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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Feb 11;177(7):1666–1676. doi: 10.1111/bph.14926

PO‐322 exerts potent immunosuppressive effects in vitro and in vivo by selectively inhibiting SGK1 activity

Yi Lai 1, Xing‐Yan Luo 2, Hui‐Jie Guo 2, Si‐Yu Wang 2, Jing Xiong 2, Shu‐Xia Yang 2, Li‐Mei Li 2, Qiang Zou 2, Chun‐Fen Mo 2,✉, Yan‐Tang Wang 2,3,✉, Yang Liu 2,3,✉
PMCID: PMC7060359  PMID: 31724152

Abstract

Background and Purpose

Immunosuppressive drugs have shown great promise in treating autoimmune diseases in recent years. A series of novel oxazole derivatives were screened for their immunosuppressive activity. PO‐322 [1H‐indole‐2,3‐dione 3‐(1,3‐benzoxazol‐2‐ylhydrazone)] was identified as the most effective of these compounds. Here, we have investigated the mechanism(s) underlying the inhibition of T‐cell proliferation in vitro by PO‐322, as well as its effects on the delayed‐type hypersensitivity (DTH) response and imiquimod‐induced dermatitis in vivo.

Experimental Approach

T‐cell proliferation and apoptosis were analysed with flow cytometry. Cell viability was assessed with a CCK‐8 assay. Protein kinase activity was assessed by SelectScreen Kinase Profiling Services. The phosphorylation of signal‐regulated molecules was measured by Western blot. Cytokine levels were determined by elisa. The effect of PO‐322 on DTH and imiquimod‐induced dermatitis was evaluated in BALB/c mice.

Key Results

PO‐322 inhibited human T‐cell proliferation with anti‐CD3/anti‐CD28 mAbs or alloantigen without significant cytotoxicity. Importantly, PO‐322 was a selective inhibitor of the serum‐ and glucocorticoid‐regulated kinase 1 (SGK1) and decreased NDRG1 phosphorylation but not p70S6K, STAT5, Akt, or ERK1/2 phosphorylation. Furthermore, PO‐322 inhibited IFN‐γ, IL‐6, and IL‐17 expression but not IL‐10 expression. Finally, treatment with PO‐322 was safe and effective for ameliorating the DTH response and imiquimod‐induced dermatitis in mice.

Conclusions and Implications

PO‐322 exerted immunosuppressive activity in vitro and in vivo by selectively inhibiting SGK1 activity. PO‐322 represents a potential lead compound for the design and development of new drugs for the treatment of autoimmune diseases.

Abbreviations

CFSE

5‐carboxyfluorescein diacetate succinimide ester

DNFB

dinitrofluorobenzene

DTH

delayed‐type hypersensitivity

mTOR

mammalian target of rapamycin

PBMCs

peripheral blood mononuclear cells

SGK1

the serum‐ and glucocorticoid‐regulated kinase 1

What is already known?

  • SGK1is a downstream target of mTOR and can be activated by IL‐2.

  • SGK1inhibitors have been reported the anticancerous activity, antihypertensive activity and preventing cardiac inflammation.

What this study adds

  • A novel compound, PO‐322, has been synthesizedand identified as the selective SGK1 inhibitor.

  • PO‐322exerts immunosuppressive activity in vitro and in vivo by inhibition of SGK1activity.

What is the clinical significance?

  • PO‐322 represents a potential lead compound for design and development of new immunosuppressive drugs.

  • PO‐322 may be used for the treatment of immunorejection of transplanted organs andautoimmune diseases.

1. INTRODUCTION

The important role of T cells in immune responses against pathogens has been clearly established and aberrant T‐cell responses play a crucial role in mediating the immuno‐rejection of transplanted organs (van Gelder, van Schaik, & Hesselink, 2014) and in the pathogenesis of autoimmune diseases, including multiple sclerosis (Sabatino et al., 2018), psoriasis (Hawkes, Yan, Chan, & Krueger, 2018), and systemic lupus erythematosus (He et al., 2016). In recent years, immunosuppressants have been widely used clinically, for treating these diseases and advances in the understanding of the mechanisms of these diseases have suggested multiple novel drug targets (Wiseman, 2016). However, there are significant differences in drug sensitivity between individual patients. Therefore, combination, rotation, or sequential therapies are often needed, especially when the response to single‐drug therapy is not satisfactory. To provide more effective treatments for patients, it is important to identify new immunosuppressants and further understand the pathogenesis of these diseases.

