International Journal of Molecular Sciences Review

ReviewRetinoid X Receptor Antagonists

RetinoidMasaki Watanabe X and Receptor Hiroki KakutaAntagonists *

Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Masaki Watanabe and Hiroki Kakuta * ID Dentistry and Pharmaceutical Sciences, 1-1-1, Tsushima-naka, Kita-ku, Okayama 700-8530, Japan; [email protected] of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical* Correspondence: Sciences, [email protected]; 1-1-1, Tsushima-naka, Kita-ku, Tel.: +81-(0)86-251-7963; Okayama 700-8530, Fax: Japan; +81-(0)86-251-7926 [email protected] * Correspondence: [email protected]; Tel.: +81-(0)86-251-7963; Fax: +81-(0)86-251-7926 Received: 12 June 2018; Accepted: 7 August 2018; Published: 10 August 2018


Received: 12 June 2018; Accepted: 7 August 2018; Published: 10 August 2018
 Abstract: Retinoid X receptor (RXR) antagonists are not only useful as chemical tools for biological Abstract:research, butRetinoid are also X receptorcandidate (RXR) drugs antagonists for the trea aretment not of only various useful diseases, as chemical including tools fordiabetes biological and research,allergies, butalthough are also no candidate RXR antagonist drugs for has the yet treatment been approved of various for diseases, clinical use. including In this diabetes review, andwe allergies,present a although brief overview no RXR antagonistof RXR structure, has yet been function, approved and for target clinical genes, use. Inand this describe review, we currently present aavailable brief overview RXR antagonists, of RXR structure, their structural function, classification, and target genes, and their and evaluation, describe currently focusing available on the latest RXR antagonists,research. their structural classification, and their evaluation, focusing on the latest research.

Keywords: Retinoid X receptor; RXR; ligands; modulators; antagonists; structural classification; classification; heterodimers; non-permissive; permissive; tRXR

1. Introduction Retinoid X receptors (RXRs; NR2B1–3) are nuclear receptors that function either as homodimers or as as heterodimers heterodimers with with other other receptors receptors such suchas peroxisome as peroxisome proliferator-activated proliferator-activated receptors receptors (PPARs; (PPARs;NR1C1–3), NR1C1–3), liver X receptors liver X (LXRs; receptors NR1H2–3), (LXRs; or NR1H2–3), farnesoid orX receptor farnesoid (FXR; X receptor NR1H4), (FXR; and others NR1H4), [1– and3]. RXR others heterodimers[1–3]. RXR that heterodimers can be activated that can by be activatedRXR agonists by RXR alone agonists are known alone as are permissive known as permissiveheterodimers heterodimers [4]. 9-cis-Retinoic [4]. 9-cis acid-Retinoic (1, Figure acid 1) (1 ,is Figure a potent1) is natural a potent agonist natural toward agonist RXRs, toward but RXRs, also butworks also as worksan activator as an activatorof retinoic of acid retinoic receptors acid receptors(RARs) [5]. (RARs) The RXR [5]. synthetic The RXR agonist synthetic bexarotene agonist bexarotene(LGD1069, (LGD1069,Targretin®, Targretin2, Figure® 1), 2 ,is Figure used 1for) is the used treatment for the treatment of cutaneous of cutaneous T cell lymphoma T cell lymphoma (CTCL) (CTCL)[6], but, [on6], the but, other on the hand, other no hand,RXR antagonist no RXR antagonist has yet entered has yet clinical entered use, clinical even though use, even anti-type though 2 anti-typediabetes [7] 2 diabetes and anti-allergy [7] and anti-allergy activities activities[8] have [been8] have found been in found animal in animalmodels. models. At present, At present, RXR RXRantagonists antagonists are mainly are mainly employed employed as analytical as analytical tools toolsin studies in studies of RXR of RXRfunction. function. In this In thisreview, review, we wewill will first first present present a brief a brief overview overview of ofthe the RXR RXR structure, structure, function, function, an andd target target genes, genes, and and then then describe currently available RXR antagonists, their structural classification,classification, and their evaluation, focusing on the latest research.

Me Me Me Me Me Me

CH2 Me Me Me

Me

CO2H 9-cis Retinoic acid (1) Bexarotene (2) CO2H

Figure 1. Chemical structures of 9- cis-retinoic acid (1) and bexarotene (2).).

2. The RXRs Among the 48 members of the nuclear receptor superfamily that have identified by sequence alignmentInt. J. Mol. Sci. and 2018, phylogenetic 19, x; doi: tree construction [3,9], 24 are ligand-binding receptors. www.mdpi These.com/journal/ijms include

Int. J. Mol. Sci. 2018, 19, 2354; doi:10.3390/ijms19082354 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 2354 2 of 20 three different subtypes of RXR, i.e., RXRα, RXRβ, and RXRγ, which are encoded by distinct genes [3] (Table1). Historically, these receptors have been named after their ligands. However, the Nuclear Receptor Nomenclature Committee has recommended a systematic nomenclature based on genome analysis [3]. Thus, RXRα is designated as NR2B1, RXRβ as NR2B2, and RXRγ as NR2B3; in this review, we retain the established nomenclature. Each RXR has two isoforms: RXRα1/α2, RXRβ1/β2, and RXRγ1/γ2 [10]. RXRα exists in liver, lung, muscle, kidney, epidermis (major subtype), and intestine, while RXRβ is distributed ubiquitously. On the other hand, RXRγ1 is expressed in the brain and muscle, while RXRγ2 is highly expressed in cardiac and skeletal muscles [10]. Most research has so far been focused on RXRα; one reason for this may be that the functions of the RXR subtypes are the same, even though their distributions are different. RXRα was the first RXR subtype to have its structure determined by X-ray crystallography [11]. RXRs, like other nuclear receptors, consist of six domains A, B, C, D, E, and F (Figure2) [ 3]. The N-terminal A/B region has a transcriptional activation function and is referred as AF-1 (Figure2, Table1). AF-1 can operate in a ligand-independent/dependent manner; i.e., it is controlled by ligand binding to the ligand-binding domain (LBD) (Figure2, Table1) in the full-length receptor, but when located outside of the receptor, it acts in a ligand-independent manner [3]. Next, the C domain acts as a DNA-binding domain (DBD) (Figure2, Table1), which contributes to the response element specificity for recognition of the target gene. Dimerization of RXR with itself or a heterodimer partner is caused by strong interactions between the LBDs of the interacting partners, as well as binding of the two DBDs. RXR homodimers bind to RXR response elements (RXREs) composed of a direct repeat of hexad half-sites (A/G)G(G/T)TCA separated by one nucleotide as a spacer (DR-1 element, direct repeat with 1 nucleotide) (Table2). RXR heterodimers also bind preferentially to specific hormone response elements (HREs), which are composed of two hexad half-sites arranged as tandem repeats. The specificity of each dimer for the target DNA is based not only on the DNA sequences of the two half-sites, but also on the geometry, spacing, and relative orientation of the half-sites in the HRE [12–15]. Other response elements with different numbers of spacer nucleotides, DR2, DR3, DR4, DR5, and others, also exist. The RXR LBD can adopt multiple conformations, providing the dimerization domain with sufficient flexibility to occupy the partner receptor [14,15]. Interestingly, RXRs can form RXR tetramers with high affinity at protein concentrations higher than about 70 nM [16]. Noy and collaborators presented evidence that binding of the apo-RXR homotetramer to two RXREs, which were separated by 250 base-pairs in a 382 base-pair sequence, permitted transcriptional regulation by DNA-looping [17]. The reason why one RXR homotetramer can bind to two different RXREs is that their DBDs are exposed. Indeed, the inhibition of mammary carcinoma cell growth by RXR ligands stems from the ability of these compounds to regulate the oligomeric state of RXR, and is independent of the direct intrinsic transcriptional activity of the receptor [18]. Compounds that trigger dissociation of RXR tetramers may comprise a novel class of anti-carcinogenic agents. Target genes of RXR heterodimers are dependent on the identity of the heterodimer partner. On the other hand, in the case of RXR homodimers, the DBDs bind to natural DR1 elements for the calcitonin receptor activity-modifying protein 2 (Ramp2), the NR subfamily 1, group D, member 1 (Nr1d1) and the glycerophospho-diester phosphodiesterase 1 (Gde1) genes, as well as the malic enzyme PPRE gene (MEp)[15,19,20]. Since both RXR and PPAR bind to DR1, RXR homodimers can bind not only RXRE, but also PPRE [20]. Domain D acts as the binder and cushion of domains C and E. Domains E/F are referred as the ligand-binding domain (LBD) (Table1, Figure2). The LBD contains four structurally distinct, but functionally linked surfaces: (1) a dimerization surface with a partner; (2) the ligand-binding pocket (LBP) for lipophilic small molecules; (3) a co-regulator binding surface; and (4) a ligand-dependent activation function helix 12 (termed AF-2) (Figure2, Table1)[ 3]. Activation of RXRs occurs when an agonist binds to the LBP and induces a conformational change in the LBD [21]. The resulting conformation allows recruitment of co-regulatory complexes, which contain chromatin-modifying enzymes required for transcription, RNA polymerase II, and general transcription factors [22,23]. RXR heterodimers with PPARs, LXRs, or FXR, which can be activated by RXR agonists alone, are known Int. J. Mol. Sci. 2018, 19, x 3 of 19 Int. J. Mol. Sci. 2018, 19, 2354 3 of 20 enzymes required for transcription, RNA polymerase II, and general transcription factors [22,23]. RXR heterodimers with PPARs, LXRs, or FXR, which can be activated by RXR agonists alone, are as permissive heterodimers [4]. In contrast, RXR heterodimers with RAR or TRs cannot be activated known as permissive heterodimers [4]. In contrast, RXR heterodimers with RAR or TRs cannot be by RXR agonists alone, and are termed non-permissive. The difference between permissive and activated by RXR agonists alone, and are termed non-permissive. The difference between permissive non-permissive heterodimers arises from the strong constitutive interaction between the unliganded and non-permissive heterodimers arises from the strong constitutive interaction between the non-permissive hetero partner and co-repressors [24]. Unliganded permissive heterodimer partners, unliganded non-permissive hetero partner and co-repressors [24]. Unliganded permissive such as PPAR or LXR, do not have a strong constitutive interaction with co-repressors [25], so their heterodimer partners, such as PPAR or LXR, do not have a strong constitutive interaction with co- RXR heterodimers can be activated by an RXR agonist alone. repressors [25], so their RXR heterodimers can be activated by an RXR agonist alone.

Figure 2. SchematicSchematic illustration of the nuclear receptor heterodimer PPARγγ/RXR/RXRα and its co-activator co-activator (NCoA-2: nuclear nuclear receptor receptor co-activator co-activator 2) 2) bound bound to to DNA. DNA. This This figure figure was created using PDB coordinates from [13] [13] (pdb: (pdb: 3DZY). 3DZY). RXR RXR is is shown shown in in red. red. The The X-ray X-ray data data was was obtained obtained using using only only a apart part of of N-CoA2, N-CoA2, consisting consisting of of EKHKILHRLLQDSY. EKHKILHRLLQDSY. (A (A)) View View from from the the side. side. The The bar bar at at the the bottom is a schematic illustration of the general domain structure of nuclear receptors; (B) View from thethe 30′-end of the DNA. RXR is shown as a CPK model in red.

Small molecules or compounds that bind reversibly to nuclear receptors into the C-terminal ligand-binding pocketpocket (LBP)(LBP) areare defineddefined asas “nuclear“nuclear receptorreceptor ligands”ligands” [[3].3]. Due to the ability to alter activity of the receptors, these are often termed “r “receptoreceptor modulators” [[26].26]. However, since there are small moleculesmolecules that that bind bind to ato different a different site from site thefrom LBP, the the LBP, definition the definition of “nuclear of receptor “nuclear modulators” receptor shouldmodulators” be broadened should asbe compoundsbroadened thatas compounds bind to nuclear that receptors bind to (Tablenuclear1). receptors Nuclear receptor (Table 1). ligands Nuclear are classifiedreceptor ligands into three are categories; classified agonists,into three inverse categories agonists,; agonists, and antagonists inverse agonists, (Table1 and). Agonists antagonists are defined (Table as1). compoundsAgonists are that defined bind as to compounds the LBP and that activate bind the to receptor.the LBP and Inverse activate agonists the receptor. are compounds Inversethat agonists bind toare the compounds LBP and result that bind in a conformational to the LBP and change result thatin a reducesconformational the basal change level of that activity reduces (reduces the basal co-activatorlevel of activity binding). (reduces In contrast, basal co-activator Antagonists binding). simply bindIn contrast, to the LBD Antagonists and prevents simply the bind conformational to the LBD changeand prevents that an the agonist conformational would cause, change thus preventing that an ag co-activatoronist would recruitment cause, thus and preventing subsequent co-activator stimulation ofrecruitment transcription. and The subsequent definition stimulat of otherion terms of istranscription. listed in Table The1. definition of other terms is listed in TableRXR 1. antagonists interfere with the binding of RXR agonists to RXRs. Although some subtype-preferentialRXR antagonists agonistsinterfere and with antagonists the binding have of RXR been agonists reported to [ 27RXRs.–29], Although their selectivities some subtype- are not sufficientpreferential to agonists allow their and useantagoni as pharmacologicalsts have been reported tools [30 [27–29],]. The their main selectivities reason for are the not difficulty sufficient in developingto allow their highly use as selective pharmacological RXR ligands tools may [30]. be thatThe themain amino reason acid for residues the difficulty of helices in developing (H) 3, 5, 7, andhighly 11, selective and the β RXR-turn, ligands which formmay be the that ligand-binding the amino acid pocket, residues are highly of helices conserved (H) 3, in 5, RXR 7, andα, β 11,, and andγ. the β-turn, which form the ligand-binding pocket, are highly conserved in RXRα, β, and γ.

