Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation

Article abstract of DOI:10.1038/nature10383

PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5 (ref. 2). Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ. 2011 Macmillan Publishers Limited. All rights reserved.

Full text of DOI:10.1038/nature10383

LETTER
doi:10.1038/nature10383
Antidiabetic actions of a non-agonist PPARc ligand
blocking Cdk5-mediated phosphorylation
Jang Hyun Choi1*, Alexander S. Banks1*, Theodore M. Kamenecka2,4*, Scott A. Busby3*, Michael J. Chalmers3, Naresh Kumar3,
Dana S. Kuruvilla3, Youseung Shin2, Yuanjun He2, John B. Bruning5, David P. Marciano3, Michael D. Cameron2,3,4, Dina Laznik1,
6
7
7
6
1
2,3,4
ˇ
´
¨
Michael J. Jurczak , Stephan C. Schurer , Dusica Vidovic , Gerald I. Shulman , Bruce M. Spiegelman & Patrick R. Griffin
PPARc is the functioning receptor for the thiazolidinedione (TZD) association between the clinical effects of rosiglitazone and the blocking
class of antidiabetes drugs including rosiglitazone and pioglitazone1. of this phosphorylation of PPARc. Thus, the contribution made by
These drugs are full classical agonists for this nuclear receptor, but classical agonism to the therapeutic effects of these drugs and to their
recent data have shown that many PPARc-based drugs have a sepa- side effects is not clear.
rate biochemical activity, blocking the obesity-linked phosphoryla-
tion of PPARc by Cdk5 (ref. 2). Here we describe novel synthetic
compounds that have a unique mode of binding to PPARc, com-
pletely lack classical transcriptional agonism and block the
Cdk5-mediated phosphorylation in cultured adipocytes and in
insulin-resistant mice. Moreover, one such compound, SR1664, has
potent antidiabetic activity while not causing the fluid retention and
weight gain that are serious side effects of many of the PPARc drugs.
Unlike TZDs, SR1664 also does not interfere with bone formation in
culture. These data illustrate that new classes of antidiabetes drugs
can be developed by specifically targeting the Cdk5-mediated phos-
phorylation of PPARc.
PPARc is a member of the nuclear receptor family of transcription
factors and is a dominant regulator of adipose cell differentiation and
development3,4. It is also the functioning receptor for the thiazolidine-
dione (TZD) class of antidiabetic drugs such as rosiglitazone and
pioglitazone1,5. These antidiabetes drugs were developed specifically
to have high affinity and full agonism towards PPARc before their
molecularmodes of action were known6. It hastherefore been assumed
that their therapeutic actions result from their functional agonism on
this receptor. From a clinical perspective, rosiglitazone (Avandia) and
pioglitazone (Actos) are both highly effective oral medications for type
2 diabetes and are well tolerated by the majority of patients7.
Unfortunately, a substantial number of patients experience side effects
from these drugs, including fluid retention, weight gain, congestive
heart failure and loss of bone mineral density8,9. Whereas some of
the non-TZD full agonists have good antidiabetic activity, they also
cause many of the same side effects, including fluid retention.
These data indicate that it might be possible to develop entirely new
classes of antidiabetes drugs optimized for the inhibition of Cdk5-
mediated phosphorylation of PPARc while lacking classical agonism.
Here we describe the development of synthetic small molecules that
bind tightly to PPARc, yet are completely devoid of classical agonism
and effectively inhibit phosphorylation at serine 273. These com-
pounds have a unique binding mode in the ligand binding pocket of
PPARc. An example from this series, SR1664, shows potent and dose-
dependent antidiabetic effects in obese mice. Unlike TZDs and other
PPARc agonists, this compound does not cause fluid retention or
weight gain in vivo or reduce osteoblast mineralization in culture.
To develop a suitable ligand, we optimized compounds for (1) high
binding affinity for PPARc, (2) blocking the Cdk5-mediated PPARc
phosphorylation and (3) lacking classical agonism. We first identified
published compounds that bind tightly to PPARc and have favourable
properties as a scaffold for extensive chemical modifications. Classical
agonism is defined here, as is standard in the nuclear receptor field, as
an increased level of transcription through a tandem PPAR response
element luciferase reporter. Of particular interest was compound 7b
describedpreviouslyasanextremelypotentandselectivePPARcpartial
agonist (30% activation compared to rosiglitazone)12. A modular syn-
thesisapproachwasusedtomakeaseriesofanaloguesofcompound7b;
these compounds were tested in vitro and in adipose cells (Sup-
plementaryFig. 1c, d). UsingaLanthaScreencompetitive bindingassay,
SR1664 (Fig. 1a) had a half-maximum inhibitory concentration (IC50)
of 80nM (Supplementary Fig. 1a, b). As shown in Fig. 1b, when com-
pared to rosiglitazone or MRL24 (a partial agonist) in a classical tran-
scriptional activity assay, SR1664 had essentially no transcriptional
The therapeutic role of classical agonism of PPARc was made agonism at any concentration. Rosiglitazone and SR1664 both effec-
somewhat confusing by the development of several compounds that tively blocked the Cdk5-mediated phosphorylation of PPARc in vitro
have less than full agonist properties (partial agonists) but retain sub- with half-maximal effects between 20 and 200 nM (Fig. 1c). In contrast,
stantial insulin-sensitizing and antidiabetic actions in experimental they had no effect on the phosphorylation of a well-characterized Cdk5
models10,11. Furthermore, we have recently shown that many anti- substrate, the Rb protein (Fig. 1d)13. This indicated that these com-
diabetic PPARc ligands have a second, distinct biochemical function: pounds do not disrupt the basic protein kinase function of Cdk5. In
blocking the obesity-linked phosphorylation of PPARc by cyclin- addition, SR1664 was also effective at blocking Cdk5-mediated phos-
dependent kinase 5 (Cdk5) at serine 273 (ref. 2). This is a direct action phorylation of PPARc in differentiated fat cells (Fig. 1e) with no mea-
of the ligands and requires binding to the PPARcligandbindingdomain surable difference in phosphorylation of Rb (Supplementary Fig. 1e).
