Polypharmacology of N6-(3-Iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) and Related A3 Adenosine Receptor Ligands: Peroxisome Proliferator Activated Receptor (PPAR) γ Partial Agonist and PPARδ Antagonist Activity Suggests Their

Article abstract of DOI:10.1021/acs.jmedchem.7b00805

A₃ adenosine receptor (AR) ligands including A₃ AR agonist, N⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (1a, IB-MECA) were examined for adiponectin production in human bone marrow mesenchymal stem cells (hBM-MSCs). In

Full text of DOI:10.1021/acs.jmedchem.7b00805

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Article
Polypharmacology of N6-(3-iodobenzyl)adenosine-5#-N-methyluronamide
(
IB-MECA) and Related A3 Adenosine Receptor Ligands: Peroxisome
Proliferator Activated Receptor (PPAR) # Partial Agonist and
PPAR # Antagonist Activity Suggests Their Anti-diabetic Potential
Jinha Yu, Seyeon Ahn, Hee Jin Kim, Moonyoung Lee, Sungjin Ahn, Jungmin Kim, Sun Hee Jin, Eunyoung
Lee, Gyudong Kim, Jae Hoon Cheong, Kenneth A. Jacobson, Lak Shin Jeong, and Minsoo Noh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00805 • Publication Date (Web): 11 Aug 2017
Downloaded from http://pubs.acs.org on August 11, 2017
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Polypharmacology of N ꢀ(3ꢀiodobenzyl)adenosineꢀ5′ꢀNꢀ
methyluronamide (IBꢀMECA) and Related A Adenosine Receptor
Ligands: Peroxisome Proliferator Activated Receptor PPAR) γ Partial
3
(
Agonist and PPARδ Antagonist Activity Suggests Their Antiꢀdiabetic
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Potential
1
, #
1,2, #
3
1,2
1,2
Jinha Yu, Seyeon Ahn,
Hee Jin Kim, Moonyoung Lee, Sungjin Ahn, Jungmin
1
,2
1,2
1,2
1
3,∥
Kim, Sun Hee Jin, Eunyoung Lee, Gyudong Kim, Jae Hoon Cheong,
Kenneth
4
1,*
1,2,*
A. Jacobson, Lak Shin Jeong, Minsoo Noh,
1
2
Pharmaceutical Research Institute, College of Pharmacy, Natural Products Research
Institute, Seoul National University, Seoul, 151ꢀ742, Republic of Korea
1
2
Pharmaceutical Research Institute, College of Pharmacy, Natural Products Research
Institute, Seoul National University, Seoul, 151ꢀ742, Republic of Korea
1
2
Pharmaceutical Research Institute, College of Pharmacy, Natural Products Research
3
Institute, Seoul National University, Seoul, 151ꢀ742, Republic of Korea, Uimyung
Research Institute for Neuroscience, Sahmyook University, 26ꢀ21 Kongreungꢀ2ꢀdong,
4
Hwarangroꢀ815, Nowonꢀgu, Seoul 139ꢀ742, Republic of Korea, Molecular Recognition
Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
2
0892ꢀ0810, United States
*Corresponding Authors
Minsoo Noh (minsoo@alum.mit.edu), Lak Shin Jeong (lakjeong@snu.ac.kr)
#
These authors contributed equally to this work.
Key words: Polypharmacophore, A3 adenosine receptor, Human bone marrow
mesenchymal stem cells, Adiponectinꢀsecreting compound, Target deconvolution
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ABSTRACT
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A3 adenosine receptor (AR) ligands including A3 AR agonist, N ꢀ(3ꢀ
iodobenzyl)adenosineꢀ5′ꢀNꢀmethyluronamide (1a, IBꢀMECA) were examined for
adiponectin production in human bone marrow mesenchymal stem cells (hBMꢀMSCs). In
this model, 1a significantly increased adiponectin production, which is associated with
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improved insulin sensitivity. However, A AR antagonists also promoted adiponectin
3
production in hBMꢀMSCs, indicating that the A AR pathway may not be directly
3
involved in the adiponectin promoting activity. In a target deconvolution study, their
adiponectinꢀpromoting activity was significantly correlated to their binding activity to
both peroxisome proliferator activated receptor (PPAR) γ and PPARδ. They functioned
as both PPARγ partial agonists and PPARδ antagonists. In the diabetic mouse model, 1a
and its structural analogues, A AR antagonists significantly decreased the serum levels
3
of glucose and triglyceride, supporting their antiꢀdiabetic potential. These findings
indicate that the polypharmacophore of these compounds may provide therapeutic insight
into their multiꢀpotent efficacy against various human diseases.
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■
Introduction
The concept of polypharmacology defines a single drug molecule that can
simultaneously modulate multiple drug targets to treat complex diseases with polygenic
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1
,2
etiology. Recently, a single drug molecule interacting with multiple kinases in a
network of dysregulated cellular pathway in cancer cells has provided experimental
3,4
evidence to demonstrate the effectiveness of the polypharmacology approach. However,
most of current polypharmacology cases in the drug discovery field have been limited to
antiꢀcancer kinase inhibitors, affecting virtually same protein families. The clinical
feasibility of polypharmacology concept should be supported by a variety of single
molecules simultaneously targeting multiple protein families with a direct causal
relationship with a multiꢀetiological complex disease.
Phenotypeꢀbased approaches have been successfully proven as a viable
4
,5
alternative to a defined molecular targetꢀbased approach in drug discovery. For
complex chronic diseases with multifactorial genetic and epigenetic etiologies, such as
type II diabetes and obesity, a phenotypeꢀbased pharmacological assay has several
advantages over targetꢀbased assays. To develop antiꢀdiabetic and antiꢀobesity drugs, a
phenotypic assay based on the adipogenesis model of human bone marrow mesenchymal
stem cells (hBMꢀMSCs) has been studied by simultaneously measuring adiponectin
6
ꢀ9
production and lipid accumulation. Adiponectin, an adipocytokine mainly produced in
the adipocytes, has been used as a diagnostic biomarker for metabolic diseases. For
example, the ratio of serum adiponectin to leptin in patients with type II diabetes is lower
10ꢀ11
than that in the healthy population.
Notably, recombinant adiponectin showed
1
0ꢀ12
therapeutic benefits in various animal models of human metabolic diseases.
. In fact,
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sulfonylureaꢀtype antiꢀdiabetic drugs and peroxisome proliferator activated receptor
(
PPAR) γ agonists increase adiponectin biosynthesis and lipid accumulation in hBMꢀ
7
,9,13,14
MSCꢀbased phenotypic assay system.
