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. 2021 May 7;12(6):1024–1029. doi: 10.1021/acsmedchemlett.1c00201

Creation of Fluorescent RXR Antagonists Based on CBTF-EE and Application to a Fluorescence Polarization Binding Assay

Maho Takioku †, Yuta Takamura †, Michiko Fujihara †,‡, Masaki Watanabe †, Shoya Yamada †,§, Mayu Kawasaki ∥, Sohei Ito ∥, Shogo Nakano ∥, Hiroki Kakuta †,*
PMCID: PMC8201752  PMID: 34141088

Abstract

graphic file with name ml1c00201_0006.jpg

Retinoid X receptor (RXR) ligands often bind in modes in which the carboxy group forms a hydrogen bond inside the ligand-binding pocket (LBP). However, our previously reported RXR antagonist, CBTF-EE (4a), binds with its carboxy group directed outside the LBP and its alkoxy side chain located inside the LBP. Here, we examined the binding modes of 4b and 4c bearing a nitrobenzoxadiazole (NBD) or boron-dipyrromethene (BODIPY) fluorophore, respectively, at the end of the alkoxy chain of 4a. Both compounds function as RXR antagonists. 4c, but not 4b, was available for a fluorescence polarization binding assay, indicating that rotation of BODIPY, but not NBD, is restricted in the bound state. The fluorescence findings, supported by docking simulations, suggest the fluorophores are located outside the LBP, so that the binding mode of 4b and 4c is different from that of 4a. The assay results were highly correlated with those of a [3H]9-cis-retinoic acid assay.

Keywords: RXR, fluorescence, NBD, BODIPY, fluorescence polarization, binding assay

The World Health Organization (WHO) predicts that lifestyle-related diseases such as ischemic heart disease, stroke, and chronic obstructive pulmonary disease (COPD) will account for a large proportion of the world’s causes of death in the 21st century.1 Such lifestyle-related diseases are related to lack of exercise as well as obesity and arteriosclerosis, which in turn are associated with excessive intake of high-fat diets and smoking.2 These diseases are generally treated with drugs that target specific proteins. For example, calcium-channel blockers and adrenaline β-blockers are used to treat ischemic heart disease, while antithrombotic drugs such as cyclooxygenase inhibitors are used to treat stroke, and anticholinergic drugs and β-stimulators that induce bronchodilation are used to treat COPD. However, as these diseases are caused by multiple factors in vivo, there is also interest in the approach used in traditional oriental medicine, such as Chinese medicine, which aims to broadly improve homeostasis in the body. We considered that if such a broadly based improvement of homeostasis could be achieved using a molecular-targeted drug, then this might be an effective approach to reduce the number of different drugs taken by patients. We call this idea “Western-style Chinese medicine”. With this idea in mind, we have focused on retinoid X receptors (RXRs),3 because it is well-known that RXRs function in concert with a variety of other nuclear receptors.

RXRs are members of the nuclear receptor superfamily and regulate the transcription of downstream genes in response to ligand binding.4 Although RXR homodimers exist, their function remains unclear.5,6 On the other hand, RXR heterodimers with other nuclear receptors, such as peroxisome proliferator-activated receptor (PPAR), thyroid hormone receptor (TR), and liver X receptor (LXR), are key players in the regulation of the internal body environment.5,6 In particular, PPAR/RXR and LXR/RXR are reported to be allosterically activated by an RXR agonist alone, a phenomenon called the permissive effect.7 Bexarotene (1, Figure 1),8 which is clinically used to treat cutaneous T-cell lymphoma (CTCL), is an RXR agonist that activates these permissive RXR heterodimers.7 The effectiveness of RXR agonists for lifestyle-related diseases has also been reported, underlining the in vivo diversity of RXR function.9−11 Interestingly, therapeutic effects of RXR antagonists in an animal model of type 2 diabetes12 and on viral infection in vitro13 have also been reported.

Figure 1.

Figure 1

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Chemical structures of RXR agonists 1 and 3 and antagonists 2, 4, and 5.