Int. J. Mol. Sci. 2018, 19, 2354 4 of 20

Table 1. Definitions of terms and abbreviations concerning nuclear receptors (adapted and modified from [3]).

Term Definition/Description/Examples Activation function-1. AF-1 consists of the N-terminal region (domains A/B), which can operate autonomously. This region can interact with cofactors such as AF-1 co-activators or other transcription factors. The activation is independent of ligand binding. The activity of AF-1 is regulated by growth factors acting through the MAP kinase pathway. Activation function-2. AF-2 is the C-terminal helix 12, located in domain E, which AF-2 mediates ligand-dependent transactivation. DNA-binding domain. Domain C of nuclear receptors. This region binds to DBD a specific DNA sequence, called the hormone response element (HRE). Ligand-binding domain. Domain E of nuclear receptors. The LBD contains (1) LBD a dimerization surface, which mediates interaction with partner LBDs; (2) the LBP; (3) a co-regulator binding surface, and 4) an activation function helix, termed AF-2. Ligand-binding pocket (LBP), which interacts with small molecules. The LBP is LBP generally located behind helix 3 and in front of helices 7 and 10, and is lined with mostly hydrophobic amino acids. Ligands for NRs Compounds that bind reversibly to NRs at the C-terminal LBP. Agonists Ligands that induce an active conformation of the receptor. Inverse agonists Ligands that can promote co-repressor recruitment. Ligands that produce a conformation and an action of the receptor distinct from Antagonists that produced by an agonist. Agonists that in a given tissue, under specific conditions, cannot elicit as large an effect (even when applied at high concentration, so that all the receptors should Partial agonists be occupied), as can another agonist acting through the same receptors in the same tissue. NR modulators Compounds that bind to NRs, which include ligands, SNuRMs, and SNuRDs [26]. Orthosteric modulators Compounds that bind to the same site of endogenous ligands. Compounds that bind to the different site of endogenous ligands. The term “allo-” Allosteric modulators means “other”. Positive allosteric Allosteric modulators that induce an amplification of the effect of the modulators (PAMs) primary ligand. Negative allosteric Allosteric modulators that reduce the effect of the primary ligand. modulators (NAMs) Silent allosteric Allosteric modulators that occupy the allosteric binding site and behave modulators (SAMs) functionally neutral; also called neutral or null modulators. Selective nuclear receptor modulators. Selective ligands with partial function-, cell-, SNuRMs and/or promoter-specific action. Selective nuclear receptor Down-regulators. Compounds that cause NR to be SNuRDs degraded and thus down-regulated. A subclass of antagonists. Fluvestrant is a selective estrogen receptor down-regulator (SERD) [31]. Selective agonists Ligands with an affinity difference (preferably greater than 100-fold) between their and antagonists primary target and other receptors. Int. J. Mol. Sci. 2018, 19, 2354 5 of 20

Table 2. Classification and nomenclature of nuclear receptors (adapted and modified from [32]). Int. J. Mol. Sci. 2018, 19, x 5 of 19 Int. J. Mol. Sci. 2018, 19, x 5 of 19

Table 2. ClassificationName and nomenclature Subtypes of Nomenclaturenuclear receptors (adapted Sequence and modified References from [32]). Table 2. Classification and nomenclature of nuclear receptors (adapted and modified from [32]). α NR1A1 [33] NameTR Subtypes Nomenclature SequenceDR4 References Name Subtypesβ NomenclatureNR1A2 Sequence References [ 33] α NR1A1 [33] TR αα NR1A1NR1B1 DR4 [33] [33–35] TR β NR1A2 DR4 [33] RAR ββ NR1A2NR1B2 DR2, DR5 [33] [33] α γ NR1B1NR1B3 [33–35] [33,36] α NR1B1 [33–35] RAR β NR1B2 DR2, DR5 [33] RAR βα NR1B2NR1C1 DR2, DR5 [33] [33] PPAR γ β/δ NR1B3NR1C2 DR1 [33,36] [33–35] γ NR1B3 [33,36] α γ NR1C1NR1C3 [33] [33–36] α NR1C1 [33] PPAR β/δ α NR1C2NR1H1 DR1 [33–35] [33–36] PPARLXR β/δ NR1C2 DR1DR4 [33–35] γ β NR1C3NR1H2 [33–36] [33–36] γ NR1C3 [33–36] VDRα NR1H1 NR1I1 DR3[33–36] [33–36] LXR α NR1H1 DR4 [33–36] LXR β NR1H2 DR4 [33–36] PXRβ NR1H2 NR1I2 DR3–5[33–36] [37] VDR  NR1I1 DR3 [33–36] LXRVDR α NR1I1NR1H1 DR3 DR4 [33–36] [33–36] PXR  NR1I2 DR3–5 [37] FXRPXR  NR1I2 NR1H4 DR3–5 IR1 *[37] [38] LXR α NR1H1 DR4 [33–36] LXR αα NR1H1NR2B1 DR4 [33–36][ 33–36] FXR NR1H4 IR1 * [38] RXRFXR β NR1H4NR2B2 IR1 *DR1 [38] [33–36] α NR2B1 [33–36] αγ NR2B1NR2B3 [33–36] [33–36] RXR β NR2B2 DR1 [33–36] Nur77RXR β NR2B2 NR4A1 DR1 DR5 [33–36] [36 ,39–41] γ NR2B3 [33–36] Nurr1γ NR2B3 NR4A2 DR5[33–36] [36,40] Nur77 NR4A1 DR5 [36,39–41] Nur77 NR4A1* IR Inverted repeat.DR5 [36,39–41] Nurr1 NR4A2 DR5 [36,40] Nurr1 NR4A2 DR5 [36,40] * IR Inverted repeat. 3. Representative RXR Antagonists * IR Inverted repeat. 3. Representative RXR Antagonists 3. RepresentativeRXR antagonists RXR are Antagonists classified into three categories; (1) compounds having a long-chain alkoxy RXR antagonists are classified into three categories; (1) compounds having a long-chain alkoxy group introducedRXR antagonists to an are RXR classified agonist into structure three catego as a scaffoldries; (1) compounds (Table3); (2) having compounds a long-chain possessing alkoxy another group introduced to an RXR agonist structure as a scaffold (Table 3); (2) compounds possessing side-chaingroup introduced group instead to an RXR of the agonist alkoxy structure group as introduced a scaffold (Table to an RXR3); (2) agonist compounds structure possessing as a scaffold another side-chain group instead of the alkoxy group introduced to an RXR agonist structure as a (Tableanother4); and side-chain (3) compounds group instead discovered of the alkoxy from gr amongoup introduced natural productsto an RXR or agonist by docking structure simulation as a or scaffold (Table 4); and (3) compounds discovered from among natural products or by docking high-throughputscaffold (Table screening4); and (3) (Table compounds5). The discovered common structure from among of RXR natural agonists products is composed or by docking of three parts: simulation or high-throughput screening (Table 5). The common structure of RXR agonists is a hydrophobicsimulation or moiety high-throughput composed ofscreening a tetramethyltetraline (Table 5). The structure, common anstructure acidic moietyof RXR composed agonists is of trienoic composed of three parts: a hydrophobic moiety composed of a tetramethyltetraline structure, an composed of three parts: a hydrophobic moiety composed of a tetramethyltetraline structure, an acid,acidic benzoic moiety acid,composed nicotinic of trienoic acid, or acid, pyrimidinecarboxylic benzoic acid, nicotinic acid, acid, and or apyrimidinecarboxylic linking moiety between acid, the two. acidic moiety composed of trienoic acid, benzoic acid, nicotinic acid, or pyrimidinecarboxylic acid, and a linking moiety between the two. andTable a linking 3. Chemical moiety between structures, the binding two. affinities, and RXR antagonistic activities of RXR antagonists havingTable 3. an Chemical alkoxy structures, side chain binding on an RXRaffinities, agonistic and RXR scaffold. antagonistic activities of RXR antagonists Table 3. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists having an alkoxy side chain on an RXR agonistic scaffold. having an alkoxy side chain on an RXR agonistic scaffold. Compounds Structures Binding Transactivity (RXRα) Ref. Compounds Structures Binding Transactivity (RXRα) Ref. Compounds Structures Binding Transactivity (RXRα) Ref.

KKi i= = 3 nM IC50 = 16 nM Ki = 333 nM IC50 = 16 nM (RXR(RXRαα,[, [ H]11)) IC50 = 16 nM LG100754 (3 (3) ) (RXRα, [3H]1) (vs. 32(vs. nM 32 2, nM 2, [42] [42] LG100754 (3) KKi i= = 8 nM (vs. 32 nM 2, [42] Ki = 8 nM CV-1 CV-1cells) cells) (RXR(RXRαα,[, [33H]22)) CV-1 cells) (RXRα, [3H]2)

Me Me Me Me O Me O Me Me Me Me Me AGN195393 (4) Me N.D. N.D. [43] AGN195393AGN195393 ( 4(4)) Me N.D.N.D. N.D. N.D. [43] [43] Me Me

CO2H CO2H

Int. J. Mol. Sci. 2018, 19, 2354 6 of 20

Table 3. Cont.

Int. J. Mol. Sci. 2018, 19, x 6 of 19 Int. CompoundsJ. Mol. Sci. 2018, 19, x Structures Binding Transactivity (RXR6α of) 19 Ref. Int. J. Mol. Sci. 2018, 19, x 6 of 19 Int. J. Mol. Sci. 2018, 19, x 6 of 19 Int. J. Mol. Sci. 2018, 19, x 6 of 19 Int. J. Mol. Sci. 2018, 19, x 6 of 19 Int. J. Mol. Sci. 2018, 19, x 6 of 19

KKi i= = 0.90.9 nM Ro26-5405 (5) i N.D. [43,44] Ro26-5405 (5) K = 0.9 nM33 N.D. [43,44] Ro26-5405 (5) (RXR(RXRKi = α0.9α,[, [nMH] 22)) N.D. [43,44] Ro26-5405 (5) (RXRα, [3H]2) N.D. [43,44] Ki = 0.9 3nM Ro26-5405 (5) (RXRKi =α 0.9, [ H]nM2 ) N.D. [43,44] Ro26-5405 (5) 3 N.D. [43,44] (RXRKi = α0.9, [ nM3H]2 ) Ro26-5405 (5) (RXRαK,i =[ H]0.92 nM) N.D. [43,44] Ro26-5405 (5) (RXRα, [3H]2) N.D. [43,44] 3 (RXRα, [ H]2)

Ki = 3 nM i KKi = 3 3 nM nM3 (RXRKi = α3 ,nM [ H] 1) IC50 = 8 nM LG101506 (6) (RXRα, [33H]1) IC50 = 8 nM [43,45] (RXRKi = 3,[ nM3 H] 1) LG101506LG101506 (6 ()6 ) (RXRKKii α== , 3 3[ nMnMH]1 ) ICIC(CV-150 = 8 8cells) nM nM (CV-1[43,45] cells) [43,45] i 3 50 LG101506 (6) (RXRKKii =α 3, 3 [nM nMH]3 1) (CV-1IC50 = 8cells) nM [43,45] (RXRKi = α3, nM[3 H] 2) LG101506 (6) (RXRK =α 3, nMi[3 H] 1) (CV-1IC50 = cells)8 nM [43,45] (RXRαK, [ 33=H] 32 nM) LG101506 (6) (RXR(RXRKi =α 3,,[ [nM3H]H] 12) ) (CV-1IC50 = 8cells) nM [43,45] (RXRKi α= ,3 [ nMH]2 3) (CV-1 cells) LG101506 (6) (RXR3 α, [ H]1) IC50 = 8 nM [43,45] LG101506 (6) (RXRKi =α 3, [nM3H] 2) (CV-1 cells) [43,45] (RXRαK, i[ =H] 3 2nM) (CV-1 cells) (RXRα, [3H]2) (RXRα, [3H]2)

Ki = 9.9 nM IC50 = 10.3 nM 7 i 50 [46] K = 9.9 nM3 IC = 10.3 nM 7 (RXRKKi = 9.9α, [nMH] 1) IC(CV-150 = 10.3 cells) nM [46] 7 (RXRi =α 9.9, [3H] nM1) (CV-1IC cells)50 = 10.3 nM [46] 7 Ki = 9.9 33nM IC50 = 10.3 nM [46] 7 (RXR(RXRKi =α 9.9,,[ [ H]nMH]1 )1 ) IC(CV-150 = 10.3 (CV-1cells) nM cells) [46] 7 3 [46] (RXRKi = α9.9, [ nM3H]1 ) IC(CV-150 = 10.3 cells) nM 7 (RXRαK,i =[ H]9.91 nM) (CV-1IC50 cells) = 10.3 nM [46] 7 (RXRα, [3H]1) (CV-1 cells) [46] 3 (RXRα, [ H]1) (CV-1 cells)