T‐cell receptor‐mediated T‐cell signalling can induce T‐cell activation and proliferation through several signalling pathways, including the calcium/calcineurin, NF‐κB, PI3K/Akt, https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2109)/p70S6K, JAK3/STAT5, and MAPK p38 signal pathways (Moulton et al., 2015). Immunosuppressants that are the inhibitors of these pathways have been approved for clinical use, such as https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1024, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6784 https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6031 (sirolimus) and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5677 (Baker et al., 2018). Recently, several new low MW inhibitors of these pathways, such as https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7372 (Nash et al., 2018), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8315 (Sands et al., 2018), and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=7792 (Wallace et al., 2018), have been advanced to late‐stage clinical trials, highlighting the potential of these immunosuppressive approaches.

The https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1534 belongs to the AGC kinase family and mediates signals of cellular growth, proliferation, and survival responses. SGK1 is activated by cAMP, IGF‐1, insulin, steroids, TGF‐β, and especially https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4985 (Talarico et al., 2016). SGK1 is a downstream target of mTOR, which integrates various signals to influence T‐cell proliferation and differentiation (Norton et al., 2014). SGK1 inhibitors, such as https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8040, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9407, and SI113, have been reported to have anti‐cancer activity (Abbruzzese et al., 2017; Yuda et al., 2018) and anti‐hypertensive activity (Du et al., 2018) and can prevent cardiac inflammation (Gan et al., 2018).

Oxazole is a privileged structural motif present in a variety of bioactive agents, including chemotherapeutic (Lu et al., 2016), antimicrobial (Zhang et al., 2011), neuroprotective (Kaushal et al., 2011), and analgesic agents (Payrits et al., 2016). Oxazole derivatives are marketed for the treatment of cardiovascular disease (Dineen et al., 2014) and as an inhibitor of carbonic anhydrase isoforms I and II (Krasavin et al., 2015). In addition, oxazole derivatives exhibit anti‐inflammatory activity by inhibiting GSK3β (Zhao et al., 2018), MAPK p38α (Kalgutkar et al., 2006), and COX‐2 and COX‐1 enzyme activities (Hashimoto, Imamura, Haruta, & Wakitani, 2002). Recently, we found an oxazole derivative, PO‐296, that could inhibit T‐cell proliferation through the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2049/STAT5 signalling pathway (Luo et al., 2019). However, the immunosuppressive activity of oxazole derivatives through SGK1 inhibition has not been reported previously.

In the present study, a series of novel oxazole derivatives (Figure 1) were tested for their immunosuppressive activity, and PO‐322 [1H‐indole‐2,3‐dione 3‐(1,3‐benzoxazol‐2‐ylhydrazone)] was the most effective lead compound. PO‐322 was identified as a selective SGK1 inhibitor and was investigated for its ability to inhibit T‐cell proliferation in vitro, as well as its effects on the delayed‐type hypersensitivity (DTH) reaction induced by dinitrofluorobenzene (DNFB) and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5003‐induced dermatitis in vivo.

Figure 1.

Figure 1

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Chemical structure of oxazole derivatives

2. METHODS

2.1. Experimental animals

All animal care and experimental procedures were approved by the Animal Ethics Committee of Chengdu Medical College on animal experiments. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology.

Mice (BALB/c, female, 8–11 weeks old, RRID:IMSR_ORNL:BALB/cRl) were purchased from Huaxi Laboratory Animal Center of Sichuan University (Chengdu, China) and kept under specific pathogen‐free conditions and provided with normal food and water.Animals were randomly assigned to treatment groups, and the experimenter was blinded to drug treatment until data analysis has been performed.

2.2. Synthesis of PO‐322

PO‐322 was synthesized in one step from commercially available 2‐indolinone and 2‐hydrazinobenzoxazole. A mixture of 2‐indolinone (1.33 g, 10 mmol), 2‐hydrazinobenzoxazole (1.49 g, 10 mmol), and acetic acid (1 ml) was stirred in ethanol (50 ml) at reflux temperature for 3 hr. After cooling to room temperature, PO‐322 (1 g, 36% yield) was collected as pale yellow powder. The compound was characterized by 1H NMR using Bruker Avance 600 Spectrometer (chemical shifts expressed in ppm with reference to tetramethylsilane peak) and electrospray ionization mass spectrum using BioTOF‐Q mass spectrometer. All reagents were obtained from J&K Chemical Co. Solvents were purchased from local suppliers.

2.3. Cell preparation

Human peripheral blood mononuclear cells (PBMCs) were isolated as previously described (Liu et al., 2013). PBMCs were cultured in RPMI 1640 supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA). T cells were isolated using Pan T cell Isolation Kit II Human (Miltenyi Biotec, Bergisch Gladbach, Germany) with negative selection. These T‐cells, with a purity of 95%, were used for the following experiments.

