Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Peroxisome proliferator-activated receptor (PPAR)
γ, one of the subtypes of PPARs, is a ligand-dependent
transcription factor of the nuclear hormone receptor
superfamily[1]. PPARγ is mainly present in the adipose tissue, skeletal muscle,
and liver[2]. Two other family members include
PPARα, which is present in the liver, kidney and heart, and the
ubiquitously-expressed PPARδ[3]. Upon ligand binding, PPARs release
relevant corepressors and form heterodimers with retinoid X
receptors (RXRs)[4]. The heterodimers bind to peroxisome
proliferator response elements (PPREs) and recruit
co-activa-tors to initiate the transcription of target
genes[5,6].
Type 2 diabetes mellitus is a heterogeneous disease
resulting from a dynamic interaction between defects in
insulin secretion and insulin action[7]. In animal models and
humans affected by this disorder, treatment with
thiazo-lidinediones decreases elevated plasma glucose, triglyceride,
and insulin concentrations[8,9]. Their actions also lead to the
stimulation of adipogenesis and the recovery of insulin
sensitivity in the target tissues[9]. The antidiabetic effects of
thiazolidinediones are directly mediated through binding to
PPARγ, thereby resulting in an active conformation of the
receptor[10].
While the clinical benefits of PPARγ agonists in treating
type 2 diabetes have been clearly demonstrated, the current
generation of glitazones is associated with undesired
side-effects, such as weight gain and
edema[11]. Thus, there has been significant interest in the design of novel
PPARγ-modulating drugs that retain efficacious insulin sensitizing
pro-perties while minimizing potential adverse effects, especially
for long-term use.
SH00012671 is a core structure discovered in a
high-throughput screening campaign directed towards
PPARγ[12]. It displayed a relatively higher binding affinity to the receptor,
but was almost inactive in cell-based assays. In this paper,
we describe the design, synthesis, and biological evaluation
of a series of analogues to SH00012671 with the aim of
identifying novel PPARγ agonists.
Materials and methods
Reagents Edetic acid, insulin, and dexamethasone were
purchased from Sigma-Aldrich (St Louis, MO, USA).
BRL49653 (rosiglitazone) was bought from Cayman
Chemical Co (Ann Arbor, MI, USA). [3H]BRL49653 (53 Ci/mmol)
was obtained from American Radiolabeled Chemicals (St
Louis, MO, USA), FlashPlate and flat-bottom Isoplate were
from PerkinElmer (Boston, MA, USA), and the
streptavidin-coated microbeads were from Amersham Biosciences UK
(Buckinghamshire, England). The plasmids of human nuclear
receptors used in this study were from Dr Xu SHEN of
Shanghai Institute of Materia Medica, Chinese
Academy of Sciences (Shanghai, China) and Dr Sai-juan CHEN of Shanghai
Institute of Hematology (Shanghai, China). Full-length
PPARγ and RXRα were produced with a baculovirus
expression system using fall armyworm (Spodoptera
frugiperda) immature ovarian (IPLB-SF-21-AE)
cells[13]. The PPRE reporter vector was purchased from Panomics (Redwood City,
CA, USA), the Steady-Glo luciferase assay system was from
Promega (Madison, WI, USA), the FuGene 6 transfection
reagent was from Roche Diagnostics (Indianapolis, IN, USA),
and the triglyceride (TG) detection kit was from Ningbo
Asia-Pacific Biotechnology Co (Zhejiang, China).
Chemistry Figure 1 depicts the syntheses of the
rho-danine derivatives designed based on SH00012671.
Esterification of aromatic acid 1 by methanol and then hydrazinolysis
of the resulting methyl ester provided the substituted
benzoyl hydrazine 2. The reaction of 2 with carbon disulphide in
ethanol in the presence of potassium hydroxide for 2 h resulted in the formation of potassium
β-acyldithiocarbazinate 3, which was used without further purification. According
to Tadashi and Masaki[14] when 3 was added to a solution of
chloroacetic acid and saturated sodium carbonate, stirred at
room temperature for 30 min, and acidified by aquous HCl,
3-benzoylamino rhodanine 4 would be separated out and then
solidify. However, we found that when acidifying with
aquous HCl, the intermediate (2-benzoyl-thiocarbazyl)
mercaptoacetic acid, instead of 3-benzoyl-amino rhodanine 4,
was separated out as evidenced by 1H-NMR (Nuclear
magnetic resonance) and ESI (Electrospray ioniza-tion). This
intermediate was thus refluxed in dioxane for 1 h to give
3-benzoylaminorhodanine 4, which could also be obtained by
dissolving 3-amino rhodanine 5 in pyridine with benzoyl
chloride. The final compounds 7_19 were obtained through
the condensation of 4 with benzoaldehyde 6 by refluxing in
glacial acetic acid in the presence of sodium acetate.
