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

Article abstract of DOI:10.1111/bph.14926

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.

Full text of DOI:10.1111/bph.14926

Liu Yang (Orcid ID: 0000-0003-4316-8885)
Title page
PO-322 has potent immunosuppressive activity in vitro and in vivo by selectively inhibiting
SGK1 activity
Running title: The immunosuppressive activity of PO-322
#
,1
#,2
#,2
2
2
2
Yi Lai , Xing-yan Luo , Hui-jie Guo , Si-yu Wang , Jing Xiong , Shu-xia Yang , Li-mei
2
2
*,2
*,2,3
and Yang Liu*,2,3
Li , Qiang Zou , Chun-fen Mo , Yan-tang Wang
1School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan, People’s
2
Republic of China; Research Center, Chengdu Medical College, Chengdu, Sichuan, People’s
3
Republic of China; Development and Regeneration Key Laboratory of Sichuan Province,
Chengdu Medical College, Chengdu, Sichuan, People’s Republic of China
#
These authors contributed equally to this work.
*
Co-corresponding author
Correspondence: Dr. Yang Liu (Research Center, Chengdu Medical College, No.783, Xindu
Avenue, Chengdu, Sichuan 610500, People’s Republic of China, Telephone:
+86-28-62739161, Fax: +86-28-62739165. E-mail: scunn519@gmail.com); Dr. Yan-tang
Wang (Research Center, Chengdu Medical College, No.783, Xindu Avenue, Chengdu,
Sichuan 610500, People’s Republic of China, Telephone: +86-28-62739161, Fax:
+86-28-62739165. E-mail: yt-wang@hotmail.com); and Dr. Chun-fen Mo (Research Center,
Chengdu Medical College, No.783, Xindu Avenue, Chengdu, Sichuan 610500, People’s
Republic of China, Telephone: +86-28-62739161, Fax: +86-28-62739165. E-mail:
mcf89@163.com)
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
1
0.1111/bph.14926
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Abstract
BACKGROUND AND PURPOSE
Immunosuppressive drugs have shown great promise in treating the 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. The purpose of the current study
was to investigate the potential mechanism of PO-322 in inhibiting T cell proliferation in
vitro, as well as its effects on the delayed-type hypersensitivity 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
delayed-type hypersensitivity and imiquimod-induced dermatitis was evaluated in a mouse
model.
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 SGK1 inhibitor and
decreased NDRG1 phosphorylation but not p70S6K, STAT5, AKT or ERK 1/2
phosphorylation. Furthermore, PO-322 inhibited IFN-γ, IL-6 and IL-17 expression but not
IL-10 expression. Finally, the administration of PO-322 was safe and effective for
ameliorating the delayed-type hypersensitivity response and imiquimod-induced dermatitis.
CONCLUSION AND IMPLICATIONS
Taken together, PO-322 exerts 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 the autoimmune diseases.
Keywords: immunosuppressive activity; T cell proliferation; SGK1; PO-322
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Abbreviations
CFSE, 5-carboxyfluorescein diacetate succinimide ester; DTH, delayed type hypersensitivity
reaction; ELISA, enzyme-linked immunosorbent assay; JAK3, Janus protein tyrosine kinase;
MAPK, mitogen activated protein kinase; mTOR, mammalian target of rapamycin; NF-κB,
nuclear factor κB; PBMCs, peripheral blood mononuclear cells; PI3K, phosphoinositide 3
kinase; RAPA, rapamycin; SGK1, the serum- and glucocorticoid-regulated kinase 1
Introduction
The important role of T cells in immune responses against pathogens has been clearly
clarified, and aberrant T cell responses play a crucial role in mediating the immunorejection
of transplanted organs (van Gelder et al., 2014) and the pathogenesis of autoimmune diseases,
including multiple sclerosis (Sabatino et al., 2018), psoriasis (Hawkes et al., 2018) and
systemic lupus erythematosus (SLE) (He et al., 2016). In recent years, immunosuppressants
have been widely used in the clinic 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 drug sensitivity differences amongst
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.
TCR-mediated T cell signalling can induce T cell activation and proliferation through several
signalling pathways, including the calcium/calcineurin, nuclear factor κB (NF-κB),
phosphoinositide 3 kinase (PI3K)/Akt, mammalian target of rapamycin (mTOR)/p70S6K,
Janus protein tyrosine kinase (JAK3)/STAT5, and mitogen activated protein kinase (MAPK)
p38 signal pathways (Moulton et al., 2015). Immunosuppressants that are the inhibitors of
these pathways have been approved for clinical use, such as cyclosporin A, FK506,
rapamycin (RAPA) and tofacitinib (Baker et al., 2018). Recently, several new small molecule
inhibitors of these pathways, such as apremilast (Nash et al., 2018), peficitinib (Sands et al.,
2
018) and baricitinib (Wallace et al., 2018) have been advanced to late-stage clinical trials,
highlighting the potential of these immunosuppressive approaches.