Ki = 3 nM IC50 = 8 nM 8 i 50 [47] K = 3 nM3 IC = 8 nM 8 (RXRKi = α3 ,nM [ H] 1) IC(CV-150 = 8 cells) nM [47] 8 (RXRα, [3H]1) (CV-1 cells) [47] KKii = 3 3 nM3 nM IC50 = IC8 nM50 = 8 nM 88 (RXRKi α= ,3 [ nMH]1 ) (CV-1IC50 = cells)8 nM [47] [47] 8 33 [47] (RXR(RXRKi =α 3,,[ [nM3H]H] 11) ) (CV-1IC50 = (CV-18cells) nM cells) 8 (RXRαK, i[ =H] 3 1nM) (CV-1IC cells)50 = 8 nM [47] 8 (RXRα, [3H]1) (CV-1 cells) [47] (RXRα, [3H]1) (CV-1 cells)

PA451 (9a) PA451 (9a) N.D. N.D. [48] PA451R = n -Pen(9a) N.D. N.D. [48] PA451R = n-Pen (9a) N.D. N.D. [48] PA451RPA451 = n-Pen (9a (9a ) ) N.D. N.D. [48] PA451R = n-Pen (9a) N.D.N.D. N.D. N.D.[48] [48] R = = PA451nn-Pen-Pen (9a) N.D. pAN.D.2 = 7.11 [48] PA452R = n-Pen (9b ) N.D. pA2 = 7.11N.D. [48] PA452R (=9b n-Pen) N.D. (vs. NEt-TMN:pA2 = 7.11 EC 50 = 5.28 [48,50] [48,50] PA452R = n -Hex(9b) N.D. (vs. NEt-TMN:pA2 = 7.11 EC 50 = 5.28 [48,50] R = n-Hex N.D. (vs. nMNEt-TMN: [49],2 COS-1 EC50 cell) = 5.28 [48,50] PA452 (9b) nM [49],pA COS-1 = 7.11 cell) RPA452 = n-Hex (9b ) N.D. (vs. NEt-TMN:pA2 = p7.11 AEC 50= = 7.11 5.28 [48,50] R = n-Hex N.D. (vs.nM NEt-TMN: [49], COS-1 2EC2 cell)50 = 5.28 [48,50] PA452PA452 (9b (9b) ) nM [49], COS-1pA = 7.11 cell) R = PA452n-Hex (9b) N.D. (vs. NEt-TMN:(vs. NEt-TMN: EC50 = 5.28 EC [48,50]50 = RR = = nn-Hex-Hex Kd = 26N.D. nM nM(vs. [49], NEt-TMN: COS-1 cell)EC50 = 5.28 [48,50] R = n-Hex Kd = 26 nM nMIC [49],50 = COS-11,100 nM cell) 5.28nM nM[49], [COS-149], COS-1 cell) cell) Bl-1003 (10a) (RXRα-LBD,K dfluorescence = 26 nM titration) IC50 = 1,100 nM Bl-1003 (10a) (RXRα-LBD,K fluorescenced = 26 nM titration) (vs.IC 150 @ = 0.1 1,100 M,nM CV -1 [51,52] X = O, Y = CH Kd = 26 nM (vs. 1 @ 0.1  M, CV-1 [51,52] Bl-1003 (10a) (RXRα-LBD,IC fluorescence50 = 46 nM titration) IC50 = cells)1,100 nM X = O, Y = CH K50d = 26 nM (vs. IC1 @50 0.1= 1,100 M, nM CV -1 [51,52] Bl-1003 (10a) (RXRα-LBD,IC fluorescence = 46 nM3 titration) cells) XBl-1003 = O, Y =(10a CH) (RXRα-LBD,(RXR fluorescence50α-LBD,Kd = 26 [ H]nM1 titration)) (vs.IC 1 50@ =0.1 1,100  M, nM CV -1 [51,52] ICK = = 2646 nM 3 cells) XBl-1003 = O, Y (=10a CH) (RXRα-LBD,(RXR fluorescenceαd -LBD, [ H]1 titration)) (vs. 1 @IC 0.150 = 1,100 M, CV nM-1 [51,52] IC50 = 46 nM3 cells) X = O,Bl-1003 Y = CH (10a ) α(RXR(RXRα-LBD,ICαd50-LBD, = fluorescence46 nM[ H] 1) titration)(vs. 1 @ 0.1 M, CV-1 [51,52] Bl-1003 (10a) (RXR -LBD,K fluorescence = 329 nM3 titration) cells)IC50 = 1100 nM X = O, Y = CH (RXRdα-LBD, [ H]1) (vs. 1 @ 0.1 M, CV-1 [51,52] ICK 50= =329 46 nMnM3 IC50 ≥cells) 10,000 nM [51,52] XBl-1005 =X O, = YO, (10b= Y CH=) CH (RXRα-LBD,(RXRICKd50 αfluorescence=-LBD, IC329=50 46 nM= nM[46H] nM1 )titration) (vs.IC50 ≥1 10,000@ 0.1 µnMM, CV-1 cells) (RXRα-LBD, [3H]1) (vs. 1 @ 0.1 cells) M, CV -1 [51,52] Bl-1005 (10b) (RXRα-LBD,K fluorescenced = 329 nM3 titration)3 IC50 ≥ 10,000 nM X = O, Y = N (RXRK(RXRαd -LBD,= 329α-LBD, nM [ H] [ 1H]) 1) (vs. 1 @ 0.1  M, CV-1 [51,52] Bl-1005 (10b) (RXRα-LBD,IC fluorescence50 = 1200 nM titration) IC50 ≥ 10,000cells) nM X = O, Y = N K50d = 329 nM (vs.IC 1 50@ ≥0.1 10,000 M, nM CV -1 [51,52] Bl-1005 (10b) (RXRα-LBD,IC fluorescence = 1200 nM3 titration) cells) XBl-1005 = O, Y ( =10b N ) (RXRα-LBD,(RXR50 fluorescenceα-LBD,Kd = 329 [ H] nM1 titration)) (vs.IC 150 @ ≥ 0.1 10,000  M, nM CV -1 [51,52] ICKd = 1200 329 nMnM3 cells) Bl-1005X = O, Y ( 10b= N) (RXRα-LBD,(RXR fluorescenceα-LBD, [ H]1 titration)) (vs. 1 @IC 0.150 ≥ 10,000 M, CV nM-1 [51,52] IC50 = 1200 nM3 cells) Bl-1005X = Bl-1005O, Y (=10b N (10b ) ) (RXRα(RXR-LBD,(RXRαIC-LBD,50 fluorescenceαd -LBD,= 1200 fluorescence [ nMH]1 ) titration) titration)(vs. 1 @ IC0.1 ≥ M,10,000 CV-1 nM[51,52] K = 15 nM3 cells)50  X = O, Y = N (RXRK50αd -LBD,= 15 nM [ H] 1) (vs. 501 @ 0.1 M, CV-1 [51,52] [51,52] X = O, Y = N ICIC = 1200 1200 nM3 nM (vs.IC1cells)@ = 67 0.1 nMµM, CV-1 cells) SR11179X = O, (10c Y )= N (RXRα-LBD,(RXR50K dαfluorescence IC=-LBD, 1550 =nM 1200 [ H] 1nM )titration) IC50 = 67 nM (RXRα-LBD, [33H]1) (vs. 1 @ 0.1 cells) M, CV -1 [51,52] SR11179 (10c) (RXRα-LBD,(RXRK αfluorescenced -LBD,= 15 nM [ H] titration)31) IC50 = 67 nM C = CH2, Y = CH (RXRKd = 15α-LBD, nM [ H]1) (vs. 1 @ 0.1  M, CV-1 [51,52] SR11179 (10c) (RXRα-LBD,IC fluorescence50 = 450 nM titration) IC50 cells)= 67 nM CSR11179 = CH2, Y ( 10c= CH) (RXRα-LBD,ICK 50fluorescenced = 15450 nM nM titration) (vs. 1 IC@ 500.1 = 67 M, nM CV -1 [51,52] 2 3 cells) C SR11179= CH , Y (=10c CH) (RXRα-LBD,(RXRICK50 dfluorescenceα =-LBD, K450 15d = nM nM15 [ H]nM 1 titration)) (vs. 1IC @50 0.1 = 67 M,nM CV -1 [51,52] 2 10c 3 (vs. 1 @ cells)0.1  M, CV-1 [51,52] CSR11179 SR11179= CH , Y ( 10c= ( CH) ) (RXRα-LBD,(RXRIC fluorescenceα50 -LBD,= 450 nM[ H] 1 titration)) IC50 = 67 nM C = CH2, Y = CH (RXRα-LBD, fluorescence3 titration) (vs. 1 @ 0.1cells)IC  M,= CV 67-1 nM [51,52] SR11179 (10c) (RXR(RXRα-LBD,ICα50-LBD, = 450fluorescence [nMH] 1) titration) cells)50 C C= CH = CH2, Y2 =, CH Y = (RXRα-LBD, [3H]1) (vs.IC50 1 = @ 0.24 0.1 μM M, CV-1 [51,52] [51,52] ICIC50 = 450 450 nM nM3 (vs.IC501 =cells)@ 0.24 0.1 μµMM, CV-1 cells) C = CH2, Y = CH (RXR50α-LBD,50 [ H]1) CH IC = 45033 nM (vs.IC IRX4204:50 = 0.24cells) ECμM50 = 0.2 UVI3003 (11) (RXR(RXRαα-LBD,-LBD,N.D. [ [H]H]1)1 ) 50 [54,55] (RXRα-LBD, [3H]1) (vs. IRX4204:IC50 = 0.24 EC μM = 0.2 UVI3003 (11) N.D. (vs.nM IRX4204:[53]IC50 @ = 100.24 ECnM, μ50M COS-7= 0.2 [54,55] UVI3003 (11) N.D. nM(vs. [53] IRX4204:IC50 @ = 10 0.24 nM, EC μM50 COS-7 = 0.2 [54,55] UVI3003 (11) N.D. nM(vs. [53] IRX4204: @ 10cells) nM, EC 50COS-7 = 0.2 [54,55] UVI3003 (11) N.D. nM [53] @ICcells) 10IC50 50 nM,= 0.24= COS-7 0.24 μMµ M[54,55] (vs. IRX4204:cells) EC 50 = 0.2 UVI3003 (11) N.D. nM [53](vs.(vs. @IRX4204: 10 IRX4204: nM, COS-7EC50 EC = 0.2[54,55] = UVI3003UVI3003 (11 ()11) N.D. means that the datum was not describedN.D.N.D. in the cited manuscript.nM [53] @cells) 10 nM, COS-7 50 [54,55] [54,55] N.D. means that the datum was not described in the cited manuscript.nM [53]cells) @ 10 nM, COS-7 N.D. means that the datum was not described in the cited manuscript.0.2cells) nM [53] @ 10 nM, N.D. means that the datum was not described in the cited manuscript. COS-7cells) cells) N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript.

