeutic applications for HNF4 ligands; however, this has not yet been carried out. Figure 17. HNF4α ligands that exhibit agonist or inverse agonist activities are structurally diverse and vary from 1–6 rings and different substituent groups [ 274 , 275 ]. HNF4α ligands that exhibit agonist or inverse agonist activities are structurally diverse and vary from 1–6 rings and different substituent groups [ 274 , 275 ].

Nuclear receptors (NRs) are ligand-activated transcription factors that play key roles in maintaining cellular homeostasis and they are also implicated in various pathological conditions [ 1–3 ]. The 48 members of the human NR superfamily are structurally related proteins which have several common domains ( Figure 1 ). These include the N-terminal A/B and DNA binding domain (C) (DBD) a hinge region (D) and an N-terminal E ligand binding domain (LBD). Activation function 1 (AF1) and AF2 are located in the N - and C-terminal domains of NRs, respectively, and their interactions with nuclear cofactors such as coactivators and corepressors are essential for the functional activities of the individual NR and the ligand-bound receptors [ 4 , 5 ]. The common structural domains observed for NRs is in contrast to their specific activities in all cell types where they perform unique and highly essential functions in both normal and diseased tissues with minimal overlap. Figure 1. Nuclear receptors. a. Structural domains of NRs include the N-terminal domain (NTD; AB), DNA-binding domain (DBD; C), a hinge region (D) and a C-terminal domain (E) which contains the ligand-binding domain (LBD). Some steroid hormone receptors such as ESR1 contain a C-terminal F domain. b. Adopted orphan receptors now have endogenous ligands which have low affinity. c. Endogenous ligands for orphan receptors have not yet been identified. Nuclear receptors. a. Structural domains of NRs include the N-terminal domain (NTD; AB), DNA-binding domain (DBD; C), a hinge region (D) and a C-terminal domain (E) which contains the ligand-binding domain (LBD). Some steroid hormone receptors such as ESR1 contain a C-terminal F domain. b. Adopted orphan receptors now have endogenous ligands which have low affinity. c. Endogenous ligands for orphan receptors have not yet been identified. Classification of NRs has been variable. For example, Evans and coworkers [ 2 ] subdivided the nuclear receptors into three major classes; namely (i) endocrine receptors which include the steroid hormone receptors and thyroid hormone, retinoic acid and vitamin D receptors, (ii) adopted orphan receptors which include receptors for which ligands were identified after their discovery, and (iii) orphan receptors for which endogenous ligands have not yet been identified. NRs that act as transcription factors have also been classified into six groups based on sequence homology [ 3 ] and also a seventh group of atypical receptors which do not have a DBD and include dosage, sex, reversal-adrenal hypoplasia critical region on chromosome X, gene1 (DAX1) and short heterodimeric partner (SHP, NR0B2). Figure 1 illustrates another possible classification of nuclear receptors [ 6 ] which include adopted orphan nuclear receptors for which exogenous ligands have been identified and may bind these receptors to modulate gene expression. In contrast, endogenous ligands for orphan nuclear receptors have not been identified even though they bind some endogenous biochemicals with low affinity. Ligand-dependent activation or inactivation of NRs is complex and requires the formation of the active complex which may be a monomer, hetero, or homodimer that exhibits sequence-specific binding to cis elements in target gene promoters. Recruitment of active NRs to promoters is also accompanied by alterations in chromatin structure and formation of transcriptionally active complexes containing cofactors, corepressors, and other nuclear cofactors including proteins associated with transcriptional machinery. The overall process is dynamic and highly tissue-specific due, in part, to variable expression of cofactors [ 4 , 6–8 ]. Figure 2 illustrates examples within the orphan NR4A family where sub-family members bind promoter DNA as monomers, dimers, and heterodimers with RXR [ 9 , 10 ]. In addition, NR4A1 and NR4A2 can act as ligand-dependent cofactors which bind Sp1 and Sp4 transcription factors (but not DNA) [ 11 ] and there is evidence that an NR4A1:NR4A2 heterodimer interacting with Sp1 activates the TWIST1 gene [ 12 ]. The NR/Sp pathway is common for most NRs [ 11 ]. In addition, there is evidence from structural studies that NRs may form more complex structures including quaternary structures [ 13 , 14 ]. Figure 2. NR4A-mediated activation/inactivation of genes can be due to NR4A monomers, homodimers, NR4A:RXR and NR4A/Sp. The receptors bind specific promoter response elements that accommodate the monomers and homodimers and the heterodimeric RXR