2.4. Drug treatment

T cells (106 cells·ml−1) were treated with rapamycin (0.1 μM, Sigma‐Aldrich, St. Louis, MO, USA), FK506 (0.1 μM, Sigma‐Aldrich), GSK650394 (10 μM, MedChem Express, Monmouth Junction, NJ, USA), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5916 (50 μM, Sigma‐Aldrich), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6004 (50 μM, Promega, Madison, WI, USA), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5676 (2 μM, Sigma‐Aldrich), or different concentrations of PO‐322 and activated by plate‐bound anti‐https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2742&familyId=852&familyType=OTHER (2 μg·ml−1, HIT3a clone, BD Pharmingen, San Diego, CA, USA) and soluble anti‐https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2863&familyId=852&familyType=OTHER (1 μg·ml−1, CD28.2 clone, BD Pharmingen) as previously described (Liu et al., 2013). Cells were randomly assigned to treatment groups, and the experimenter was blinded to drug treatment until data analysis has been performed.

2.5. CFSE labelling assay

T‐cell proliferation was measured by flow cytometry (Acurri C6, Becton Dickinson, San Jose, USA) with 5‐carboxyfluorescein diacetate succinimide ester (CFSE, Molecular Probes, Eugene, OR, USA) labelling as previously described (Liu et al., 2015).

2.6. Cell apoptosis assay

Fluos‐labelled Annexin V and PI dual staining kit (Roche, Indianapolis, IN, USA) was used to assess the cell apoptosis following the protocol and analysed by flow cytometry (Acurri C6) as previously described (Luo et al., 2019).

2.7. Cell viability assay

The CCK‐8 assay kit (Dojindo, Kumamoto, Japan) was used to assess the viability of cells by detecting the OD values at 450 nm in a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) as previously described (Liu et al., 2013).

2.8. Protein kinase profiling

The effect of PO‐322 against almost 100 kinases was assessed by SelectScreen Kinase Profiling Services (Thermo Fisher Scientific, Madison, WI, USA). PO‐322 was dissolved in DMSO at 100 μm, and a final concentration of 1 μm was used for screening. Then, the inhibitory activity of PO‐322 against SGK1 was measured with different concentrations of PO‐322.

2.9. Western blot analysis

The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

Cell pellets were treated with lysis buffer and clarified by centrifugation. Cell lysate proteins (20 μg per lane) were separated on 10% SDS‐PAGE and transferred to Immobilon PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked by PBS containing 5% BSA and stained with antibodies against N‐Myc downstream‐regulated gene 1 (NDRG1), phospho‐NDRG1 (Cell Signaling Technology, Cat# 5482, RRID:AB_10693451), Akt, phospho‐Akt (Ser473), p70S6K, phospho‐p70S6K, STAT5, phospho‐STAT5, ERK1/2, or phospho‐ERK1/2 (CST Inc., Danvers, MA, USA) overnight at 4°C followed by HRP‐conjugated second antibodies (Santa Cruz Biotechnology, Santa Cruz, USA) incubation. Finally, proteins were visualized by enhanced chemiluminescence (Millipore). All Western blotting procedures and analysis were conducted in accordance with current guidelines (Alexander et al., 2018).

2.10. Cytokine elisa assay

The levels of IL‐2, IL‐6, IL‐10, IL‐17A, and IFN‐γ were determined by elisa kits (eBioscience, San Diego, CA, USA) as previously described (Liu et al., 2015).

2.11. DNFB‐induced DTH reaction in mice

The experiments of DNFB‐induced DTH reaction in mice were performed. Individual BALB/c mice were sensitized topically with 20 μl of 0.5% (v/v) DNFB (Sigma‐Aldrich) in acetone : olive oil (4:1) onto each hind foot of mice on Days 0 and 1. These mice were injected i.p. with different doses of PO‐322 (2.5, 10, and 40 mg·kg−1), rapamycin (2 mg·kg−1), or vehicle alone beginning on Day 6 for three consecutive days. The mice were challenged topically with 10 μl of 0.5% (v/v) DNFB on the inner and outer surfaces of the right ear on Day 7. The thickness of both left and right ears and the weight of ear patches (8‐mm punches) were measured 48 hr post‐challenge.