Int. J. Mol. Sci. 2018, 19, 2354 7 of 20

Int. J. Mol. Sci. 2018, 19, x 7 of 19 Table 4. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists Int. J. Mol. Sci. 2018, 19, x 7 of 19 havingTable a non-alkoxy 4. Chemical side structures, chain or binding another affinities, structure and on RXR an RXR antagonistic agonistic activities scaffold. of RXR antagonists Int. J. Mol. Sci. 2018, 19, x 7 of 19 Int. J. Mol.having Sci. a2018 non-alkoxy, 19, x side chain or another structure on an RXR agonistic scaffold. 7 of 19 Table 4. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists Compounds Structures Binding Transactivity (RXRα) Ref. Compounds Structures Binding Transactivity (RXRα) Ref. Tablehaving 4. aChemical non-alkoxy structures, side chain binding or anothe affinities,r structure and RXRon an antagonistic RXR agonistic activities scaffold. of RXR antagonists Int. J. Mol.having Sci. 2018a non-alkoxy, 19, x side chain or another structure on an RXR agonistic scaffold. 7 of 19 Int. J. Mol.Compounds Sci. 2018, 19 , x Structures Binding Transactivity (RXRα) 7 of 19Ref. Compounds Structures Binding TransactivityICIC50= =1.0 1.0 µμ(RXRM α) Ref. Compounds Structures Binding Transactivity50 (RXRα) Ref. TableHX531HX531 4. ( (12Chemical12)) structures, binding affinities, and RXRN.D.N.D. antagonistic(vs.(vs. IRX4204: IRX4204: activities EC50 EC =of 0.2 RXR= nM 0.2 antagonists[53] nM @ 10 [55 [55,56],56] Table 4. Chemical structures, binding affinities, and RXR antagonistic activities of50 RXR antagonists having a non-alkoxy side chain or another structure on an RXR agonistic[53] @ nM,scaffold. 10 nM,IC COS-750 = COS-7 1.0 cells) μM cells) having a non-alkoxy side chain or another structure on an RXR agonistic scaffold. HX531 (12) N.D. (vs. IRX4204:IC50 EC = 1.050 = μ0.2M nM [53] @ 10 [55,56] Compounds Structures Binding TransactivityIC50 = 1.0(RXR μMα ) Ref. HX531 (12) N.D. (vs. IRX4204:nM, EC COS-750 = 0.2 cells) nM [53] @ 10 [55,56] CompoundsHX531 (12) Structures BindingN.D. (vs. IRX4204:Transactivity EC50 (RXR= 0.2 nMα) [53] @ 10 Ref.[55,56] 13a nM, COS-7 cells) 13a1 nM, COS-7 cells) R1 1 = Et, IC50 = 0.095 μM R = Et, N.D. ICIC50 == 1.0 0.095 μM µM [57] R2 = NHSO13a2-(3-CF 3)Ph, (vs. 1 @ 5020 nM, HEK-293 cells) 2R = NHSO2-(3-CF3)Ph, N.D. (vs. 1 @IC 2050 = nM, 1.0 μHEK-293M cells) [57] R = NHSOHX53121 -(3-CF (12) 3)Ph, N.D. (vs.(vs. IRX4204:1 @ 20 EC nM,50 = 0.2 HEK-293 nM [53] cells)@ 10 [55,56] XR13a = =H Et, IC50 = 0.095 μM HX531X13a = H ( 12 ) N.D.N.D. (vs. IRX4204: EC50 = 0.2 nM [53] @ 10 [55,56][57] 2 X = H nM, COS-7 cells) R = NHSOR113b = Et,2 -(3-CF 3)Ph, (vs.nM, 1 @IC COS-7 2050 =nM, 0.095 cells) HEK-293 μ M cells) R1 = Et, N.D. IC50 = 0.095 μM [57] 2 1 R = NHSOR113b =X 2n -(3-CF=-Pr, H 3)Ph, N.D. (vs. 1 @ IC2050 nM, = 0.076 HEK-293 μM cells) [57] R2 = NHSO2-(3-CF3)Ph, N.D. (vs. 1 @ 20 nM, HEK-293 cells) [57] 2 1 N.D. [57] R2 = NHSOX =13b2 -(3-CFH 3)Ph, (vs. 1 @ 20 nM, HEK-293µ cells) R = NHSOR X=13a =n2 -(3-CFH-Pr, 3)Ph, (vs. 1IC @ 5020 =nM, 0.076 HEK-293M cells) 2 1 N.D. [57] R = NHSORX113b13a ==-(3-CF Hn -Pr, )Ph, (vs. 1 @ 20IC nM,50 = HEK-2930.076 μM cells) RX13b =2= Et,H 3 N.D. IC50 = 0.095 μM [57] 2 R1 1 = Et,2 3 N.D. IC50 = 0.095 μM [57] R2 = NHSORX = 13c 2n H-Pr, -(3-CF 3 )Ph, (vs. 1 @IC 2050 =nM, 0.076 HEK-293 μM cells) R = NHSO1 -(3-CF )Ph, N.D. (vs. 1 @ 20 nM,5050 HEK-293 cells) [57] 2 R = n-Pr, N.D. ICIC50 = = 0.076 0.50 μ μMM [57] R2 = NHSO1 X2 -(3-CF=2 H 3)Ph, N.D. (vs. 1 @ 20 nM, HEK-293 cells) [57] R2 =R NHSO = Et,X = R2 -(3-CFH = CN,3)Ph, N.D. (vs. 1 @ 20 nM, HEK-293 cells) [58] R = NHSO2-(3-CF3)Ph, (vs. 1(vs. @ 20 1, nM,HEK-293 HEK-293 cells) cells) X13c = H (vs. 1, HEK-293 cells) 1 XX13b ==13c 2 HF IC50 = 0.50 µM R = Et,X R= H= CN, N.D. IC50 = 0.50 μM [58] 1 13b 2 R R =1 Et,=13c n -Pr,R = CN, N.D. (vs.IC150, = HEK-293 0.076 μM cells) [58] X13c = F (vs. 150, HEK-293 cells) R1 = n-Pr, N.D. IC50IC = 0.076 = 0.50 μM μ M [57] 2 R1 = Et,X 2R =2 =F CN,3 N.D. IC50 = 0.50 μM [58] R =R NHSO1 = Et, R-(3-CF2 = CN,)Ph, N.D.N.D. (vs. 1 @ 20 nM, HEK-293 cells) [57][58] 2 142 3 (vs. 1, HEK-293 cells) R = NHSOXX 14= = -(3-CFH F )Ph, N.D. (vs. 1 @(vs. 20 nM,1, HEK-293 HEK-293N.D. cells) cells) [59] X = F X13c = H IC50 = 0.50 μM 1 1413c142 N.D.N.D. N.D.N.D. [59][59] R = Et, R = CN, N.D. IC50 = 0.50 μM [58] 1 15a2 50 R = Et,15a14 R = CN, N.D.N.D. (vs. 1, HEK-293IC50 N.D.= 4.1 cells) μM [58][59] X 14= F N.D. (vs. 1, HEK-293N.D. cells) [59][29] XX == ClF (vs. 2 @ 10 nM, COS-1 cells) 15a IC50 = 4.1 μM 15a N.D. IC50 = 4.1 µM [29] 15aX14 = Cl N.D.N.D. (vs. 2 @ICN.D. 1050 = nM, 4.1 μCOS-1M cells) [59][29 ] X =15a Cl N.D. (vs. 2 @ 10IC nM,50 = 4.1 COS-1 μM cells) [29] X 14= Cl N.D.N.D. (vs. 2 @ 10N.D. nM, COS-1 cells) [59][29] X15b = Cl (vs. 2 @IC 1050 nM, = 3.2 COS-1 μM cells) N.D. [29[29]] X = CF3 (vs. 2 @ 10 nM, COS-1 cells) 15a IC50 = 4.1 μM 15a15b N.D. IC50 IC= 4.150 = μ 3.2M μM [29] X = Cl N.D.N.D. (vs. 2 @ 10 nM, COS-1 cells) [29] [29] X15bX15b = = Cl CF 3 (vs. (vs.2 @ IC102 @IC 50nM, 1050= = nM,COS-1 3.23.2 µμCOS-1M cells) cells) 15b N.D.N.D. IC50 = 3.2 μM [29] XX = = CFCF33 N.D. (vs.(vs.2 @2 @ 10 10 nM, nM, COS-1COS-1 cells) [29] X = CF3 (vs. 2 @ 10 nM, COS-1 cells)

15b IC50 = 3.2 μM 15b N.D. IC50 = 3.2 μM [29] X = CF3 (vs. 2 @ 10 nM, COS-1 cells) N.D. [29] X = CF3 (vs. 2 @ 10 nM,pA2 COS-1= 8.23 cells) 16 N.D. (vs. NEt-TMN: EC50 = 5.28 nM [49], [50] COS-1pA2 =cells) 8.23

16 N.D. (vs. NEt-TMN:pA2 =EC 8.2350 = 5.28 nM [49], [50] pApA2 2= = 8.238.23 16 N.D. (vs. NEt-TMN:COS-1 EC50 cells)= 5.28 nM [49], [50] 1616 N.D.N.D. (vs.(vs. NEt-TMN: NEt-TMN: EC50 EC = 5.2850 = nM 5.28 [49], [50[50]] COS-1 cells) nM [p49ACOS-12], = COS-1 8.23 cells) cells) N.D. means that the datum was not described in the citedpA manuscript.2 = 8.23 16 N.D. means that the datum was notN.D. described (vs. in NEt-TMN:the cited manuscript.EC50 = 5.28 nM [49], [50] 50 16 N.D. (vs. NEt-TMN:COS-1 EC cells) = 5.28 nM [49], [50] N.D. means that the datum was not described in the citedCOS-1 manuscript. cells) Table 5. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists N.D. means that the datum was not described in the cited manuscript. from natural productsN.D.N.D. means means or others. that that the the datum datum was was not not described described in in the the cited cited manuscript. manuscript. Table 5. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists

CompoundsTablefrom 5.natural Chemical productsN.D. structures, means orStructures thatothers. binding the datum affinities, was not and described RXR Binding antagonistic in the cited manuscript. activitiesTransactivity of RXR antagonists(RXRα) Ref. Table 5. ChemicalN.D. structures, means that binding the datum affinities, was not and described RXR antagonistic in the cited manuscript.activities of RXR antagonists Table 5. Chemical structures, binding affinities, and RXRKd =antagonistic 6.2 μM activities of RXR antagonists from natural products or others. 50 from natural products or others. IC50 = 0.11 μM DanthronTableCompounds 5. ( 17aChemical) structures,Structures binding affinities, and (RXRRXR αantagonistic Binding-LBD, SPR) activities Transactivityof RXR antagonists (RXRα) Ref. from natural products or others. (vs. 1 @ 0.1 μM, HEK-293T [60] CompoundsTableR = H5. Chemical structures,Structures binding affinities, and RXRKKd Binding =antagonisticd =7.5 6.2 μ μM M activitiesTransactivity of RXR antagonists (RXRα) Ref. Compoundsfrom natural products or others.Structures Binding TransactivityIC50cells) = 0.11 (RXR μM α) Ref. Danthronfrom natural (17a )products or others. (RXR(RXRKdα =α-LBD, 6.2-LBD, μM ITC) SPR) Compounds Structures BindingKd = 6.2 μM Transactivity(vs. 1IC @50 0.1 = (RXR 0.11μM, μ HEK-293TαM) [60] Ref. DanthronCompoundsR = H(17a ) Structures (RXR BindingKαd-LBD, = 7.5 μ SPR)M TransactivityIC50 = 0.110.75 (RXR μMα) Ref. DanthronCompoundsRhein (17b (17a) ) Structures (RXR Bindingα-LBD, SPR) Transactivity(vs. 1 @ 0.1 μcells) M,(RXR HEK-293T α) Ref. [60] R = H K(RXRdK =d 6.2N.D.=µα 7.5 -LBD,μM μ* M ITC) (vs. 1 @ 0.1 μM, HEK-293T [60][61] Kd = 6.2d N.D.M * (vs. 1 @ 0.1 μM, HEK-293T [61] R =R CO= H2 H KdK = 6.2= 7.5 μM μ M IC50 = 0.11cells) μM DanthronDanthron (17a ()17a) (RXR(RXRα(RXR-LBD,α-LBD,α-LBD, SPR) SPR) ITC) ICIC50= =IC 0.11 0.1150cells) =µ μ0.75MM μM DanthronRhein ((17b17a) (RXR(RXRα-LBD,α-LBD, SPR) ITC) (vs. 1 @50 0.1 μM, HEK-293T [60] [60] R = H Kd = 7.5N.D. μM * (vs. 1 @ 0.1 μM, HEK-293T [61] R = H Kd = 7.5 µM (vs. 1 (vs.@ 0.1IC 1 50@µ value0.1M,IC μ50 HEK-293TM, =is 0.75notHEK-293T describedμM cells) [60] Rheinβ-Apo-13-RR = = CO(H17b 2H) Kd = 7.5 μM IC50 valueICcells)50 =is 0.75 not describedμM Rheinβ-Apo-13- (17b) (RXR(RXRα-LBD,α-LBD,N.D.N.D. ITC) ITC)* (vs.(vs. 1 @1 cells)0.1@ 0.01~1000 μcells) M, HEK-293T nM, [61][62] R = CO2H (RXRα-LBD,N.D.N.D. ITC)* (vs.(vs. 1 @1 0.1@ 0.01~1000 μM, HEK-293T nM, [61][62] carotenoneR = CO2H (18 ) IC50 = 0.75 μM IC50 valueCOS-7cells) is notcells) described RheinRhein (17b ()17b) IC50IC50= COS-7= 0.75 0.75cells)µ μ cells)MM Rheinβ-Apo-13- (17b) N.D.N.D. * * (vs. 1 @ 0.1 μM, HEK-293T [61] [61] R = CO2H N.D. IC50(vs.µ value 1 @ is 0.01~1000 not described nM, [62] R =carotenone CO2H (18) N.D. * (vs. 1 (vs.@ 0.1 1 50@ 0.1M, μ HEK-293TM, HEK-293T cells) [61] βR-Apo-13- = CO2H IC valuecells) is not described β-Apo-13- N.D. (vs. 1 cells)@COS-7 0.01~1000 cells) nM, [62] carotenone (18) IC50 ≈N.D. 200 μM (vs. 1 @ 0.01~1000 nM, [62] carotenoneR-Etodolac (1819) IC50ICvalue50 value is COS-7is not notN.D.described described cells) [63] β-Apo-13- (RXRα-LBD, [3H]1) IC50 value COS-7is not described cells) β-Apo-13-carotenoneβ-Apo-13- (18) (RXRN.D.N.D.α-LBD, [ H]1) (vs.(vs.1 @1 @ 0.01~1000 0.01~1000 nM, [62] [62] carotenone (18) ICN.D.50 ≈ 200 μM (vs. 1 @ 0.01~1000 nM, [62] carotenoneR-Etodolac (18 (19) ) COS-7COS-7 cells) cells)N.D. [63] 3 (RXRIC50 α≈-LBD, 200 μ M[ H] 1) COS-7 cells) R-Etodolac (19) IC50 ≈ 200 μM N.D. [63] R-Etodolac (19) 3 N.D. [63] (RXRα-LBD, [3H]1) (RXRIC50 ≈α 200-LBD, μM [ H]1) R-Etodolac (19) N.D. [63] IC50 ≈ 200 μ3M R-Etodolac (19) (RXRα-LBD, [ H]1) N.D. [63] (RXRα-LBD, [3H]1)

Int. J. Mol. Sci. 2018, 19, x 7 of 19

Table 4. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists having a non-alkoxy side chain or another structure on an RXR agonistic scaffold.