and NR4A also act as a cofactor to Sp transcription factor. These interactions can be modulated by ligands and their formation is gene promoter and cell context dependent. NR4A-mediated activation/inactivation of genes can be due to NR4A monomers, homodimers, NR4A:RXR and NR4A/Sp. The receptors bind specific promoter response elements that accommodate the monomers and homodimers and the heterodimeric RXR and NR4A also act as a cofactor to Sp transcription factor. These interactions can be modulated by ligands and their formation is gene promoter and cell context dependent. This review is primarily focused on ligands that target orphan nuclear receptors and activate or inactivate patterns of gene expression that have clinical potential. These will primarily be small molecules that exhibit agonist, antagonist or inverse agonist activities and in some cases partial agonist/antagonist effects are observed and these ligands are called selective receptor modulators (SRMs) [ 6–8 ]. For example, in humans the antiestrogen tamoxifen is a selective estrogen receptor modulator (SERM) that exhibits antiestrogenic activity in the mammary gland and in ER positive tumors tamoxifen has been a major drug clinically used for treatment of early-stage ER-positive breast cancer [ 15 , 16 ]. However, the antagonist/agonist activities of tamoxifen are tissue specific ( Figure 3 ) and although tamoxifen as an agonist enhances bone health, the ER agonist activity of tamoxifen in the uterus is a risk factor for uterine and endometrial cancer in women [ 17 ]. The mechanisms associated with the SERM- and SRM-like activity of NR ligands are linked to several factors including ligand-induced conformational changes in the receptor and the tissue-specific expression of cofactors/corepressors and other nuclear proteins [ 1 , 4 , 6–8 ]. Figure 3. Tamoxifen, a selective ER modulator exhibits tissue-specific receptor agonist/antagonist activities and acts as an ERα agonist in the uterus bone and liver and an antagonist in breast cancer [ 15 , 16 ]. Tamoxifen, a selective ER modulator exhibits tissue-specific receptor agonist/antagonist activities and acts as an ERα agonist in the uterus bone and liver and an antagonist in breast cancer [ 15 , 16 ]. The classical NR ligands are those that directly interact with the LBD of the receptor, and these will be discussed in this review; however, there are also multiple examples of alternative NR ligands that influence receptor-mediated gene expression [ 18–22 ]. Alternative sites or surfaces on NRs that can be targeted by ligands include coactivator/corepressor and cofactor sites, surfaces required for the formation of NR homo or heterodimers and for NR-DNA interactions. Proteolysis Targeting Chimera (PROTAC) molecules are potential receptor antagonists that incorporate a NR ligand and a linker that connects with a substrate for an E3-ligase. These dual functional agents bind the targeted NR and also recruit the 26S proteasome complex to induce degradation of the receptor. A recent study [ 23 ] reported that a novel PROTAC targeted to NR4A1 decreased NR4A1 (proteasome-dependent) but not NR4A2 or NR4A3 proteins in melanoma cell lines. The PROTAC (NR-VO4) contained the NR4A1 ligand celastrol covalently bound to polyethylene glycol linker, which was in turn bound to a VHL E3 ligase ligand ( Figure 4 ). NR-VO4 enhanced immune surveillance in vivo and in tumor infiltrating lymphocytes NR-VO4 induced memory CD8 + T cells, decreased myeloid-derived suppressor cells and induced tumor infiltrating B cells and enhanced immune surveillance [ 23 ]. Figure 4. NR-VO4 contains the NR4A1 ligand celastrol which is bound through its carboxyl group to polyethyleglycol linkers to a VHL E3 ubiquitin ligase [ 23 ]. NR-VO4 contains the NR4A1 ligand celastrol which is bound through its carboxyl group to polyethyleglycol linkers to a VHL E3 ubiquitin ligase [ 23 ]. Orphan NRs have emerged as important drug targets for multiple diseases [ 24–28 ] and studies have focused on both understanding the ligand-dependent mechanisms, their transcriptional activation of orphan NRs and potential clinical applications. The steroid hormone receptors bind their endogenous hormone ligands with high affinity with K D values in the low nM range. However, these receptors [ 29 ] and orphan receptors also bind structurally diverse endogenous compounds, natural products and synthetic compounds and including pharmaceuticals with lower affinity and their structural diversity is illustrated throughout this review. At present, the number of clinically used orphan NR ligands is low but varies among receptors and this review will highlight some of the recent advances in this field. Structures of ligands for orphan NRs will also be featured in this review to demonstrate that most orphan receptors bind to structurally diverse compounds similar to those observed for steroid hormone receptors such as ERα.

