Crystal structure of the peroxisome proliferator-activated receptor γ (PPARγ) ligand binding domain complexed with a novel partial agonist: A new region of the hydrophobic pocket could be exploited for drug design

Article abstract of DOI:10.1021/jm800733h

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiaz

Full text of DOI:10.1021/jm800733h

7768
J. Med. Chem. 2008, 51, 7768–7776
Crystal Structure of the Peroxisome Proliferator-Activated Receptor γ (PPARγ) Ligand
Binding Domain Complexed with a Novel Partial Agonist: A New Region of the Hydrophobic
Pocket Could Be Exploited for Drug Design
Roberta Montanari,† Fulvio Saccoccia,† Elena Scotti,‡ Maurizio Crestani,‡ Cristina Godio,‡ Federica Gilardi,‡ Fulvio Loiodice,§
Giuseppe Fracchiolla,§ Antonio Laghezza,§ Paolo Tortorella,§ Antonio Lavecchia,| Ettore Novellino,| Fernando Mazza,†,
Massimiliano Aschi, and Giorgio Pochetti*,†
Consiglio Nazionale delle Ricerche, Roma 00016, Italy, UniVersita` degli Studi di Milano, Milano 20133, Italy, UniVersita` degli Studi di Bari,
Bari 70125, Italy, UniVersita` degli Studi di Napoli “Federico II”, Napoli 80131, Italy, and UniVersita` di L’Aquila, L’Aquila 67010, Italy
ReceiVed June 17, 2008
The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating
glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect
to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired.
We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue,
respectively complexed with the ligand-binding domain (LBD) of PPARγ, and provide an explanation on
a molecular basis for their different potency and efficacy against PPARγ. The more potent S-enantiomer is
a dual PPARR/PPARγ agonist which presents a partial agonism profile against PPARγ. Docking of the
S-enantiomer in the PPARR-LBD has been performed to explain its different subtype pharmacological profile.
The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists,
is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the
PPARγ-LBD never sampled before by other ligands.
Introduction
PPARsa belong to the nuclear receptors superfamily,1,2 and
they are transcription factors activated by specific ligands, which
are usually lipophilic small molecules.3 Binding of these ligands
results in conformational changes of the receptors that facilitate
their interaction with coactivator proteins in the nucleus.2,4,5 The
resulting protein complexes activate the transcription of specific
target genes, resulting in the induction of intracellular signalling
cascades that mediate the physiological effects of the ligands.6,7
Figure 1. Chemical structures of (S)-1, (R)-1, and metaglidasen.
So far, three PPAR subtypes have been described in mam-
mals: PPARR, PPARγ, and PPARꢀ/δ. All of these are targets
for treatment of the metabolic syndrome, a cluster of risk factors
for cardiovascular disease and diabetes including obesity,
atherogenic dyslipidemia, hypertension, insulin resistance, and
elevated fasting blood glucose.2 The fibrate class of lipid-
lowering drugs (e.g., fenofibrate and gemfibrozil) are PPARR
ligands.8-10 The marketed TZD class of antidiabetic agents
(rosiglitazone and pioglitazone) activates PPARγ.11,12 They
enhance insulin sensitivity in target tissues and lower glucose
and fatty acid levels in type 2 diabetic patients. However, despite
their proven benefits, these drugs have been plagued by certain
adverse effects such as weight gain, higher rate of bone fractures,
fluid accumulation, and pulmonary edema, leading to increased
frequency of congestive heart failure.13-15 A new class of dual
agonists, glitazars, have elicited high hopes and deep disap-
pointment as potential new drugs. Also known as PPARR/
PPARγ dual agonists, glitazars are designed to treat both insulin
resistance and key aspects of dyslipidemia that contribute to
the high risk of cardiovascular diseases in diabetics. The two
lead compounds in this class are muraglitazar and tesaglitazar,
but both drugs were discontinued because of important side
effects.14,15 One of the key challenges for the development of
a dual agonist is identifying the optimal receptor subtype
selectivity ratio. The intrinsic potencies at each receptor subtype
will ultimately determine the overall efficacy with respect to
metabolic effects and minimized side effects. All failed PPAR
agonists to date are apparently pure PPARγ or PPARγ-
preferential dual agonists. Consequently, most safety issues that
led to development discontinuations are rather associated with
overactivation of PPARγ than with action on the R subtype.
