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Potent antagonists of ROR#t, cardenolides from Calotropis gigantea, exhibit discrepant effects on the differentiation of T lymphocyte subsets Juan Liu, Li-Ping Bai, Fen Yang, Xiaojun Yao, KAWAI LEI, Christopher Wai Kei Lam, QIBIAO WU, Yuxin Zhuang, Riping Xiao, Kangsheng Liao, Hioha Kuok, Ting Li, and Liang Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01063 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on December 31, 2018

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Potent antagonists of RORγt, cardenolides from Calotropis gigantea, exhibit

2

discrepant effects on the differentiation of T lymphocyte subsets

3 4

Juan Liu1, Li-Ping Bai1, Fen Yang1, Xiaojun Yao1, Kawai Lei1, Christopher Wai Kei

5

Lam1, Qibiao Wu1, Yuxin Zhuang1, Riping Xiao1, Kangsheng Liao1, Hioha Kuok1,

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Ting Li1,*, Liang Liu1,*

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1. State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute

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for Applied Research in Medicine and Health, Macau University of Science and

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Technology, Macau, People's Republic of China.

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* Corresponding authors

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Address for correspondence: State Key Laboratory of Quality Research in Chinese

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Medicines/Macau Institute for Applied Research in Medicine and Health, Macau

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University of Science and Technology, Avenida Wai Long, Taipa, Macau, China

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E-mail addresses: [email protected]; [email protected]

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Tel: +853 8897-2401; +853 8897-2799

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Fax: +853 2882-5886; +853 2882-7222

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Keywords:

Uscharin;

Calactin;

Calotropin;

T

1


cell

subsets;

RORγt

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Abstract

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RORγt is the master transcription factor of IL-17 cytokine expression and Th17

21

lymphocyte differentiation, which are responsible for the induction of many

22

autoimmune diseases. Recently, RORγt has become an attractive target for drug

23

development to treat these types of diseases, and the field of RORγt antagonist

24

research is now extremely competitive. In our current study, molecular docking was

25

applied to demonstrate that cardenolides, including uscharin, calactin and calotropin

26

derived from Calotropis gigantea, probably directly bound to RORγt. Therefore, the

27

inhibitory effect was further validated using a luciferase reporter assay. Because

28

RORt is the key transcriptional factor for Th17 differentiation, the effects of these

29

compounds on Th17 differentiation were studied by flow cytometry. The results

30

showed that uscharin, calactin and calotropin inhibited Th17 differentiation from 100

31

to 500 nM. Furthermore, uscharin had a better effect than digoxin, a well-known

32

inverse agonist of RORt, in reducing Th17 polarization. Additionally, the effects of

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the cardenolides on the differentiation of other Th lineages, including Th1, Th2 and

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Treg, were investigated. Uscharin suppressed Th1, Th2 and Treg cell differentiation,

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while calactin suppressed the differentiation of Th1 cells and calotropin did not

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influence the other T cell subsets, indicating that calactin suppressed Th1 and Th17

37

differentiation and calotropin selectively quenched Th17 polarization. Structural

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analysis of the three compounds showed that the selectivity of uscharin, calactin and

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calotropin on the suppression of the different subsets of T cells is correlated to the

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minor differences in their chemical structures. Collectively, calactin and calotropin

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have greater potential to be developed as lead compounds than uscharin to treat

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autoimmune diseases mediated by Th17 and/or Th1 cells.

43

2



45

1. Introduction

46

The expression of retinoic acid-related orphan receptors γt (RORγt, also termed

47

RORC2) is predominantly found in immune organs, such as the thymus, immature

48

double-positive thymocytes, and Th17 lymphocytes [1]. Th17 cells are absent in

49

RORγt-deficient mice, and the differentiation of Th17 cells in vitro and

50

Th17-mediated inflammatory disease in vivo require the induction of RORγt,

51

indicating that RORγt is the key transcription factor for Th17 differentiation [2]. Th17

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cells are crucial effector cells by producing numerous cytokines, including IL-17 and

53

IL-23, to orchestrate the pathology of various autoimmune diseases, including

54

rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD)

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and systemic lupus erythematosus (SLE). Therefore, inhibition of Th17 cell

56

differentiation through direct suppression of RORγt is a promising strategy to treat

57

autoimmune diseases.