2.12. Imiquimod‐induced dermatitis in mice

The backs of female BALB/c mice (RRID:IMSR_ORNL:BALB/cRl) were shaved with an electric clipper 1 day prior to treatment. Mice were injected i.p. with 10 or 40 mg·kg−1 of PO‐322 or vehicle 1 day prior to application of 62.5 mg of 5% imiquimod cream (Aldara, 3M Pharmaceuticals, St. Paul, MN) or control cream (Curél, Kao, Japan). Then, 10 or 40 mg·kg−1 of PO‐322 or vehicle was given 0.5 hr before treating with 62.5 mg of 5% imiquimod cream or control cream, and this treatment was repeated for six consecutive days. Each day, the mice were assessed by the same researcher for redness and scaling on a 0–4 scale prior to handling, according to previously published guidelines (van der Fits et al., 2009). On the sixth day, mice were killed by cervical dislocation, and photographs were taken. Back skin (3‐mm diameter) was isolated and fixed in 10% formaldehyde. Then, fixed skin was paraffin embedded, sliced at 6 mm using a microtome (RM2235, Leica, Nußloch, Germany), and stained with haematoxylin and eosin. Haemotoxylin and eosin slides were coded and an overall score of severity was assessed (0–4) by a trained researcher. Sections of skin were photographed at an objective magnification of 20× using a microscope (BX63, Olympus, Tokyo, Japan). Spleen was isolated, and spleen mass was determined. Then, splenic mononuclear cells were prepared and resuspended in PBS. T cells in splenic mononuclear cells were stained with PE‐anti‐CD3 (BD Pharmingen) and analysed on a flow cytometry (Acurri C6).

2.13. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology. Results are expressed as mean ± SEM, and the inhibitory concentration of the compound that reduced cell proliferation by 50% (IC50 values) was calculated using GraphPad Prism 6 (GraphPad Prism, RRID:SCR_002798). The sample size was n = 8 per group in animal experiments and n = 5 per group in other experiments. One‐way ANOVA with Dunnett comparisons on post‐tests was used to analyse data and compare groups. The post hoc tests were run only if F achieved P < .05 and there was no significant variance inhomogeneity. In each experiment, n represents the number of separate experiments (in vitro) and the number of mice (in vivo). Technical replicates were used to ensure the reliability of single values. A P < .05 was considered to be statistically significant.

2.14. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Fabbro et al., 2019; Alexander, Kelly et al., 2019).

3. RESULTS

3.1. Synthesis and characterization of PO‐322

PO‐322 was synthesized in one step from 2‐indolinone (1) and 2‐hydrazinobenzoxazole (2), with 36% yield (Figure 2). 1H NMR (300 MHz, DMSO‐d 6): δ (ppm) 12.64 (brs, 1H), 10.57 (brs, 1H), 8.34 (d, 1H, J = 6.9 Hz), 7.66 (m, 1H), 7.18–7.68 (m, 4H), 7.04 (m, 1H), 6.86 (m, 1H). ESI‐MS: m/z 277 [M − H]−.

Figure 2.

Figure 2

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Synthesis of PO‐322. A mixture of compound 1 (2‐indolinone, 1.33 g, 10 mmol), compound 2 (2‐hydrazinobenzoxazole, 1.49 g, 10 mmol), and acetic acid (1 ml) was stirred in ethanol (50 ml) at reflux temperature for 3 hr. After cooling to room temperature, PO‐322 (1 g, 36% yield) was collected as a pale yellow powder

3.2. PO‐322 inhibits human T‐cell proliferation without obvious cytotoxicity in vitro

PO‐322 and its analogues were screened for their immunosuppressive activity. Among these chemical compounds, PO‐322, PO‐324, PO‐326, PO‐327, PO‐335, PO‐341, and PO‐342 showed significant inhibitory effects on T‐cell proliferation following anti‐CD3 and anti‐CD28 stimulation (Table 1). The most potent inhibitor, PO‐322, was selected for further studies. PO‐322 was found to inhibit human T‐cell proliferation after anti‐CD3 or anti‐CD28 stimulation with an IC50 value of 0.7 ± 0.2 μM (Figure 3a,b) and alloantigen stimulation with an IC50 value of 0.6 ± 0.3 μM (Figure 3c). T‐cell proliferation was also inhibited by PO‐322 following Phytohaemagglutinin (PHA) or PMA/ionomycin stimulation (Figure S1).

Table 1.

Immunosuppressive activity of oxazole derivatives

Compound IC50 (μM)a Compound IC50 (μM)
PO‐322 0.7 ± 0.2 PO‐342 28.9 ± 3.9
PO‐324 31.6 ± 2.3 PO‐343 >100
PO‐325 >100 PO‐345 >100
PO‐326 25.3 ± 3.1 PO‐346 >100
PO‐327 15.1 ± 3.9 PO‐348 >100
PO‐329 >100 PO‐349 >100
PO‐333 >100 PO‐350 >100
PO‐335 32.4 ± 4.2 PO‐353 >100
PO‐341 24.5 ± 3.5 PO‐355 >100
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Note. In the experiment, CFSE‐labelled T cells were treated with a series of oxazole derivatives following anti‐CD3 and anti‐CD28 mAbs stimulation for 72 hr. Cell proliferation was analysed on a flow cytometry using proliferation index. The results are presented as the mean ± SEM, n = 5 for each experimental group.

a

IC50 of inhibitory effect on cell proliferation.