New drugs, which act as partial agonists of PPARγ, have been
developed with the goal of retaining the beneficial effects while
diminishing the adverse effects. Metaglidasen (see Figure 1), a
PPARγ partial agonist, is the most advanced insulin sensitizer
that is currently in phase III clinical trials. The results of phase
II clinical trials showed that metaglidasen, a prodrug ester that
is rapidly and completely modified in vivo to its mature
circulating free acid form, significantly improved metabolic
parameters without the side effects of fluid retention/edema or
weight gain.14 Recently, we reported the synthesis and activity
of some chiral clofibric acid analogues16 whose structures are
* Towhomcorrespondenceshouldbeaddressed.Phone:0039.06.90672627.
Fax: 0039.06.90672630. E-mail: giorgio.pochetti@ic.cnr.it.
†
Consiglio Nazionale delle Ricerche.
Universita` degli Studi di Milano.
Universita` degli Studi di Bari.
Universita` degli Studi di Napoli “Federico II”.
‡
§
|
Universita` di L’Aquila.
a Abbreviations: PPAR, peroxisome proliferator-activated receptor; TZD,
thiazolidinediones; LBD, ligand-binding domain; PDB, Brookhaven Protein
Data Bank; MD, molecular dynamics; RMSF, root mean square fluctuation.
10.1021/jm800733h CCC: $40.75
2008 American Chemical Society
Published on Web 11/18/2008

PPARγ Partial Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7769
Scheme 1. Synthesis of (S)-1a
(Table 2). The R-enantiomer is not active on both human
PPARR (Figure 2B and Table 2) and mouse PPARR (Table 2).
Both the R and S-enantiomers activate PPARγ (Figure 2C),
although their potency is significantly lower than that of the
reference PPARγ agonist rosiglitazone (Table 2). Interestingly,
transfected cells treated with (S)-1 and (R)-1 do not achieve
full activation of PPARγ-dependent transcription (Figure 2C
and Table 2), suggesting that these two compounds may be
partial agonists of PPARγ.
a (a) PPh3-PS, DIAD, toluene; (b) THF, 1 N NaOH (1:1).
related to that of the active metabolite of metaglidasen. These
compounds show dual activity toward PPARR and PPARγ with
the stereochemistry playing a crucial role in the receptor
activation. In the search for a well balanced dual agonist, we
focused our attention on full agonist (S)-1 (Figure 1) displaying
a selectivity ratio equal to 1. A more in-depth investigation of
PPARR/PPARγ agonist activity of this ligand and its enantiomer
revealed that the former has a lower efficacy on PPARγ
compared to that previously reported16 and is about 10 times
more potent than the latter. On the other hand, the R-enantiomer
is completely devoid of activity on PPARR. Here, we report
the X-ray structures of the PPARγ-LBD in the complex with
(S)-1 and (R)-1, respectively, and compare their structures,
providing a molecular explanation for their different potency
against PPARγ. Moreover, we compare the structure of the more
potent S-enantiomer with that of the same ligand docked in the
hydrophobic pocket of PPARR and provide an explanation at
the molecular level for its different behavior as full and partial
agonist of PPARR and PPARγ, respectively. Noteworthy, we
also characterize a new region of the PPARγ internal hydro-
phobic pocket never sampled before by other PPARγ agonists
that could be exploited in the design of new PPAR ligands.
Binding of (S)-1 to PPARγ-LBD. Figure 3A shows the
unambiguous positioning of the S-enantiomer fitted into the
electron density map calculated in the hydrophobic pocket of
PPARγ. Figure 4 summarizes the binding interactions between
the polar head of the S-enantiomer and the surrounding residues.