Next, we changed the substitutions on
R4 or R5 while R1 or
R2 was fixed with chloro (Cl), Br (bromo), or H (hydrogen;
Figure 1). We used p-hydroxy benzaldehyde or
m-hydroxy benzaldehyde 43 as the raw material. It was alkylated with
different halogenated aralkyl 44 of which the chain length
was changed from 1 to 3. Compounds 20_37 were obtained
through the condensation of 4 with benzoaldehyde 45 by
refluxing in glacial acetic acid in the presence of sodium
acetate. Compounds 38_42 were made by substituting thio
on the rhodanine with oxygen, in which the former rhodanine
was refluxed with excessive chloroacetic acid in ethanol and
water (Figure 1).
Receptor-binding assay Biotinylated PPRE (2 µL from a
stock solution of 10 g/L) was mixed with the assay buffer (10
mL) containing fish sperm DNA (Shanghai Sangon
Biotechnology Co, Shanghai, China; 10 µL from a 10 g/L stock
solution) and 4 mg streptavidin-coated microbeads in a
conical polypropylene centrifuge tube (Corning, Corning, NY,
USA) and incubated overnight at 4 °C. The mixture was
centrifuged for 10 min at 1 500×g. The supernatant was then
removed and washed 3 times with 10 mL of the assay buffer.
The reaction solution 10 mL containing 700 µg human
PPARγ extract protein (70 mg/L), 47 µg human
RXRα extract protein (4.7 mg/L), 10 nmol/L
[3H]BRL49653, and various
concentrations of BRL49653 or test compounds were distributed to
each well of the Isoplate (100 µL/well) and incubated at 4°C
for 4 h before counting by the MicroBeta counte (PerkinElmer,
USA).
Cotransfection assay The CV-1 cells
(1×106 per well) were inoculated into 6 cm culture plates and incubated in 5%
CO2/air at 37 °C for 6 h. Dulbecco's modified Eagle's medium
(DMEM; Gibco, Grand Island, NY, USA) containing 10%
fetal bovine serum (FBS) and 10 mL/L
penicillin-streptomycin (5 000 IU/mL and 5 000 mg/mL, respectively; Gibco, USA)
was used as the culture medium. The cells were transiently
transfected with PPRE, PPARγ, and RXRα expression
vectors using FuGene 6. Sixteen hours after transfection, the
cells were inoculated into 96-well plates, and following 2 h
incubation in 5% CO2/air at 37°C, the test compounds were
added and the cells were further incubated for 48 h.
Lucifer-ase assay substrate (Promega, USA) was introduced to each
well and the intensity of emitted luminescence was
determined using EnVision (PerkinElmer, USA).
PPARγ trans- criptional activity was expressed as the relative luminescence
intensity to that of the control. For antagonist effects, the
test compounds were added to the cells 30 min before the
addition of BRL49653 (1 µmol/L) and incubated for 24 h.
Luciferase activity was determined as above.
Adipogenesis assay 3T3-L1 cells (20 000 cells/well) were
planted onto 24-well culture plates and maintained for 2 d
after reaching confluence. The culture medium (DMEM,
Gibco, USA) was then changed with the differentiation
medium (DMEM containing 10% FBS, 1 µmol/L dexamethasone,
and 1 µg/mL insulin). The cells were treated with BRL49653
(0.5 µmol/L) or the test compounds (5 µmol/L) for 2 d.
Following the change of the medium containing the compounds,
the cells were cultured for an additional 3 d. The
differentiation medium was replaced by the adipocyte growth medium
(DMEM supplemented with 10% FBS and 1 µg/mL insulin)
and incubated for a further 2 d. The cells were washed with
phosphate-buffered saline and lysed by 0.1% Nonidet P-40
(Sigma-Aldrich, USA). The supernatant of the preparation
was examined by the TG detection kit at a wavelength of 500
nm on VERSAmax (Molecular Devices, Sunnyvale, CA,
USA).