Figure 3.

Figure 3

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PO‐322 inhibits human T‐cell proliferation without obvious cytotoxicity. CFSE‐labelled T cells were treated with PO‐322 (0.125, 0.5, 2, and 8 μM) and activated with (a, b) anti‐CD3/anti‐CD28 or (c) allogeneic peripheral blood mononuclear cells (PBMCs) for 72 hr. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). T cells were treated with PO‐322 (0.5, 2, and 8 μM), rapamycin (RAPA; 0.1 μM), or vehicle and activated with anti‐CD3/anti‐CD28 for 24 or 48 hr. (d) Cell apoptosis was assessed by flow cytometry with Annexin V and PI dual staining. (e) Resting T cells, (f) IL‐7‐treated activated T cells, and (g) PBMCs were treated with PO‐322 (10, 20, 40, and 80 μM) or vehicle for 72 hr. The CCK‐8 assay kit was used to assess cell viability. Cells without drug treatment served as controls (100%). “+” and “−” indicate with or without anti‐CD3/anti‐CD28 stimulation, respectively. The results are presented as the mean ± SEM, n = 5 for each experimental group

To investigate the potential cytotoxicity of PO‐322, activated human T cells treated with PO‐322 were measured for apoptosis by flow cytometry. Resting T cells and IL‐7‐treated activated T cells both survived but did not proliferate under these conditions (Rathmell, Farkash, Gao, & Thompson, 2001). Additionally, PBMCs treated with PO‐322 were assessed for cell viability with a CCK‐8 assay. PO‐322 treatment did not induce activated T‐cell apoptosis at 24 or 48 hr (Figure 3d) and had no significant effects on the relative viability of resting T cells (Figure 3e), IL‐7‐treated activated T cells (Figure 3f), or PBMCs at 72 hr (Figure 3g), indicating that the activity of PO‐322 was immunosuppressive, rather than cytotoxic.

3.3. PO‐322 is a highly selective inhibitor of SGK1

To identify the targets of PO‐322, a protein kinase activity screen was carried out. Among 100 protein kinases screened, PO‐322 (1,000 nM) showed a very high inhibitory activity towards SGK1 (98% inhibition) but not other kinases (inhibition <50%; Figure 4a). It was interesting that PO‐322 inhibited SGK1 activity with an IC50 value of 54 ± 6 nM (Figure 4b). These results suggested that PO‐322 was a highly selective SGK1 inhibitor.

Figure 4.

Figure 4

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PO‐322 is a highly selective inhibitor of SGK1. The effect of PO‐322 against almost 100 kinases was assessed by SelectScreen Kinase Profiling Services. (a) PO‐322 was dissolved in DMSO at 100 μM, and a final concentration of 1 μM was used for screening. (b) Then, the inhibitory activity of PO‐322 against SGK1 was measured with different concentrations of PO‐322. T cells were incubated alone for 6 hr after 72 hr of anti‐CD3/anti‐CD28 stimulation. T cells were then treated with PO‐322 (0.5, 2, or 8 μM), GSK650394 (10 μM), rapamycin (RAPA; 0.1 μM), AG‐490 (50 μM), LY294002 (50 μM), PD184352 (2 μM), or vehicle for another 6 hr. Subsequently, T cells were induced by IL‐2 for 30 min, and the relative phosphorylation and expression levels of (c) NDRG1, (d) p70S6K, (e) STAT5, (f) Akt, and (g) ERK1/2 were assessed by Western blot analysis. “+” and “−” indicate experiments with or without IL‐2, respectively. The results are presented as the mean ± SEM, n = 5 for each experimental group. *P < .05, significantly different from the activated group without drug

To further reveal the signalling pathway affected by PO‐322, the effects of PO‐322 on the related kinase phosphorylation levels was analysed by western blot. The results showed that NDRG1 phosphorylation, but not its expression, was significantly inhibited by PO‐322 (Figure 4c). Notably, the expression of p70S6K and STAT5 did not significantly change, but their phosphorylation levels were increased by PO‐322 (Figure 4d,e). Finally, the Akt (Figure 4f) and ERK1/2 (Figure 4g) expression and phosphorylation levels were not affected by PO‐322. Phosphorylation of NDRG1 has previously been shown to be a specific target of SGK1 activity (Inglis et al., 2009), indicating that PO‐322 inhibited T‐cell proliferation through the SGK1/NDRG1 signalling pathway.