58

According to the literature, digoxin and its derivatives can suppress Th17 cell

59

differentiation by antagonizing RORt activity without affecting the differentiation of

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other T cell lineages [3], though its cardiotoxicity constrains its clinical application.

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Synthesized compounds, including T0901317 and its derivative SR1001, also act as

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inverse agonists of RORt and in turn inhibit the differentiation of Th17 cells in vitro

63

and in vivo [4, 5]. However, the moderate efficacy of the two compounds does not

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fulfill the requirements of clinical application. Therefore, antagonists of RORγt

65

remain unavailable and desirable.

66

Cardenolides, including uscharin, calactin and calotropin, have been isolated

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from Calotropis gigantea, which is a common plant in Africa, Eastern Asia and

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Southeast Asia. This plant is a rich source of cardenolides [6, 7]. Flowers, latex, roots

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and leaves of this plant reportedly have biological activity against arthritis, as well as

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bacteria, fungi and influenza [8-11]. However, the effects of these compounds on

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Th17 cells and other subsets of the T lymphocyte population have not been

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investigated. 3


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Th1 cells are the quintessential cell type involved in inflammation. The signature

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cytokine of the Th1 subset, IFN-, is associated with the pathogenesis of several

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autoimmune diseases. According to several different observations in humans, Th2

76

cells have a protective effect in autoimmune conditions via secretion of IL-4, the

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signature cytokine of Th2 lymphocytes. Another T cell subset, regulatory T cells

78

(Treg), also mediates immune suppression through the expression of inhibitory

79

receptors and secretion of anti-inflammatory cytokines.

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In the current study, we have for the first time demonstrated that uscharin,

81

calactin and calotropin significantly inhibited Th17 cell differentiation by

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antagonizing RORγt activity. After determining the effects of these compounds on

83

other subsets of T lymphocytes and analyzing their chemical structures, we found that

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the discrepant effects of these compounds on the different T lymphocyte subsets are

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most likely attributed to their differences in chemical structure.

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2. Material and methods

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2.1 Reagents and animals

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Anti-mouse CD3, CD28, IL-4, IFN-γ, recombinant mouse IL-6, IL-23, IL-2,

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IL-12, IL-4, recombinant human TGF-β1, APC anti-mouse IL-4, PE anti-mouse

90

IL-17A, FITC anti-mouse IFN-γ, PerCP/Cy5.5 anti-mouse CD4, monensin solution,

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brefeldin A solution, and red blood cell lysis buffer were obtained from BioLegend

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(San Diego, CA, USA). A primary antibody against β-actin was obtained from Santa

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Cruz (Dallas, Texas, USA). APC anti-mouse Foxp3 and RORγt antibodies were

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obtained from eBioscience (San Diego, CA, USA). Phorbol-12-myristate-13-acetate

95

(PMA) was obtained from Sigma (Santa Clara, CA, USA). Ionomycin was obtained

96

from Millipore (Billerica, Massachusetts, USA). CytofixTM Fixation buffer and stain

97

buffer were obtained from BD Bioscience (Franklin, NJ, USA). CD4+ T cell isolation

98

kits were purchased from Miltenyi Biotec (Cologne, Germany). Lipofectamine™

99

LTX Reagent with PLUS™ Reagent was obtained from Invitrogen (Carlsbad, CA, 4



100

USA). RPMI-1640, DMEM, trypsin and fetal bovine serum (FBS) were provided by

101

GIBCO (Grand Island, NY, USA).