Data analysis Data were analyzed using GraphPad Prism
software (GraphPad, San Diego, CA, USA). Non-linear
regression analyses were performed to generate dose_response
curves. Ki values were calculated from the half maximal
inhibitory concentration (IC50)
using the equation of Cheng and
Prusoff[15]:
Results
A group of 16 analogues to SH00012671 were
synthesized initially (Figure 1) with various substitutions at
R1_R5, of which only compounds 13, 15, and 17 showed
submicromolar binding affinities to PPARγ, but not as
potent as the initial hit (Table 1). However, they displayed
some agonist activities with less than 50% efficacies when
compared to rosiglitazone in the cotransfection assay (see
Table 4 below). We next synthesized another group of 18
rhodanine derivatives by making small modifications on the
R1 or R2 (substitution of H with Cl or Br in
meta- or para-site) and with different chain lengths. In this case (Table 2),
significant improvement in PPARγ binding was observed among
one-half of these compounds (21, 23, 25, 27, 29, 31, 33, 35,
and 37), to the extent that some of them displayed
Ki values similar to that of rosiglitazone (21, 27, 31, and 37). In the
cotransfection assay, compounds 21, 25, 31, and
37 exhibited similar half maximal effective concentration
(EC50) values compared to rosiglitazone with lower efficacies
(23%_69%; see Table 4 below). When tested in the antagonist
mode, compounds 27, 31, 29, and 37, with good binding affinities and modest transactivation efficacies, were unable
to suppress luciferase activity induced by rosiglitazone (1 µmol/L).
To reduce intrinsic cytotoxicities associated with this
class of molecules, the atom thio on the rhodanine was
changed to oxygen (Figure 1). This alteration led to a
decrease in receptor-binding affinities (Table 3) while the
modified analogues maintained efficacies in the cotransfection
assay, with the exception of compound 42 (42
vs 37; Table 4).
Representative compounds from each of the above 3
groups were evaluated in the pre-adipocyte differentiation
assay. All of the 8 compounds tested were able to induce
adipogenesis in 3T3-L1 cells with compound 31 being the
best inducer relative to BRL49653, consistent with its
performance in both the PPARγ-binding and cotransfection
assays (Figure 2; Tables 2, 4).
Discussion
In a previous study[12], we reported the discovery of 12
hit compounds with selective PPARγ binding properties, of
which 2 belong to the thiazodinedione class, including
SH00012671 with a relatively lower
IC50 value (0.2 µmol/L). When tested for its effects on cells, it acted either as a poor
activator in the cotransfection assay (Table 4) or was
inactive in stimulating pre-adipocyte (3T3-L1) differentiation. We
thus started some limited chemistry efforts by modifying the
rhodanine core structure to better understand the molecular
basis of this discrepancy. Three compounds synthesized
initially (4i, 4ii, and 4iii) did not bind to
PPARγ at all (Table 1), suggesting that benzylidene is required for
receptor-binding properties. Therefore, the 5-substituted benzylidene
group was introduced, and simple substitutions at
R3, R4 and R5 with methoxy, ethoxy, 2-furancarboxy, or
aminocarbonyl-methoxy did not change the binding situation (Table 1).
How-ever, when R4 or R5 was substituted with benzyloxy or
benzeneethoxy, 4 derivatives (13, 15, 17, and 19) possessed
both receptor-binding and PPARγ agonist properties (Tables
1, 4). Hydroxy in R2 (14) abolished the receptor-binding
ability of 13, while chloride in R1 may be responsible for
PPARγ agonist activity (15 vs 16 and 17
vs 18; Tables 1, 4). Cytotoxicity was noted in the cells treated with 19 (data not shown)
and this might have led to the lower agonist efficacy
observed (Table 4).
In the receptor-binding assay, we found that the removal
of methoxy in 5-substituted benzylidene would markedly
improve binding activity (Table 2). Among the 18 compounds
in this group, meta-substitution behaved better than
para-substitution (21 vs 20, 23 vs
22, 25 vs 24, 27 vs 26, 29
vs 28, 31 vs 30, 33 vs 32,
35 vs 34, and 37 vs 36; Table 2). The chain
length also affected the receptor-binding activity.