The representative RXR antagonist PA452 (2) inhibits not only RXR homodimers but also RXR heterodimers activated by partner receptor agonists.14−16 This means that RXR antagonists of this type induce an “allosteric effect” that influences the partner receptor in the permissive RXR heterodimers. We were interested in RXR antagonists that do not show such allosteric inhibition but that effectively inhibit RXR homodimers. Based on the observation that allosteric activation of PPARγ/RXR, a permissive RXR heterodimer, by CBTF-PMN (3) is weak,17 we designed a nonallosteric RXR antagonist, CBTF-EE (4a), and confirmed that it does not show allosteric inhibition of permissive RXR heterodimers, in contrast to 2.18 One reason for this difference is thought to be the binding mode of 4a in the human RXRα ligand-binding pocket (hRXRα-LBP). Most reported RXR ligands bind in modes in which the carboxy group enters the LBP, whereas our X-ray cocrystal analysis indicated that the carboxy group of 4a is directed outside the hRXRα-LBP, and instead, the alkoxy chain is located inside the LBP.18 In addition, an AutoDock docking simulation of our previously reported fluorescent RXR antagonist NEt-C343 (5) suggested that 5 has a similar binding mode to 4a, i.e., that the fluorophore coumarin 343 (C343), not the carboxy group, enters the LBP.19

Consequently, we were interested to see whether the introduction of a fluorescent group at the end of the alkoxy chain of 4a would afford fluorescent RXR-antagonistic derivatives whose fluorophore would enter the LBP. Compounds that show changes in fluorescence properties in response to a change in the environment or a decrease in molecular motility are expected to be applicable for RXR ligand-binding assays. Thus, in this study, we designed and synthesized derivatives of 4a bearing a fluorescent group at the end of the alkoxy side chain and evaluated their RXR antagonist activities and fluorescence properties.

As fluorophores, we selected nitrobenzoxadiazole (NBD) and boron-dipyrromethene (BODIPY), which have fairly similar excitation and fluorescence wavelengths20,21 that are longer than those of fluorescence amino acids such as tyrosine and tryptophan present in RXR and can be detected by a fluorescent plate reader using readily available filters. Furthermore, NBD is a relatively small molecule, while BODIPY is sterically bulky. The size of the fluorophore is likely to affect its ability to enter the LBP.

Compounds 4b (bearing NBD) and 4c (bearing BODIPY) were obtained by linking NBD-Cl or carboxy-BODIPY via an amino group (SI Schemes S1 and S2). A reporter gene assay using COS-1 cells revealed that both 4b and 4c act as RXR antagonists, although they are less potent than 4a (Figure 2B). The fluorescence properties in MeOH solution were evaluated, and the excitation (λEx)/fluorescence (λEm) maxima of 4b and 4c were 464 nm/530 nm and 499 nm/509 nm, respectively (Figure 2C,D and Table 1). The molar extinction coefficient (ε) and the fluorescence quantum yield (Φ) of 4c were both slightly larger than those of 4b. A similar tendency was also observed in EtOH (Table 1).

Figure 2.

Figure 2

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RXR-antagonistic activity and fluorescence properties of 4b and 4c. (A) Chemical structure of NEt-TMN (6), an RXR agonist.22 (B) Antagonistic activities of 4a–4c toward hRXRα homodimer in COS-1 cells. Transactivation of 6 (open circles) relative to DMSO in the absence or presence of 1 μM 4a (green), 4b (blue), or 4c (pink). Data are mean ± SD (n = 3). (C,D) Fluorescence excitation spectra (broken line) and emission spectra (solid line) of 1 μM 4b (C) and 4c (D) in MeOH solution, respectively.

Table 1. Fluorescence Properties of 4b and 4c.

compd solvent ε [L/(mol·cm)] λEx [nm] λEm [nm] Φa
4b MeOH 6.7 × 103 464 530 0.224 ± 0.05
EtOH 8.4 × 103 461 527 0.237 ± 0.03
4c MeOH 2.8 × 104 499 508 0.349 ± 0.01
EtOH 2.8 × 104 499 509 0.471 ± 0.04
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a

Data are mean ± SD (N = 3).

b

Standard compound: coumarin 6 (λEx = 459 nm, Φ = 0.78).23

Ligand binding to the hRXRα-LBD quenches the fluorescence around λEm = 330 nm due to Trp282 and Trp305 in the LBD via FRET, and binding assays based on this phenomenon can be used to determine the Kd values.24,25 Concentration-dependent quenching around λEm = 330 nm was observed at λEx = 290 nm (Figure 3A,B), and the Kd values of 4b and 4c were obtained as 0.629 ± 0.074 and 0.761 ± 0.057 μM (N = 3, mean ± SD), respectively. As regarding the fluorescence derived from each fluorophore, the fluorescence intensity of 4b at 550 nm and that of 4c at 520 nm increased in a concentration-dependent manner (Figure 3A,B). The fluorescence intensity also increased in response to excitation at the maximum excitation wavelength of each compound (SI Figure S1). 4c showed a high correlation between quenching at 330 nm and increasing fluorescence intensity near 520 nm, but 4b did not (Figure 3C–F).