One of the carboxylate oxygens can form a bifurcated H-bond
with the Y473 OH and the H449 Nε2 groups; the latter group
engages the ligand ether oxygen in a further H-bond. The other
carboxylate oxygen is at H-bonding distances from H323 Nε2,
Y473 OH, and S289 OH. The residues H323, Y473, H449, and
S289 are generally involved in the canonical intermolecular
H-bonding network in the presence of carboxylate containing
ligands. Figure 5A shows the cavity where the diphenyl group
of the S-enantiomer is deeply inserted, there forming several
favorable hydrophobic interactions. The bottom of the cavity
is delimited by the loop 11/12 and is contoured sidewise by H3
and H11.The diphenyl group there acts as a stabilization pivot
between these helices and the loop 11/12. Inside the cavity, the
diphenyl group interacts on one side with L453 and more weakly
with I456 of H11 and interacts on the other side with the Q286
of H3. Favorable van der Waals contacts are also realized by
the benzyl group and the asymmetric carbon atom of the
S-enantiomer with the S289 OH group. At the bottom of the
cavity, the terminal end of the diphenyl group faces the M463
side chain of the loop 11/12. The short contact between the
distal aromatic ring and M463 is well evidenced by continuous
electron density between the two interacting groups (see
Supporting Information Figure1).
Results and Discussion
Chemistry. The two enantiomers (S)-1 and (R)-1 were
obtained as previously reported16 by a two-step procedure
including the condensation of 4-phenylphenol with the optically
active methyl phenyllactates under Mitsunobu conditions fol-
lowed from alkaline hydrolysis (in Scheme 1 the synthesis of
(S)-1 is depicted). However, the employment of triphenylphos-
phine supported on cross-linked styrene/divinylbenzene copoly-
mer resin (PPh3-PS) allowed us to simplify the workup of the
first reaction and to improve the overall yield from 39% to
77-81%, leaving unchanged the enantiomeric excess of g98%.
Transcriptional Activation of PPARr and γ Subtypes
by (S)-1 and (R)-1. In previous studies we had shown that new
enantiomeric compounds were active toward both the PPARR
and γ subtypes.16 To gain more insight on the activity of one
of these ligands displaying a selectivity ratio equal to 1, we
first determined the effect of its enantiomeric forms, (S)-1 and
(R)-1, on the transcriptional activity of a panel of nuclear
receptors. As shown in Figure 2A, (S)-1 activates both PPARR
and PPARγ but does not affect the transcriptional activity of
the other tested nuclear receptors. Conversely, (R)-1 activates
only PPARγ. We next compared the potency and efficacy of
the two enantiomers to those of the known PPARR and γ
agonist, Wy 14,643 and rosiglitazone, respectively. The
concentration-response curves obtained from cell-based assays
indicate that (S)-1 is a more potent PPARR agonist than Wy
14,643, here used as the reference (Figure 2B). The EC50 of
(S)-1 is about 1 order of magnitude lower than that of Wy 14,643
(Table 2), whereas the efficacy is comparable, suggesting that
this compound is a full PPARR agonist. We also tested these
compounds with the murine PPARR, and in this case Wy 14,643
is more potent than (S)-1 (Table 2). The potency of (S)-1 on
the murine PPARR is comparable to that of the human, while
the EC50 of Wy 14,643 with the murine receptor is much lower
Conformational Change Induced by (S)-1 in the
Hydrophobic Pocket of PPARγ. A comparison between the
crystal complex PPARγ/(S)-1 and PPARγ complexes (PDB
codes 1FM9, 1K74, 1ZEO, 2GOG, 2GOH)17-20 including
ligands occupying the “benzophenone pocket”, a contiguous
region of the hydrophobic pocket formed by H3, H7, and H11,
reveals a conformational change induced by (S)-1 in the PPARγ-
LBD. Figure 6 reports the superposition between the crystal
complex of the S-enantiomer with that of farglitazar (PDB code
1FM9). There, it can be seen that the rigid and straight diphenyl
group of the S-enantiomer induces a switching of the F282 side
chain, from the extended conformation (ꢁ1 ) 172°), adopted in
the PDB files cited above, to the folded g* conformation (ꢁ1 )
-58°). As a consequence, the F282 side chain turns to the
benzophenone pocket, leaving free its previous position that is
replaced by the distal ring of the S-enantiomer. In this way, the
F282 side chain could function as a gate-keeper, rendering
accessible a new region for accommodation of long and straight
substituents of the ligand. The size and shape of this region
have been evaluated by the program VOIDOO21 (see Figure
7). The region is “L” shaped and the diphenyl group occupies
the first branch, corresponding to about 50% of its entire volume
(∼1016 Å3). We propose to name this branch “diphenyl pocket”
in analogy with the “benzophenone pocket”. The diphenyl
pocket is delimited at the bottom by the central part of the loop
11/12. In this way the distal aromatic ring of the ligand plays
the key role, normally played by F282, in stabilizing the region

7770 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Montanari et al.