(LBD), causing a conformational change that interferes with the ability Additional analogues were synthesized and four compounds were
of Cdk5 to phosphorylate serine 273. Rosiglitazone and MRL24 (a selec- identified that have similar in vitro profiles (Supplementary Fig. 1b).
tive partial agonist towards PPARc) both modulate serine 273 phos- SR1824 (Fig. 1a) was further characterized for its ability to block Cdk5-
phorylation at therapeutic doses in mice. Furthermore, a small clinical dependent phosphorylation of PPARc (Fig. 1b–e). These data demon-
trial of newly diagnosed type 2 diabetics showed a remarkably close strate that ligands can be made that potently block Cdk5-dependent
1Department of Cancer Biology and Division of Metabolismand Chronic Disease, Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
2Translational Research Institute, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, USA. 3Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida,
Jupiter, Florida 33458, USA. 4The Scripps Research Molecular Screening Center (SRMSC), The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, USA. 5Department of Biochemistry and
Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA. 6Howard Hughes Medical Institute, Departments of Internal Medicine and Cellular & Molecular Physiology, Yale University
School of Medicine, New Haven, Connecticut 06510, USA. 7Center for Computational Science, University of Miami, Miami, Florida 33136, USA.
*These authors contributed equally to this work.
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RESEARCH LETTER
a
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C/EBPα
Glut4
Vehicle Rosiglitazone SR1664 (1 μM)
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–12 –11 –10 –9 –8 –7
–6 –5
–4
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CDK5
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IB: Anti-PPARγ
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pRb
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H12
pPPARγ
PPARγ
H12
H11
IB: Anti-CDK substrate
Rb
IB: Anti-pSer273
H11
S273
IB: Anti-Rb
H3
IB: Anti-PPARγ
H3
Figure 1
|
Novel PPARc ligands lack classical agonism, block
phosphorylation at Ser 273. a, Chemical structures of SR1664 and SR1824.
b, Transcriptional activity of a PPAR-derived reporter gene in COS-1 cells
following treatment with rosiglitazone, SR1664 or SR1824 (n 5 3). c, d, In vitro
Cdk5 assay with rosiglitazone, SR1664 or SR1824 with PPARc or Rb substrates.
IB, immunoblot; NT, not treated; pPPARc, phosphorylated PPARc; pRb,
phosphorylated Rb. e, TNF-a-induced phosphorylation of PPARc in
differentiated PPARc knock-out MEFs expressing wild-type PPARc treated
with rosiglitazone, SR1664 or SR1824. Error bars are s.e.m.
Figure 2
|
Structural and in vitro functional analysis of SR1664. a, Lipid
accumulation in differentiated 3T3-L1 cells treated with rosiglitazone or
SR1664 following Oil Red O staining. b, Expression of adipocyte-enriched
genes in these cells was analysed by qPCR (n 5 3). c, Mineralization of MC3T3-
E1 osteoblast cells as determined by Alizarin Red-S. Error bars are s.e.m.;
*P , 0.05, **P , 0.01, ***P , 0.001. NT, no treatment. d, Overlay of
differential HDX data onto the docking model of 2hfp bound to SR1664 (see
Supplemental Fig. 3). This overlay depicts the difference in HDX between
ligand-free and SR1664 bound PPARc LBD. Perturbation data are colour
coded and plotted onto the backbone of the PDB file according to the key. n.s.,
not significant. Observed changes in HDX were statistically significant
(P , 0.05) in a two-tailed t-test (n 5 3).
phosphorylation of PPARc in cells while demonstrating little to no
classical agonism.
Of the four compounds identified as non-agonist inhibitors of
Cdk5-mediated PPARc phosphorylation, SR1664 had adequate phar-
macokinetic properties to move forward to biological and therapeutic
assays. Adipogenesis was the first known biological function of
PPARc3 and agonist ligands for PPARc have been shown to stimulate
potently the differentiation of pre-adipose cell lines; this response has
beenwidely used as a sensitive cellular test for PPARc agonism1,14,15. As
shown in Fig. 2a, rosiglitazone potently stimulated fat cell differenti-
ation, as evidenced by Oil Red O staining of the cellular lipid. In
contrast, SR1664 did not stimulate increased lipid accumulation or
changes in morphology characteristic of differentiating fat cells. The
stimulation of fat cell gene expression was also apparent with rosigli-
tazone, as illustrated by an increased expression of genes linked to
adipogenesis. In contrast, SR1664 induced little or no change in the
expression of these genes (Fig. 2b).
hydrogen bonds within the C-terminal helix (H12) of the receptor18–21
.