In addition, nonꢀsteroidal antiꢀinflammatory
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drugs (NSAIDs) such as aspirin, ibuprofen, and indomethacin also increase adiponectin
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,15
production and lipid droplet development during adipogenesis in hBMꢀMSCs.
At
higher concentrations, ibuprofen and indomethacin directly bind to PPARγ, which
1
6
explains their pharmacological effect on adiponectin production during adipogenesis. In
contrast, aspirin does not directly bind to PPARγ, and the molecular targets associated
with the effect of aspirin on adipogenesis are not fully understood. Therefore, chemical
compounds discovered from phenotypic assays require additional study to identify and
validate their direct molecular targets; this process is defined as drug target
3ꢀ5
deconvolution.
Extracellular adenosine regulates various biological functions by acting on a Gꢀ
proteinꢀcoupled receptor (GPCR) family of adenosine receptors (ARs), which consists of
17
four subtypes: A , A , A , and A ARs. The downstream effectors of ARs are coupled
1
2A
2B
3
with the cyclic adenosine monophosphate (cAMP)ꢀmediated intracellular signaling
pathway. A and A ARs inhibit intracellular cAMP signaling, whereas A and A2B
1
3
2A
receptors promote it. Adenosine plays a role in many physiological functions such as
circulation, renal blood flow, cardiac rhythm, lipolysis, immune function, and
angiogenesis. Importantly, the modulation of AR functions by their specific agonists or
antagonists has pharmacological significance in inflammatory diseases,
neurodegenerative diseases, metabolic diseases, and cancers. Adenosine and its chemical
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derivatives are also used clinically in the diagnosis of supraventricular tachycardia or
1
8,19
administered as antiarrhythmic agents.
Several reports indicate that adenosine plays a role in mammalian adipogenesis
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and osteogenesis. Caffeine, a major ingredient of coffee, tea, and many commercial soft
2
0
drinks, is classified as an AR antagonist. Caffeine inhibits adipogenesis in adipose
2
1
tissueꢀderived MSCs and adenosine affects the differentiation lineage commitment of
2
2,23
hMSCs into adipocytes or osteoblasts.
The results of studies in AR knockout mice
2
4
indicate that AR modulators have therapeutic potential for diabetes and obesity. In a
highꢀfat diet mouse model, A2B AR upregulation was correlated with insulin receptor
substrate 2 (IRSꢀ2) expression, indicating that A AR is a potential drug target in human
2B
2
5
metabolic diseases. Currently, the role of ARs in adipogenesis in hMSCs is not fully
understood.
Figure 1. Structures of A AR ligands used in this study
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In the present study, we evaluated the effect of various AR agonists, 1aꢀd and
aꢀd and antagonists 3aꢀd on adipogenesis in hBMꢀMSCs to elucidate the subtype
2
specific roles of ARs (Figure 1). From this study, we discovered that a specific A AR
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agonist, N ꢀ(3ꢀiodobenzyl)adenosineꢀ5′ꢀNꢀmethyluronamide (1a, IBꢀMECA)
and
related A AR ligands promoted adiponectin production during adipogenesis in hBMꢀ
3
MSCs. In the structureꢀactivity relationship (SAR), the adiponectin promoting activity of
A AR ligands tested was not correlated to their A AR binding affinities. It was found
3
3
that 1a and related A AR ligands have polypharmacological characteristics of an A AR
3
3
modulator, a PPARγ partial agonist, and a PPARδ antagonist in the target deconvolution
of their adiponectin promoting activity in hBMꢀMSCs. Herein, we report the
polypharmacology of A AR ligands acting as PPARγ partial agonists and PPARδ
3
antagonists.
■
RESULTS AND DISCUSSION
Synthesis of A AR agonists 2aꢀd and A AR antagonists 3aꢀd.
3
3
A AR agonists 2aꢀd were synthesized as shown in Scheme 1, according to our
3
2
7
previously published procedure. 2,3ꢀIsoproplylideneꢀ
to 2,3ꢀisoproplylideneꢀ ꢀlyxonolactone (5) via the mesylation followed by intramolecular
relactonization of the product of aqueous potassium hydroxide (KOH) cleavage of the
Dꢀribonolactone (4) was converted
L
D
ꢀ
2
8
ribonolactone ring. Benzoylation of 5 followed by reduction with sodium borohydride
(
NaBH ) afforded diol 6, which was converted to 4ꢀthiosugar 7 by mesylation and
4
cyclization of resulting dimesylate with sodium sulfide (Na S).
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HO
O
O
OH
O
ref. 27
O
a,b
OH
HO
BzO
BzO
O
O
O
O
83%
for 2 steps
O
O
4
5
6
10
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42%
for 2 steps
HN
N
HN
c,d
N
N
N
N
I
I
HO
N
BzO
N
N
S
S
e,f,g
S
h,i,j
4
4%
82%
TBSO OTBS
O
O
O
O
for 3 steps
for 3 steps
9
8
7
k,l,m
HN
N
N
N
I
O
2a (R = ethyl, 66% for 3 steps)
2b (R = cyclopropyl, 62% for 3 steps)
2c (R = cyclopropylmethyl, 61% for 3 steps)
2d (R = cyclobutyl, 53% for 3 steps)
RHN
N
S
HO OH
Reagents and conditions: a) BzCl, pyridine, CH Cl , rt, 12 h, 92%; b) NaBH , MeOH, 0
2
2
o
4
o
.
C to rt, 3 h, 90%; c) MsCl, Et N, DMAP, CH Cl , 0 C, 30 min; d) Na S 9H O, DMF, 100
3
2
2
2
2
o
o
C, 15 h, 42% for 2 steps from 6; e) m-CPBA, CH Cl , -78 C, 1 h, 95%; f) 6-chloropurine,
2
2
o
Et N, TMSOTf, CH CN, DCE, rt to 80 C, 4 d, 53%; g) R NH , Et N, EtOH, rt, 24 h; h)
3
3
2
2
3
o
o
80% AcOH, 70 C, 12 h; i) TBSOTf, pyridine, 50 C, 5 h; j) NaOMe, MeOH, rt, 4 h, 82%
for 3 steps from 8; k) PDC, DMF, rt, 20 h; l) RNH , EDC, HOBt, DIPEA, CH Cl , rt, 15 h;
2
2
2
m) TBAF, THF, rt, 1 h.