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C57BL/6 mice (8-14 weeks old) were supplied by the Laboratory Animal

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Services Center, The Chinese University of Hong Kong (Hong Kong, China). Mice

104

were separately housed under a 12:12 h light cycle in rooms maintained at 20 ± 0.5 °C.

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Animal care and experiments were conducted in accordance with Macau University

106

of Science and Technology regulations.

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2.2 Compounds, plasmid and cell lines

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Uscharin, calactin, and calotropin (>98% purity, verified by HPLC) were

109

isolated from Calotropis gigantea according to our previous report [12]. The purity of

110

these compounds was validated by HPLC (Fig. 1A). These compounds were

111

dissolved in DMSO at a concentration of 100 mM and stored at -80°C for future study.

112

Digoxin was purchased from Sigma. Human ROR/RORC/NR1F3 luciferase stable

113

reporter cell line and RORt plasmid was purchased from Novus Biologicals

114

(Centennial, CO, USA). HEK293 cell line was provided by American Type Culture

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Collection (ATCC, Manassas, VA, USA).

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2.3 Molecular docking

117

Molecular docking calculations were employed to study the interaction between

118

the small molecules and RORγt through the Induced Fit Docking module in

119

Schrodinger software (Schrodinger, Inc., New York, NY, USA, Year 2009). The 3D

120

structures of uscharin, calactin and calotropin were constructed and optimized in the

121

LigPrep module. The 3D structure of RORγt co-crystallized with digoxin was derived

122

from the PDB database (PDB ID: 3B0W) and prepared using the Protein Preparation

123

Wizard during the induced fit docking. The water molecules within 5 Angstrom from

124

digoxin were kept, and the centroid of the digoxin was used to define the active site in

125

the molecular docking calculations.

5


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2.4 Luciferase reporter assay

127

ROR t LUCPorterTM Stable Reporter cells and LUCPorterTM Vector Control

128

HEK293 cells were plated (5×104/well) in 96-well plates in triplicate in RPMI 1640

129

medium with 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100

130

μg/ml) and puromycin (3 μg/ml) overnight. The cells were incubated with different

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concentrations of uscharin, calactin, calotropin or digoxin for an additional 16 h. One

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hundred microliters of assay solution was added to the wells. The mixture was

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incubated in the dark at room temperature for 30 min and transferred into a white

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96-well plate. Finally, the luciferase activity was assessed with a Sirius luminometer

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(Bertold Detection System GmbH, Pforzheim, Germany).

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2.5 Transfection

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The transfection assay was performed according to the manufacturer’s

138

instruction for Lipofectamine LTX. HEK293 cells were seeded in DMEM with 10%

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FBS at 4×105 cells per well. Five hundred microliters of OPTI-MEM Reduced Serum

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Media containing 2 μg of DNA was added to the cells to be transfected, and then 2 μL

141

of PLUS was added into the above diluted OPTI-MEM and DNA solution, followed

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by gentle mixing and incubation for an additional 5 min at room temperature.

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Subsequently, Lipofectamine LTX™ Reagent was added to the above solution and

144

then mixed gently and incubated for 30 minutes at room temperature to form

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DNA-Lipofectamine

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DNA-Lipofectamine LTX Reagent complexes was added directly to each well

147

containing the cells, mixed gently, and incubated at 37°C in a CO2 incubator for 24 h.

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2.6 Protein extraction and western blotting analysis

LTX

Reagent

complexes.

Finally,

500

μL

of

the

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HEK293 cells transfected with were transfected with 2g RORγt plasmid were

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incubated with the indicated compounds at different concentrations for 6 h. The cells

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were lysed with RIPA buffer containing 1× protease inhibitor mix to harvest total

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cellular proteins, which were measured using a bicinchoninic acid (BCA) assay. The

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total cellular extracts were then subjected to electrophoresis in 10% SDS/PAGE and

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transferred to nitrocellulose membranes. After blocking with 5% non-fat milk in a 6



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Tris-buffered saline-0.1% Tween 20 buffer, the membrane was subsequently

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incubated with specific primary antibodies and HRP-conjugated secondary antibodies.