Compounds with n=1 or 3 in the chain length bound to
PPARγ more potently than compounds with n=2 in the chain length
(21, 25 vs 23 and 27, and 31 vs 29; Table 2). When
R1 or R2 was substituted with halogen, the receptor-binding activity was
also improved (27 vs 21; 29, 35 vs 23; 31, and 37
vs 25; Table 2). In the PPARγ cotransfection assay, both the chain length
and halogen substitution affected the agonist properties.
The activities of compounds with n=3 in the chain length
were better than compounds with n=1 or 2 in the chain length
(25, 31, 37, and 39; Table 4). The halogen (eg Cl) group also
increased the cotransfection activity (27, 29, and 31; Table 4).
To reduce intrinsic cytotoxicities, the atom thio on the
rhodanine was changed to oxygen. This alteration led to a
decrease in receptor-binding affinities while modified
analogues generally maintained efficacies in cell-based assays,
except compound 42 (42 vs 37; Table 4). The underlining
mechanism of this phenomenon deserves further
investi-gation.
According to Amit and colleagues[16], a 3-D quantitative
structure_activity relationship model of PPARγ agonist
thiazolidine-2,4-dione has 3 biophoric centers, A, B, and C,
corresponding to carbonyl oxygen of thiazolidine-2,4-dione,
sulphur atom of thiazolidine-2,4-dione, and the oxygen atom
attached to the phenyl ring, respectively. These are
electron-rich sites capable of donating electrons and may be
involved in electrostatic, ionic, and p-p interactions. Site C is
essential to the agonistic action of PPARγ, whereas the
carbonyl group of thiazolidinediones (site A) may be
responsible for the formation of hydrogen bonds with 2 histidine
residues, His 323 and 449, of PPARγ. This information could
certainly help us understand why the benzyloxy,
phenyl-ethoxy, and phenylpropoxy groups are important to the
PPARγ agonists described in the present study.
It is conceivable that the modest transactivation
efficacies observed with these compounds (68.8% maximum
compared to rosiglitazone) were due to partial agonist effects.
The fact that they did not behave as antagonists in the same
assay system suggests that their potencies at
PPARγ should be further improved. Likewise, we noted some
inconsistencies relative to transactivation versus adipogenesis assays
in terms of agonist effects. Although some agonists have
relatively potent agonistic activities in the cotransfection
assay (eg 39), they acted poorly on pre-adipocyte
differen-tiation. This is in agreement with previous studies on a
group of PPARγ agonists, including KR-62980, GW0072,
FMOC-L-leucine, PAT5A and nTZDpa[17]. Biochemical and
structural studies with PPARγ revealed that different ligands
may occupy the PPARγ ligand-binding pocket in different
manners, thereby inducing different allosteric changes in its
conforma-tion. Such alterations would result in different
kinds of interactions between receptor and cofactor proteins,
such as steroid receptor co-activator 1 (SRC1), nuclear
receptor co-activator TIF2, cAMP response element (CRE)
binding protein (CBP), TRAP220/DRIP205, nuclear receptor
co-repressor (NcoR), and the silencing mediator of the
retinoid and thyroid hormone receptors
(SMRT)[18]. Therefore, the effect of
PPARγ modulators on the downstream adipogenesis and glucose uptake will depend on the context of
interaction between the ligand and the receptor.
In summary, 5-benzylidene was required for the
receptor-binding properties. Simple substitutions on benzylidene
with methoxy, ethoxy, 2-furancarboxy, or
amino-carbonyl-methoxy did not change the binding affinity. However, when
benzylidene was substituted with aralkyl, both
receptor-binding and PPARγ agonist activities were demonstrable.
Meta-substitution was better than that of
para. The chain length also affected the
PPARγ-binding and agonist activities. The best chain length studied was 3. Finally, the substitution of
halogen in R1 or R2 improved binding activity. Of the
analogues studied, compound 31 exhibited about 70% the
efficacy exerted by BRL49653 in both the cotransfection and
pre-adipocyte differentiation assays, in addition to its weak
agonist action on hPPARα (data not shown). High receptor
binding potency (Ki =47.4 nmol/L) and less than optimal
agonist efficacy (EC50=700 nmol/L) relative to rosiglitazone
create an opportunity to use rhodanine derivatives as a new
scaffold in further understanding the molecular mechanism
of agonism at PPARs.