6
27
Scheme 1. Synthesis of N ꢀ(3ꢀiodobenzyl)ꢀ4′ꢀthioadenosine derivatives 2aꢀd
Oxidation of 7 with metaꢀchloroperbenzoic acid (mCPBA) followed by Pummererꢀtype
condensation of the resulting sulfoxide with 6ꢀdichloropurine in the presence of
6
trimethylsilyl trifluoromethanesulfonate (TMSOTf) afforded the N ꢀ(3ꢀiodobenzyl)amino
derivative 8 after treating the condensed product with 3ꢀiodobenzylamine. Hydrolysis of
2
,3ꢀacetonide of 8, protection of resulting diol with tꢀbutyldimethylsilyl (TBS) group, and
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removal of the benzoyl group yielded 9. Oxidation of 9 with pyridinium dichromate
PDC) in DMF yielded the acid, which was coupled with various amines such as
(
ethylamine, cyclopropylamine, 1ꢀcyclopropylmethamine, and cyclobutylamine in the
presence of 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDC) and 1ꢀ
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hydroxybenzotriazole (HOBt) afforded various 5′ꢀuronamides 2aꢀd after desilylation.
OH
OH
MsO
a,b,c
2%
d,e
S
MsO
D-Mannose
O
O
8
63%
for 2 steps
O
O
O
O
for 3 steps
10
11
f
60%
NHR
N
Cl
N
N
N
N
N
S
N
N
Y
Y
S
i
g,h
OAc
S
O
O
HO OH
HO OH
3a (R = 3-I-Bn, Y = Cl, 84%)
13a (Y = H, 71% for 2 steps)
13b (Y = Cl, 76% for 2 steps)
12
3
3
3
b (R = 3-Cl-Bn, Y = Cl, 82%)
c (R = 2-Cl-Bn, Y = Cl, 81%)
d (R = 3-I-Bn, Y = H, 88%)
Reagents and conditions: a) 2,2-dimethoxypropane, camphosulfonic acid, acetone, rt,
15 h, 95%; b) NaBH EtOH, rt, 2 h, 92%; c) MsCl, Et N, CH Cl , rt, 1 h, 94%; d) Na S,
4
,
3
2
2
2
o
DMF, 80 C, 15 h, 78%; e) 60% AcOH, rt, 2 h, 81%; f) Pb(OAc) , EtOAc, rt, 15 h, 60%; g)
4
o
6
-chloropurine for 13a, 2,6-dichloropurine for 13b, ammonium sulfate, HMDS, 170 C, 15
o
h, then TMSOTf, DCE, rt to 80 C, 3 h; h) 2 N HCl, THF, rt, 15 h; i) RNH , Et N, EtOH, rt,
2
3
1
-3 d.
2
9
Scheme 2. Synthesis off truncated 4′ꢀthioadenosine derivatives 3aꢀd
A AR antagonists 3aꢀd were synthesized from
D
ꢀmannose according to our
3
29
previously published procedure, as illustrated in Scheme 2. Treatment of
Dꢀmannose
with 2,2ꢀdimethoxypropane under acidic conditions gave the diacetonide, which was
reduced with NaBH followed by mesylation of resulting diol afforded the dimesylate 10.
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Cyclization of 10 with Na S in DMF followed by selective hydrolysis of 5,6ꢀacetonide
2
yielded diol 11. Treatment of 11 with excess lead(IV) tetraacetate gave the glycosyl
donor 12. Condensation of 12 with 6ꢀchloropurine and 2,6ꢀdichloropurine in the presence
of TMSOTf as a Lewis acid afforded 6ꢀchloropurine derivative 13a and 2,6ꢀ
dichloropurine derivative 13b, respectively after the removal of the isopropylidene group.
Treatment of 13a with 3ꢀiodoꢀ, 3ꢀchloroꢀ, and 2ꢀchlorobenzylamines yielded the final 3aꢀ
c, respectively, while 13b was converted to 3d by treating with 3ꢀiodobenzylamine.
Effects of 1a on adiponectin production during adipogenesis in hBMꢀMSCs. To
determine whether AR signaling affects adipogenesis in hBMꢀMSCs, an endogenous
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ligand adenosine, A AR agonist, 2ꢀchloroꢀN ꢀcyclopentyladenosine (14, CCPA) , a
1
3
1
nonꢀspecific AR agonist, 5′ꢀ(Nꢀethylcarboxamido)adenosine (15, NECA) , A AR
2
A
agonist,
2ꢀpꢀ(2ꢀcarboxyethyl)phenethylaminoꢀ5′ꢀNꢀethylcarboxamidoadenosine
32
26
hydrochloride hydrate (16, CGS21680) , or A AR agonist 1a were added to the cells
3
along with the IDX adipogenesisꢀinducing medium (Figure 2A). Caffeine, a nonꢀspecific
AR antagonist, was evaluated in parallel. A AR agonist 1a significantly promoted
3
adiponectin production during adipogenesis in hBMꢀMSCs compared to that of the
insulin dexamethasone and isobutylmethylxanthine (IDX) control whereas adenosine, 14,
2
1
1
5, or 16 had no significant effect. Consistent with a previous report in the literature, 1
mM of caffeine inhibited adipogenesis in hMSCs. Regarding lipid accumulation, 1a
increased the number and size of lipid droplets in differentiated adipocytes compared
with that in the IDX control, whereas caffeine decreased them (Figure 2B). Compound 1a
upregulated adiponectin production during adipogenesis in hBMꢀMSCs in a
concentrationꢀdependent manner (Figure 2C).
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Figure 2. Effects of AR signaling modulators on adipogenesis in hBMꢀMSCs. (A)
hBMꢀMSCs were grown under IDX conditions and/or coꢀtreated with adenosine, caffeine,
th
1
4, 15, 16, or 1a. (B) ORO staining was performed to estimate lipid droplets on the 7
th
day in culture. (C) On the 7 day in culture, the supernatant was harvested and ELISA
was performed to measure the levels of adiponectin accumulated in the supernatants over
8 hours. Results are the mean ± SD of three measurements using hBMꢀMSCs from three
4
independent donors (n = 3, three independent experiments). * p ≤ 0.05 and ** p ≤ 0.01.
In preadipocyte studies in the human ARꢀtransfected murine osteoblast precursor
2
2
cell line 7F2, the AR agonists 14 and 15 increased adipocyte differentiation by 20ꢀ30%.
However, in the adipogenesis model of hBMꢀMSCs, both 14 and 15 did not significantly
promote adipogenesis in comparison with that in the control (Figure 1), suggesting that
the AR signaling pathways differ between hMSCs and the murine 7F2 cell line. AR
subtypes show transitional expression profile changes after the induction of adipocyte
3
3
differentiation from preadipocytes. In the human ARꢀtransfected murine 7F2 system, A
1
AR overexpression promotes adipogenesis whereas A AR overexpression suppresses
2
22
it.