Compounds Structures Binding Transactivity (RXRα) Ref.

IC50 = 1.0 μM HX531 (12) N.D. (vs. IRX4204: EC50 = 0.2 nM [53] @ 10 [55,56] nM, COS-7 cells)

13a R1 = Et, IC50 = 0.095 μM N.D. [57] R2 = NHSO2-(3-CF3)Ph, (vs. 1 @ 20 nM, HEK-293 cells) X = H 13b R1 = n-Pr, IC50 = 0.076 μM N.D. [57] R2 = NHSO2-(3-CF3)Ph, (vs. 1 @ 20 nM, HEK-293 cells) X = H 13c IC50 = 0.50 μM R1 = Et, R2 = CN, N.D. [58] (vs. 1, HEK-293 cells) X = F

14 N.D. N.D. [59]

15a IC50 = 4.1 μM N.D. [29] X = Cl (vs. 2 @ 10 nM, COS-1 cells)

15b IC50 = 3.2 μM N.D. [29] X = CF3 (vs. 2 @ 10 nM, COS-1 cells)

pA2 = 8.23 16 N.D. (vs. NEt-TMN: EC50 = 5.28 nM [49], [50] COS-1 cells)

N.D. means that the datum was not described in the cited manuscript.

Table 5. Chemical structures, binding affinities, and RXR antagonistic activities of RXR antagonists from natural products or others.

Compounds Structures Binding Transactivity (RXRα) Ref. Kd = 6.2 μM IC50 = 0.11 μM Danthron (17a) (RXRα-LBD, SPR) (vs. 1 @ 0.1 μM, HEK-293T [60] R = H Kd = 7.5 μM cells) Int. J. Mol. Sci. 2018, 19, 2354 (RXRα-LBD, ITC) 8 of 20 IC50 = 0.75 μM Rhein (17b) N.D. * (vs. 1 @ 0.1 μM, HEK-293T [61] R = CO2H cells) Table 5. Cont. IC50 value is not described β-Apo-13- N.D. (vs. 1 @ 0.01~1000 nM, [62] carotenone (18) Compounds Structures Binding TransactivityCOS-7 cells) (RXR α) Ref.