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A chemiluminescence (ECL) detection system was used to detect the antibody-bound

158

proteins on the membrane.

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2.7 Purification of CD4+ T cells and differentiation of T cell subsets

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CD4+ single T lymphocyte were sorted by magnetic bead (Miltenyi Biotec,

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Cologne, Germany). Briefly, the splenocytes were isolated from C57BL/6 mice, and

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the red blood cells were lysed using lysis buffer. The isolated cells were labeled using

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biotin-antibody cocktail and anti-biotin microbeads and sorted by magnetic separation

164

in MS columns. The sorted CD4+ T cells were further utilized for differentiation into

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different T-helper cell subsets.

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Different T-helper cell subsets were polarized according to the following

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conditions. Briefly, 2 μg/ml anti-CD3 was coated in a plate and 5 μg/ml anti-CD28

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in the presence of 50 ng/ml IL-6, 1 ng/ml TGF-β1, 10 μg/ml anti-IL-4 and 5 ng/ml

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IL-23 were supplied to polarize Th17 cells. For the polarization of Th1 cells, 1 μg/ml

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anti-CD28 in the presence of 2 ng/ml IL-2 and 5 ng/ml IL-12 were provided. For the

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differentiation of Th2 cells, 1 μg/ml anti-CD28 in the presence of 2 ng/ml IL-2, 10

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ng/ml IL-4, and 10 μg/ml anti-IFN- were applied. For the differentiation of Treg

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cells, 1 μg/ml anti-CD28 in the presence of 5 ng/ml IL-2 and 5 ng/ml TGF-β1 were

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applied. The cells were collected and intracellular staining indicated antibodies for

175

analysis of Treg. For Th17 polarization, cells were re-stimulated with 50 ng/ml

176

phorbol-12-myristate-13-acetate (PMA), 1 μg/ml ionomycin and 1 μg/ml brefeldin A

177

for 5 hours after three days. For Th1 and Th2 differentiation, cells were re-stimulated

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with 50ng/ml PMA, 1 μg/ml ionomycin for 1 h followed by incubation of 2 μM

179

Monensin for additional 4 h before intracellular staining.

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2.8 Flow cytometric analysis

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For the analysis of the T-cell subsets with or without indicated treatments, the

182

cells were stained with the indicated fluorophore-conjugated monoclonal antibodies. 7


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PerCP/Cy5.5 anti-CD4 was used to stain the cytokines on the membrane surface of

184

cells. After the cells were permeabilized, FITC anti-IFN-, APC anti-IL-4, PE

185

anti-IL-17A and APC anti-Foxp3 were used to stain intracellular cytokines. The

186

expression levels of these cytokines were determined by BD FACSAriaTM III (BD

187

Bioscience) and analyzed by FlowJo software.

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2.9 Statistical analysis

189

Data are expressed as the mean ± S.E.M. Significant differences were analyzed

190

by one-way analysis of variance (ANOVA) using IBM SPSS Statistics 20 software.

191

Values of p < 0.05, p < 0.01 and p < 0.001 were considered statistically significant.

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3 Results

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3.1 Uscharin, calactin and calotropin have the potential to be RORt antagonists

194

The three non-classical cardenolides, uscharin, calactin and calotropin, were

195

isolated from Calotropis gigantea as previously described [12], and the purities of

196

these compounds used in current study are approximately 98% according to the

197

results of LC-MS (Fig. 1B). These cardenolides share a similar steroidal core with

198

digoxin, a classical cardenolide, and digoxin can inhibit Th17 differentiation via

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directly targeting RORt. We therefore hypothesized that uscharin, calactin and

200

calotropin have the potential to inhibit Th17 differentiation. Due to the key role of

201

RORt in regulating Th17 differentiation, the effects of these compounds on RORt

202

were studied by molecular docking. As shown in Table 1, the standard precision (SP)

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docking score of digoxin on RORt was -13.51 kcal/mol, and the scores of uscharin,

204

calactin and calotropin were -14.13, -13.6 and -13.15 kcal/mol, respectively,

205

suggesting that uscharin, calactin and calotropin have the potential to bind directly to

206

RORt and uscharin most likely has better binding affinity on RORt than the other

207

two compounds.