Figure 3.

Figure 3

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Fluorescence spectra and intensity ratios of 4b or 4c in the absence and presence of hRXRα-LBD. (A,B) Fluorescence titration emission spectra of hRXRα-LBD (0.5 μM) upon addition to (A) 4b or (B) 4c (0–16 μM, black–brown) in HEPES buffer at λEx = 290 nm. (C,D) Fluorescence intensity ratios [1 – F330 nm/F0330 nm] (λEx = 290 nm/λEm = 330 nm, open circles) and [F550 nm/F0550 nm] (4b; λEx = 290 nm/λEm = 550 nm, open triangles; λEx = 478 nm/λEm = 550 nm, closed diamonds) or [F520 nm/F0520 nm] (4c; λEx = 290 nm/λEm = 520 nm, open triangles; λEx = 502 nm/λEm = 520 nm, closed diamonds). F0 and F are the fluorescence intensity in the absence of 4b or 4c at each wavelength and the observed fluorescence intensity, respectively. (E,F) Replots of selected data from (C,D) on a linear scale.

Since the fluorescence intensity of 4c increased in response to binding to the hRXRα-LBD, it was expected that the fluorescence intensity would decrease due to competition in the presence of RXR ligands. However, although there appeared to be competition between 4c and 1 after incubation for 1 h, there was significant variability in the data (Figure 4A). A similar tendency was seen upon incubation for 0 or 2 h (SI Figure S2A). Therefore, we decided to examine the fluorescence polarization instead. Fluorescence polarization is considered to be due to a reduction of the mobility of the fluorescence label upon binding to a receptor having a large molecular weight.26,27 When changes in the fluorescence polarization of 4c in the presence of various concentrations of hRXRα-LBD were investigated, stable and reproducible results were obtained (Figure 4B). Interestingly, competition between 4b and 1 could not observed (SI Figure S2C). The fact that the fluorescence polarization of 4b was unaffected in the presence of the hRXRα-LBD indicates that the motility of the NBD fluorophore is not affected by binding of 4b to the hRXRα-LBD. This in turn suggests that the fluorophore at the end of the alkoxy chain of 4b exists outside the LBP. Since NBD is structurally small and has low lipophilicity, it seems likely that a “propeller effect”28 occurs, i.e., NBD can rotate freely like a propeller in the solvent outside the LBP. The BODIPY moiety of 4c is larger than NBD and so should also be located outside the hRXRα-LBD. However, the larger size of BODIPY compared to NBD may mean that the BODIPY moiety of 4c can nevertheless not rotate freely, and consequently, fluorescence polarization is observed. Docking simulations on AutoDock Vina29 were performed using an X-ray structure of the RXR-antagonistic form. For 4c, since AutoDock vina does not support the B atom, the structure other than the fluorophore (S1, SI Figure S3) was separated from the fluorophore (S2, SI Figure S3) using Chem3D. These structures and 4b were energetically minimized by means of molecular mechanics (MM) and semiempirical molecular orbital calculations (MOPAC, PM3 for S1 and 6c, and AM1 for S2). When we used the hRXRα-LBD coordinates (PDB code: 7CFO(18)) obtained from the X-ray analysis of the cocrystal with 4a, docking simulation with S1 or 4c failed. The reason for this is thought to be that the space occupied by the alkoxy side chain of 4a is too narrow to accept the benzamide moiety of S1 or NBD. Docking simulation using the hRXRα-LBD coordinates obtained from the X-ray analysis of the cocrystal with RXR antagonist LG100754 (S3, SI Figure S3),30 which has a wider ligand-binding pocket (LBP), was successful. For 4c, the lowest-energy structure of S1 was combined with S2. The obtained binding mode seems reasonable, although a cocrystal structure analysis will be needed to enable detailed discussion. The docking data show that 4b or 4c binds in modes in which the carboxy group forms a hydrogen bond inside the LBP and each fluorophore is located outside the LBP, whereas the carboxylic acid moiety of 4a is located outside the LBP (Figure 4C,D and SI movies S1 and S2). These results support the binding modes of 4b and 4c inferred from the fluorescence findings.