Acknowledgements
We are indebted to Xin HUI and Meng-meng NING for
technical assistance.
References
1 Wilson TM, Wahli W. Peroxisome proliferators-activated
receptor agonists. Curr Opin Chem Biol 1997; 1: 235_41.
2 Braissant O, Foufelle F, Scotto C, Dauca M, Wahli W.
Differential expression of peroxisome proliferator-activated receptors
(PPARs): tissue distribution of PPAR-α, -β, and -γ in the adult
rat. Endocrinology 1996; 137: 354_66.
3 Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U,
Mangelsdorf DJ, et al. Differential expression and activation of
a family of murine peroxisome proliferator-activated receptors.
Proc Natl Acad Sci USA 1994; 91: 7355_9.
4 Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM.
Convergence of 9-cis retinoic acid and peroxisome proliferator
signaling pathways through heterodimer formation of their
receptors. Nature 1992; 358: 771_4.
5 Ijpenberg A, Jeannin E, Wahli W, Desvergne B. Polarity and
specific sequence requirements of PPAR-RXR heterodimer
binding to DNA: a functional analysis of the malic enzyme gene
PPRE. J Biol Chem 1997; 272: 20108_17.
6 Aubry CJ, Pernin A, Favez T, Burger AG, Wahli W, Meier
CA, et al. DNA binding properties of peroxisome
proliferator-activated receptor subtypes on various natural peroxisome
proli-ferator response elements: importance of the 5' flanking region.
J Biol Chem 1997; 272: 25 252_9.
7 Keller JK, Collet P, Bianchi A, Huin C, Kremarik PB, Becuwe
P, et al. Implications of peroxisome proliferator activated
receptors (PPAR) in development, cell life status and disease. Int J
Dev Biol 2000; 44: 429_42.
8 Sohda T, Momose Y, Meguro K, Kawamatsu Y, Sugiyama Y,
Ikeada H. Studies on antidiabetic agents. Synthesis and
hypoglycemic activity of
5-[4-(pyridylalkoxy)benzyl]-2,4-thiazolidine-diones. Arzneimittelforschung 1990; 40: 37_42.
9 Kletzien RF, Clarke SD, Ulrich RG. Enhancement of adipocyte
differentiation by an insulin-sensitizing agent. Mol Pharmacol
1992; 41: 393_8.
10 Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO,
Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a
high affinity ligand for peroxisome proliferators-activated
receptor gamma (PPARγ). J Biol Chem 1995; 270: 12 953_6.
11 Javiya VA, Patel JA. The role peroxisome
proliferator-activated receptors in human disease. Edu Forum 2006; 38: 243_53.
12 Wu B, Gao J, Wang MW. Development of a complex
scintillation proximity assay for high-throughput screening of
PPARγ modulators. Acta Pharmacol Sin 2005; 26: 339_44.
13 Gearing KL, Göttlicher M, Teboul M, Widmark E, Gustafsson J.
Inter-action of the peroxisome proliferator-activated receptor
and retinoid X receptor. Proc Natl Acad Sci USA 1993; 90:
1440_4.
14 Tadashi S, Masaki O. Studies on sulfur-containing heterocyclic
compounds. II. Reaction of potassium 3-benzoyldithiocarbazate
and monochloroacetic acid and its ester. J Pharm Soc Japan
1955; 75:1535_9.
15 Cheng Y, Prusoff WH. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes 50
percent inhibition (IC50) of an enzymatic reaction. Biochem
Pharmacol 1973; 22: 3099_108.
16 Amit K, Sushil KK, Anil KS. QSAR and molecular modeling
studies in imidazopyridinethiazolidine-2,4-diones:
PPARγ agonists. Med Chem Res 2004; 13: 770_80.
17 Kwang RK, Jeong HL, Seung JK, Sang DR, Won HJ, Sung-Don Y,
et al. KR-62980: A novel peroxisome proliferator-activated
receptor γ agonist with weak adipogenic effects. Biochem
Pharmacol 2006; 72: 446_54.
18 Béatrice D, Walter W. Peroxisome proliferator-activated
receptors: nuclear control of metabolism. Endocr Rev 1999; 20:
649_88.
|