This interaction stabilized the AF2 surface (helix 3-4 loop, C-terminal
end of H11 and H12) of the receptor facilitating co-activator interac-
tions. Interestingly, high affinity partial agonists have been identified
that do not make these interactions yet still possess some level of
classical agonism, and several of these have been shown to bind the
backbone amide of S342 (S370 in PPARc2) within the b-sheet of the
LBD18. More recently, we demonstrated that the proximity of ligand to
theamide of S342 correlated with increased stabilityof thehelix 2-helix
29loop,theregionofthereceptorcontainingS273 (S245inPPARc1)as
determined by HDX2. Surprisingly, HDX analysis of SR1664 and
SR1824 increased the conformational mobility of the C-terminal end
of H11, a helix that abuts H12 (Fig. 2d); in contrast, the full and partial
agonists stabilized the same region of H11 (Supplementary Fig. 3).
In silicodocking studies were carriedout to understand the structural
basis of SR1664 interactions in the PPARc1 ligand binding domain
(Supplementary Fig. 4). In this model, the phenyl-substituted nitro
Another well-known effect of both rosiglitazone and pioglitazone is
that they decrease bone formation and bone mineral density leading to
an increase in fracture risk8,16. TZDs have also been shown to decrease
bone mineralization in cultured osteoblasts17. As shown in Fig. 2c,
rosiglitazone treatment reduced the mineralization of mouse osteoblastic
cells, as measured by Alizarin red staining. Moreover, the expression of
genes involved in the differentiation of these cells was impaired (see group of SR1664 clashes with hydrophobic side chains of H11 such
Supplementary Fig. 2). Importantly, treatment with SR1664 did not affect as Leu 452 and Leu 453 (Leu 480 and Leu 481 in PPARc2, respectively)
the extent of calcification or the expression of this osteoblast gene set in as well as Leu 469 and Leu 465 (corresponding to Leu 497 and Leu 493
MC3T3-E1 cells.
in PPARc2) of the loop N-terminal to H12. This potentially explains
Co-crystallography, mutagenesisandhydrogen/deuteriumexchange the lack of stabilization of H12 and the destabilization of the region
(HDX) have all demonstrated that full agonists of PPARc affect critical of H11 near His 449 as seen by HDX. Despite the altered mode of
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LETTER RESEARCH
binding, SR1664 and rosiglitazone both bind to the same core residues computed by HOMA-IR, showed a clear and dose-dependent improve-
mentwithSR1664 (Fig. 3b). Thesechanges occurred without significant
differences in body weight compared to vehicle-treated mice (Sup-
plementary Fig. 5).
withinthePPARcLBD. ThisisdemonstratedbytheabilityofSR1664to
attenuate the transcriptional activity of rosiglitazone on PPARc in the
context of a competitive ligand binding assay (Supplementary Fig. 4b).
To determine whether the altered transcriptional activity of SR1664
maybeattributedtodifferencesinDNAbinding orcoactivatorrecruit-
ment, we compared the chromatin association of PPARc or steroid
receptor co-activator-1 (SRC1) within the aP2 promoter. As expected,
rosiglitazone significantlyincreasedSRC1 occupancywithout affecting
PPARc occupancy. However, SR1664 treatment did not influence the
occupancy of PPARc or SRC1 recruitment to the aP2 promoter, indi-
cating that SR1664 has a very different activity of co-regulator recruit-
ment (Supplementary Fig. 4c).
We next asked whether SR1664 had antidiabetic properties in vivo.
Wild-typemicefedahigh-fathigh-sugardietbecomeobeseandinsulin-
resistant, with activation of Cdk5 in their adipose tissues2. Figure 3a
demonstrates that SR1664, injected twicedailyfor5 days, causedadose-
dependent decrease in the Cdk5-mediated phosphorylation of PPARc
at serine 273 in adipose tissue. Moreover, SR1664 treatment also caused
a trend towards lowered (and normalized) glucose levels, and a sig-
nificant reduction in the fasting insulin levels. Insulin resistance, as
The most accurate method for measuring changes in insulin sensi-
tivity in vivo is the hyperinsulinaemic-euglycaemic clamp22. As shown
in Fig. 3c and in Supplementary Fig. 6, the glucose infusion rate (GIR)
needed to maintain euglycaemia in the mice treated with SR1664 was
significantly greater than in animals treated with the vehicle alone,
indicating improved whole-body insulin sensitivity. Suppression of
hepatic glucose production (HGP), an important component of
insulin action, was improved by SR1664. Whereas no difference in
whole-body glucose disposal was detected from calculations of
3H-glucose turnover, analysis of tissue-specific 14C-2-deoxyglucose
transport demonstrated improved insulin-stimulated glucose disposal
in adipose tissue of SR1664-treated mice. Similarly, reductions in both
basal and clamped plasma free fatty acids levels, as well as a 20%
greater suppression of lipolysis in response to insulin, indicated
improved adipose tissue insulin sensitivity in SR1664-treated mice.
Together, these data indicate that SR1664 improves insulin sensitivity.