Figure 4.

Figure 4

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Fluorescence change upon binding with hRXRα-LBD and docking study. (A) Fluorescence intensity and (B) fluorescence polarization data for 4c (0.3 μM) at λEx = 485 nm/λEm = 535 nm. The saturation curve (pink circles) was calculated by subtracting “with 10 μM 1 (open triangles)” from “without 1 (closed triangles)”. Data are mean ± SD (N = 3). The Kd value of 4c for the hRXRα-LBD was obtained as 0.375 ± 0.134 μM from curve fitting of the dose-dependent saturation curves (pink). (C) Comparison of the binding mode of 4c (pink) to hRXRα-LBD (PDB code: 3A9E,30 gray) predicted by AutoDock with that in the X-ray cocrystal structure of 4a (green) with the hRXRα-LBD (PDB code: 7CFO,18 pale orange). (D) Predicted binding modes of 4b (blue) and 4c (pink) in the antagonistic form with hRXRα-LBD (PDB code: 3A9E(30)). The fluorophore is located outside the LBP in both cases.

Since the above results indicate that 4c could be used for a fluorescence-polarization-based RXR ligand-binding assay, we set out identify suitable conditions. Figure 4B shows the results obtained with a fixed concentration of 0.3 μM 4c. We also tried 0.1 μM 4c, but the baseline was higher, and the change in fluorescence polarization (ΔmP) was smaller (SI Figure S2B). There was no significant difference in incubation time between 1 and 2 h (SI Figure S2B). We also examined the time-dependent change of the Z′ factor as a measure of the quality of the screening31 and found that it was >0.6 at both time points, indicating that this assay is stable. As regarding the receptor concentration, a receptor concentration at which 50–80% of the receptors are in the bound state is appropriate for the ligand-binding assay.32 Thus, the hRXRα-LBD concentration should be 0.5 to 1 μM when 0.3 μM 4c is used (SI Figure S2B). Finally, we selected 0.3 μM 4c, 0.5 μM hRXRα-LBD, and a 1 h incubation time as the optimum conditions and examined the change in fluorescence polarization at various concentrations of test compound 1. A concentration-dependent decrease of fluorescence polarization was observed (Figure 5A), and the IC50 was obtained as 632 ± 103 nM (mean ± SD, N = 3). The Ki value was calculated as 350 nM using the Cheng–Prusoff equiation33 (the Kd value of 4c is 375 nM as mentioned in Figure 4). The Ki value of 1 determined with the widely used [3H]9-cis retinoic acid assay was 201 nM. Therefore, we next evaluated the binding abilities of various other RXR ligands (Figure 5C) under the same conditions. There was a high correlation between the obtained Ki values and those in the [3H]9-cis retinoic acid assay (R2 = 0.9614) (Figure 5B, SI Table S1, and SI Figure S4).

Figure 5.

Figure 5

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(A) Fluorescence polarization plot for 1 using 4c at λEx = 485 nm/λEm = 535 nm. Data are mean ± SD (N = 3). (B) Correlation of Ki values between this assay and the [3H]9-cis retinoic acid assay. Light blue, bexarotene (1); red, PA452 (2); green, CBTF-PMN (3); blue, CBTF-EE (4a); yellow, NEt-TMN (6); white, CBt-PMN (7); pink, NEt-3IB (9); black, NEt-SB (11); brown, NEt-BA (12); and orange, TBTCl (13). (C) Chemical structures of the test compounds.