Mammalian adipogenesis involves the lineage commitment of MSCs to
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preadipocytes, establishment of the adipogenic lineage, and terminal differentiation into
3
4
functional adipocytes. Each AR subtype may have different roles in adipogenesis
regulation depending on the differentiated stage of MSCs. Therefore, the difference
between the pharmacological effects of AR agonists on hBMꢀMSCs and those on the
human ARꢀtransfected murine osteoblast precursor cell line 7F2 may be partly explained
by different lineage commitment stages for adipogenesis or osteogenesis.
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Independency of A AR signaling on 1aꢀinduced upregulation of adiponectin
3
3
5
production in hBMꢀMSCs. A AR agonists, such as 1a and its 2ꢀchloro derivative 1b
3
have been studied as novel therapeutics to treat rheumatoid arthritis or myocardial
1
7,36
ischemiaꢀreperfusion injury.
We have reported the results of structureꢀactivity
relationship on novel A AR agonists and antagonists, whose pharmacophore was
3
2
7,29
structurally related to 1a (Table 1).
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2
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3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
Table 1. Adiponectinꢀsecreting activity of 1a and related A AR ligands
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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2
3
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5
6
7
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0
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2
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#
#
#
##
K (hA AR) nM
K (hA AR) nM
K (hA AR) nM
Adiponectin (pg/ml)
i
1
i
2A
i
3
Compound
X
Y
R
or % displacement or % displacement or % displacement
2
0 ꢁM
4 ꢁM
at 1 ꢁM
51.2 ± 5.1
222 ± 22
20.2 ± 2.9
193 ± 46
5.4 ± 0.3
9.27 ± 0.83
159 ± 40
23.6 ± 4.2
2490 ± 940
38%
at 1 ꢁM
2910 ± 580
5360 ± 2470
475 ± 144
223 ± 36
57.6 ± 6.9
15.2 ± 2.6
1600 ± 80
122 ± 62
at 1 ꢁM
1.8 ± 0.7
1.4 ± 0.3
0.3 ± 0.1
0.38 ± 0.07
0.42 ± 0.22
3.03 ± 0.23
2.16 ± 0.29
1.17 ± 0.16
4.16 ± 0.5
1.66 ± 0.9
25.8 ± 6.3
1.5 ± 0.4
1
1
1
a
b
c
O
O
S
S
S
S
S
S
S
S
S
S
H
Cl
H
Cl
H
H
H
H
Cl
Cl
Cl
H
CH
CH
CH
CH
3
3
3
3
216 ±12**
603 ± 10**
343 ± 20**
738 ± 59**
242 ± 4**
249 ± 26**
490 ± 16**
633 ± 39**
857 ± 69**
442 ± 5**
268 ± 14**
247 ± 7**
117 ± 20
221 ± 10**
126 ± 14
131 ± 10
118 ± 38
174 ± 5**
242 ± 11**
323 ± 30**
192 ± 19*
196 ± 1**
107 ± 6
1d
2a
2b
A AR agonists
3
CH CH
2
3
Cyclopropyl
CyclopropylꢀCH
Cyclobutyl
3ꢀIꢀBn
2
c
2
2
3
d
a
341 ± 75
3b
3c
3d
3ꢀClꢀBn
2ꢀClꢀBn
3ꢀClꢀBn
18%
A AR antagonists
3
13%
860 ± 210
1600 ± 135
440 ± 110
163 ± 12*
IDX control
Glibenclamide
100 ± 6
1000 ± 193**
838 ± 67**
27,29
# Binding affinities of 1a and related A AR ligands to human A , A , and A AR were previously reported . ## In the IDX medium, 1a and related A AR
3
1
2A
3
3
ligands were included to induce adipogenesis in hBMꢀMSCs. On the 7th day in culture, cell culture supernatants were harvested and ELISA was performed to
measure levels of adiponectin. Results are the mean ± SD of three measurements using hBMꢀMSCs from three independent donors (n = 3, three independent
experiments). * p ≤ 0.05 and ** p ≤ 0.01.
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Figure 3. Evaluation of adiponectinꢀpromoting activity of 1a and related A AR ligands. (A)
3
th
1a, 1b, 1c, or 3a was coꢀtreated with the IDX medium in hBMꢀMSCs. On the 7 day in culture,
cell culture supernatants were harvested and ELISA was performed to measure levels of
adiponectin. (B) The effects of A AR agonists 1a, 1c and 17, and A AR antagonist 18 on
3
3
adiponectin production were evaluated. The pharmacological correlation between A AR (C),
1
A2A AR (D), A AR (E) binding K value at 1 ꢁM and adiponectin levels were analyzed. Values
3
i
represent mean ± SD (n = 3, three independent experiments). * p ≤ 0.05 and ** p ≤ 0.01.
In order to confirm the specific association of the A AR signaling pathway with the
3
regulation of adipogenesis in hBMꢀMSCs, we investigated the effects of both A AR agonists
3
and antagonists on adiponectin production. A AR agonists 1aꢀc and 2aꢀd significantly promoted
3
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adiponectin production in hBMꢀMSCs (Table 1). Notably, A AR antagonists 3aꢀd also
3
increased adiponectin production in the same phenotypic assay. At a concentration of 20 ꢀM,
compound 3a was the most potent promoter of adipogenesis among the tested A AR agonists
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and antagonists (Figure 3A and Table 1). These results suggest that A AR signaling is not
3
associated with adiponectinꢀpromoting activity during adipogenesis in hBMꢀMSCs. To address
this question, we evaluated the effects of the A AR ligands, (1S,2R,3S,4R,5S)ꢀ4ꢀ[6ꢀ[[(3ꢀ
3
chlorophenyl)methyl]amino]ꢀ2ꢀ[2ꢀ(3,4ꢀdifluorophenyl)ethynyl]ꢀ9Hꢀpurinꢀ9ꢀyl]ꢀ2,3ꢀdihydroxyꢀ
3
7
Nꢀmethylbicyclo[3.1.0]hexaneꢀ1ꢀcarboxamide (17, MRS5698) and Nꢀ[2ꢀ(2ꢀfuranyl)ꢀ8ꢀpropylꢀ
Hꢀpyrazolo[4,3ꢀe][1,2,4]triazolo[1,5ꢀc]pyrimidinꢀ5ꢀyl]ꢀN'ꢀ(4ꢀmethoxy phenyl)urea 18
MRE3008F20) , which are chemically different from 1a, on adipogenesis in hBMꢀMSCs
Figure 3B). Unlike 1a and related A AR ligands, the A AR agonist 17 and A AR antagonist
8
( ,
3
8
(
3
3
3
18 had no effect on adipogenesis in hBMꢀMSCs. Next, we determined whether a
pharmacological correlation existed between the adiponectinꢀpromoting activity and A AR
3
binding affinity values of 1a and related A AR ligands. The correlation coefficient between the
3
adiponectinꢀpromoting activity at 20 ꢀM and K values of 1aꢀrelated A AR ligands was 0.04 (p
i
3
=
0.54), which indicates no statistically significant association between the two variables (Figure
3C). When comparing the A or A AR binding affinity of these compounds, no significant
1
2A
correlation was observed (Figures 3D and 3E). In this regard, the lack of structureꢀactivity
relationship between AR binding affinity and the adiponectinꢀpromoting activity of 1a and its
related A AR ligands suggests that other molecular targets are associated with the effect of 1a
3
on adipogenesis in hBMꢀMSCs.