Using cells expressing the S273A mutant of PPARc, we previously
defined a gene set in cultured adipose cells that was most sensitive to
the phosphorylation at this site2. Treatment of mice with SR1664
caused changes in the expression of 11/17 (65%) of these genes, all
in the direction predicted for the inhibition of the PPARc S273 phos-
phorylation (Fig. 3d). Adiponectin and adipsin, genes long recognized
as being reduced in obesity23,24, are both induced by SR1664. We also
defined a separate set of genes reflective of a full agonist (rosiglitazone)
on cultured fat cells. SR1664 caused changes in expression of 6/19
genes in this ‘agonist’ gene set; importantly, three of these changes
were in the same direction as expected for an agonist, but three were
changed in the opposite direction (Fig. 3e). Taken together, these data
show that SR1664 has an insulin-sensitizing effect with preferential
regulation of the gene setsensitive to the phosphorylation of PPARc by
Cdk5.
A more severe model of obesity is the leptin-deficient ob/ob mouse.
These animals are very obese and insulin-resistant, with substantial
compensatory hyperinsulinaemia. Preliminary pharmacokinetic and
pharmacodynamic experiments showed comparable drug exposures
at 40 mg kg21 for SR1664 and 8 mg kg21 for rosiglitazone, both
injected twice daily (Supplementary Fig. 7). Functional analyses were
performed at days 5 and 11 after the start of treatments. As shown in
Fig. 4a, both drugs caused a similar reduction in PPARc phosphoryla-
tion at S273. After 5 days of treatment, there were no overt differences
in fasting body weight or glucose levels (Fig. 4b). Control mice receiv-
ing only the vehicle remained hyperinsulinaemic, but both rosiglita-
zone and SR1664 substantially reduced these insulin levels (Fig. 4b).
Glucose tolerance tests were markedly improved with both rosiglita-
zone and SR1664, and the areas under these glucose excursion curves
were statistically indistinguishable, without changing body weight
(Fig. 4c).
a
Vehicle
5 mg kg–1
10 mg kg–1
20 mg kg–1
1.2
0.8
0.4
0
pPPARγ
PPARγ
**
IB: Anti-pSer273
***
***
IB: Anti-PPARγ
b
6
2.0
1.5
1.0
0.5
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150
100
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Vehicle SR1664
Vehicle
Vehicle SR1664
Vehicle SR1664
4
3
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Rosiglitazone
SR1664
*
**
**
*
*
*
*
**
*
**
*
*
*
*
*
*
*
*
**
e
Vehicle
4
Rosiglitazone
SR1664
*
*
3
2
1
0
**
*
*
*
**
*
Weight gain and fluid retention caused by TZD drugs like rosiglita-
*
**
*
*
*
*
*
zone are suspected to be key factors in their increased cardiac risk9,25
.
**
*
**
After recovering from the glucose tolerance test on day 5, rosiglitazone-
treated mice began to show an increase in body weight (Fig. 4d). This
increased mass is accounted for primarily by fluid retention, quantified
by a decrease in haematocrit seen with haemodilution (Fig. 4f).
However, an increase in body fat was also observed by magnetic res-
onance imaging (Fig. 4e, f). Importantly, SR1664 treatment did not cause
the weight gain seen with the rosiglitazone treatment. Furthermore,
SR1664 treatment showed no decrease in the haematocrit or change
in body adiposity. These results were confirmed by measurements
showing a decreased concentration of haemoglobin in the mice treated
with rosiglitazone, but not in those treated with SR1664 (Supplemen-
tary Fig. 8). Taken together, these data indicate that SR1664, a non-
agonist PPARc ligand, has antidiabetic actions in two murine models
Figure 3
|
Antidiabetic activity of SR1664 in high-fat diet (HFD) mice.
a, Dose-dependent inhibitionof phosphorylation of PPARc by SR1664 in white
adipose tissue (WAT). Quantification of PPARc phosphorylation compared to
total PPARc (right). b, Ad libitum-fed glucose (P 5 0.062 at 10 mg kg21),
insulin and HOMA-IR in HFD mice. c, Glucose infusion rate (GIR),
suppression of hepatic glucose production (HGP), whole body glucose disposal
and WAT 2-deoxyglucose tracer uptake during hyperinsulinaemic-
euglycaemic clamps. d, Expression of a gene set regulated by PPARc
phosphorylation in WAT. e, Expression of an agonist gene set (see Methods) in
WAT. Error bars are s.e.m.; *P , 0.05, **P , 0.01.
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RESEARCH LETTER
a
rather they seem to function as partial agonists18,21,27. This strongly
suggests that when engaged by ligands, other structural features of
the AF2 surface such as H3, H3-H4 loops and the C-terminal end of
H11 contribute to partial agonism of the receptor. As expected SR1664
and SR1824 do not interact with H12 in any detectable way, but
unexpectedly both ligands cause an increase in the conformational
mobility of H11, which is part of the AF2 surface and directly abuts
H12. Hence, it seems likely that the destabilization of H11 distorts the
AF2 surface enough to block partial agonism. Whether there are other
alternative modes of ligand binding that would lead to a complete lack
of classical agonism remains to be determined.