Given the pivotal and varied roles that NR4As play in the broader context of tumor development and other inflammatory diseases, these receptors have emerged as an important target for developing therapeutics [ 78 , 79 ]. NR4As are categorized as orphan nuclear receptors, implying that they have no known endogenous ligands that control NR4A function. However, various exogenous ligands, have been discovered and developed as either NR4A agonists, antagonists, inhibitors or inverse agonists [ 80 , 81 ]. This section will detail the current understanding of developments in drug/ligand discovery that differentially modulate NR4A function and can be used appropriately for various context-dependent treatments. This section will be divided into four sections, namely potential endogenous ligands, natural products, and their analogs, and synthetic ligands and those derived from library screens. NR4As do not have any known or confirmed endogenous ligands that regulate their functional activity; however, NR4As bind multiple biochemicals and natural products. A recent study reported that unsaturated fatty acids, rather than saturated fatty acids, were enriched in NR4A1 protein elutions from brain tissue extracts [ 82 ]. Two unsaturated fatty acids, arachidonic acid (AA) and docosahexaenoic Acid (DHA), bind NR4A1 LBD, resulting in a conformational change [ 82 ]. Further evidence determined that AA stabilized NR4A1 dimers and modulated their transcriptional activity [ 82 ]. Prostaglandin A2 covalently binds the LBDs of NR4A1, NR4A2, and NR4A3, and stimulates their transcriptional activities [ 83–85 ]. PGA2 binding to NR4A2 helps rescue behavioral phenotypes such as locomotor deficits and neurodegeneration in a Parkinson’s Disease fly model [ 83 ] and in rat models [ 86 ]. When bound to NR4A3, PGA2 sensitized C2C12 myocytes to insulin through NR4A3, leading to increased glucose uptake and AKT phosphorylation [ 86 ]. PGA2 displays pro-apoptotic and anti-proliferative effects in MCF-7 and HeLa cancer cell lines [ 87 ]. PGA2, a dehydrated metabolite of PGE2 also binds NR4A (LBD) [ 83 ]. PGE2 is a COX-2 derived gene product that is pro-oncogenic in colon cancer, induces NR4A2 in both in-vivo and in-vitro models, and enhances fatty acid oxidation [ 88–90 ]. Furthermore, PGE2-mediated induction of NR4A2 increases expression of prolactin, which initiates prostate cancer through tumor-stromal prolactin signaling [ 91 ]. Prostaglandins A1 and E1 covalently bind both NR4A1 and NR4A2 at their LBDs [ 92 ]. Interestingly, PGA1 and PGE1 have differentially induce transcriptional activity of NR4A2. PGE1 mediates NR4A2 functional activation through both direct ligand binding and the EP2-mediated pathway, whereas PGA1 only acts through direct binding [ 92 ]. These prostaglandins exhibit neuroprotective functions through NR4A2, protecting dopaminergic neurons from MPP+-induced toxicity and preventing neural degeneration in the midbrain. PGE1 improves peripheral neuropathies in diabetic patients, but the role that NR4A might play in this phenotype rescue is currently unknown [ 93 ]. Cytosporone B is a naturally occurring octaketide, isolated from the endophytic fungus Dothiorella sp. HTF3 [ 94 ] ( Figure 8 ). This ligand was first characterized as an NR4A1 agonist through testing the promoter binding and transcriptional activity of NR4A1 with a luciferase-reporter gene that contained a NurRE element in its promoter region. Results indicate that CsnB treatment specifically induced NR4A1-driven transcriptional activity [ 95 ], and CsnB binds the LBD of NR4A1, specifically at the residue Y453 with a KD of 8.52x10 −7 M. The binding causes a conformational change in the receptor while not affecting protein stability. Figure 8. Cytosporone B and related synthetic analogs-NR4A1 ligands that exhibit a wide range of activities [ 94–101 ]. Cytosporone B and related synthetic analogs-NR4A1 ligands that exhibit a wide range of activities [ 94–101 ]. NR4A1 plays an important role in the upregulation of gluconeogenic activity. CsnB, in its role as a NR4A1 agonist, increases blood glucose levels in wild type mice, and increases expression of various gluconeogenic genes, including G6PC and FBP1. CsnB also reverses the effect of insulin mediated reduction of blood glucose levels [ 95 ]. Additionally, CsnB induces NR4A1 mediated apoptosis by promoting nuclear export of NR4A1 to the mitochondria [ 95 , 96 ]. CsnB has been extensively utilized and shown to be effective in multiple disease models [ 86–88 ]. In addition, several synthetic CsnB analogs including TMPA (ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl] acetate), THPN (1-(3, 4, 5-trihydroxyphenyl)-1-nonanone), and PDPNA (n-pentyl-2-(nonanoyl) phenyl acetate) ( Figure 8 ), and their NR4A-dependent antidiabetic, autophagic, and anti-inflammatory activities, respectively, have been extensively investigated [ 99–101 ]. Many studies have also shown that flavonoids are anti-cancer and chemo-preventive compounds that might have immune-modulator activities that