3.4. PO‐322 inhibits pro‐inflammatory cytokines but does not affect anti‐inflammatory cytokines

To understand the effects of PO‐322 on pro‐inflammatory and anti‐inflammatory cytokine expression, the levels of IL‐17, IFN‐γ, IL‐6, and IL‐10 were measured by elisa in the supernatants of activated T cells. The results showed that PO‐322 significantly inhibited IL‐17, IFN‐γ, and IL‐6 expression but did not affect IL‐10 (Figure 5), indicating potential anti‐inflammatory effects of PO‐322.

Figure 5.

Figure 5

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PO‐322 inhibits pro‐inflammatory cytokine expression and does not affect anti‐inflammatory cytokine expression. T cells were treated with PO‐322 (0.125, 0.5, and 2 μM), LY‐294002 (50 μM), or vehicle and activated with anti‐CD3/anti‐CD28 for 48 hr. The supernatants were collected, and the levels of (a) IL‐17A, (b) IFN‐γ, (c) IL‐6, and (d) IL‐10 were measured by elisa. “+” and “−” indicate experiments with or without anti‐CD3/anti‐CD28, respectively. The results are presented as the mean ± SEM, n = 5 for each experimental group. *P < .05, significantly different from the activated group without drug

3.5. PO‐322 significantly mitigates DNFB‐induced DTH reaction and ameliorates imiquimod‐induced dermatitis in mice

Th1/Th17 responses are clearly related to the DTH reaction (Sido, Jackson, Nagarkatti, & Nagarkatti, 2016). We explored the activity of PO‐322 on DNFB‐induced DTH reaction in BALB/c mice. Administration of PO‐322 markedly reduced ear swelling, including increases in ear thickness and patch weight, in a dose‐dependent manner (Figure 6).

Figure 6.

Figure 6

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PO‐322 mitigates DNFB‐induced delayed‐type hypersensitivity (DTH) reaction in mice. BALB/c mice were sensitized with DNFB on Days 0 and 1 and injected i.p. with different doses of PO‐322 (2.5, 10, and 40 mg·kg−1), rapamycin (RAPA; 2 mg·kg−1), or vehicle alone beginning on Day 6 for three consecutive days. These mice were challenged with DNFB on Day 7. The thickness of both the left and right ears and the ear patch weights were measured 48 hr post‐challenge. Ear swelling was calculated as an increase in (a) ear thickness and (b) ear patch weight between the left (DNFB untreated) and right (DNFB treated) ears. The results are presented as the mean ± SEM, n = 8 for each experimental group. *P < .05, significantly different from the group without drug

Finally, we explored the activity of PO‐322 in imiquimod‐induced dermatitis, a mouse model that closely resembles human psoriatic lesions (van der Fits et al., 2009). Treatment with PO‐322 markedly reduced scaling and redness compared with the vehicle‐treated group (Figure 7a). In addition, the histopathological assessment of the skin on the back indicated that PO‐322‐treated mice showed significantly reduced histological scores, splenic mass, and total T‐cell number in the spleen (Figure 7b–d), demonstrating that PO‐322 can substantially reduce disease severity.

Figure 7.

Figure 7

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PO‐322 relieves imiquimod‐induced psoriasis‐like dermatitis. BALB/c mice were injected i.p. with 10 or 40 mg·kg−1 of PO‐322 or vehicle 1 day prior to the application of imiquimod or control cream. Then, PO‐322 or vehicle was given 0.5 hr before treatment with imiquimod cream, and this treatment was repeated for six consecutive days. Clinical scores for (a) redness and scaling and (b) haematoxylin and eosin‐stained slides were scored (0–4) for disease severity. (c) Mice were killed, and splenic masses were determined. (d) The number of T cells was measured by flow cytometry. “+” and “−” indicate runs with or without imiquimod, respectively. The results are presented as the mean ± SEM, n = 8 for each experimental group. *P < .05, significantly different from the imiquimod‐induced group without drug

Furthermore, drug‐related deaths, sickness, or abnormal food and water intake were not observed in our experiments. These results suggested that PO‐322 was safe and effective in ameliorating T‐cell‐mediated DTH reaction and imiquimod‐induced dermatitis in vivo.