208

We were then interested in determining whether the binding affinity discrepancy 8



209

of uscharin and digoxin on RORt was attributable to the different binding pockets.

210

Hence, the binding sites were further elucidated by a molecular docking assay. As

211

shown in Fig. 2A, the binding pocket for digoxin was surrounded by a series of

212

hydrophobic residues, including Trp317, Leu324, Phe377, Phe378, Phe388, Leu391

213

and Ile400. Three water molecules were found to stabilize the pocket by forming a

214

hydrogen network with the surrounding residues. One water molecule along with

215

Phe377 formed hydrogen bonds with the hydroxyl group of digoxin. At the interior of

216

the pocket, Arg367 forms a hydrogen bond with the carbonyl of 2(5H)-furanone. Fig.

217

2B shows that the pocket for uscharin encompassed almost the same residues as

218

digoxin while the polar interactions for uscharin were different. Two water molecules

219

formed hydrogen bonds with a hydroxyl of uscharin; Leu287 and Arg367 both formed

220

hydrogen bonds with the carbonyl of 2(5H)-furanone at the interior of the pocket.

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His479 formed an extra hydrogen bond with the other of uscharin outside the pocket.

222

These differences can account for the better binding affinity of uscharin on RORt

223

than digoxin.

224

3.2 Uscharin, calactin and calotropin inhibit RORγt luciferase reporter activity

225

and protein expression

226

Because uscharin, calactin and calotropin showed the potential to bind RORγt by

227

molecular docking assay, the RORγt luciferase reporter was employed to determine

228

whether these compounds have inhibitory effects on RORγt. Consistent with the

229

molecular docking results, uscharin, calactin and calotropin strongly inhibited RORγt

230

luciferase activity from 100 to 250 nM (Fig. 3A-C), suggesting that these compounds

231

suppress RORt luciferase activity most likely via directly binding to RORt.

232

Because uscharin, calactin and calotropin could significantly inhibit the RORγt

233

luciferase reporter, we were interested in investigating the effect of these compounds

234

on the protein expression level of RORγt. RORγt plasmid was prepared and

235

transfected into HEK293 cells to validate the effect of uscharin, calactin and

236

calotropin on RORγt protein expression. Consistent with the inhibitory effect of these

237

compounds on RORγt luciferase activity, all three compounds could significantly and 9


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dose-dependently inhibit RORγt expression. Of note, the effective concentrations of

239

uscharin, calactin and calotropin were from 100 to 500 nM and the effective

240

concentration of digoxin was 10 M (Fig. 3D-F), implying that uscharin, calactin and

241

calotropin are more potent than digoxin in suppressing Th17 differentiation.

242

3.4 Uscharin, calactin and calotropin inhibit Th17 differentiation

243

Before determine the effect of uscharin, calactin and calotropin on Th17

244

differentiation, we investigated the cytotoxicity of these compounds on CD4+ T

245

lymphocytes. The CD4+ T lymphocytes were isolated and sorted from spleen and

246

incubated with indicated compounds for 72h. The MTT results showed that the three

247

compounds have not obvious cytotoxicity on CD4+ T lymphocytes up to 72 h (Fig.

248

4A-C).