That classical agonism is not required for strong antidiabetic actions
of a PPARc ligand is now clear. In both diet-induced and genetically
obese animals, SR1664 has strong antidiabetic actions. The ability to
improve adipose tissue insulin sensitivity is similar to the effects shown
for rosiglitazone28. SR1664 has inferior pharmacokinetic properties
compared to rosiglitazone, so an absolute quantitative comparison of
their efficacy is difficult. However, using our best calculations to get
approximately equal exposure to the two drugs in vivo, SR1664 has
very robust antidiabetic activity, roughly equivalent to rosiglitazone in
the experiments shown here. The unfavourable pharmacokinetic
properties of SR1664 strongly suggest that this compound will never
be administered to patients but it proves that non-agonist compounds
can have robust therapeutic effects.
Analysis of the side effects of PPARc ligands can be difficult because
some of these (like cardiovascular disorders) do not occur in mice
whereas others (like loss of bone mineral density) take many months
of treatment to manifest. However, weight gain and fluid retention
occur rapidly in both humans and mice. Increased body weight,
increased accretion of fat tissues and increased fluid retention all occur
in mice within 11 days of treatment with rosiglitazone (Fig. 4). The
non-agonist SR1664 shows none of these side effects, even as it effec-
tively improves glucose homeostasis. Unlike rosiglitazone, SR1664
does not affect bone cell mineralization in culture (Fig. 2c). Taken
together, these data indicate that many of the known side effects of
the TZD drugs occur as a consequence of classical agonism on target
genes. Whether ligands directed at the Cdk5-mediated phosphoryla-
tion have their own problems remains to be determined. Still, these
studies illustrate that the development of entirely new classes of
PPARc-targeted drugs is feasible.
1.2
1
Vehicle
Rosiglitazone
SR1664
pPPARγ
PPARγ
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0.6
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IB: Anti-PPARγ
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SR1664
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SR1664
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Treatment day
Figure 4
|
SR1664 has potent antidiabetic activity and does not promote
fluid retention in ob/ob mice. a, Phosphorylation of PPARc in WAT (left).
Quantification of PPARc phosphorylation compared to total PPARc (right).
b, c, Fasting body weight, blood glucose and insulin levels before glucose-
tolerance tests (GTT) in ob/ob mice treated with vehicle, rosiglitazone or
SR1664 (n 5 8). Whole-body weight(d) andfatchange(e) with continueddrug
administration following the GTT. f, Packed cell volume (PCV) in whole blood
from ob/ob mice treated with vehicle, rosiglitazone or SR1664. Error bars are
s.e.m.; *P , 0.05, **P , 0.01, ***P , 0.001. n.s., not significant.
of insulin-resistance. Furthermore, this non-agonist doesnot stimulate
two of the best documented side-effects of the PPARc agonist drugs
in vivo.
METHODS SUMMARY
The TZD class of drugs has been important for the treatment of type
2 diabetes26. Whereas these drugs function as full agonists for PPARc,
the role of agonism in their therapeutic effects has been called into
question recently. Rosiglitazone and partial agonists like MRL24 both
block the obesity-linked phosphorylation of PPARc at serine 273
(ref. 2). The tight correlation between inhibition of this phosphoryla-
tion and the therapeutic effects of these drugs in both mouse and man
suggested that it might be possible to create new classes of non-agonist
ligands for PPARc which are effective for the treatment of diabetes
and cause fewer side effects. Hence, this paper addresses three key
questions: first, is it possible to create novel PPARc ligands that
block Cdk5-mediated PPARc phosphorylation yet have no classical
agonism? Second, would such compounds have robust antidiabetic
activity? Finally, would non-agonist compoundshave fewerside effects
than classical full agonists like rosiglitazone?
We show here that it is possible to create new ligands that have high
affinity for PPARc, block the Cdk5-mediated phosphorylation and
completely lack classical agonism. SR1664 does not function as an
agonist and has no adipogenic action in vitro. The structural require-
ments for the non-agonist actions of SR1664 and SR1824 are particu-
larly interesting. Ligands that function as classical full agonists, like
rosiglitazone, have been shown to alter the conformation and HDX
kinetics of H12, the major agonist helix. Surprisingly, ligands that do
not affect the conformational dynamics of H12 are not non-agonists,
Cell culture. Adipocytedifferentiation in 3T3-L1or PPARc-null mouse embryonic
fibroblasts (MEFs) expressing PPARc2 was induced by treating cells with 1 mM
dexamethasone, 0.5 mM isobutylmethylxanthine, and 850nM insulin for 48h and
cells were switched to the maintenance medium containing 850 nM insulin for
6days.
Gene expression analysis. Total RNA was isolated from cells or tissues using
TRIzol reagent (Invitrogen). The RNA was reverse-transcribed using ABI reverse
transcription kit. Quantitative PCR (qPCR) reactions were performed with SYBR
green fluorescent dye using an ABI9300 PCR machine. Relative mRNA expression
was determined by the DD-Ct method using TATA-binding protein (TBP) levels.
Animals. All animal experiments were performed according to procedures
approved by Beth Israel Deaconess Medical Center’s Institutional Animal Care
and Use Committee. Male C57BL/6J and C57BL/6J-Lepob/ob mice (4- to 5-week-
old) were obtained from the Jackson Laboratory. C57BL/6J mice were fed a high-
fat, high-sucrose diet (60% kcal fat, D12492, Research Diets Inc.). For glucose
tolerance tests, mice were injected intraperitoneally (i.p.) with rosiglitazone or
SR1664 for 5 days, and fasted overnight before i.p. injection of 1 g kg21 D-glucose.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 16 May; accepted 22 July 2011.