resemble those observed for NR4A1 ligands ( Figure 9 ) [ 115–117 ]. The flavonoid quercetin, primarily found in onions and broccoli, inhibits growth of the human metastatic ovarian cancer cell line PA-1 cells by downregulating anti-apoptotic Bcl-2 and upregulating pro-apoptotic caspace-3 and CytC, which are NR4A regulated genes [ 118 ]. Another common flavonoid, kaempferol, suppressed Bcl-2 protein and expression of survivin in endometrial cancer cells and G9a in gastric cancer cells [ 119 , 120 ]. A study from our laboratory discovered that both quercetin and kaempferol bind NR4A1 at the LBD with low μM KD values (3.1 and 0.93 µM respectively). Both flavonoids decreased NR4A1-dependent transactivation in Rh30 and Rh41 rhabdomyosarcoma cell lines, highlighting their roles as NR4A1 inverse agonists [ 121 ]. Furthermore, the effects of quercetin and kaempferol in endometriosis cells were similar to those observed in cancer cells where these flavonoids exhibited anti-endometriotic activity acting as NR4A1 inverse agonists [ 102 ]. Subsequent studies have demonstrated the NR4A1 activity of structurally diverse hydroxy flavonoids resveratrol [ 103–106 ], tetrandrine [ 107 ], the xanthene CCE9 [ 108 , 109 ], ligustilide [ 110 , 111 ] and celastrol [ 112 , 113 ]. All of these natural products are NR4A1 ligands and induce NR4A1-dependent anticancer activities; however, celastrol also induces nuclear export of NR4A1 to the mitochondria where it forms a pro-apoptotic complex with Bcl-2 [ 114 ]. Figure 9. Celastrol and the polyphenolics quercetin, kaempferol and resveratrol natural products that bind NR4A1 and act as inverse agonists to NR4A1-dependent pro-oncogenic activities [ 102–114 ]. Celastrol and the polyphenolics quercetin, kaempferol and resveratrol natural products that bind NR4A1 and act as inverse agonists to NR4A1-dependent pro-oncogenic activities [ 102–114 ]. Indole-3-carbinol (I3C) is a bioactive compound found in most cruciferous vegetables, such as cabbage and broccoli and is known for having antioxidant and anti-cancer properties ( Figure 10 ) [ 133 ]. The dimeric metabolite of I3C, namely 1,1-bis (3’-indolyl) methane (DIM) exhibits similar but more potent anticancer activities. DIM was used as a precursor for the synthesis of a series of triarylmethane derivatives, 1,1-bis(3’-indolyl)-1-(substituted phenyl) methane (CDIM) compounds with variable substituent groups (position and number) ( Figure 10 ). The first set of 4’-substituted phenyl ligands containing up to 12 different substituents showed that some of these compounds bound NR4A1 (e.g. 4-hydroxy:DIM-4-OH, 4-carbomethoxy:DIM-4-CO 2 Me) and others (eg. 4-chloro: DIM-4-Cl, 4-bromo:DIM-4-Br) bound NR4A2. DIM-4-OH (DIM8) has been shown in solid tumor-derived cell lines to inhibit growth, survival, migration/invasion [ 122 , 123 ]. This was associated with downregulation of EGFR, survivin, β1-integrin, other integrins, G9a and PD-L1 [ 124–127 ]. In contrast, DIM-4-Cl binds to the cofactor site of NR4A2 and in cancer cells, inducing several pathways and some genes in common with DIM-4-OH/NR4A1 [ 128–130 ]. Structure–activity relationships have identified a second and third generation of CDIM compounds [ 128 , 131 , 132 , 134 ]. These include 3,5-disubstituted-4-hydroxyphenyl (DIM8–3,5) and 3,5-substituted phenyl (DIM-3,5) analogs [ 128 ]. In athymic nude mouse xenograft studies, the IC 50 values for tumor growth inhibition by the DIM-3,5 compounds were <1 mg/kg/day [ 128 ]. Subsequent studies with DIM3,5 and DIM8–3,5 analogs indicate that they bind both NR4A1 and NR4A2, and act as dual receptor inverse agonists in cancer cells and tumors to inhibit pro-oncogenic NR4A1 and NR4A2-dependent pathways and genes [ 132 ]. Figure 10. Structures of NR4A2 (DIM-4-CI), NR4A1 (DIM-4-OH) and dual NR4A1/2 second generation (DIM8–3,5) and third generation (DIM-3,5) ligands that bind both NR4A1 and NR4A2 [ 122–132 ]. Structures of NR4A2 (DIM-4-CI), NR4A1 (DIM-4-OH) and dual NR4A1/2 second generation (DIM8–3,5) and third generation (DIM-3,5) ligands that bind both NR4A1 and NR4A2 [ 122–132 ]. In addition to compounds derived from natural sources, the NR4A receptor subfamily have equally been attractive targets for the development of synthetic ligands. The following sections detail the known progress in developing ligands that specifically bind to NR4A1, NR4A2, or NR4A3. IMCA (2-imino-6-methoxy-2 h-chromene-3-carbothioamide) was first identified as a potential NR4A1 ligand through virtual screening of the Specs compounds database using the known NR4A1 crystal structure. The study indicated that IMCA bound NR4A1 at its LBD. IMCA inhibits proliferation with an IC 50 of 13.18 μM and induces apoptosis in thyroid carcinoma cells. Treatment with IMCA increased nuclear export of NR4A1 and formation of the pro-apoptotic NR4A1/Bcl-2 mitochondrial complex [ 134 ]. Compound 10E, a 5-((8-methoxy-2-methylquinolin-4-yl)amino)-1 h-indole-2-carbohydrazide derivative, was recognized as a potential NR4A1 ligand due to its anti-tumor effects in