249

As mentioned, Th17 lymphocytes have been implicated in the pathogenesis of

250

most common autoimmune diseases, and the differentiation of Th17 is mainly

251

regulated by RORt. Our results indicated that uscharin, calactin and calotropin

252

directly bind to RORt and suppress the luciferase activity and protein expression

253

level of RORt. We therefore hypothesized that uscharin, calactin and calotropin

254

could inhibit Th17 differentiation. In our current study, CD4+ T cells were sorted and

255

polarized under Th17 conditions with or without the cardenolide compounds. As

256

shown in Fig. 4, Th17 was successfully differentiated in our current study. With the

257

addition of 10 M digoxin, Th17 differentiation was significantly decreased, which is

258

consistent with pervious literature reports. Consistent with the molecular docking

259

prediction, uscharin, calactin and calotropin effectively and does-dependently

260

inhibited Th17 differentiation from 100 to 500 nM (Fig. 4), and the IC50 of uscharin,

261

calactin and calotropin are 267.5 nM, 191.5nM and 393.9nM, respectively.

262

3.5 Uscharin and calactin rather than calotropin suppress Th1 differentiation

263

In addition to Th17, Th1 lymphocytes also play a crucial role in autoimmune

264

diseases and both subsets synergistically mediate disease progression [13-16].

265

Therefore, we determined whether these cardenolides could affect Th1 differentiation. 10



266

CD4+ T cells were isolated from the spleens of C57BL/6 mice and were cultured

267

under Th1-polarizing conditions for 3 days with or without cardenolide treatments.

268

The cells were then harvested and intracellular cytokine levels were detected using

269

flow cytometry with intracellular staining for IFN-, the signature cytokine of Th1. As

270

shown in Fig. 5, digoxin did not have a significant effect on Th1 differentiation.

271

Interestingly, although uscharin, calactin and calotropin share a similar chemical

272

structure, there was a discrepancy among these compounds in their inhibition of Th1

273

differentiation. Uscharin and calactin decreased IFN- production, while calotropin

274

showed no obvious effect in suppressing IFN- production (Fig. 5), implying that

275

uscharin and calactin have the potential to reduce Th1 polarization.

276

3.6 Uscharin rather than calactin or calotropin inhibits Th2 differentiation

277

Th2 is another important subset of T lymphocytes. In autoimmune diseases, Th2

278

cells were initially described as anti-inflammatory subsets based on their ability to

279

suppress cell-mediated or Th1 models of disease [17-19]. Considering the critical role

280

of Th2 in the progression of autoimmune diseases, we investigated the effects of these

281

cardenolides on Th2 differentiation. Th2 was prepared from CD4+ T lymphocytes

282

sorted from the splenocytes of C57BL/6 mice and differentiated in polarizing

283

conditions for 3 days with or without cardenolides. As shown in Fig. 6, Th2 cells were

284

successfully induced and digoxin could not prevent the differentiation of Th2.

285

Uscharin dose-dependently decreased IL-4 production, indicating that uscharin

286

reduced Th2 polarization. However, neither calactin nor calotropin reduced Th2

287

differentiation.

288

3.7 Uscharin, calactin and calotropin inhibit Treg differentiation

289

Treg lymphocytes were identified and designated in the mid-1990s and they have

290

important immunoregulatory activity in many autoimmune disease conditions [20, 21].

291

Increasing Treg numbers or enhancing their suppressive function is beneficial for

292

treating autoimmune diseases [22-24]. We therefore investigated the effects of the 11


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293

cardenolides on Treg differentiation. As shown in Fig. 7, digoxin showed no obvious

294

effect on Treg differentiation, while uscharin and calactin intensively inhibited Treg

295

differentiation compared to calotropin at 500nM. Interestingly, all of these

296

compounds even slightly increased Treg differentiation at 100 and 250 nM.

297

Collectively, the results indicated that uscharin non-selectively inhibited the

298

differentiation of T cell subsets, including Th1, Th2 and Th17 from 100 to 500nM.

299

Calactin inhibited Th17 and Th1 differentiation and calotropin selectively suppressed

300

Th17 differentiation.