Published online 4 September 2011.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We are grateful for support from S. Novick for assistance in the
HDX studies andfromR. D. Garcia-Ordonez inprotein production and mutagenesis. We
are grateful for support from B. Pascal andS. Willis for software analysing the HDX data.
We are grateful to R. Gupta and P. Cohen for their critical comments on the manuscript.
This work was supported in part by the Intramural Research Program of the National
Institutes of Health (NIH), National Institute of Mental Health (grant U54-MH074404,
H. Rosen principal investigator), the National Institute of General Medical Sciences
(grant R01-GM084041, P.R.G.) and the NationalInstituteofDiabetes and Digestiveand
Kidney Diseases (grant 1RC4DK090861, B.M.S.). This work also supported by NIH
DK31405 to B.M.S.
Author Contributions B.M.S. and P.R.G. conceived the project and designed research;
J.H.C., A.S.B., T.M.K., S.A.B., M.J.C., N.K., D.S.K., Y.S., Y.H., J.B.B., D.P.M., M.D.C., D.L., M.J.J.,
S.C.S., and D.V. performed research; J.H.C., A.S.B., T.M.K., S.A.B., M.J.C., N.K., D.K., J.B.B.,
D.M., M.D.C., D.L., M.J.J., S.C.S., D.V., G.I.S., B.M.S and P.R.G. analysed data; and B.M.S.,
A.S.B. and P.R.G. wrote the paper with contributions from all authors.
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osteoblast differentiation by PPARc2. J. Cell. Biochem. 74, 357–371 (1999).
18. Bruning, J. B. et al. Partial agonists activate PPARc using a helix 12 independent
mechanism. Structure 15, 1258–1271 (2007).
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to B.M.S. (bruce_spiegelman@dfci.harvard.edu) or P.R.G.
(pgriffin@scripps.edu).
19. Hamuro, Y. et al. Hydrogen/deuterium-exchange (H/D-Ex) of PPARc LBD in the
presence of various modulators. Protein Sci. 15, 1883–1892 (2006).
2 2 S E P T E M B E R 2 0 1 1
| V O L 4 7 7 | N A T U R E | 4 8 1
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RESEARCH LETTER
Ensemble docking. PPARc co-crystal structures (68 in total) with unique ligands
were identified in the Protein Data Bank (PDB) (as of 3 January 2011). Four
structures were selected based on the maximum similarity of the co-crystal ligands
to SR1664; specifically 3kmg (ligand 538, 0.98 similarity), 2hfp (ligand NSI,
similarity of 0.91), 1fm9 (ligand 570, 0.90 similarity), 2pob (ligand GW4, 0.88
similarity). SR1664 was prepared using Schrodinger LigPrep generating tautomers
and ionization states (pH range 7 6 2). Flexible ligand docking of SR1664 against
the four structures was performed using Schrodinger Glide. At least one of the two
constraints Arg288 and Ser 342 (Arg 316 and Ser 370 in PPARc2) was required to
score docking poses. The best docking score (Glide docking scores are meant to
correspond to binding affinity) of 29.21 was achieved with the PPARc structure
2hfp and SR1664 forms a hydrogen bond to Ser 342 (shown in Fig. 2).
Unconstrained docking produced almost the same docking pose with the pre-
served hydrogen bonding to Ser 342 and a slightly less favourable docking score of
28.99 indicating Ser 342 as a critical ligand binding element.
Differentiation of MC3T3-E1. After reaching confluence, cells were grown in
a-MEMsupplementedwith10%FBS,1%penicillin-streptomycin, 200 mMascorbic
acid and 10mM b-glycerophosphate. The cells were treated with either rosiglita-
zone (10 mM) or SR1664 (10 mM) or left in vehicle at the start of differentiation. The
cells were collected 7 days post-differentiation for gene expression analysis and
21days post-differentiation for mineralization. The mineralization of MC3T3-E1
cellswasdeterminedbyAlizarinredSstaining(Milliporecatalogueno.ECM815)as
per manufacturer’s instructions.
Preparation of cell or tissue lysates and immunoblotting. Differentiated
adipocytes were pre-treated with PPARc ligands for 45 min, and incubated with
TNF-a for 30 min. For tissue lysates, WAT from mice was homogenized in RIPA
buffer (50mM Tris pH 7.5, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS with protease and phosphatase inhibitors). For western blotting, a
phospho-specific antibody against PPARc Ser 273 was used2. Total tissue lysates
were analysed with an anti-PPARc antibody (Santa Cruz).
Gene expression analysis. Total RNA was isolated from cells or tissues using
TRIzol reagent (Invitrogen). The RNA was reverse-transcribed using the ABI
reverse transcription kit. Quantitative PCR reactions were performed with
SYBR green fluorescent dye using an ABI9300 PCR machine. Relative mRNA
expression was determined by the DD-Ct method normalized to TATA-binding
protein (TBP) levels. The sequences of primers used in this study are found in
Supplementary Table 1.