hepatocellular carcinomas. It was reported that Compound 10E, which contains both quinoline and naphthalene moieties, binds to the LBD of NR4A1 with a KD value of 2.25 μM [ 135 ]. In addition, several other structural classes of compounds are being investigated. NR4A1, NR4A2, and NR4A3 have remarkable structural similarities but different expression patterns. NR4A2 is predominantly expressed in the brain and has been implicated in several neuroinflammatory and neurodegenerative diseases like Parkinson’s disease. Among those compounds identified as NR42 ligands that impact neurotoxicities include several isoxazole pyridines and more complex pyridine derivatives, amodiaquine and chloroquine [ 81 ]. Isoxazole pyridine compounds bind to the LBD of NR4A2 with an EC 50 of 78 nM and were shown to be NR4A2 activators through a luciferase reporter assay with the promoter of tyrosine hydroxylase (TH), a gene sensitive to NR4A2 activation [ 136 , 137 ]. SA00025 (2-{3-[2-(4-chlorophenyl)imidazo[1,2-a]-pyridin-6-yl]phenyl}propan-2-ol) displayed neuroprotective effects in mouse models of Parkinson’s Disease. SA00025 increased the expression of NR4A2 and known NR4A2-modulated dopaminergic genes such as TH, dopamine active transporter (DAT) and vesicular monoamine transporter (VMAT) but evidence for binding NR4A2 has not been reported [ 138 ]. Amodiaquine (AQ) and chloroquine (CQ), both FDA approved anti-malarial drugs, and glafenine, an FDA approved pain-relief drug, were all found to bind NR4A2 and stimulate its transcriptional activity in rat models of PD [ 87 , 139 ]. Several other studies on identifying and characterizing NR4A2 ligands are ongoing. NR4A3 ligands have been studied less than NR4A1 and NR4A2 ligands despite evidence that this receptor is a potential druggable target for multiple diseases. One research group is attempting to identify NR4A3-specific ligands using a structure–activity relationship (SAR) experimental model and testing in-vitro activity. Library screening produced two hits with potential inverse agonist activities, Compounds 1 and 19, that are carboxymethyl analogs of indole-3-carbinol. These compounds have IC 50 values of 47 to 8 μM and increased mRNA levels of MYC, a gene that is transcriptionally repressed by NR4A3, in a dose-dependent manner [ 140 ]. Several FDA-approved drugs currently being used clinically for treating multiple diseases could be repositioned for treating diseases that could benefit from either inactivating or inducing NR4A ( Figure 11 ). DHE, an ergot alkaloid, is an FDA approved drug used for treating migraines and cluster headaches since 1946 [ 149 ]. DHE activates NR4A1-mediated transcriptional activity and generates an NR4A1-dependent gene signature that is anti-leukemic [ 150 ]. Nilotinib is an FDA-approved chemotherapy drug used for the treatment of chronic myeloid leukemia (CML) [ 141 ]. A recent study has identified Nilotinib as a potential therapeutic for non-small cell lung cancer (NSCLCs), specifically for nonsmoking female patients [ 142 ]. Nilotinib binds to the LBD of NR4A1, reduces cell proliferation, and increases senescence in H1975 cells in a dose-dependent manner. 6-Mercaptopurine (6-MP) is a purine biosynthesis inhibitor that is an FDA approved drug used to treat many blood cancers, such as acute childhood leukemia and CML, as well as several intestinal inflammatory diseases. 6-MP was first shown to be NR4A2 and NR4A3 agonists that bind the AF-1 domain of these receptors to mediate its transactivation function [ 143 , 144 ]. This finding provided evidence that antiproliferative and anti-cancer properties of 6-MP were due to modulation of cofactor recruitment to the AF-1 domain of these receptors [ 145 ]. Amodiaquine (AQ) and Chloroquine (CQ), which are FDA-approved anti-malarial drugs, are also NR4A2 ligands that stimulate NR4A2-dependent pathways. There is also evidence that metformin, a widely used antidiabetic drug that is also associated with other health benefits, binds near the C-terminal domain of NR4A2. Metformin-induced changes in gene expression might be NR4A1/NR4A2-dependent [ 146 , 147 ]. A recent study discovered that several FDA-approved statins, including fluvastatin, simvastatin, lovastatin, atorvastatin, and rosuvastatin exhibit NR4A2 agonist activities, induce expression of NR4A2-dependent neuroprotective genes and repress expression of pro-inflammatory genes, although NR4A2 binding has not been confirmed [ 148 ]. Thus, there is ample evidence that new NR4A ligands coupled with FDA-approved drugs are excellent candidates for treating several diseases where NR4As are druggable candidate receptors. Figure 11. Structures of clinically approved drugs that could be repositioned to target NR4A1 or NR4A2 [ 141–148 ] and in multiple diseases including cancer and non-cancer endpoints such as Parkinson’s disease. Structures of clinically approved drugs that could be repositioned to target NR4A1 or NR4A2 [ 141–148 ] and in multiple diseases including cancer and non-cancer endpoints such as Parkinson’s disease.