301

4 Discussion

302

T helper (Th) lymphocytes play critical roles in orchestrating adaptive immune

303

responses. The initial discovery of the existence of specialized Th effector populations

304

demonstrated that differentiated CD4+ T cells can be classified into two groups,

305

designated Th1 and Th2 cells, based on their cytokine production. With significant

306

technical advances, Th17 cells have been recognized as playing critical roles in the

307

pathogenesis of some autoimmune diseases [25-28]. According to literature reports,

308

digoxin can suppress Th17 differentiation and in turn ameliorate the symptoms of

309

EAE, indicating that compounds with a cardioglycoside chemical structure can have

310

an inhibitory effect on Th17 cells. In our previous study, uscharin, calactin and

311

calotropin, isolated from Calotropis gigantea, were found to have a steroidal

312

core similar to digoxin, a typical cardenolide. We therefore hypothesized that these

313

compounds could inhibit Th17. Accumulating studies have reported that RORγt is a

314

master regulator in Th17 differentiation and hence can be a promising therapeutic

315

target for autoimmune diseases [2, 4, 29]. Before our investigation on the effects of

316

cardenolide compounds on Th17 polarization, molecular docking assay was applied to

317

predict the affinity of these compounds on RORt.

318

According to previous reports, digoxin and its derivatives have been identified as

319

effective therapeutic RORt inhibitor candidates that attenuate autoimmune diseases

320

[3]. Therefore, digoxin was used as a reference compound in our current study. The 12



321

results implied that the binding affinities of our cardenolides on RORt are similar to

322

that of digoxin. Furthermore, uscharin has greater potential than digoxin to inhibit

323

Th17 according to the molecular docking results. The binding sites were also explored

324

using the assay. Although the binding pocket of uscharin for RORt has almost the

325

same residues as digoxin, the polar interactions for uscharin were different, which

326

most likely accounted for the better binding affinity of uscharin for RORt than

327

digoxin.

328

To further identify the effects of the three compounds on RORt activity and

329

expression, we employed a luciferase assay and over-expressed RORt. We found that

330

all three compounds significantly inhibited RORt luciferase activity. Interestingly,

331

we found all of the three compounds could suppress RORt overexpression in a

332

dose-dependent manner. It was reported that transcription factors upstream

333

stimulatory factor 1 (USF-1) and USF-2 regulate the expression of RORγt in human

334

lymphocytes, and upregulation of USF-1 and USF-2 was found during the

335

differentiation of Th17 cells from naive CD4+cells[30]. Due to the indispensable role

336

of USF-1 and USF-2 for the transcription of RORγt in lymphocytes, uscharin, calactin

337

and calotropin suppressed RORγt expression probably resulting from regulation of

338

USF-1 and USF-2. Furthermore, the effective concentration of these compounds was

339

500 nM, lower than digoxin, indicating that uscharin, calactin and calotropin have

340

more potential than digoxin to prevent Th17 polarization. Consistent with these

341

results, the three compounds also exhibited significant inhibition on Th17 polarization

342

at 500 nM.

343

Abnormal activation of Th1 cells is thought to be the critical event in most

344

organ-specific autoimmune diseases [31, 32]. In contrast, Th2 cells play a protective

345

role or at least a neutral role [30, 33, 34]. Furthermore, it is thought that some

346

autoimmune diseases result from an imbalance between Treg cells and Th17 cells [20,

347

21]. Therefore, regulating the balance between Treg cells and Th17 cells may be

348

developed as a therapeutic strategy to treat autoimmune disorders. Hence, we

349

intensively investigated the effect of the three compounds on the different T cell

350

subpopulations. although uscharin had the best inhibitory effect on Th17 13


Page 14 of 26

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351

differentiation among the three compounds, as it simultaneously suppressed the

352

differentiation of Th1, Th2 and Treg cells, suggesting that uscharin has no selective

353

effect in inhibiting T lymphocyte subsets.