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Serum- and glucocorticoid-regulated kinase 1 (SGK1) 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 IL-2 (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 GSK650394,
EMD638683 and SI113, have been reported to have anticancerous activity (Abbruzzese et al.,
2
017; 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 structure 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 cyclooxygenase-2 and cyclooxygenase-1 enzyme
activities (Hashimoto et al., 2002). Recently, we found an oxazole derivative, PO-296, that
could inhibit T cell proliferation through the JAK3/STAT5 signalling pathway (Luo et al.,
2
018). 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 DNFB-induced delayed type
hypersensitivity (DTH) reaction and imiquimod-induced dermatitis in vivo.
Methods
Experimental animals
Mice (BALB/c, 8-11 weeks, RRID:IMSR_ORNL:BALB/cRl) were purchased from Huaxi
Laboratory Animal Center of Sichuan University (Chengdu, China) and kept under specific
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pathogen-free conditions and provided with normal food and water. All experiments were
approved by the Animal Ethics Committee of Chengdu Medical College on animal
experiments. Animals were randomly assigned to treatment groups and the experimenter was
blinded to drug treatment until data analysis has been performed.
Synthetic method of PO-322
PO-322 was synthesized in one step from commercially available 2-indolinone and
2
2
-hydrazinobenzoxazole.
A
mixture of 2-indolinone (1.33 g, 10 mmol),
-hydrazinobenzoxazole (1.49 g, 10 mmol) and acetic acid (1 mL) was stirred in ethanol (50
mL) at reflux temperature for 3 hours. After cooled down to room temperature, PO-322 (1 g,
6% yield) was collected as pale yellow powder. The compound was characterized by 1H
3
NMR using Bruker Advance 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.
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. The T cell
purity of 95% was used for the following experiments.
Drug treatment
6
-1
T cells (10 cells ml ) were treated with RAPA (0.1 μM, Sigma-Aldrich, St Louis, MO, USA),
FK506 (0.1 μM, Sigma-Aldrich), GSK650394 (10 μM, MedChem Express, Monmouth
Junction, NJ, USA), AG-490 (50 μM, Sigma-Aldrich), LY-294002 (50 μM, Promega,
Madison, WI, USA), PD184352 (2 μM, Sigma-Aldrich) or different concentrations of
-1
PO-322 and activated by plate-bound anti-CD3 (2 μg ml , HIT3a clone, BD PharMingen,
-1
San Diego, CA, USA) and soluble anti-CD28 (1 μg ml , CD28.2 clone, BD PharMingen) as
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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.
CFSE labeling 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)-labeling as previously described (Liu et al., 2015).
Cell apoptosis assay
Fluos-labeled Annexin V and PI dual staining kit (Roche, Indianapolis, IN, USA) was used to
assess the cell apoptosis following the protocol and analyzed by flow cytometry (Acurri C6)
as previously described (Luo et al., 2018).
Cell viability assay
The CCK-8 assay kit (Dojindo, Kumamoto, Japan) was used to assess the viability of cell by
detecting the O.D. values at 450 nm in a SpectraMax M5 microplate reader (Molecular
Devices, Sunnyvale, CA, USA) as previously described (Liu et al., 2013).
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.
Western blot analysis
Cell pellets were treated with lysis buffer and clarified by centrifugation. Cell lysate proteins
-1
(
20 g lane ) were separated on 10 % SDS-polyacrylamide gel electrophoresis and
transferred to immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA,
USA). The membranes were blocked by PBS containing 5 % BSA and stained with
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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-ERK 1/2 (CST
Inc., Danvers, MA, USA) overnight at 4 °C followed by horseradish peroxidase
(
HRP)-conjugated second antibodies (Santa Cruz Biotech, 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).
Cytokine ELISA assay
The levels of IL-2, IL-6, IL-10, IL-17A and IFN-γ were determined by enzyme-linked
immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA, USA) as previously
described (Liu et al., 2015).
DNFB-induced DTH reaction in mice
The experiments of DNFB-induced DTH reaction in mice were performed. Individual BAL
b/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 treated
-1
-1
intraperitoneally with different doses of PO-322 (2.5, 10 and 40 mg kg ), RAPA (2 mg kg )
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 h post challenge.