In conclusion, based on our previous finding that the alkoxy chain of RXR antagonist 4a is located inside the RXR-LBP, we designed 4b and 4c bearing the fluorophores NBD and BODIPY, respectively, at the end of the alkyl chain. Although 4b and 4c both showed RXR antagonist activity, an increase in fluorescence polarization was observed only for 4c in response to binding to the hRXRα-LBD, despite the fact that NBD is smaller than BODIPY. The fluorescence findings suggest that the fluorophore of 4b and 4c lies outside the LBD, unlike the alkyl chain of 4a. Thus, the binding mode of 4b or 4c is different from that of 4a. Docking calculations were consistent with this conclusion. Nevertheless, presumably because of restricted rotation of the large BODIPY moiety outside the LBP, 4c could be used to evaluate the binding abilities of RXR ligands by means of a fluorescence polarization assay. The Ki values of various RXR ligands obtained in the optimized assay using 4c were highly correlated with those obtained with the widely employed radioisotope method. Our assay enables convenient screening of rexinoids within a few hours, without the need for processes such as filtration or precautions associated with the use of a radiolabel.

Acknowledgments

The authors are grateful to the Division of Instrumental Analysis, Okayama University, for the NMR and MS measurements.

Glossary

Abbreviations

BODIPY

boron-dipyrromethene

COPD

chronic obstructive pulmonary disease

CTCL

cutaneous T-cell lymphoma

Em

emission

Ex

excitation

FITC

fluorescein isothiocyanate

FRET

fluorescence resonance energy transfer

LBP

ligand-binding pocket

LXR

liver X receptor

NBD

nitrobenzoxadiazole

PPAR

peroxisome proliferator-activated receptor

RXR

retinoid X receptor

hRXRα-LBD

human retinoid X receptor α ligand-binding domain

SD

standard deviation

TR

thyroid hormone receptor

Trp

tryptophan

WHO

World Health Organization

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00201.

  • Experimental section, chemistry (preparation of RXR ligands, compound data), in vitro assay (luciferase reporter gene assay, UV–vis and fluorescence spectra measurements, determination of fluorescence quantum yield, cloning, expression, and purification of ligand-binding domain of RXRα, fluorescence titration measurements, fluorescence competition measurements, fluorescence polarization binding assay using 4c, calculation of Z′-factor), docking simulation using AutoDock vina, synthetic schemes, figures (fluorescence spectra of 4b and 4c, fluorescence binding assay data, chemical structures for prediction of binding mode of 4c, fluorescence polarization ratio curve of each compound using 4c), table for Ki values of test compounds, NMR charts, MS charts, HPLC charts, and supporting movie legends (PDF)

  • Structural comparison of hRXRα-LBD/4a (PDB code: 7CFO) and a binding model of 4c in the antagonistic form with hRXRα-LBD (PDB code: 3A9E) obtained with AutoDock vina (MOV)

  • Binding models of 4b and 4c in the antagonistic form with hRXRα-LBD (PDB code: 3A9E) obtained with AutoDock vina (MOV)

  • Molecular formula strings (XLSX)

Author Contributions

# M.T. and Y.T. contributed equally.

Author Contributions

S.Y. and H.K. conceived and designed the project. M.T. and M.W. synthesized compounds. M.T., Y.T., and M.W. analyzed NMR and fluorescence data. M.T. and Y.T. evaluated the fluorescence properties. Y.T. and H.K. performed the docking simulation. M.T. and M.F. performed the fluorescence polarization binding assay. M.K., S.I., and S.N. produced hRXRα-LBD. The manuscript was written by M.T., Y.T., and H.K.

This work was partially supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) 12J06716 (to S.Y.), grants from the Okayama Foundation for Science and Technology (to H.K.), The Tokyo Biochemical Research Foundation (TBRF) (to H.K.), the Kobayashi Foundation (to H.K.), and scholarship support from the Shoshisha Foundation (to Y.T.)

The authors declare the following competing financial interest(s): This research was partially performed in collaboration with AIBIOS Co., Ltd. M.F. was an employee of AIBOS. No other author reports any potential conflict of interest relevant to this article.

Supplementary Material

ml1c00201_si_001.pdf (1.5MB, pdf)
ml1c00201_si_002.mov (8.2MB, mov)
ml1c00201_si_003.mov (9.6MB, mov)
ml1c00201_si_004.xlsx (9.6KB, xlsx)

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ml1c00201_si_001.pdf (1.5MB, pdf)
ml1c00201_si_002.mov (8.2MB, mov)
ml1c00201_si_003.mov (9.6MB, mov)
ml1c00201_si_004.xlsx (9.6KB, xlsx)

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