IC IC≈50200 ≈ 200µ MμM R-EtodolacR-Etodolac (19) (19) 50 N.D.N.D. [63] [63] (RXR(RXRα-LBD,α-LBD, [3H] [3H]1)1) Int. J. Mol. Sci. 2018, 19, x 8 of 19 Int.Int. J. Mol.J. Mol. Sci. Sci. 2018 2018, 19, 19, x, x 8 of8 of19 19 Int. J. Mol. Sci. 2018, 19, x 8 of 19 Int. J. Mol. Sci. 2018, 19, x 8 of 19 Int. J. Mol. Sci. 2018, 19, x 8 of 19 Int. J. Mol. Sci. 2018, 19, x 8 of 19 Int. J. Mol. Sci. 2018, 19, x 8 of 19 Int. J. Mol. Sci. 2018, 19, x IC50IC=50 80 = 80µM μM 8 of 19 Sulindac sulfide (20) IC50 = 80 μM N.D. [64] SulindacInt. J. Mol. sulfide Sci. (201820) , 19, x 50 3 3 N.D.8 of 19 [64] Sulindac sulfide (20) IC(RXRα = 80α50-LBD, μM [ H]1) N.D. [64] Sulindac sulfide (20) (RXR -LBD,IC50 = 80 [ 3 μH]M1 ) N.D. [64] Int.Sulindac J. Mol. Sci.sulfide 2018 (20, 19) , x (RXRα-LBD,3 [ H]1) N.D. 8[64] of 19 Sulindac sulfide (20) (RXRα-LBD,IC50 = [ 80H] μ1M)3 N.D. [64] Sulindac sulfide (20) (RXRICα50-LBD, = 80 μ [M3H] 1) N.D. [64] Sulindac sulfide (20) (RXRα-LBD, [3H]1) N.D. [64] IC50 = 80 μ3M Sulindac sulfide (20) (RXRα50-LBD, [ H]1) N.D. [64] K-80003 (21a) IC = 80 μM3 Sulindac sulfide (20) (RXRIC50α -LBD,= 80 μ [MH] 1) N.D. [64] K-80003K-80003 (21a (21a) ) Me (RXRIC50α -LBD,= 2.4 μ [M3H] 1) K-80003Sulindac (X21a sulfide= F,) (20) Me IC50IC =50 2.4 = 80 μM μ M N.D. [64,65][64] K-80003 (21a) Me ICIC(RXR5050 = =2.4α 2.4-LBD, μMµ M [3H]1) SulindacX = F,sulfide (20) Me (RXRICICα5050-LBD, == 2.480 μ [MH] 1) N.D.N.D. [64,65][64] XXK-80003 = F, (21a) MeMe (RXRICα50-LBD, = 2.433 μ [3MH] 1) N.D.N.D. [64,65] [64,65] SulindacK-80003R =X COsulfide= F, (221a H )( 20) Me (RXR(RXRαα-LBD,IC-LBD,50 = 2.43 [[ H]μM11) ) N.D. [64,65][64] R = CO2H Me (RXRα-LBD, [ H]1)3 R = COX2 H = F, Me Me Me (RXRICα50-LBD, = 2.4 μ [MH] 1) N.D. [64,65] R =K-8008 K-80003COR = HCO ( 21b2(H21a ) ) Me 3 R =X CO= F,2 H MeMe Me (RXRICα50-LBD, = 2.4 μ[ MH] 1) N.D. [64,65] K-8008K-80003 (21b 2()21a ) Me Me 3 K-8008R ( =21bX CO =) F, H Me Me (RXRICIC50α50 -LBD,= =16.8 2.4 μ[ MH] 1) IC50 =N.D. 13.2 μM [64,65] K-8008K-80003K-8008 (X21b = H( )(21b221a )) Me R µ 3 K-8008R =X CO = ( F,21bH ) Me Me ICIC(RXR5050= =50 16.8α16.8-LBD, μMM [ H]1) IC50 = 13.2N.D. μM [64,65] K-80003K-8008X = H ( (21b21a)) Me R IC50 =IC 16.8 = μ 2.4M μM3 IC50 = 13.2 μM X =R H X= CO= F, 2H Me R Me TR-FRET,(RXRIC GST-RXR50α =-LBD, 16.8 μ [αMH]-LBD, 1) 1 @ (vs. 1 IC@ 10050 =N.D. 13.2nM, μHCT-116M [65,66][64,65] XK-80003 = HX = H (21a ) R TR-FRET,ICIC50 = = 16.8 2.4 μ μ3MM ICIC = = 13.2 13.2µ μMM K-8008RX = =CO HF,(21b 2H ) MeMe Me TR-FRET,TR-FRET, GST-RXR(RXR GST-RXRIC50α -LBD,= 16.8α-LBD,α -LBD,[μH]M 1 ) @ 1 @ (vs.(vs. 1 @1 @100 10050IC nM,50 nM, =N.D. 13.2HCT-116 HCT-116 μM [65,66] [65,66][64,65] K-8008X = H (21b ) Me R TR-FRET,IC GST-RXR5010 = nM)2.4 μ 3αM-LBD, 1 @ (vs. 1 @ 100cells) nM, HCT-116 [65,66][ 65,66] R X= CO= HF,2 H X MeR TR-FRET,GST-RXR(RXRIC10 GST-RXR50α nM)-LBD,=α 16.8-LBD, μ[αMH]-LBD, 1) (vs. 1 @ 1(vs.@ 100 1 IC@ nM,cells)10050 =N.D. 13.2nM, HCT-116 μHCT-116M cells) [65,66][64,65] R =K-8008R = CO (221bH ) Me X Me TR-FRET,(RXR10IC nM)GST-RXR50α -LBD,= 16.8 [μ3αH]M-LBD, 1) 1 @ (vs. 1 cells)@IC 10050 = nM,13.2 HCT-116μM [65,66] R = K-8008X = (H21b ) Me X X R TR-FRET, GST-RXR10 nM) α-LBD, 1 @ (vs. 1 @ 100cells) nM, HCT-116 [65,66] R = R =X CO = H2H X Me R 1 @IC 105010 = nM) nM)16.8 μM IC50 cells)= 13.2 μM RTriptolide K-8008= (21b (22a) ) X Me TR-FRET,IC GST-RXR50 = 16.8 μαM-LBD, 1 @ (vs. 1IC @ 50100 = 13.2 nM, μ HCT-116M [65,66] TriptolideR K-8008= X = ( H22a(21b ) ) X R TR-FRET, GST-RXR10N.D. nM) α-LBD, 1 @ (vs. 1 @ 100cells)N.D. nM, HCT-116 [65,66][67] TriptolideTriptolideXR ( 22a= H )( 22a) R ICN.D.50 10= 16.8 nM) μ M ICN.D.50 =cells) 13.2 μM [67] TriptolideRTriptolide = (22a ()22a) X TR-FRET,ICN.D. GST-RXR50 = 16.8 μαM-LBD, 1 @ (vs. 1 ICN.D.@ 10050 = 13.2nM, μHCT-116M [67] [65,66] TriptolideR =R H=X H = H ( 22a) R TR-FRET, N.D.GST-RXR10N.D. nM) α-LBD, 1 @ (vs. 1 @ 100 N.D.N.D.cells) nM, HCT-116 [65,66][67] [67] TRC4R = (H22b ) X 10N.D. nM) cells)N.D. [67] TRC4RTriptolideR == HR ( 22b= H ) ( 22a) X TR-FRET, GST-RXRα-LBD, 1 @ (vs. 1 @ 100 nM, HCT-116 [65,66] TRC4RTriptolide = (22bR =) H (22a) 10N.D. nM) cells)N.D. [67] TRC4RR = =(H22b ) X N.D. N.D. [67] TRC4RTriptolide =TRC4 (R22b = ()22b (22a) ) X 10 nM) cells) TriptolideR = HClRR = = H ( 22a) N.D.N.D. N.D.N.D. [68][67] R =TRC4HCl RR ==( 22b H ) OH N.D.N.D. N.D.N.D. [68][67] TriptolideHClTRC4RN =( 22b (22aOH) ) N.D. N.D. [68] TriptolideNRHCl H= HOH (22a) N.D.N.D. N.D.N.D. [68][67] [68] NTRC4RHClR = = H( 22b )OH N.D. N.D. [68] O H NR = OH N.D. N.D. [67] R = O HTRC4RHClH N= (H22b ) N.D. N.D. [68] O OTRC4HR ( =22b ) OH N.D. N.D. [68] O HClRNH = OH OTRC4HClHN (22b) N.D. N.D. [68] O HClR = OH N.D. N.D. [68] RHN = OH N.D. N.D. [68] O HClNH OH O HClH Ki = N.D.15.7 μM N.D. [68] NSC-640358N (23OH) i N.D. [69] O i K = 15.7 μM 3 NSC-640358HN (23) K(RXR = 15.7iα-LBD, μM [ H]1) N.D. [69] NSC-640358O (23) Ki =K 15.7i = 15.7µM3 μM N.D. [69] NSC-640358H (23) (RXRα-LBD,3 [ H]1) N.D. [69] NSC-640358NSC-640358 (23) (23) (RXRα-LBD,Ki = 15.7 [ H]3 μ1M)3 N.D.N.D. [69] [69] NSC-640358O (23) (RXR(RXRα-LBD,α-LBD, [ H] [3H]1)1) N.D. [69] (RXRKiα =-LBD, 15.7 μ [M3H] 1) NSC-640358 (23) (RXRKαi =-LBD, 15.7 μ [MH] 1) N.D. [69] NSC-640358 (23) (RXRα-LBD, [3H]1) N.D. [69] Ki = 15.7 μM3 NSC-640358 (23) (RXRKi α= -LBD,15.7 μ M[ H] 1) N.D. [69] NSC-640358 (23) (RXRα-LBD, [3H]1) N.D. [69] Ki = 15.7 μM3 NSC-640358 (23) (RXRKiα =-LBD, 0.2815.7 μ [MH] 1) N.D. [69] 24 i 3 N.D. [70] NSC-640358 (23) (RXRi K = 0.28α-LBD, μM [ 3H]1) N.D. [69] 24 K(RXR = 0.28iα-LBD, μM [3H]1) N.D. [70] 24 (RXRKi α=-LBD, 0.283 μ [MH] 1) N.D. [70] 24 (RXRα-LBD,3 [ H]1) 24 (RXRKα-LBD,=K 0.28i = 0.28 [ µH]M μ1M)3 N.D. [70] 24 (RXRi α-LBD, [3H]1) N.D. [70] 24 (RXRKiα =-LBD, 0.283 μ [M3H] 1) N.D. [70] 24 (RXR(RXRα-LBD,Kαi =-LBD, 0.28 [ H]μ [MH]1 )1) N.D. [70] 24 (RXRα-LBD, [3H]1) N.D. [70] Ki = 0.28 μM3 24 (RXRi α-LBD, [ H]1) Ki = 0.810.28 μM3 N.D. [70] 2524 (RXRi Ki =α -LBD,0.28 μ M[ H] 1) N.D. [70] i K = 0.81 μM 3 25 24 K(RXR = 0.81iα-LBD, μM [ H]1) N.D.N.D. [70][70] 25 Kii = 0.810.283 μM3 N.D. [70] 2524 (RXR(RXRα-LBD,iα-LBD,3 [ H] [ H]1) 1) N.D. [70] 25 (RXRα-LBD,K = 0.81 [ H] μ1M)3 N.D. [70] 25 K(RXR=K 0.81iα =-LBD, 0.81µM μ [M3H] 1) N.D. [70] 25 (RXRi α-LBD, [3H]1) N.D. [70] 25 Ki = 0.813 μ3M N.D. [70] 25 (RXR(RXRα-LBD,αi -LBD, [ H] [ H]1)1) N.D. [70] K = 0.81 μM3 IC50 = 2 μM 25 (RXRKi α= -LBD,0.81 μ M[ H] 1) N.D. [70] 3 IC50 = 2 μM 25 (RXRKi =α -LBD,0.81 μ M[ H] 1) IC50 = 2 μN.D.M [70] 26 N.D. 3 (vs. 1 @ IC0.150 μ =M, 2 μ HEK-293TM [71] 26 25 (RXRKN.D.iα =-LBD, 0.81 μ [MH] 1) (vs. 1 @ 0.1IC μM,N.D. = HEK-293T2 μ M [71][70] 26 25 (RXRN.D.α-LBD, [3H]1) (vs. 1 @ 0.1 μICM,50cells) N.D.HEK-293T = 2 μ M [71] [70] 26 N.D. 3 (vs. 1 @ IC0.150 μ =M, 2 μ HEK-293TM [71] 26 (RXRα-LBD,N.D. [ H]1) (vs. 1 @ 0.1cells) μM, HEK-293T [71] cells)IC50cells) = 2 μM 26 N.D. (vs. 1 @ 0.150cells) μM,µ HEK-293T [71] IC50IC=cells) = 2 2 μMM 26 26 N.D.N.D. (vs. 1 IC@IC 0.15050 = μ=2.45 M,2 μ HEK-293TμMM [71] [71] Kd = 488 nM 50µ cells) 26 N.D. (vs. 1 @(vs. 0.1IC50 1 @ IC= M,0.1 2.4550 HEK-293T=μ M,2 μ μM HEK-293TM cells) [71] 27 Kd = 488 nM (vs.IC 1 @= 2.450.150 cells)μ μM,M HEK-293T [71] 26 d N.D. (vs. 1 @IC 0.150 50= μ 2.45M, HEK-293T μM [71] 27 K(RXR = 488dα -LBD,nM SPR) (vs. 1 @ 0.1IC μM, = HEK-293T2 μM 27 26 Kd =N.D. 488 nM (vs. 1(vs. @ 0.1 1 @ ICμ 0.1M,50 = μcells)HEK-293T 2.45M, HEK-293T μM [71] [71] [71] 27 (RXRαK-LBD,d = 488 SPR) nM (vs. 1 @ 0.1cells) μM, HEK-293T [71] 2726 (RXR(RXRα-LBD,αN.D.-LBD, SPR) SPR) (vs. 1 @IC 0.1cells)50 = μ 2.45M, HEK-293T μM [71] 27 (RXRKdα =-LBD, 488 nM SPR) (vs. 1 @cells)IC 0.150 cells) = μ 2.45M, HEK-293T μM [71] 27 (RXRα-LBD, SPR) (vs. 1 @ 0.1cells) μM, HEK-293T [71] Kd = 488 nM IC50 cells)= 2.45 μM 27 (RXRdα-LBD, SPR) (vs. 1 @ 0.1cells) μM, HEK-293T [71] Kd =K 488 = 488 nM nM IC50IC50= =cells) 2.45 2.45 µ μMM 27 27 (RXRKd α= -LBD,488 nM SPR) (vs. 1 @ 500.1 μM, HEK-293T [71] [71] (RXRα-LBD, SPR) IC =cells) 2.45 μM 27 (RXRαK-LBD,d = 488 SPR) nM (vs. 1IC(vs.@50 0.1 1value @ICµ 0.1M,50 is= μ HEK-293T 2.45notM, HEK-293T described.μM cells) [71] (RXRKd =α 11.04-LBD, μ SPR)M 50 cells) 27 Kd = 488 nM IC50 (vs. value 1 @ is0.1 not μM, described. HEK-293T [71] Fluvastatin (28) d IC value(vs.50 is1 @not 100cells) described. nM, MCF-7 [72] 27 dK(RXR = 11.04α-LBD, μM SPR) IC(vs.50 1value @ 0.1 is μ notM, HEK-293Tdescribed. [71] Fluvastatin (28) K (RXR= 11.04d α-LBD, μM SPR) (vs. 1 @ 100 nM, MCF-7 Fluvastatin (28) (RXRKd =α 11.04-LBD, μ SPR)M (vs.IC 1 50@ value100 nM, iscells) not MCF-7 described. [72] [72] Fluvastatin (28) (RXRKαd-LBD, = 11.04 SPR) μM (vs.50 1 @ 100cells) nM, MCF-7 [72] Fluvastatin (28) (RXR(RXRα-LBD,α-LBD, SPR) SPR) IC(vs. value 1 @cells) 100 is not nM, described. MCF-7 [72] Fluvastatin (28) (RXRKd =α 11.04-LBD, μ SPR)M IC(vs.50 value 1cells) @ 100 is not nM, described. MCF-7 [72] Fluvastatin (28) (RXRα-LBD, SPR) (vs. 1 @ 100cells) nM, MCF-7 [72] Kd = 11.04 μM IC50 value cells)is not described. Fluvastatin (28) (RXRKd α= -LBD,11.04 μSPR)M (vs.50 1 @ 100 nM, MCF-7 [72] (RXRα-LBD, SPR) IC value iscells) not described. Fluvastatin (28) Kd =K 11.04d = 11.04µM μM IC50ICvalue(vs.50 value 1 @ is 100is not not nM, described.described. MCF-7 [72] FluvastatinFluvastatin (28) (28) (RXRKd =α 11.04-LBD, μ MSPR) (vs. 1 @ 100cells) nM, MCF-7 [72] [72] (RXRα α-LBD, SPR) IC50 value iscells) not described. Fluvastatin (28) (RXR K-LBD,d = 13.3011.04 SPR) μM (vs. 501(vs.@ 100 1 @ nM,100 nM, MCF-7 MCF-7 cells) [72] (RXRα-LBD, SPR) IC50 value is notcells) described. PitavastatinFluvastatin (2829)) Kd = 13.30 μM IC value(vs.(vs.50 is 11 @not@ 10010 described. nM, nM, MCF-7 MCF-7 [72] Kd = 13.30 μM IC50 value iscells) not described. Pitavastatin (29) (RXRKd =α 13.30-LBD, μ SPR)M (vs. 1 @ 10 nM, MCF-7 [72] Pitavastatin (29) Kd = 13.30 μM (vs.IC 150 @ value 10 nM, iscells) notMCF-7 described. [72] Pitavastatin (29) (RXRKαd-LBD, = 13.30 SPR) μM IC(vs.50 value 1 @ 10is notnM, described. MCF-7 [72] Pitavastatin (29) (RXR(RXRα-LBD,α-LBD, SPR) SPR) (vs. 1 @cells) 10 nM, MCF-7 [72] (RXRKd =α 13.30-LBD, μ SPR)M IC50 valuecells) cells)is not described. Pitavastatin (29) (RXRKd =α -LBD,13.30 μSPR)M IC(vs.50 value 1 @ 10 cells)is nM,not described.MCF-7 [72] Pitavastatin (29) (RXRα-LBD, SPR) (vs. 1 @ 10cells) nM, MCF-7 [72] Kd = 13.30 μM IC50 value iscells) not described. Pitavastatin (29) (RXRKd =α 13.30-LBD, μ SPR)M IC50(vs. value 1 @ is10 not nM, described. MCF-7 [72] (RXRα-LBD, SPR) 50 cells) Pitavastatin (29) K =K 13.30d = 13.30µM μM IC ICvalue(vs.50 value 1 @ is 10is not notnM, described.described. MCF-7 [72] d(RXRKd α= -LBD,5.12 μ SPR)M 5050IC value iscells) not described. PitavastatinPitavastatin (29) (29) Kd = 13.30 μM IC50 value(vs. 1 is@ not10 nM, described. MCF-7 [72] [72] 30 Kd = 5.12 μM IC value(vs.50 is1 @not 100cells) described. nM, MCF-7 [72] Pitavastatin (29) (RXRKd(RXR =α 5.12-LBD,α -LBD,μM SPR) SPR) (vs.IC1(vs.50@ value 10 1 @ nM, 10is notnM, MCF-7 described. MCF-7 cells) [72] 30 (RXRKd α= -LBD,5.12 μ SPR)M (vs.50 1 @ 100 cells)nM, MCF-7 [72] 30 (RXRKd α= -LBD,5.12 μ SPR)M (vs.IC 1 @ value100 nM, iscells) not MCF-7 described. [72] 30 (RXR(RXRα-LBD,Kα-LBD,d = 5.12 SPR) SPR) μ M IC(vs.50 value 1 @ 100 iscells) not nM, described. MCF-7 [72] 30 (RXRd α-LBD, SPR) (vs. 1 @cells) 100 nM, MCF-7 [72] (RXRK α=-LBD, 5.12 μ SPR)M IC50 valuecells) cells)is not described. 30 (RXRKdα =-LBD, 5.12 μ SPR)M IC(vs.50 value 1 @ 100 cells)is notnM, described. MCF-7 [72] 30 (RXRα-LBD, SPR) (vs. 1 @ 100cells) nM, MCF-7 [72] Kd = 5.12 μM IC50 value iscells) not described. 30 N.D. means that the datum was not described(RXRKd =α -LBD,5.12 inμ MSPR) the cited manuscript.IC(vs.50 value 1 @ 100is not nM, described. MCF-7 [72] 30 N.D. means that the datum was not described(RXRKd =α 5.12-LBD, in μthe MSPR) cited manuscript.(vs. 1 @ 100cells) nM, MCF-7 [72] N.D.N.D. means means that thatthe datumthe datum was wasnot describednot described(RXRα -LBD,in the in SPR) citedthe cited manuscript. manuscript.IC50 value iscells) not described. 30 N.D. means that the datum was notK described=Kd 5.12 = 5.12µ M inμM the citedIC manuscript.(vs.value 1 @ is100 not nM, described. MCF-7 [72] 30 30 N.D. means that the datum was not (RXRdescribedd α-LBD, in SPR) the cited manuscript.50 (vs. 1 @ 100cells) nM, MCF-7 [72] [72] N.D. means that the datum was not(RXR described(RXRα-LBD,α-LBD, SPR) in SPR) the cited(vs. manuscript.1 @ 100 nM,cells) MCF-7 cells) N.D. means that the datum was not described in the cited manuscript.cells) N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript. N.D. means that the datum was not described in the cited manuscript.