Imiquimod-induced dermatitis in mice
The backs of female BALB/c mice (RRID:IMSR_ORNL:BALB/cRl) were shaved with an
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electric clipper 1 d prior to treatment. Mice were treated intraperitoneally with 10 or 40
mg/kg PO-322 or vehicle 1 day prior to application of 62.5 mg 5 % imiquimod cream (Aldara,
3
M Pharmaceuticals, St Paul, Minn) or control cream (Curel, Kao, Japan). Then, 10 or 40
mg/kg PO-322 or vehicle was given 0.5 h before treating with 62.5 mg 5 % imiquimod cream
or control cream and this treatment was repeated for 6 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 sacrificed 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 haemotoxylin and eosin (H&E). H&E slides were blinded, 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 analyzed on a flow cytometry (Acurri C6).
Statistical analysis
Results are expressed as mean ± S.E.M. and the inhibitory concentration of the compound
that reduced cell proliferation by 50% (IC50) values were 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 analysis of variance
ANOVA) with Dunnett comparisons on post-tests were used to analyze data and compare
(
groups. The post hoc tests were run only if F achieved P < 0.05 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 < 0.05 was considered to be statistically significant. The data
and statistical analyses comply with the recommendations and requirements on experimental
design and analysis in pharmacology (Curtis et al., 2018).
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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 2017/18 (Alexander et al., 2017).
Results
Synthesis and characterization of PO-322
PO-322 was synthesized in one step from 2-indolinone (1) and 2-hydrazinobenzoxazole (2),
1
with 36% yield (Figure 2). H NMR (300 MHz, DMSO-d6) 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,4 H), 7.04 (m, 1H), 6.86 (m,
H). ESI-MS: m/z 277 [M - H]-.
1
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/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 PHA or PMA/ionomycine stimulation (Figure S1).
To investigate the potential cytotoxicity of PO-322, human activated 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 the conditions (Rathmell et al.,
2
001). 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 h or 48 h
Figure 3D) and had no significant impact on the relative viability of resting T cells (Figure
E), IL-7-treated activated T cells (Figure 3F), or PBMCs at 72 h (Figure 3G), indicating that
the activity of PO-322 was immunosuppressive rather than cytotoxic.
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(
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
1
00 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.
To further reveal the signalling pathway affected by PO-322, the impact of PO-322 on the
related kinase phosphorylation levels were 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 ERK 1/2 (Figure 4G) expression and phosphorylation levels were not
(
affected by PO-322. Phosphorylation of NDRG1 has previously been shown to be a specific
SGK1 target (Inglis et al., 2009), indicating that PO-322 inhibited T cell proliferation through
the SGK1/NDRG1 signalling pathway.
PO-322 inhibits pro-inflammatory cytokines but does not affect anti-inflammatory
cytokines
To understand the impact 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.
PO-322 significantly mitigates DNFB-induced DTH reaction and ameliorates
imiquimod-induced dermatitis in mice
Th1/Th17 responses are clearly related to DTH reaction (Sido et al., 2016). We explored the
activity of PO-322 on DNFB-induced DTH reaction in BAL b/c mice. We found that the
administration of PO-322 markedly reduced ear swelling, including increases in ear thickness
and patch weight, in a dose-dependent manner (Figure 6).
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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). We found that
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 cells numbers in the spleen (Figure 7B, C and D),
demonstrating that PO-322 can substantially reduce disease severity.
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.
Discussion and Conclusions
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/ionomycine stimulation. Notably,
PO-322 did not induce activated T cells 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
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phosphorylation was increased by PO-322. The mTOR/SGK1 pathway inhibition may
+
selectively expand regulatory T cells and promote de novo generation of Foxp3 regulatory T
cells through the upregulation 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 cytokines 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 well 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 et al., 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 implicates the anti-psoriatic
properties of PO-322 in vivo. However, the potential mechanism of PO-322 in treating DTH
and psoriasis remains 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, small-molecule chemical therapies can
be administered orally and are often less expensive to manufacture, resulting 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
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new drugs and would ultimately benefit patients with safer and more effective therapeutic
options for these diseases.
In summary, we synthesized and identified 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 vivo. Further structure and activity relationship (SAR) studies and the
optimization of PO-322 are warranted to improve the potency and help 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. W. conceived and designed the experiments; Y. L., X. L., H. Z., S. W. and J. X.
performed the experiments; Y. L. and X. L. analyzed the data; C. M., H. G., Y. W., S. Y. and L.
L. contributed reagents/materials/analysis tools; Y. L. and Y. L. wrote the paper.
Acknowledgments
The authors declare that this work was supported by National Natural Science Foundation of
China (No.81302786, 81871300, 81402944), State Key Laboratory of Phytochemistry and
Plant Resources in West China (No.P2018-KF03), Scientific Research Fund of Sichuan
Provincial Education Department (No.18ZA0143), Sichuan Science and Technology Program
(
No.2018JY0440, 2018JY0481).
Conflicts of Interest
The authors state no conflict 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
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Design & Analysis, and as recommended by funding agencies, publishers and other
organizations engaged with supporting research.