ChIP. Differentiated 3T3-L1 adipocytes were treated on day 6 with 1 mM of
compounds or vehicle for 24 h. The samples were prepared using manufacturer’s
protocol (ChampionChIP One-Day Kit, Qiagen). Briefly, cross-linked chromatin
was sonicated and 5 mg of antibody was used to immunoprecipitate the pre-cleared
samples. The following antibodies were used: normal rabbit IgG, PPARc (Santa
Cruz), SRC-1 (Abcam). The promoter region of aP2 for PPAR-c binding was
amplified using PCR with reverse transcription (RT–PCR). The primers used
for aP2 were aP2 forward 59-AAATTCAGAAGAAAGTAAACACATTATT-39;
aP2 reverse 59-ATGCCCTGACCATGTGA-39.
Gene sets from microarray. We performed a microarray with total RNA isolated
from PPARc-null fibroblasts expressing wild-type or S273A mutant of PPARc or
WT cells treated with 1 mM rosiglitazone for 24 h (ref. 2). To create refined gene
sets regulated by phosphorylation of PPARc or rosiglitazone, we first calculated
P-values as well as fold-change of gene expression in wild-type versus S273A
mutant cells or wild-type versus wild-type /Rosiglitazone cells, and we plotted
2log P-value versus log2 fold-change. From this list of genes, we selected genes
which were changed in magnitude ($1.4 fold difference) and statistical signifi-
cance (P , 0.05). The selected genes were validated in cells by using qPCR, the
resulting gene sets (phosphorylation-dependent or agonist-dependent gene sets)
were analysed in WAT of mice using qPCR.
METHODS
SR1664. (S)-49-((5-((1-(4-nitrophenyl)ethyl)carbamoyl)-1H-indol-1-yl)methyl)-
[1,19-biphenyl]-2-carboxylic acid. Commercially available ethyl 2,3-dimethyl-1H-
indole-5-carboxylate was N-alkylated with commercially available tert-butyl
49-(bromomethyl)biphenyl-2-carboxylateusing NaH in DMF. The corresponding
ethyl ester was hydrolysed using aqueous NaOH in ethanol to give the acid, which
was coupled to (S)-1-(4-nitrophenyl)ethanamine using 2-(3H-[1,2,3]triazolo
[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (HATU)
and diisopropylethylamine in CH2Cl2 to give the amide. Final deprotection of the
tert-butyl ester using 30% trifluoroacetic acid in CH2Cl2 and purification by flash
chromatography (ethyl acetate/hexanes 10–100%) afforded SR1664. Electrospray
ionisation coupled with mass spectrometry (ESI-MS; m/z): 576 [M1H]1; 1H NMR
(400 MHz, dimethylsulphoxide (DMSO)-d6): d (p.p.m.) 8.83 (d, J57.6 Hz, 1H),
8.25 (m, 1H), 8.16 (d, J 51.2 Hz, 1H), 7.74-7.68 (m, 4H), 7.57 (dt, J51.6, 7.2 Hz,
1H), 7.51 (d, J 5 8.4 Hz, 1H), 7.46 (dt, J51.2, 7.2 Hz, 1H), 7.36 (dd, J 50.8, 7.6 Hz,
1H), 7.28 (m, 2H), 7.03 (m, 2H), 5.52 (s, 2H), 5.32 (quint, J57.2 Hz, 1H), 2.36 (s,
3H), 2.34 (s, 3H), 1.57 (d, J 56.8 Hz, 3H); 13C NMR (400MHz, DMSO-d6): d
(p.p.m.) 170.5, 167.9, 154.5, 147.2, 141.5, 140.7, 138.7, 138.2, 135.1, 133.2, 131.8,
131.5, 130.0, 129.6, 128.6, 128.2, 128.1, 126.8, 125.8, 124.4, 121.4, 118.8, 109.7, 108.3,
49.4, 46.7, 22.9, 11.0, 9.7.
SR1824. (S)-49-((5-(1-(4-bromophenyl)ethylcarbamoyl)-2,3-dimethyl-1H-indol-1-yl)
methyl)biphenyl-2-carboxylic acid (1824) was synthesized in the same manner
using (S)-1-(4-bromophenyl)ethanamine. ESI-MS (m/z): 581/583 [M1H]1;
1H NMR (400 MHz, DMSO-d6): d (p.p.m.) 1.48 (d, J 5 J 5 6.8 Hz, 3H, CH3
(4-bromophenyl)ethylcarbamoyl), 2.28 (s, 3H, CH3 indole), 2.32 (s, 3H, CH3
indole), 5.17 (quintuplet, J 5 7.6Hz, 1H, CH (4-bromophenyl)ethylcarbamoyl),
5.47 (s, 2H, CH2-biphenyl), 6.99 (d, J 5 8 Hz, 2H, H7 and H9 biphenyl), 7.24
(d, J 5 J 5 8 Hz, 2H, H6 and H10 biphenyl), 7.31 (d, J 5 7.6Hz, 1H, H7 indole),
7.36–7.55 (m, 7H, H2, H3 and H4 biphenyl, H6 indole and H 4-bromophenyl), 8.10
(d, J 5 J 5 1.6 Hz, 1H, H4 indole), 8.65 (d, J 5 8 Hz, 1H, NH amide). 13C NMR
(400 MHz, DMSO-d6): d (p.p.m.) 169.5, 166.7, 144.9, 140.5, 139.7, 137.6, 137.3,
134.0, 132.2, 131.0, 130.8, 130.4, 129.0, 128.6, 128.4, 127.6, 127.2, 125.9, 125.0,
120.3, 119.4, 117.7, 108.7, 107.3, 47.9, 45.7, 22.1, 10.1, 8.6.