Int. J. Mol. Sci. 2018, 19, 2354 9 of 20

3.1. RXR Antagonists Having a Long-Chain Alkoxy Group The chemical structures of RXR antagonists in this category are illustrated in Table3. LG100754 ( 3) was reported as the first RXR antagonist in 1996 [42]. Prior to that, in 1994, Boehm et al. had noted that some compounds having RXR binding affinity, but not showing RXR agonist activity, might exhibit RXR antagonistic activity [73]. Compound 3 was designed by introducing an n-propoxy group into the 30-position of the backbone of tetrahydrotetramethylnaphthyl octatrienoic acid, whose chemical structure is similar to that of 9-cis-retinoic acid (1) (Figure3). A similar compound, AGN195393 (4)[43], was also reported. Compound 3 showed IC50 = 16 nM against 32 nM 2 (EC50 = 33 nM) [73] in reporter assay for RXRα in CV-1 cells. Although initially identified as an RXR homodimer antagonist, subsequent experiments revealed that 3 acts as an agonist toward RAR/RXR [74], PPARα/RXR [75], and PPARγ/RXR [76,77]. Although 3 has been reported to act as a ‘phantom ligand’ activating RAR via allosteric control through the binding to RXR [74], it is revealed that the activation of RAR/RXR by 3 is caused by a direct binding of 3 to RAR that stabilizes co-activator interactions [78]. Int. J. Mol. Sci. 2018, 19, x 10 of 19

Structures of RXR agonists Me Me Me Me Me Me Me Me Me Me Me N Me Me Me Me CO H Me 2 NN Me Me Me HO CO2H CO2H CO2H 9-cis retinoic acid (1) PA024 (31) 32 CD3254 (33)

Structures of RXR antagonists Me Me Me Me Me Me Me Me O O OR X Me Me Me CO H Me Me 2 N Me Me Me Me Me Me Me Me HO NN Y Me UVI3003 (11)

CO H CO H LG100754 (3) CO H 2 2 O Me 2 Me Me 9a: R = nPen Bl-1003 (10a): X = O, Y = CH 9b: R = nHex Bl-1005 (10b): X = O, Y = N SR11179 (10c): X = CH2, Y = CH CO2H Me Me HO 34

FigureFigure 3. 3.Chemical Chemicalstructures structures ofof RXRRXR agonistsagonists and RXR antagonists having having a a long-chain long-chain alkoxy alkoxy group. group.

3.2. RXR Antagonists Possessing Another Side Group Ro26-5450 (5)[44] and LG101506 (6)[45] have a (2E,4E,6Z)-7-(2-alkoxy-3,5-di-alkylbenzene)-3- methylocta-2,4,6-trienoicRXR antagonists possessing acid scaffold. another Compound side group6 binds instead to RXRof theα atalkoxy low concentrationschain are summarized and shows in RXRFigure antagonist 4 and Table activity, 4. but a synergistic effect with an agonist of PPARγ was also found. Subsequently, 7, whichHX531 has a(12 ring) was structure designed at the 6by and introducing 7 positions ofa thenitr trienoico group acid into structure the ofstructure6, and 8,of which the hasdiazepinylbenzoic another ring structure acid derivative at the 4RXR and agonist 5 positions HX600 of (735, were) [56]. created Compound [47,48 12]. showed Compound IC50 =8 1.0shows μM 3 moreagainst potent 10 nM RXR IRX4204 antagonist in a reporter activity thanassay6 toward[47]. Their RXRKαi valuesin COS-7 for cells RXR α[55].in theCompound presence 12 of has [ H]9- beencis retinoicreported acid to show are 3 nMantagonism (6), 9.9 nM towards (7), and not 3 nM only (8 ).RXR, Although but also the RAR IC50 values[56]. It towardalso shows RXR αantagonisticin reporter assayactivity using against CV-1 RAR/RXR cells were or alsoPPAR reportedγ/RXR asheterodimers 8 nM (6), 10.3 [7]. nMCompound (7), and 812 nM shows (8), thea hypoglycemic RXR agonist andeffect the in concentrationan animal model used of were type not2 diabetes, mentioned and [45is –thought47]. Since to improve these RXR insulin ligands resistance activate through specific heterodimers,antagonism to the the authors PPARγ refer/RXR to heterodimer the compounds [7]. as An “selective improvemen RXR modulators”t of leptin resistance [45]. was also reportedPA451 [81]. (9a However,) and PA452 the ( 9bCmax) are value RXR at antagonists 100 mg/kg havingoral administration a pentoxy or of a hexoxy12 to mice group was at 4.1 the μ orthog/mL position(8.5 μM). of Two-week the amino group administration on the benzene of diet ring containing forming the 12 tetramethyltetraline at 0.1% weight showed structure a ofhypoglycemic an N-methyl derivativeeffect [7]. For of RXR the agonistpurpose PA024 of im (proving27). These the compounds oral availability inhibit of RXR/RAR 12, 13a, and heterodimers 13b were [created48]. The [57].pA2 When they were orally administered to rats at 1 mg/kg, the Cmax values were 468 nM and 519 nM, respectively. Further development of these structures yielded 13c, which was reported to show a hypoglycemic effect in KK-Ay mouse, a type 2 diabetes model [58]. Compound 14 has a boron cluster (carborane) at the hydrophobic site instead of a tetramethyltetraline structure [59]. At 1 μM, 14 completely represses RXRα transcription induced by 10 nM RXR agonist PA024 (31). Morishita and colleagues produced new RXR antagonists, 15a and 15b, having a sulfonamide on an amino linking group instead of the N-ethyl group of NEt-TMN (36) [29]. However, their RXR antagonist activity was weaker than that of HX531 (12). To reduce the lipid solubility of existing RXR agonists, the RXR full agonist NEt-3IB (37, EC50 = 19 nM), which has an isobutoxy group at a hydrophobic site, was designed [27,82]. The para position to the isobutoxy group on the benzene ring is electron-rich because this position is also at the ortho position relative to the nitrogen atom of the amino linking group. Therefore, it is easily halogenated. A new RXR antagonist 16, which has a stilbene structure, was created by transformation of an iodine precursor using a palladium catalyst [50]. The pA2 value of 16 toward RXRα agonist NEt-TMN (EC50 = 5.28 nM) [49] was 8.23 based on a Shild plot, while that of PA452 (9b) was 7.11; thus, 16 is one of the strongest RXR antagonists discovered thus far.

Int. J. Mol. Sci. 2018, 19, 2354 10 of 20

value of 9b in the presence of RXR agonist NEt-TMN (36, EC50 = 5.28 nM) [49] was determined as 7.11 from a Shild plot [50]. Bl-1003 (10a)[51] is a propoxy derivative of RXR agonist 28 [79]. Compounds 10b and 10c were designed by replacing the benzoic acid of 10a with nicotinic acid and the propoxy group of 10a with a butyl group, respectively. Reporter assay toward RXRα using 0.1 µM 1 in CV-1 cells gave IC50 = 1100 nM (10a), >10,000 nM (10b), and 67 nM (10c), respectively [52]. Interestingly, although 10c showed a 10-times-greater Kd value than 10a in a competition test using tritium-labeled 1, the antagonism in the reporter assay was 20 times more potent. UVI3003 (11) is an RXR antagonist obtained by converting the 30-methyl group of RXR agonist CD3254 (33)[54] to a pentoxy group. In this study, the authors synthesized analogs with an alkyl chain ranging from C1 to C6 in length, and evaluated RXR agonistic and antagonistic activities. Compounds having a short alkoxy side chain act as partial or weak RXR agonists, but when the number of carbons is more than 3, they show RXR antagonist activity. Among them, 11 shows potent RXR antagonistic activity. Since 34, the positional isomer of 11, shows only weak RXR antagonist activity, the position of the alkoxy group is important for the activity [80]. Compound 11 showed IC50 = 0.24 µM against 10 nM IRX4204 (formerly designated AGN194204 and NRX 194204, RXR agonist) [53] in a reporter assay for RXRα in COS-7 cells [55].

3.2. RXR Antagonists Possessing Another Side Group RXR antagonists possessing another side group instead of the alkoxy chain are summarized in Int.Figure J. Mol.4 andSci. 2018 Table, 194, x. 11 of 19

Structures of RXR agonists Me Me Me Me Me Me N Me SO Me Et N 2 O N N Me Me Me Me Me N N Me HX600 (35) 36

CO2H NEt-3IB (37) CO2H HO2C

Structures of RXR antagonists X Me Me Me N Me NO2 Me Me Me N Me Me SO Et N 2 O N Me Me Me HX531 (12) N 16 N 15a; X = Cl Me HO2C 15b; X = CF3 CO2H CO2H

Figure 4. Chemical structures of RXR agonists and RXR antagonists possessing another side group instead of the alkoxy group on an RXR agonistagonist structure.structure.

3.3. RXR Antagonists Discovered among Natural Products or by Docking Simulation or High-Throughput HX531 (12) was designed by introducing a nitro group into the structure of the diazepinylbenzoic Screening acid derivative RXR agonist HX600 (35)[56]. Compound 12 showed IC50 = 1.0 µM against 10 nM IRX4204The chemical in a reporter structures assay and toward assay RXR dataα ofin RXR COS-7 antagonists cells [55]. classified Compound in this12 categoryhas been are reported shown into Table show 5. antagonism towards not only RXR, but also RAR [56]. It also shows antagonistic activity againstDanthron RAR/RXR (17a or), a PPAR componentγ/RXR of heterodimers rhubarb, used [7]. in Compound Chinese medicine,12 shows showed a hypoglycemic RXR antagonist effect in activityan animal with model IC50 of= 0.11 type μ 2M diabetes, for 1 μM and 1 in is a thoughtreporter to assay improve for Gal4-RXR insulin resistanceα-LBD inthrough HEK293T antagonism cells [60]. The Kd value for RXRα is 6.2 μM. Compound 17a shows antagonist activity toward not only RXR homodimer, but also heterodimers such as PPARγ/RXRα and LXRα/RXRα. Compound 17a has also been evaluated in vivo and was found to improve insulin resistance in DIO mice. Rhein (17b), another compound derived from rhubarb, likewise shows RXR antagonist activity with IC50 = 0.75 μM for 1 in the same assay system [61]. β-Apo-13-carotenone (18), which is produced by β-carotene cleavage, antagonizes RXRα activation by 1 through receptor tetramerization, which stabilizes the inactive state [62]. Though competition assay against 1 in a reporter assay in COS-7 cells has been investigated, the IC50 value was not described. R-Etodolac (19), a non-steroidal anti-inflammatory drug (NSAID), induces apoptosis of tumor cells in a mouse model of prostate cancer [63]. Zhang et al., reported that 19 acts as an antagonist of RXRα and down-regulates RXR. A competition assay with 38.1 nM [3H]1 revealed that the IC50 value of 19 is about 200 μM. After this study, sulindac (20), another NSAID, was also found to bind to RXRα and induce apoptosis [64]. The IC50 value of 20 in competition assay for [3H]1 is 82.9 μM. K-80003 (21a) was created to improve the affinity for RXR (IC50 = 2.4 μM),, and to eliminate COX inhibition [65,66]. Though K-8008 (22b), which has a tetrazole instead of the carboxylic acid moiety of 21a, showed a slightly decreased affinity for RXRα (IC50 = 16.8 μM), crystal structure analysis showed that it binds at the RXRα interface and stabilizes the tetramer of RXR [65]. Zhang et al., also discovered triptolide (22a) [67], which has antagonistic activity against RXRα and induces apoptosis, as well as NSC-640358 (23) [69], by virtual screening. The Kd value of 23 for RXRα is 15.7 μM. Furthermore, they conducted a one hybrid assay using their in-house compound library and identified 24 and 25, which are nitrostyrene derivatives, as RXRα modulators [70]. They detected RXR agonistic activity in the mammalian one-hybrid assay using Gal4-DBD-RXRα-LBD, and antagonistic activity in reporter assay using the full-length RXR homodimer. Zhang et al.,

Int. J. Mol. Sci. 2018, 19, 2354 11 of 20 to the PPARγ/RXR heterodimer [7]. An improvement of leptin resistance was also reported [81]. However, the Cmax value at 100 mg/kg oral administration of 12 to mice was 4.1 µg/mL (8.5 µM). Two-week administration of diet containing 12 at 0.1% weight showed a hypoglycemic effect [7]. For the purpose of improving the oral availability of 12, 13a, and 13b were created [57]. When they were orally administered to rats at 1 mg/kg, the Cmax values were 468 nM and 519 nM, respectively. Further development of these structures yielded 13c, which was reported to show a hypoglycemic effect in KK-Ay mouse, a type 2 diabetes model [58]. Compound 14 has a boron cluster (carborane) at the hydrophobic site instead of a tetramethyltetraline structure [59]. At 1 µM, 14 completely represses RXRα transcription induced by 10 nM RXR agonist PA024 (31). Morishita and colleagues produced new RXR antagonists, 15a and 15b, having a sulfonamide on an amino linking group instead of the N-ethyl group of NEt-TMN (36)[29]. However, their RXR antagonist activity was weaker than that of HX531 (12). To reduce the lipid solubility of existing RXR agonists, the RXR full agonist NEt-3IB (37, EC50 = 19 nM), which has an isobutoxy group at a hydrophobic site, was designed [27,82]. The para position to the isobutoxy group on the benzene ring is electron-rich because this position is also at the ortho position relative to the nitrogen atom of the amino linking group. Therefore, it is easily halogenated. A new RXR antagonist 16, which has a stilbene structure, was created by transformation of an iodine precursor using a palladium catalyst [50]. The pA2 value of 16 toward RXRα agonist NEt-TMN (EC50 = 5.28 nM) [49] was 8.23 based on a Shild plot, while that of PA452 (9b) was 7.11; thus, 16 is one of the strongest RXR antagonists discovered thus far.