Specific binding of 1a and related A AR ligands to PPARγ and PPARδ. Nuclear
3
receptors (NRs) like PPARα, PPARγ, PPARδ, liver X receptor (LXR)α, LXRβ, and
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glucocorticoid receptor (GR) play roles in mammalian adipogenesis. In order to identify
molecular targets directly associated with the phenotypic activity of 1a and related A AR
3
ligands in promoting adiponectin production during adipogenesis of hBMꢀMSCs, we
investigated the effects on NR binding or coactivation (Figure 4A).
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11
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14
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17
18
19
20
21
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59
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Figure 4. TRꢀFRET NRs binding or coactivator assays with 1a and related A AR ligands.
3
(A) TRꢀFRET competitive binding assays with 1a and 1b at 4 ꢁM as ligands of PPARα, PPARγ,
PPARδ, and GR were performed. In TRꢀFRET LXRα and LXRβ coactivator assays, 1a and 1b
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at 4 ꢁM were evaluated to determine whether transactivation occurred. The positive controls
were 19 for PPARα, 21 for PPARγ, 22 for PPARδ, dexamethasone for GR, and 20 for LXRα
and LXRβ. DMSO in buffer was used as a blank control. (B) The kinase activity was evaluated
by measuring the γꢀ PꢀATP incorporation to CDK complexes. The inhibitory effects of 1a and
1c on the phosphorylation of CDK5/p25 and CDK5/p35 were tested at each Km ATP
concentration. DMSO was included in each negative control. Values were expressed in terms of
percentage compared to each positive control. The TRꢀFRETꢀbased competitive binding
activities of troglitazone, 1a, 1b, 1c, and 3a to PPARα (C), PPARγ (D), and PPARδ (E) were
evaluated. Pearson’s correlation coefficients (r ) between the binding affinities to PPARγ at 20
M (F) or 4 ꢀM (G) and relative adiponectin levels in the cell culture supernatants were
calculated with RStudio software. Correlation coefficients between the binding affinities to
PPARδ at 2 ꢀM (H) or 0.4 ꢀM (I) and relative adiponectin levels were calculated in the same
way. Results are the mean ± SD of three independent experiments. * p ≤ 0.05 and ** p ≤ 0.01.
3
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
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0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
2
ꢀ
®
Compounds 1a and 1b were evaluated in the preliminary target identification study
because they have been clinically investigated for various diseases such as cancers and
1
7,36
rheumatoid arthritis.
In the timeꢀresolved fluorescence resonance energy transfer (TRꢀ
FRET)ꢀbased receptorꢀbinding assay, 4 ꢀM of 1a and 1b competitively replaced the binding of
the labeled PPARδ ligand by 26% and 75%, respectively (Figure 4A). Compound 1b also
replaced the binding of the labeled PPARγ ligand by 59%. Both compounds had no significant
effects on PPARα, LXRα/β, or GR, compared to their positive controls, 2ꢀ(4ꢀ(2ꢀ(1ꢀ
4
0
cyclohexanebutyl)ꢀ3ꢀcyclohexylureidoethyl)phenylthio)ꢀ2ꢀmethylpropionic acid (19, GW7647) ,
Nꢀ(2,2,2ꢀtrifluoroethyl)ꢀNꢀ[4ꢀ[2,2,2ꢀtrifluoroꢀ1ꢀhydroxyꢀ1ꢀ
4
1
(trifluoromethyl)ethyl]phenyl]benzenesulfonamide (20, T0901317)
and dexamethasone,
respectively (Figure 4A). In addition, cyclinꢀdependent kinase 5 (CDK5) was recently reported
4
2
to regulate adipogenesis by affecting PPARγ phosphorylation. We also evaluated the effect of
1
a and 1b on CDK5 activity because purine nucleoside structure of these compounds may affect
kinase activity. Neither 1a nor 1b affected CDK activity at concentrations up to 20 ꢀM (Figure
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B). Therefore, both PPARγ and PPARδ may contribute to the ability of 1a and related A AR
3
ligands to promote adiponectin production, which can be used as the measure of insulin
1
2
sensitivity. Compound 1a and related A AR ligands can bind to PPARs at higher
3
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17
18
19
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43
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47
48
49
50
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53
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concentrations. Next, we analyzed the concentration–response relationship of 1a and related A3
AR ligands in terms of their binding activity to PPARα, PPARγ, and PPARδ (Figures 4Cꢀ4E,
Table 2). In the PPARα binding assay, 1a, 1b, 1c, and 3a did not replace over 50% of the
binding activity of the labeled PPARα ligand up to 20 ꢀM, which was much less potent than 19
(Figure 4C, Table 2).
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1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
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3
3
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Table 2. TRꢀFRET PPAR binding activity of 1a and related A AR
3
#
##
###
PPARα
K (PPARγ )
K (PPARδ
)
i
i
Compound % displacement
or % displacement
at 20 ꢁM
or % displacement
at 20 ꢁM
at 20 ꢁM
1
1b
1
1d
2a
a
16.0 %
46.5%
13.5%
37.8 %
29.3%
37.6%
16.4%
11.7%
35.6%
4.50%
19.2%
18.5%
22.2%
2.18 ± 0.32 nM
46.9%
4.60 ± 0.26 nM
37.1%
48.3%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.43 ± 0.03 nM
2.54 ± 0.15 nM
0.16 ± 0.09 nM
3.15 ± 0.47 nM
2.98 ± 0.39 nM
2.37 ± 0.66 nM
2.61 ± 0.72 nM
0.00483 ± 0.00023 nM
0.0102 ± 0.0091 nM
0.62 ± 0.14 nM
c
A AR
agonists
3
2b
49.1%
2
c
1.83 ± 0.25 nM
0.17 ± 0.06 nM
3.42 ± 0.47 nM
40.8%
2d
3a
3b
3c
3d
A AR
3
antagonists
19.2%
18.5%
49.1%
##
#
The K of the positive control 19 was 0.0541 ± 0.0089 in the parallel study. The K of the
i
i
positive control 21 in the parallel experiment was 0.0497 ± 0.0115. Troglitazone and
#
##
glibenclamide showed 96.2% and 77.5% binding to PPARγ at 10 ꢀM respectively. The K of
i
the positive control 22 was 0.0480 ± 0.0014 in the parallel experiment. Troglitazone and
glibenclamide bound to PPARδ 23.4% and 21.5% at 10 ꢀM respectively, compared to 22.