4. DISCUSSION

In this study, a series of new oxazole derivatives, including PO‐322, were tested for immunosuppressive activity. PO‐322 was identified as the lead molecule with the highest activity among these derivatives. PO‐322 significantly inhibited T‐cell proliferation after anti‐CD3/anti‐CD28 mAbs, alloantigen, PHA, or PMA/ionomycin stimulation. Notably, PO‐322 did not induce activated T‐cell apoptosis and had no significant cytotoxicity on resting T cells, IL‐7‐treated activated T cells, or PBMCs, indicating that the activity of PO‐322 was primarily immunosuppressive rather than cytotoxic. Therefore, further exploration of the possible mechanism of action for PO‐322 and its immunosuppressive effects in vivo is warranted.

Next, to identify the targets of PO‐322, a protein kinase activity screen was carried out. We found that PO‐322 was a selective SGK1 inhibitor. Several pathways are involved in T‐cell proliferation, such as the mTOR/p70S6K, JAK3/STAT5, PI3K/Akt, and p38 MAPK pathways. Furthermore, IL‐2 engagement with its receptor can induce SGK1 activation, which is a downstream target of mTOR (Talarico et al., 2016). Western blotting results of PO‐322 on these signalling pathways showed that the PI3K/Akt and p38 MAPK pathways were not affected by PO‐322 treatment, but the SGK1/NDRG1 pathway was significantly inhibited under the same conditions in IL‐2‐induced T cells. Therefore, PO‐322 inhibited activated T‐cell proliferation by targeting SGK1 activity. Interestingly, p70S6K and STAT5 phosphorylation was increased by PO‐322. Inhibition of the mTOR/SGK1 pathway may selectively expand regulatory T cells and promote de novo generation of Foxp3+ regulatory T cells through the up‐regulation of the STAT5 phosphorylation (Shan et al., 2015). This implies a potential role of PO‐322 in inducing tolerance of immune responses though regulating the balance between regulatory T cells and effector T cells.

SGK1 is activated by IL‐2 in T cells and induces T‐cell proliferation and differentiation. Furthermore, SGK1 regulates TH1 and TH2 cell differentiation, as well as TH17 cell development, and improves pro‐inflammatory cytokine levels (Norton et al., 2014). The loss of SGK1 in T cells leads to a selective defect in pathogenic Th17 differentiation (Heikamp et al., 2014). SGK1 has been shown to be critical for the development of autoimmune diseases, such as experimental autoimmune encephalomyelitis (Wang et al., 2017) and ulcerative colitis (Spagnuolo et al., 2018). PO‐322, as a SGK1 inhibitor, significantly inhibited IFN‐γ, IL‐6, and IL‐17 release from activated T cells. It has been established that pro‐inflammatory cytokines, including IFN‐γ, IL‐6, and IL‐17, play a critical role in autoimmune diseases (Hawkes et al., 2018; McGeachy, Cua, & Gaffen, 2019). Therefore, PO‐322 may have beneficial anti‐inflammatory effects by regulating Th‐cell differentiation in treating autoimmune diseases.

The mouse model experiments also showed that PO‐322 was effective and safe in inhibiting T‐cell‐mediated inflammation in vivo. We found that the administration of PO‐322 significantly reduced DNFB‐induced DTH reaction and substantially reduced disease severity in an imiquimod‐induced dermatitis mouse model, which would imply anti‐psoriatic properties of PO‐322 in vivo. However, the mechanism(s) involved in treating DTH and psoriasis with PO‐322 remain to be investigated in further studies.

While highly specific and potent biological drugs targeting the components of the immune system have received much attention in recent years, treatments involving low MW compounds can be administered orally and are often less expensive to manufacture, can result in better patient compliance and affordability. Nevertheless, the structure of PO‐322 is significantly different from other known inhibitors of SGK1, as well as from other immunosuppressants. It would be interesting to identify the mechanism of PO‐322 in autoimmune diseases, which could help to further elucidate the pathogenesis of autoimmune diseases and to guide the design of new drugs and would ultimately benefit patients with safer and more effective therapeutic options for these diseases.

In summary, we have synthesized and characterised a novel oxazole derivative, PO‐322, as a potent immunosuppressant. PO‐322 inhibits T‐cell proliferation by selectively inhibiting SGK1 activity and relieving both the DNFB‐induced DTH reaction and imiquimod‐induced dermatitis in mice, in vivo. Further structure–activity relationship studies and the optimization of PO‐322 are warranted to improve the potency and to elucidate the mechanism of action for this promising lead compound for the treatment of the immunorejection of transplanted organs and autoimmune diseases.