3.3. RXR Antagonists Discovered among Natural Products or by Docking Simulation or High-Throughput Screening The chemical structures and assay data of RXR antagonists classified in this category are shown in Table5. Danthron (17a), a component of rhubarb, used in Chinese medicine, showed RXR antagonist activity with IC50 = 0.11 µM for 1 µM 1 in a reporter assay for Gal4-RXRα-LBD in HEK293T cells [60]. The Kd value for RXRα is 6.2 µM. Compound 17a shows antagonist activity toward not only RXR homodimer, but also heterodimers such as PPARγ/RXRα and LXRα/RXRα. Compound 17a has also been evaluated in vivo and was found to improve insulin resistance in DIO mice. Rhein (17b), another compound derived from rhubarb, likewise shows RXR antagonist activity with IC50 = 0.75 µM for 1 in the same assay system [61]. β-Apo-13-carotenone (18), which is produced by β-carotene cleavage, antagonizes RXRα activation by 1 through receptor tetramerization, which stabilizes the inactive state [62]. Though competition assay against 1 in a reporter assay in COS-7 cells has been investigated, the IC50 value was not described. R-Etodolac (19), a non-steroidal anti-inflammatory drug (NSAID), induces apoptosis of tumor cells in a mouse model of prostate cancer [63]. Zhang et al., reported that 19 acts as an antagonist 3 of RXRα and down-regulates RXR. A competition assay with 38.1 nM [ H]1 revealed that the IC50 value of 19 is about 200 µM. After this study, sulindac (20), another NSAID, was also found to bind 3 to RXRα and induce apoptosis [64]. The IC50 value of 20 in competition assay for [ H]1 is 82.9 µM. K-80003 (21a) was created to improve the affinity for RXR (IC50 = 2.4 µM)„ and to eliminate COX inhibition [65,66]. Though K-8008 (22b), which has a tetrazole instead of the carboxylic acid moiety of 21a, showed a slightly decreased affinity for RXRα (IC50 = 16.8 µM), crystal structure analysis showed that it binds at the RXRα interface and stabilizes the tetramer of RXR [65]. Zhang et al., also discovered triptolide (22a)[67], which has antagonistic activity against RXRα and induces apoptosis, as well as NSC-640358 (23)[69], by virtual screening. The Kd value of 23 for RXRα is 15.7 µM. Furthermore, they conducted a one hybrid assay using their in-house compound library and identified 24 and 25, which are nitrostyrene derivatives, as RXRα modulators [70]. They detected RXR Int. J. Mol. Sci. 2018, 19, 2354 12 of 20 agonistic activity in the mammalian one-hybrid assay using Gal4-DBD-RXRα-LBD, and antagonistic activity in reporter assay using the full-length RXR homodimer. Zhang et al., demonstrated that nitrostyrene derivatives 24 and 25 could inhibit the TNFα/NFκB signaling pathway by binding to N-terminally truncated RXRα (tRXRα), leading to TNFα and tRXRα-dependent apoptosis of cancer cells. Moreover, Zhang et al., identified 26 and 27 as RXR antagonists by means of virtual screening using the structure of RXRα-LBD in the complex with CD3254 (33) and a coactivator peptide (PDB code, 3FUG) [71]. These compounds do not bind to the ligand-binding pockets, but bind at the surface of the co-regulator binding site and inhibit co-regulator binding there. Reporter assay using 0.1 µM 1 toward RXRα in MCF-7 cells yielded IC50 values of 2 µM for 26 and 2.45 µM for 27. Zhang and colleagues also found that the statin drugs fluvastatin (28) and pitavastatin (29) are RXR antagonists by virtual screening of an FDA-approved drug database [72]. Further structure optimization of 28 afforded 30, whose Kd value for RXRα is 5.1 µM, which is lower than that of danthron (17a).

4. Evaluation of RXR Antagonistic Activity Though various RXR antagonists have been reported so far, their antagonistic activity has been evaluated in various ways, i.e., in terms of the dissociation constant (Ki value) using a tritium-labeled ligand such as 9-cis-retinoic acid (1), the binding constant obtained by the SPR method, the Kd value, the IC50 value, and pA2 against an RXR agonist in reporter assays (Tables3–5). The dissociation constant has been measured by using radioisotopes. However, this technique is complicated and requires special laboratory equipment, as well as disposal arrangements for radioactive waste. So far, no method using a fluorescent ligand has been established. Additionally, even if the binding ability to the receptor is detected, poor membrane permeability of the compound may influence the actual activity, as in the cases of 10a and 10c [52]. Antagonistic activity of LG100754 (3), the first reported RXR antagonist, was evaluated in terms of the IC50 value on transcriptional activation by 2 in reporter gene assays using CV-1 cells [42]. Similarly, PA452 (9b)[48] and UVI3003 (11)[54] were evaluated using PA024 (31) and CD3254 (33) as agonists, respectively. Since the activity differs depending on the coexisting RXR agonist, it is difficult to compare the observed potencies. The most widely used RXR agonist for reporter gene assays is 1 at the concentration of 0.1 µM. Therefore, it may be better to use this method as one index of activity in screening for new RXR antagonists. The pA2 value is used as an index of competitive antagonist activity. It is the negative logarithm of the molar concentration of the competitive antagonist required to shift the agonist’s EC50 to two-fold higher concentration. The pA2 value is also consistent with the affinity constant for the receptor [83]. Thus, it is desirable to include this method in a more rigorous evaluation of antagonist activity. However, in order to obtain these data, it is necessary to obtain a capacity activity curve of the agonist at three different antagonist concentrations at minimum. Compounds 9b and 16 have been evaluated using the pA2 value as an indicator of competitive antagonist activity [50]. RXR forms not only RXR homodimers, but also heterodimers with various nuclear receptors [2]. Therefore, it is interesting to know whether RXR antagonists act as homodimer antagonists and/or heterodimer antagonists. Though 3 was found as an RXR homodimer antagonist, subsequent experiments revealed that it also acts as an agonist toward RAR/RXR [74], PPARα/RXR [75], and PPARγ/RXR [76,77]. Compound 6 has been found to show a synergistic effect in the presence of an agonist of PPARγ [45]. Compound 9b selectively antagonizes RXR in RXR/RAR heterodimer [48]. One micromole of 12 suppressed the activity of 100 nM rosiglitazone (PPARγ agonist) toward PPARγ/RXR to about a half [7]. Compound 17a has antagonistic activity not only towards the RXR homodimer, but also towards heterodimers such as PPARγ/RXRα, FXR/RXRα, LXRα/RXRα, etc. [60]. However, there was no description of the concentration of each agonist for partner receptors. Int. J. Mol. Sci. 2018, 19, 2354 13 of 20

Among them, for LXR/RXR, T0901317 [84] with an EC50 of 20 nM for LXRα was used at 5 µM. Based on these facts, it seems necessary to standardize assay systems for heterodimers.

5. Latest Research on RXR Antagonists Here, we will briefly summarize research on RXR antagonists reported in the last five years, and then consider the prospects for RXR antagonists. LG100754 (3) was reported to have a protective effect against oxidative stress in retinal pigment epithelial cells [85]. This effect is thought to be caused by activation of PPARγ/RXR. PA452 (9b) was reported to decrease an infection marker concentration-dependently in an HBV infection model using human hepatic stem cells [86]. It is considered that 9b suppresses transcription of viral RNA in HBV-infected hepatocyte-like cells by antagonizing RXR. Teratogenicity of UVI3003 (11) was studied using zebrafish and Xenopus [87,88]. A difference in gene expression in Xenopus eggs was found depending on the exposure time to 11 [89]. In 2017, 11 was found to activate PPARγ in a reporter assay using Xenopus embryos. Moreover, studies using Xenopus treated with RXR agonist bexarotene (2) or 11 revealed that T3-dependent gene expression was altered during transformation of tadpoles [90]. Ro26-5405 (5) is reported to block T helper 2 differentiation and to prevent allergic lung inflammation [8]. The mechanism was suggested to be inhibition of Th2 differentiation by antagonizing RXR. In addition, in an atopic dermatitis model mouse, 11 was used as a tool to investigate the expression of thymic stromal lymphopoietin (TSLP), which is triggered in atopic dermatitis and is involved in suppression [91]. TSLP is an IL-7-like cytokine and was shown to be a master switch of allergic inflammation at the epithelial cell–dendritic cell interface, leading to allergic sensitization. It is reported that the expression of TSLP involves RARγ/RXR. Huang et al. used 12 as a tool to show that activation of RXR has a protective effect against hypoxia-reoxygenation disorder in H9c2 cardiomyocytes [92]. Franklin and colleagues revealed that phagocytosis and remyelination of myelin debris accompanying aging progressed upon activation of RXR using 12 [93]. Kajta et al. reported that apoptotic neurotoxic activity of 4-para-nonylphenol occurs simultaneously with RXR activation and a decrease in classical estrogen receptor signaling. They found that the effect of 4-para-nonylphenol on mitochondrial membrane potential was canceled by 12, indicating that this neurotoxicity involves activation of RXR [94]. Compound 12 is also reported to decrease both mobility and growth of Trichuris muris (a parasite) in vitro, indicating its potential as an anthelmintic drug [95]. RXR is negatively regulated by 1 and 12 through a nongenomic effect on platelets and thrombus formation [96]. Compound 12 is also used as a tool to investigate the influence of environmental hormones on RXR. For example, the mechanism of neurotoxicity by dichlorodiphenyldichloroethylene (DDE) [97], the effect of tributyltin on osteogenesis [98], and the toxicity of organotin [99] were found to involve transcriptional activation of RXR. Zhang and colleagues found that R-etodolac (19), a NSAID, induces an antitumor effect via antagonistic activity toward RXRα, and also induces degradation of RXRα via the ubiquitin-proteasome system [63]. Subsequently, they also found RXR antagonist activity of sulindac (20), another NSAID. They suggested that nongenomic action of an N-terminally truncated RXRα (tRXRα) could play a role in the crosstalk with TNFα signaling in cancer cells [64,100]. tRXRα, which is produced by proteolytic cleavage of full-length RXRα, is highly expressed in a variety of tumor cells and tissues [101,102]. Furthermore, 20 was structurally developed to afford compounds 21a and 21b [64,65]. Crystal structure analysis of 21b in RXRα revealed that it binds to the RXR interface rather than the ligand-binding pocket, stabilizing RXR tetramers [65]. Similarly, Zhang et al., discovered triptolide (22a) in a natural product library [67]. Compound 22a regulates the survival of tRXRα-dependent cancer cells by apoptosis induction. Furthermore, 22a was structurally converted to TRC4 (22b), and 22b showed tRXRα-selective antagonism without transcriptional activation of RXRα [68]. In addition, NSC-640358 (23), which was discovered by virtual Int. J. Mol. Sci. 2018, 19, 2354 14 of 20

screening (Kd = 15.7 µM), induces apoptosis of cancer cells [69]. Compound 23 has been reported to inhibit the transcriptional activation of RXR homodimer by 1, but the IC50 value was not given. In addition, Zhang et al., carried out one-hybrid assay with a compound library and found nitrostyrene derivatives 24 and 25 as RXR modulators [70]. Although these compounds showed RXR activity in mammalian one-hybrid assay using Gal4-DBD-RXRα-LBD, they showed antagonist activity in reporter assays using full-length RXR homodimer. Interestingly, 24 and 25 stabilize the RXR homodimer, unlike 21b. Size-exclusion chromatography indicated that the structure of the homodimer differs from the activated structure. These compounds have no activity to down-regulate tRXRα. Compounds 26, 27 were also discovered by virtual screening [71].

6. Important Points in the Use of RXR Antagonists Some RXR antagonists reported to date show agonistic activity on RXR heterodimers. For example, LG100754 (3), in addition to antagonism of the RXR homodimer [43], shows agonist activity toward RAR/RXR [74], PPARα/RXR [75], and PPARγ/RXR. [76,77] UVI3003 (11) also shows agonistic activity for PPARγ/RXR [55]. HX531 (12), the most widely used RXR antagonist in vivo, has also been reported to antagonize RAR. [7] Chen et al. reported that down-regulation of RXRα leads to cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in aged macrophages [103]. These data were obtained by administering 12 to mice. However, 12 was administered at a high concentration of 10 mg/kg i.p., every 24 h for seven days. The Cmax of 12 in mice after 100 mg/kg oral administration was only 4.1 µg/mL (8.5 µM) [7]. In order to improve oral absorption, 13a, 13b and 13c were created [57,58]. However, although 13a and 13b give Cmax values of approximately 500 nM after oral administration to rats at 1 mg/kg, there is no report as yet on their activities toward RXR heterodimers.

7. Conclusions RXR antagonists are of increasing interest because of their therapeutic effects, i.e., hypoglycemic effect in type 2 diabetes models and anti-tumor effect via tRXRα. However, currently available RXR antagonists require high dosages in vivo when orally administered because of their poor absorption, and some of them activate heterodimers. Thus, there is still a need to develop new RXR antagonists to overcome these problems, and such compounds would be promising drug candidates, as well as useful experimental tools for biological studies on the roles of nuclear receptors.

Funding: This paper was supported by Okayama University. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations i.p. Intraperitoneal injection NFκB Nuclear factor-kappa B NR Nuclear receptor Th2 T helper type 2 TNFα Tumor necrosis factor alpha TR Thyroid hormone receptor VDR Vitamin D receptor PXR Pregnane X receptor

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