Values represent the mean expression ± SD (three independent experiments). ND: not
determined. * p ≤ 0.05 and ** p ≤ 0.01.
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In the PPARγ analysis, 1b and 3a displayed significant competitive binding activity in a
concentrationꢀdependent manner (Figure 4D, Table 2), while 1c showed very weak binding
activity. The K values of 1b and 3a were 2.18 and 3.42, respectively, but not as potent as the
i
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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PPARγ
agonist,
Nꢀ(2ꢀbenzoylphenyl)ꢀOꢀ[2ꢀ(methylꢀ2ꢀpyridinylamino)ethyl]ꢀLꢀtyrosine
4
3
13
hydrochloride (21, GW1929) (Figure 4D). Consistent with the literature, glibenclamide, a
sulfonylurea antidiabetic drug, showed PPARγ binding activity in the TRꢀFRETꢀbased assay
(Figure 4D). Compared to PPARα and PPARγ binding activities, 1a and most related A AR
3
ligands competitively displaced the labeled PPARδ ligand in a concentrationꢀdependent manner
Figure 4E, Table 2). Importantly, compounds 3a and 3b exhibited maximal PPARδ binding
activity, which was compared to that of a PPARδ agonist, 2ꢀ[2ꢀmethylꢀ4ꢀ[[[4ꢀmethylꢀ2ꢀ[4ꢀ
(
4
4
(trifluoromethyl)phenyl]ꢀ5ꢀthiazolyl]methyl]thio]phenoxy]acetic acid (22, GW501516) (Figure
4E). The K values for PPARδ binding of compounds 3a and 3b, which were identified as A AR
i
3
antagonists, were 4.83 and 10.2 nM, respectively (Figure 4E, Table 2). Notably, a significant
correlation was observed between the level of PPARγ ligand replacement at 20 and 4 ꢀM and the
adiponectinꢀpromoting activity in hBMꢀMSCs (Figures 4F and 4G). Regarding the correlation
coefficients between PPARδ binding affinity and adiponectinꢀpromoting activity, significant
associations were observed with 1a and related A AR ligands at both 2 and 0.4 ꢀM
3
concentrations (Figures 4H and 4I). Therefore, in affecting adipogenesis of hBMꢀMSCs, the
effects of 1a and related A AR ligands on PPARδ occurred at lower concentrations than those
3
for PPARγ. These results suggest that 1a and related A AR ligands promote adiponectin
3
production in hBMꢀMSCs by modulating the activity of PPARγ and PPARδ, not A AR.
3
Structureꢀactivity relationships for PPAR activity were also analyzed. Among A AR agonists
3
tested, 2ꢀCl substitution (1b and 1d) showed better binding activities at PPARγ and PPARδ than
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the 2ꢀH substitution (1a and 1c), while more bulky alky or cycloalky substituent at the 5'ꢀ
uronamides increased the binding activity at PPARγ. For PPARδ binding activity, compound 1d
was the most potent inhibitor among A AR agonists. Extension of the 5'ꢀuronamide group with
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alkyl or cycloalkyl substituents did not reduce the inhibitor potency at PPARδ compared to 1c.
For A AR antagonists 3aꢀd, they generally displayed poor binding activity at PPARα and
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PPARγ, but showed very strong binding activity at the PPARδ, among which N ꢀ3ꢀiodobenzyl
derivative 3a was the most potent. All tested A AR agonists and antagonists were devoid of
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binding activity at PPARα. Structurally, this polypharmacophore encompasses adenosineꢀ5'ꢀ
uronamides and its 4'ꢀtruncated derivatives that also have bulky hydrophobic substitutions at the
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N position, such as halobenzyl.
Polypharmacophore of 1a to bind A AR, PPARγ and PPARδ. Next, we investigated
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whether 1a and related A AR ligands were PPAR agonists or antagonists. To determine a
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functional outcome for the effect of 1a, 1c, and 3a on PPARγ, we performed a luciferaseꢀreporter
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PPARγ transactivation assay. We observed that 10 ꢀM of 1a, 1c, and 3a increased PPARγ
transactivation by 29.9%, 46.9%, and 54.3%, respectively (Figure 5A).
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Figure 5. PPARγ and PPARδ transactivation activity of 1a, 1c, and 3a. (A) For PPARγ
transactivation assay, CVꢀ1 cells were transiently cotransfected with the PPARγ expression
vector and the PPARγ responsive elements (PPRE)ꢀluciferase reporter, and then treated with
troglitazone, 1a, 1c, or 3a. (B) TRꢀFRET PPARδ coactivator assay was performed using
fluoresceinꢀC33 coactivator peptide. 22 and 23 were used as PPARδ antagonist and PPARδ
agonist, respectively. Results are the mean ± SD of three measurements (n = 3, three independent
experiments). * p ≤ 0.05 and ** p ≤ 0.01.
Compound 3a (30 ꢀM) did not achieve maximal PPARγ transactivation activity
compared to troglitazone, a PPARγ full agonist; this result was similar to that of a PPARγ
binding assay (Figure 4D). PPARγ partial agonists can upregulate adiponectin production and
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also improve glucose homeostasis. The adiponectinꢀpromoting activity of 1a and related A AR
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ligands was partially contributed by PPARγ partial agonism. To elucidate a functional
consequence for PPARδ, we performed a TRꢀFRET PPARδ coactivator assay, which measures
the level of interaction between the PPARδ ligandꢀbinding domain and a fluoresceinꢀlabeled
coactivator peptide. A selective PPARδ agonist 22 increased the signal associated with the
activated PPARδ in a concentrationꢀdependent manner (Figure 5B). Like a PPARδ antagonist 3ꢀ
[[[2ꢀmethoxyꢀ4ꢀ(phenylamino)phenyl]amino]sulfonyl]ꢀ2ꢀthiophenecarboxylic acid methyl ester
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23, GSK0660) , 1a, 1c, and 3a had no effect on the recruitment of the labeled coactivator
peptide, suggesting that 1a and related A AR ligands are PPARδ antagonists (Figure 5B).
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Figure 6. Validation of 1a and related A AR ligands as PPARδ antagonists. (A) The effects
of 1a, 1c, 3a, and 23 (1 ꢀM) on the 22 (0.03 ꢀM)ꢀinduced interaction between fluoresceinꢀC33
coactivator peptides and PPARδ LBD in a TRꢀFRET PPARδ coactivator assay were evaluated.