Rev-Erb nuclear receptors, including Rev-Erbα (NR1D1) and Rev-Erbβ (NR1D2), are key regulators of transcription, particularly in circadian rhythms, metabolism, and inflammation. These orphan receptors function as transcriptional repressors by recruiting corepressor complexes to target genes that influence lipid metabolism, glucose homeostasis, and mitochondrial biogenesis [ 51–57 ]. Endogenous ligands, including heme and other metabolic intermediates, modulate Rev-Erb activity, linking cellular metabolism to gene expression. This section explores the biochemical landscape of endogenous Rev-Erb ligands and their role in transcriptional regulation, emphasizing the integration of metabolic signals into nuclear receptor activity. The orphan receptor known as germ cell nuclear factor (GCNF), also known as NR6A1, belongs to the nuclear receptor superfamily. During embryonic development, its expression is limited to formation of the nervous system. In adulthood, however, GCNF is also present at specific stages of germ cell maturation in the ovary and testis [ 54–58 ]. This suggests that GCNF may play a role in regulating both neurogenesis and reproductive functions. Liver Receptor Homolog-1 (LRH-1), also called NR5A2, is an orphan nuclear receptor that plays a key role in controlling embryonic development, cholesterol transport, bile acid balance, and steroid production. Its functions are important in endoderm-derived tissues such as the intestine, liver, pancreas, and ovary [ 59 ]. Nuclear receptors are transcription factors that regulate gene expression in response to physiological and environmental stimuli. Unlike classical ligand-activated nuclear receptors, Rev-Erbα and Rev-Erbβ function primarily as transcriptional repressors. They bind directly to Rev-Erb response elements (RevREs) in target gene promoters and exert their effects by recruiting repressor complexes, including NCoR1 and HDAC3 [ 51 ]. Rev-Erb activity is tightly regulated by endogenous biochemicals, with heme being the most well-characterized ligand [ 52 ]. Other metabolites, including NAD+, bile acids, and ketone bodies, have also been implicated in modulating Rev-Erb-mediated transcriptional repression. Heme, an iron-containing porphyrin binds the ligand-binding domain (LBD) of Rev-Erbs, modulating their transcriptional activity [ 52 ]. The interaction between heme and Rev-Erbs enhances recruitment of NCoR1/HDAC3 to promote gene repression [ 51 ]. Additionally, heme levels fluctuate in a circadian manner, positioning Rev-Erbs as metabolic sensors that couple cellular heme availability with rhythmic transcriptional control [ 53 ]. Although NAD+ does not directly bind Rev-Erbs, it influences their activity through SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase. SIRT1 modulates circadian gene expression by deacetylating core clock proteins, indirectly affecting Rev-Erb-mediated repression [ 60 ]. The NAD+/SIRT1 axis links cellular energy status to Rev-Erb transcriptional regulation, integrating metabolic signals into gene expression networks. Emerging evidence suggests that bile acids regulate Rev-Erb activity, connecting lipid metabolism to transcriptional control [ 61 ]. Additionally, ketone bodies, such as β-hydroxybutyrate (BHB), have been implicated in modulating Rev-Erb function, particularly in metabolic adaptations during fasting and ketogenic states [ 62 ]. These interactions highlight the role of endogenous metabolites in fine-tuning Rev-Erb-mediated transcriptional repression. Retinoic acid (RA) is a key regulator of embryogenesis and germ cell development, that involves GCNF [ 54 ]. While GCNF does not directly bind RA, its expression is tightly regulated by RA signaling pathways. During embryonic development, RA signaling induces the expression of GCNF, which in turn represses Oct4 , leading to the exit from pluripotency [ 55 ]. Additionally, RA metabolites, such as 9-cis-retinoic acid and 4-oxo-retinoic acid, modulate GCNF activity indirectly by influencing its recruitment of corepressors [ 56 ]. Several orphan nuclear receptors are regulated by lipid-based ligands, raising the possibility that GCNF may interact with phospholipids, fatty acids, or cholesterol derivatives [ 57 ]. Structural analysis of GCNF’s ligand-binding domain (LBD) suggests that it could accommodate hydrophobic molecules, though direct ligand binding but this has not yet been confirmed [ 55 ]. Potential candidates include fatty acid derivatives, which modulate other nuclear receptors involved in metabolism, and sterol-based molecules, which may act as weak agonists or antagonists for GCNF-mediated transcriptional repression. Recent studies suggest that intracellular metabolites, such as NAD+ and acetyl-CoA, may influence nuclear receptor function [ 58 ]. Although no direct evidence links these molecules to GCNF activity, these metabolic intermediates could regulate its transcriptional role indirectly through post-translational modifications or cofactor recruitment. Phospholipids have been identified as possible endogenous ligands for LRH-1 ( Figure 6 ). Structural studies indicate that phosphatidylcholine and other phospholipids can occupy the ligand-binding pocket of LRH-1, stabilizing its active conformation and influencing transcriptional activity [ 63 ]. Phosphatidylcholine enhances LRH-1 transcriptional activity by stabilizing the receptor’s active conformation. Phosphatidylethanolamine may function as a weaker LRH-1 ligand, modulating its interaction with coregulators. Certain lysophospholipids, including lysophosphatidylcholine (LPC), may modulate LRH-1 activity by altering its LBD. These phospholipids facilitate the ability of LRH-1 to regulate genes involved in lipid metabolism, steroidogenesis, and cellular differentiation. Cholesterol metabolism is tightly linked to LRH-1 function, and several cholesterol derivatives and bile acids have been proposed as possible endogenous modulators of its activity [ 64 ]. Cholesterol sulfate functions as an endogenous modulator of LRH-1, potentially influencing its role in cholesterol transport and metabolism. Certain bile acids such as CDCA, LCA and DCA can interact with LRH-1 and regulate genes involved in bile acid homeostasis, suggesting a feedback mechanism in hepatic metabolism [ 65 ]. These interactions suggest that LRH-1 integrates metabolic signals from lipid and bile acid metabolism to regulate transcription. LRH-1 plays a key role in steroid hormone biosynthesis by regulating the expression of CYP11A1 , a gene required for conversion of cholesterol to pregnenolone [ 66 ]. While not direct ligands, pregnenolone and other steroid precursors are regulated by LRH-1 activity, linking LRH-1 to endocrine function. RA signaling pathways intersect with LRH-1 activity, although direct binding of retinoids to LRH-1 has not been confirmed [ 67 ]. These findings highlight the complex network of biochemical signals that influence LRH-1 function in steroidogenesis. Figure 6. Examples of endogenous LRH-1 ligands include phospholipids phosphatidyl choline and ethanolamine [ 63 ]. Examples of endogenous LRH-1 ligands include phospholipids phosphatidyl choline and ethanolamine [ 63 ]. Several synthetic ligands enhance Rev-Erb activity by mimicking heme’s effects on transcriptional repression. SR9009 and SR9011 are widely studied synthetic Rev-Erb agonists that enhance transcriptional repression ( Figure 7 ) [ 68 ]. Newer Rev-Erb agonists, such as GSK2945 and ARN5187, have demonstrated improved bioavailability and selectivity, making them promising candidates for clinical applications [ 71 ]. Although most research has focused on agonists, Rev-Erb antagonists have potential applications in restoring circadian rhythm disorders and neurodegenerative diseases. SR8278 is a selective Rev-Erb antagonist that disrupts corepressor recruitment and restores BMAL1 expression [ 72 ]. The development of potent and selective antagonists remains an area of ongoing research. Figure 7. Synthetic compounds that bind Rev-Erb, LRH-1 and GCNF. SR9009 and related compounds that bind Rev-Erb [ 68 ], RJW100, an LRH-1 agonist [ 69 ] and SLA-12 a partial GCNF agonist [ 70 ]. Synthetic compounds that bind Rev-Erb, LRH-1 and GCNF. SR9009 and related compounds that bind Rev-Erb [ 68 ], RJW100, an LRH-1 agonist [ 69 ] and SLA-12 a partial GCNF agonist [ 70 ]. Plant-derived compounds may influence the activity of GCNF [ 73 ] and synthetic ligands have been developed to enhance the repressor activity of GCNF, particularly in stem cell differentiation. GCNF-S1 (experimental agonist) is a synthetic analog designed to stabilize the repressive function of GCNF by enhancing corepressor interactions [ 74 ]. Thiazolidinedione derivatives originally developed as PPAR modulators, interact with orphan nuclear receptors, including GCNF [ 75 ]. Benzodiazepine-based compounds are experimental molecules designed to interfere with the DNA binding of GCNF, thus preventing transcriptional repression. Selective ligand analogs (SLA-12) are partial agonists that modulate GCNF activity in a tissue-specific manner [ 70 ]. These synthetic analogs provide valuable tools for studying GCNF function and its transcriptional effects. Several synthetic agonists enhance LRH-1 activity, with implications in metabolic and endocrine regulation. BL001 and BL002 are first-generation synthetic agonists that enhance LRH-1 activity by mimicking endogenous phospholipid binding [ 76 ]. These small-molecule agonists increase transcription of bile acid synthesis genes, potentially benefiting patients with metabolic disorders. RJW100 is a selective LRH-1 agonist that enhances CYP7A1 transcription, promoting bile acid synthesis [ 69 ]. This agonist demonstrates potential for treating metabolic dysfunction, including nonalcoholic fatty liver disease (NAFLD). ML-180 is a synthetic LRH-1 modulator that enhances pancreatic β-cell proliferation by activating insulin transcriptional networks and ML-80 may have applications for treating diabetes [ 77 ]. These synthetic agonists are valuable tools for studying LRH-1 function and its transcriptional effects on metabolism and endocrine signaling. Antagonists that inhibit LRH-1 activity have been explored as potential therapeutic agents in treating hormone-dependent cancers. SR1848 is a selective LRH-1 antagonist that suppresses LRH-1-driven transcriptional activation and induces apoptosis in breast and pancreatic cancer models [ 59 ]. NCoR peptide mimetics are designed to disrupt LRH-1 coactivator interactions, reducing gene activation in proliferative tissues and have potential applications for treating hormone-sensitive cancers such as breast and ovarian cancer. These findings highlight the potential of LRH-1 inhibitors in controlling aberrant transcriptional activation in cancer. Structurally diverse ligands bind Rev-Erb, GCNF and LRH-1 and despite the different scaffolds there are no FDA-approved drugs specifically targeting these receptors. This absence presents a unique opportunity for drug repositioning strategies, to identify existing drugs that can modulate these NRs for treating various diseases.