Cell culture. COS-1, 3T3-L1 and HEK-293 cells were obtained from ATCC.
Adipocyte differentiation in 3T3-L1 or PPARc-null mouse embryonic fibroblasts
(MEFs) expressing PPARc2 was induced by treating cells with 1 mM dexamethasone,
0.5mM isobutylmethylxanthine, and 850 nM insulin with 10% FBS in DMEM for
48 h and cells were switched to the maintenance medium containing 10% FBS and
850 nM insulin. Lipid accumulation in the cells was detected by Oil Red O staining.
All chemicals for cell culture were obtained from Sigma unless otherwise indicated.
In vitro kinase assay. Active Cdk5/p35 was purchased from Millipore. In vitro
CDK kinase assay was performed according to the manufacturer’s instructions
(Cell Signaling Technology). Purified PPARc (0.5 mg; Cayman Chemicals) were
incubated with active CDK kinase in assay buffer (25 mM Tris-HCl pH 7.5, 5 mM
beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM
MgCl2) containing 20 mM ATP for 15 min at 30 uC. PPARc ligands were pre-
incubated with the specified substrates for 30 min before the assay was performed.
Rb (Cell Signaling Technology) was used as a positive control.
LanthaScreen. PPARccompetitive binding assay (Invitrogen) was performed accord-
ingto the manufacturer’s protocol. A mixture of 5 nM glutathione S-transferase fused
with the PPARc ligand binding domain (GST–PPARc–LBD ), 5 nM Tb-GST-anti-
body, 5 nM Fluormone Pan-PPAR Green, and serial dilutions of SR1664 beginning at
10 mM downwards was added to wells of black 384-well low-volume plates (Greiner)
to a total volume of 18 ml. All dilutions were made in TR-FRET assay buffer C. DMSO
at 2% final concentration was used as a no-ligand control. Experiments were per-
formed in triplicate and incubated for 2 h in the dark before analysis in Perkin Elmer
ViewLux ultra HTS microplate reader. The FRET signal was measured by excitation
at 340 nm and emission at 520 nm for fluorescein and 490 nm for terbium. The fold
change over DMSO was calculated by 520 nm/490 nm ratio. Graphs were plotted as
fold change of FRET signal for each compound over DMSO-only control.
Cell-based transactivation assay. COS-1 cells were cotransfected in batch by
adding 4.5 mg full-length murine PPARc2-pSV Sport or full-length human
PPARc2-pSport6, with 4.5 mg 33 multimerized PPRE-luciferase reporter and
Animals. All animal experiments were performed according to procedures
approved by Beth Israel Deaconess Medical Center’s Institutional Animal Care
and UseCommittee. Male C57BL/6Jor C57BL/6J-Lepob/ob mice (4- to 5-week-old)
were obtained from the Jackson Laboratory. C57BL/6J mice were fed a regular diet
(10% kcal fat, D12450B, Research Diets Inc.) or a high-fat, high-sugar diet (60%
kcal fat, D12492, Research Diets Inc.) for either 8, 10 or 18 weeks. The mice were
27 ml X-treme Gene 9 transfection reagent in serum-free Opti-mem reduced intraperitoneally (i.p.) injected twice daily with 4 mg kg21 rosiglitazone or 20 mg
serum media (Gibco). After 18-h incubation at 37 uC in a 5% CO2 incubator, kg21 SR1664 for 6 days before gene expression analysis or hyperinsulinaemic-
transfected cells were plated in triplicate in white 384-well plates (Perkin Elmer) euglycaemic clamp experiments. Clamps were performed essentially as previously
at a density of 10,000 cells per well. After replating, cells were treated with either described with one exception to the standard protocol29. As the mice were fed a
DMSO vehicle only or the indicated compounds in increasing doses from 2 pM– high-fat diet for 8 weeks before the clamp studies, a higher insulin infusion rate of
10 mM for mouse receptor or 220pM–2 mM for the human receptor. After 18-h 4 mU (kg-min)21 was used instead of the typical 3 mU (kg-min)21 for standard
incubation, treated cells were developed with Brite Lite Plus (Perkin Elmer) and chow studies. For glucose tolerance tests, 6-week-old male C57BL/6J-Lepob/ob mice
read in 384-well Luminescence Perkin Elmer EnVision Multilabel plate reader. were i.p. injected twice daily with 8 mg kg21 rosiglitazone or 40 mg kg21 SR1664
Graphs were plotted in triplicate as fold change of treated cells over DMSO-treated for 6 days, and fasted overnight before i.p. injection of 1 g kg21 D-glucose. Glucose
control cells.
was measured by tail vein bleeds at the indicated intervals using a Truetrack
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LETTER RESEARCH
29. Kim, H. J. et al. Differential effects of interleukin-6 and -10 on skeletal muscle and
liver insulin action in vivo. Diabetes 53, 1060–1067 (2004).
glucometer. Serum insulin concentrations were determined by ELISA (Crystal
Chem).
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