B) In a TRꢀFRET PPARδ corepressor assay with SMRTꢀID2 peptides, the activities of 23, 24,
and 3a (1 ꢀM) were assessed in the condition that 22 (0.1 ꢀM) existed or not. (C) 23, 24, 3a, and
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c were evaluated in a TRꢀFRET PPARδ corepressor assay with SMRTꢀID2 peptides at various
concentration. For functional validation assay for PPARδ antagonism, hBMꢀMSCs were
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differentiated in the IDX condition, and coꢀtreated with 23, 24, or 3a in the medium. On the 3
day in culture, total RNA was extracted and QꢀRTꢀPCR was performed for ANGPTL4 (D) and
PDK4 (E). GAPDH was used as an internal control for QꢀRTꢀPCR standardization. Values
represent mean ± SD (n = 3, three independent experiments). * p ≤ 0.05 and ** p ≤ 0.01.
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To confirm the PPARδ antagonism, we evaluated whether 1a, 1c, and 3a competitively
inhibited the 22ꢀinduced coactivator recruitment to PPARδ (Figure 6A). Compound 3a (1 µM)
significantly decreased the effect of 22 on PPARδ coactivator recruitment by 57%. At 1 ꢀM
concentration, both 1a and 1c tended to inhibit the effect of 22 by 14% and 22%, respectively,
although the inhibition was not statistically significant. PPARδ agonists interfere with the
interaction between PPARδ and its corepressors such as silencing mediator of retinoid and
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thyroid hormone receptors (SMRT). Therefore, we analyzed whether compound 3a affected the
interaction between PPARδ and the labeled corepressor peptide, which was derived from the
interaction domain 2 (ID2) of SMRT (Figure 6B). As expected, 22 decreased the interaction
between PPARδ and the corepressor peptide (Figure 6B). The PPARδ antagonists, 23 and 4ꢀ
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chloroꢀNꢀ[2ꢀ[[5ꢀ(trifluoromethyl)ꢀ2ꢀpyridinyl]sulfonyl]ethyl]benzamide
(24,
GSK3787)
significantly promoted the recruitment of the corepressor peptide by PPARδ, an effect that was
antagonized by 22. Importantly, similar to the effects of PPARδ antagonists 23 and 24, the
promotion of the interaction between PPARδ and corepressor peptide by 3a, an A AR
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antagonist, was concentrationꢀdependent (Figure 6C). The A AR agonist 1c also enhanced the
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interaction of PPARδ with the corepressor peptide (Figure 6C). In mammalian cells, PPARδ
agonists increase the gene transcription of angiopoietinꢀlike 4 (ANGPLT4) and pyruvate
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dehydrogenase kinase 4 (PDK4).
To confirm that compound 3a is a PPARδ antagonist, we
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measured ANGPLT4 and PDK4 mRNA levels during adipogenesis in hBMꢀMSCs (Figures 6D
and 6E). Consistent with previous reports, the PPARδ agonist 22 significantly increased
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ANGPTL4 and PDK4 gene transcription, as measured on the 3 day after the induction of
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adipogenesis in hBMꢀMSCs (Figures 6D and 6E). Similar to the other PPARδ antagonists, 23
and 24, compound 3a did not affect ANGPTL4 and PDK4 mRNA levels in hBMꢀMSCs.
Recently, it has been reported that the overexpression or transcriptional activation of PPARδ
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inhibits PPARγ activity, suggesting the regulatory role of PPARδ in PPARγ functions.
Therefore, the adiponectinꢀpromoting activity of 1a and related A AR ligands during
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adipogenesis in hBMꢀMSCs is associated with both PPARγ partial agonism and PPARδ
antagonism.
Effects of 1a and related A AR ligands on insulin sensitivity in streptozotocin
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STZ)ꢀinduced diabetic mice. Antiꢀdiabetic drugs like PPARγ agonists and sulfonylureas
increase adiponectin production during adipogenesis in hBMꢀMSCs, which correlates with
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improved insulin sensitivity.
Compound 1a and related A3 AR ligands have a
polypharmacophore to bind A AR, PPARγ and PPARδ. However, whether 1a and related A3
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AR ligands promote insulin sensitivity has not been experimentally tested under in vivo
conditions. Therefore, we evaluated the insulinꢀsensitizing effect of these compounds in the
STZꢀinduced diabetes model in C57BL/6J mice (Figure 7A).
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Figure 7. Effects of 1a, 1c, and 3a on STZꢀinduced diabetic mice. STZꢀinduced diabetic male
C57BL/6J mice were orally administered potential antiꢀdiabetic drugs at 20 mg/ kg for 5 days.
Eight mice were used in each group. Glibenclamide was used as a positive control. Serum
glucose (A), triglyceride (B) and lactate (C) levels were measured. Results are the mean ± SD of
three independent experiments. Statistical analyses were performed using oneꢀway ANOVA
followed by Tukey’s postꢀtest. (D) Serum glucose concentrations were measured just before drug
administration (0 hour), and at 1 and 4 hours after drug administration. Each symbol represents
the mean ± SD of the difference in the time spent after the drugꢀ or vehicleꢀtreatments (8 animals
per each group). * p ≤ 0.05 and ** p ≤ 0.01, significantly different from the vehicleꢀtreated STZꢀ
induced diabetic mouse group
Compounds 1c and 3a significantly decreased serum glucose levels in the STZꢀinduced
diabetic mice, suggesting insulinꢀsensitizing activity. Compound 1a tended to decrease serum
glucose levels, although the effect was not statistically significant. These compounds also
downregulated serum triglyceride levels in the mouse model (Figure 7B). Compound 3a tended
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to decrease the serum lactate levels in this model, but the effect was not significant (Figure 7C).
The glucoseꢀlowering effect of 3a in diabetic mice was doseꢀdependent (Figure 7D). Thus, a
compound with a polypharmacological profile of an A AR modulator, a PPARγ partial agonist,
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and a PPARδ antagonist has insulinꢀsensitizing activity.
It is known that partial PPARγ agonists improve pathologic parameters in various human
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metabolic diseases.
The insulinꢀsensitizing effects of PPARδ antagonists are still
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controversial. PPARδ itself may not affect insulin sensitivity but can competitively inhibit the
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transactivation of cellular PPARγ. Recent reports showed that the overexpression or
transcriptional activation of PPARδ inhibits PPARγ activity, suggesting a regulatory role for
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PPARδ in PPARγ functions. Therefore, the PPARδ antagonist activity of 1a and related A AR
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ligands may improve insulin sensitivity by blocking the inhibitory effect of PPARδ on PPARγ
transactivation. Notably, the adiponectinꢀpromoting activity of 1a and its related A AR ligands
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correlated more significantly with their binding affinity to PPARδ than that to PPARγ (Figure 4).