AUTHOR CONTRIBUTIONS

Y.L. and Y.‐T.W. conceived and designed the experiments; Y.L., X.‐Y.L., Q.Z., S.‐Y.W., and J.X. performed the experiments; Y.L. and X.‐Y.L. analysed the data; C.‐F.M., H.‐J.G., Y.‐T.W., S.‐X.Y., and L.‐M.L. contributed the reagents/materials/analysis tools; and Y.L. and Y.L. wrote the paper.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14206 and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1. PO‐322 inhibits human T cell proliferation following PHA or PMA/ionomycine stimulation. CFSE‐labeled T cells were treated with PO‐322 (0.125, 0.5, 2 and 8 μM) and activated with PHA (A) or PMA/ionomycine (B) for 72 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Figure S2. PO‐324, PO‐326, PO‐327, PO‐335, PO‐341 and PO‐342 inhibit human T cell proliferation. CFSE‐labeled T cells were treated with PO‐324 (A), PO‐326 (B), PO‐327 (C), PO‐335 (D), PO‐341 (E) or PO‐342 (F) following anti‐CD3 and anti‐CD28 mAbs stimulation for 72 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Figure S3. Treatment with exogenous IL‐10 had no obvious inhibition of T cell proliferation in our system. The CFSE‐labeled T cells were treated with or without exogenous IL‐10 (5 ng/ml) and PO‐322 (2 μM) and activated with anti‐CD3/anti‐CD28 for 72 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative control (0%), while the cells with stimulation but without PO‐322 treatment served as the positive control (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Figure S4. The inhibition of PO‐322 for T cells proliferation is reversible. T cells were cultured with or without PO‐322 (0.125, 0.5 or 2 μM) and stimulated with anti‐CD3/anti‐CD28 for different span (24 h and 96 h). At 24 h, the cultured cells were collected to remove the medium by centrifugation and re‐culture with medium containing IL‐2 (100 IU/ml) for total 96 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Click here for additional data file. (207.8KB, pdf)

Data S1 Supporting Information

Click here for additional data file. (398B, csv)

ACKNOWLEDGEMENTS

The authors declare that this work was supported by the National Natural Science Foundation of China (81302786, 81871300, and 81402944), State Key Laboratory of Phytochemistry and Plant Resources in West China (P2018‐KF03), Scientific Research Fund of Sichuan Provincial Education Department (18ZA0143), Sichuan Science and Technology Programme (2018JY0440 and 2018JY0481).

Lai Y, Luo X‐Y, Guo H‐J, et al. PO‐322 exerts potent immunosuppressive effects in vitro and in vivo by selectively inhibiting SGK1 activity. Br J Pharmacol. 2020;177:1666–1676. 10.1111/bph.14926

Yi Lai, Xing‐Yan Luo, and Hui‐Jie Guo contributed equally to this work.

Contributor Information

Chun‐Fen Mo, Email: mcf89@163.com.

Yan‐Tang Wang, Email: yt-wang@hotmail.com.

Yang Liu, Email: scunn519@gmail.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. PO‐322 inhibits human T cell proliferation following PHA or PMA/ionomycine stimulation. CFSE‐labeled T cells were treated with PO‐322 (0.125, 0.5, 2 and 8 μM) and activated with PHA (A) or PMA/ionomycine (B) for 72 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Figure S2. PO‐324, PO‐326, PO‐327, PO‐335, PO‐341 and PO‐342 inhibit human T cell proliferation. CFSE‐labeled T cells were treated with PO‐324 (A), PO‐326 (B), PO‐327 (C), PO‐335 (D), PO‐341 (E) or PO‐342 (F) following anti‐CD3 and anti‐CD28 mAbs stimulation for 72 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Figure S3. Treatment with exogenous IL‐10 had no obvious inhibition of T cell proliferation in our system. The CFSE‐labeled T cells were treated with or without exogenous IL‐10 (5 ng/ml) and PO‐322 (2 μM) and activated with anti‐CD3/anti‐CD28 for 72 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative control (0%), while the cells with stimulation but without PO‐322 treatment served as the positive control (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Figure S4. The inhibition of PO‐322 for T cells proliferation is reversible. T cells were cultured with or without PO‐322 (0.125, 0.5 or 2 μM) and stimulated with anti‐CD3/anti‐CD28 for different span (24 h and 96 h). At 24 h, the cultured cells were collected to remove the medium by centrifugation and re‐culture with medium containing IL‐2 (100 IU/ml) for total 96 h. Cell proliferation was measured by flow cytometry. Cells without stimulation and PO‐322 treatment served as negative controls (0%), while the cells with stimulation but without PO‐322 treatment served as positive controls (100%). The results are presented as the mean ± S.E.M., n = 5 for each experimental group.

Click here for additional data file. (207.8KB, pdf)

Data S1 Supporting Information

Click here for additional data file. (398B, csv)

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