354

Alternatively, calactin simultaneously inhibited Th1, Th17 and Treg cells without

355

an obvious effect on Th2 cells, indicating that calactin could be developed to treat

356

autoimmune diseases, more so than uscharin. Furthermore, calotropin selectively and

357

intensively suppressed Th17 differentiation rather than other T cell subsets.

358

Collectively, uscharin, calactin and calotropin are cardenolides with similar chemical

359

structures, and their discrepant effects on different T cell subsets are most likely due

360

to the subtle differences in chemical structure.

361

Uscharin, calactin and calotropin share an identical chemical structure except for

362

their functional groups at the C-3' position. A thiazoline ring plays a key role in

363

uscharin’s strongest inhibitory effect on Th17 differentiation but without selectivity

364

for the T cell subsets. A β-hydroxyl group contributes to calactin’s selectivity for

365

suppressing differentiation of both Th1 and Th17 cells while an α-oriented

366

counterpart in calotropin results in its specificity for Th17 cell differentiation. The

367

above structure-activity relationship suggests the importance of the C-3'

368

stereochemistry of nonclassical cardenolides for their inhibitory effects on the

369

differentiation of T cell subsets.

370

In summary, calotropin selectively inhibited Th17 differentiation rather than

371

other T cell subsets, and it should be preferred in treating autoimmune diseases

372

mediated by Th17, indicating that calotropin could be developed as therapeutic

373

compounds to treat autoimmune diseases. The differences in the chemical structures

374

of uscharin, calactin and calotropin are correlated with their selective inhibitory

375

effects on T cell subsets. This finding may lead to strategic modifications of chemical

376

structures in the future.

377 378

Conflict of interests

379

The authors declare that they have no conflicts of interest. 14



380

381

Acknowledgments

382

This work was supported by Macau Science and Technology Development Fund

383

(project code 0017/2018/A1).

15


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385

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469


Figures

470

Fig. 1 Chemical structures and purity of uscharin, calactin and calotropin. (A) The

471

chemical structures of uscharin, calactin, calotropin and digoxin. (B) The purity of

472

uscharin, calactin and calotropin was analyzed by LC-MS.

18



474 475 476

Table 1 Value of the SP docking score for uscharin, calactin, calotropin and digoxin on RORγt.

Ligand

Docking score (Kcal/mol)

Digoxin

-13.51

Uscharin

-14.13

Calactin

-13.61

Calotropin

-13.15

19


Page 20 of 26

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478 479

A

B

480 481 482 483 484 485 486 487 488

Fig. 2 Identification of the interaction between the compounds and RORγt via molecular docking calculations. (A) Schematic diagram of the interaction mode between the RORγt binding site (yellow) and digoxin (green). The red spheres represent water. Hydrogen bonds are highlighted with a red dashed line. (B) Schematic diagram of the interaction mode between the RORγt binding site (yellow) and uscharin (cyan). The red spheres represent water. Hydrogen bonds are highlighted with a red dashed line. 20



490 491 492 493 494 495 496 497 498 499 500 501 502

Fig. 3 Effect of uscharin, calactin and calotropin on RORγt luciferase reporter activity and protein expression level. (A-C) The effect of uscharin, calactin, calotropin and digoxin on RORγt luciferase reporter activity. The RORγt LUCPorter™ Stable Reporter Cells were incubated with uscharin, calactin, calotropin and digoxin at the indicated concentration for 16 h. The cell lysates were prepared and used for luciferase activity analysis. (D-F) The effect of uscharin, calactin, calotropin and digoxin on the protein expression of RORγt. The HEK293 cells transfected with RORγt plasmid were incubated with the indicated compounds at different concentrations for 6 h and then the total lysates were prepared and the expression of RORγt and β-actin were analyzed by western blotting. Data of three independent experiments are presented as mean ± SEM, *p

Potent Antagonists of RORÎ³t, Cardenolides from - American Chemical

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