The polypharmacological outcome of 1a and related A AR ligands for adiponectin production
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during adipogenesis in hBMꢀMSCs is primarily dependent on the binding activity to the nuclear
transcription factors PPARγ and PPARδ. Regardless of being A AR agonists or antagonists, 1a
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and the related A AR ligand 3a showed significant glucoseꢀlowering effects in STZꢀinduced
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diabetic C57BL6/J mice. The antiꢀdiabetic potential of 1a and related A AR ligands may be due
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to their effect on both PPARγ and PPARδ.
Implications of polypharmacology in the pleiotropic activities of 1a on cancer and
inflammatory diseases. The polypharmacology profile of 1a and its related A AR ligands is
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consistent with their pleiotropic activities in clinical trials to treat cancer and autoimmune
inflammatory diseases. The effects of 1a and 1b in their clinical development, have been
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evaluated in many human diseases such as cancer, psoriasis, rheumatoid arthritis, and dry eye
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syndrome.
However, the A ARꢀmediated pharmacology alone may not fully explain these
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diverse clinical activities of 1a and 1b against various human diseases. Compounds 1a and 1b
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have been studied for their antiꢀcancer effects on various human cancerous tumors such as
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melanoma, lymphoma, colon carcinoma and hepatocellular carcinoma. In fact, A AR has been
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reported to be upregulated in different tumor cell types, generating interest in A AR as an antiꢀ
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cancer drug target. Compound 1a inhibits colon carcinoma growth in syngeneic and xenograft
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murine models. Compound 1b has been shown to suppress the growth of hepatocellular
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carcinoma (HCC) with upregulated A AR expression. However, the results of studies with
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various cancer cell lines indicated that 1a and 1b have A ARꢀindependent antiꢀcancer
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mechanisms. Interaction of 1a with estrogen receptor (ER) α in a breast cancer cell line was
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reported as a potential A ARꢀindependent mechanism for its antiꢀcancer activity.
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The polypharmacologlical profile of 1a demonstrated in this study suggests the existence
of an alternative A ARꢀindependent antiꢀcancer mechanism for 1a and 1b. Interaction with both
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PPARγ and PPARδ can account for the antiꢀcancer activity of 1a and 1b. Recently, it was
reported that the ligandꢀinduced PPARγ activation could lead to apoptosis of cancer cells,
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suggesting that PPARγ agonists have antiꢀcancer potential in some cancer subtypes. In this
regard, the PPARγ partial agonist activity of 1a and 1b may contribute to their diverse antiꢀ
cancer activity. PPARδ is upregulated in colorectal cancers and associated with the direct
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promotion of colorectal tumorigenesis. In addition, a PPARδ antagonist inhibits the cell growth
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of human carcinoma lines whereas the PPARδ agonist 22 promotes the tumorigenesis of some
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cancer cell lines in animal models. The tumorꢀpromoting effects of 22 were significantly
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attenuated in intestinal PPARδꢀdeleted Apc(+/ꢀ) mice. The PPARδ antagonist activity of 1a and
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b may be beneficial to prevent tumorigenesis in human cancers associated with PPARδ
dysregulation. Therefore, the antiꢀcancer activity of 1a and 1b must be explained with their
polypharmacological characteristics of an A AR agonist, a PPARγ partial agonist, and a PPARδ
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antagonist. The molecular characterization of human cancers in the polypharmacological context
of A AR, PPARγ, and PPARδ may provide therapeutic benefits in the future clinical
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development of 1a and related A AR ligands as antiꢀcancer drugs.
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In addition to their antiꢀcancer potential, 1a and 1b have been clinically evaluated for the
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treatment of human inflammatory diseases such as rheumatoid arthritis and psoriasis.
The
polypharmacology profile of 1a and related A AR ligands provides a good mechanistic
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explanation for their antiꢀinflammatory activity. PPARγ agonists inhibit proꢀinflammatory
cytokine production from monocytes or macrophages, whose PPARγ expression is correlated
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with the disease severity in rheumatoid arthritis. In a randomized clinical trial, the PPARγ
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agonist pioglitazone mildly improved the pathological symptoms of rheumatoid arthritis.
Therefore, the antiꢀinflammatory activity of 1a and related A AR ligands can be explained by
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both A ARꢀ and PPARγꢀmediated mechanisms in various clinical conditions. Recently,
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inhibition of PPARδ by selective antagonists such as 23 and 24 was reported to improve
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inflammatory psoriatic conditions in animal models. Because 1a and related A AR ligands
3
have PPARδ antagonist activity, it is necessary to address the possible involvement of PPARδꢀ
mediated pathways in their in vivo antiꢀinflammatory outcomes under various clinical conditions.
■
CONCLUSION
This study demonstrated that the antiꢀdiabetic potential of 1a and related A AR ligands
3
is associated with previously undetected interactions, i.e. both PPARγ partial agonism and
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PPARδ antagonism. In order to develop these compounds to treat human metabolic diseases,
further studies will be necessary because clinical outcomes associated with efficacy or toxicity
have not yet been clearly addressed depending on their A AR agonist or A AR antagonist
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activity. In addition, when 1a and related A AR ligands are clinically developed as A AR
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modulators to treat A ARꢀassociated clinical conditions, the adverse effects or clinical benefits
3
associated with PPARγ partial agonism and PPARδ antagonism should be considered. So far, the
molecular targets of polypharmacology case studies have been limited to a structurally similar
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protein family, such as an inhibitor against multiple tyrosine kinases. In this study, 1a provides
a good case study for a single drug molecule simultaneously targeting different protein families:
the GPCR and NR families. Defining polypharmacology in the context of the accurate prediction
of clinical efficacy and safety is one of the technical goals of systems pharmacology and future
medicinal chemistry. To accomplish this, more evidence on a single drug molecule
simultaneously targeting multiple targets that belong to different protein families must be
presented. In this regard, the polypharmacological feature of 1a and related A AR ligands may
3
provide therapeutic insight into their multiꢀpotent efficacy against various human diseases such
as cancers, rheumatoid arthritis, psoriasis, and dry eye syndrome.
■ EXPERIMENTAL SECTION
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General Methods. H NMR (400 MHz) and C NMR (100 MHz) spectra were measured in
CDCl , CD OD or DMSOꢀd and chemical shifts are reported as parts per million (δ) relative to
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the solvent peak. Coupling constants (J) are reported in hertz (Hz). Melting points are
uncorrected. Elemental analyses (C, H, and N) were used to determine the purity of all
synthesized compounds, and the results were within ± 0.4% of the calculated values, confirming
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