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Figure 1. Chemical structure of oxazol derivatives.
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Figure 2. 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 hours. After cooled down to room temperature,
PO-322 (1 g, 36% yield) was collected as pale yellow powder.
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Figure 3. 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
anti-CD3/anti-CD28 (A, B) or allogeneic PBMCs (C) 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%). T cells were treated with PO-322 (0.5, 2 and 8 μM), Rapa (0.1
μM) or vehicle and activated with anti-CD3/anti-CD28 for 24 h or 48 h. Cell apoptosis was
assessed by flow cytometry with Annexin V and PI dual staining (D). Resting T cells (E),
IL-7-treated activated T cells (F) and PBMCs (G) were treated with PO-322 (10, 20, 40 and
8
0 μM) or vehicle for 72 h. 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. RAPA is an abbreviation for rapamycin. The
results are presented as the mean ± S.E.M., n = 5 for each experimental group.
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Figure 4. 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. PO-322 was
dissolved in DMSO at 100 μM, and a final concentration of 1 μM was used for screening (A).
Then, the inhibitory activity of PO-322 against SGK1 was measured with different
concentrations of PO-322 (B). T cells were incubated alone for 6 h after 72 h of
anti-CD3/anti-CD28 stimulation. T cells were then treated with PO-322 (0.5, 2 or 8 μM),
GSK650394 (10 μM), RAPA (0.1 μM), AG-490 (50 μM), LY294002 (50 μM), PD184352 (2
μM) or vehicle for another 6 h. Subsequently, T cells were induced by IL-2 for 30 min, and
the relative phosphorylation and expression levels of NDRG1 (C), p70S6K (D), STAT5 (E),
Akt (F) and ERK 1/2 (G) were assessed by western blot analysis. “+” and “-” indicate
experiments with or without IL-2, respectively. The results are presented as the mean ±
S.E.M., n = 5 for each experimental group. *P < 0.05 vs. the activated group without drug.
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Figure 5. 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 h. The
supernatants were collected and the levels of IL-17A (A), IFN-γ (B), IL-6 (C) and IL-10 (D)
were measured by ELISA. “+” and “-” indicate experiments with or without
anti-CD3/anti-CD28, respectively. The results are presented as the mean ± S.E.M., n = 5 for
each experimental group. *P < 0.05 vs. the activated group without drug.
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Figure 6. PO-322 mitigates DNFB-induced DTH reaction in mice. BALB/c mice were
sensitized with DNFB on days 0 and 1 and treated intraperitoneally with different doses of
-1
-1
PO-322 (2.5, 10 and 40 mg kg ), RAPA (2 mg kg ) 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 h post challenge. Ear
swelling was calculated as an increase in ear thickness (A) and ear patch weight (B) between
the left (DNFB-untreated) and right (DNFB-treated) ears. The results are presented as the
mean ± S.E.M., n = 8 for each experimental group. *P < 0.05 vs. the group without drug.
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Figure 7. PO-322 relieves imiquimod-induced psoriasis-like dermatitis. BALB/c mice
were treated intraperitoneally with 10 or 40 mg/kg PO-322 or vehicle 1 day prior to the
application of imiquimod or control cream. Then, PO-322 or vehicle was given 0.5 h before
treatment with imiquimod cream, and this treatment was repeated for 6 consecutive days.
Clinical scores for redness and scaling (A) and H&E-stained slides (B) were scored (0 - 4) for
disease severity. Mice were sacrificed and splenic masses were determined (C). The numbers
of T cells were measured by flow cytometry (D). “+” and “-” indicate runs with or without
imiquimod, respectively. The results are presented as the mean ± S.E.M., n = 8 for each
experimental group. *P < 0.05 vs. the imiquimod-induced group without drug.
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Table 1. Immunosuppressive activity of oxazole derivatives
a
IC (μM)
Compound
IC (μM)
Compound
5
0
5
0
PO-322
PO-324
PO-325
PO-326
PO-327
PO-329
PO-333
PO-335
PO-341
0.7 ± 0.2
31.6 ± 2.3
> 100
PO-342
PO-343
PO-345
PO-346
PO-348
PO-349
PO-350
PO-353
PO-355
28.9 ± 3.9
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
25.3 ± 3.1
15.1 ± 3.9
> 100
> 100
32.4 ± 4.2
24.5 ± 3.5
a
IC50 of inhibitory effect on cell proliferation. In the experiment, CFSE-labeled T cells were
treated with a series of oxazole derivatives following anti-CD3 and anti-CD28 mAbs
stimulation for 72 h. Cell proliferation was analyzed on a flow cytometry using proliferation
index. The results are presented as the mean ± S.E.M., n = 5 for each experimental group.
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