Orphan nuclear receptors clearly play essential roles in maintaining cellular homeostasis and pathophysiology and there is clear evidence that these receptors bind structurally diverse ligands and some of these chemicals bind more than one orphan NR. It is likely that future studies will identify functional endogenous ligands for some orphan NRs and convert them into adopted orphans. It is questionable whether the new “endogenous” ligands for orphan NRs will exhibit nM K D values similar to that observed for ligands binding the steroid hormone NRs. It is probable that the functionality of these lower affinity endogenous ligands will be highly cell-type specific. Based on the increasing sophistication of methods for screening chemical libraries for receptor ligands, the number of active compounds and scaffolds for subsequent chemical modifications will also increase in the future. This will ensure that the current disappointing number of clinical applications of orphan NR ligands will increase and be available for targeting these receptors as clinically acceptable agents for treating multiple diseases.

The Retinoid-Related Orphan Receptors (RORs) are a family of orphan nuclear receptors, with no known endogenous ligands. Comprised of three members: RORα, RORβ, and RORγ (NR1F1–3), the RORs exhibit modular structures with highly conserved DNA-binding domains and moderately conserved ligand-binding domains, which is common amongst all NRs, that primarily differ at the amino terminus [ 30 ]. Each of the RORs bind as monomers to ROR response elements (ROREs) and are known to interact with various coactivators and corepressors [ 30 ]. Despite these similarities, ROR isoforms exhibit a unique tissue expression patterns, resulting in regulatory roles in vastly different biological processes. There are no ROR-specific drugs with FDA-approval, however development of ROR-specific ligands is ongoing and promising. RORα, RORβ, and RORγ are key regulators of circadian rhythms, metabolism, and immune responses through the transcriptional regulation of specific genes. RORα regulates circadian rhythms by positively influencing BMAL1 and competing with REV-ERBα at their shared binding site, while also playing a role in lipid and glucose metabolism by activating Cyp7b1, G6Pase, and FGF21 [ 31–35 ]. In RORα knockout mice, decreased Cyp7b1 and impaired glucose metabolism were observed, suggesting its involvement in metabolic diseases [ 31 ]. RORγ, expressed primarily in immune tissues, regulates immune responses by promoting the differentiation of CD4+ cells into TH17 cells, which secrete cytokines like IL-17, IL-21, and IL-22 [ 36–38 ]. RORγ −/− mice displayed impaired TH17 differentiation, while a double knockout of RORα and RORγ resulted in resistance to autoimmune diseases, highlighting the potential of targeting these receptors in, autoimmune disorders [ 39–43 ]. These findings suggest RORα and RORγ as promising drug targets for metabolic and immune-related diseases. Despite their accepted status as orphan nuclear receptors, recent studies have implicated several endogenous compounds as potential high-affinity ligands for RORs ( Figure 5 ). Most notable of these compounds are the oxygenated sterols, which are also ligands of other NRs including the liver X receptors (LXRs). Specifically, the 7-oxygenated sterols: 7α-OHC (7α-hydroxycholesterol), 7β-OHC, and 7-ketocholesterol, serve as inverse agonists for both RORα and RORγ and inhibit gene transcription and expression of their downstream gene products in a receptor-dependent manner [ 44 ]. An additional potential endogenous inverse agonist of RORα and RORγ is 24S-hydroxycholesterol (24S-OHC), and similar to the 7-oxygenated sterols it exhibits receptor-dependent and dose-dependent inhibition of downstream gene products, notably those involved in circadian rhythms [ 45 ]. There are also several RORγ-specific potential endogenous ligands including: 24S,25-epoxycholesterol (24,25-epoC) and 24 R-cholesterol (24 R-OHC), which exhibit inhibitory effects similar to the 7-oxygenated sterols and 24S-OHC [ 45 ]. RORγ also has several putative ligands, but these are not yet validated as RORγ ligands and are only known to affect their co-effector peptides [ 46 ]. Figure 5. Endogenous and synthetic molecules that bind ROR include hydroxylated steroidal compounds [ 43 , 44 ] and LY-55716 [ 45 ]. Endogenous and synthetic molecules that bind ROR include hydroxylated steroidal compounds [ 43 , 44 ] and LY-55716 [ 45 ]. There are several RORγ-specific synthetic ligands in active clinical trials, however development of a RORα and/or RORβ inhibitors is limited to the preclinical setting. The first of the RORγ-specific synthetic ligands is LYC-55716 that is designed for use in cancer ( Figure 5 ) [ 47 ]. Activation of T-cells in the tumor microenvironment has been speculated as a potential target for cancer therapies and since LYC-55716 is an RORγ agonist, induces TH17 cell differentiation and has proceeded to Phase1/2 clinical trials for the treatment of solid tumors [ 48 ]. The remaining synthetic ligands in Phase1/2 clinical trials are all RORγ-specific inverse agonists for the treatment of autoimmune disorders due to their inhibition of IL-17 expression [ 49 , 50 ]. The majority of these compounds are being used for the treatment of several forms of psoriasis. Although the hydroxylated steroids have similar structures, there are considerable differences with the structure of the synthetic LYC-55716 RORγ ligand.