Figure 2. Transcription activity of 1: (A) activity of (S)-1 and (R)-1 was tested toward a panel of nuclear receptor LBDs in a Gal4-based assay;
(B, C) concentration-response curves of (S)-1 and (R)-1 with human (B) PPARR and (C) PPARγ. Results are expressed as a percentage of efficacy,
and each point is the mean ( SEM of three to five independent experiments each performed in triplicate wells.
Table 1. Statistics of Crystallographic Data and Refinement
PPARγ/(S)-1
PPARγ/(R)-1
PPARγ/(S)-2
wavelength (Å)
temperature (K)
space group
no. of mol in the au
cell axes (Å)
ꢀ angle (deg)
resolution range (Å)
Rmerge (%)
multiplicity
I/σ(I)
completeness (%)
Rfactor (%)
1.2
100
C2
2
0.933
100
C2
0.933
100
C2
2
93.14, 61.57, 118.80
103.40
93.54, 61.05, 119.24
103.49
93.25, 60.98, 118.54
102.83
20.00-2.60 (2.69-2.60)a
4.5 (36.6)a
20.00-2.40 (2.53-2.40)a
9.4 (32.7)a
20.00-2.65 (2.79-2.65)a
8.4 (48.6)a
4 (4)a
3.5 (3.3)a
3.7 (3.8)a
25.6 (3.4)a
4.8 (2.0)a
7.7 (1.4)a
99.8 (99.7)a
24.8
98.4 (97.3)a
24.8
98.3 (98.1)a
24.1
a The values in parentheses refer to the outer shell.
Table 2. Potency and Efficacy of (S)-1 and (R)-1 toward PPARR and PPARγ As Determined in Gal4-Based Assaysa
hPPARγ hPPARR
mPPARR
tested ligand
EC50 (µM)
efficacy (%)
100
EC50 (µM)
efficacy (%)
EC50 (µM)
efficacy (%)
rosiglitazone
Wy 14,643
(S)-1
0.04 ( 0.02
1.62 ( 0.34
0.22 ( 0.02
na
100
100(9.5
na
0.04 ( 0.002
0.26 ( 0.06
na
100
120(11
na
0.48 ( 0.08
5.93 ( 2.60
35 (6.5
24 (4.7
(R)-1
a Values are the mean ( SEM. Efficacy values were calculated as percentage of rosiglitazone (100%) for PPARγ and as percentage of Wy 14,643 (100%)
for human and mouse PPARR. Values were calculated from three to five independent experiments each performed in triplicate. na: not active at the tested
concentrations.
including this loop. It is well known that differences in the
hydrophobic packing of this loop may contribute to different
H12 dynamics.22,23 This is the first structure of PPARγ in which
such conformational change of the F282 side chain has been
observed. A similar conformational change induced by the
ligand is also present in the crystal complex between the
PPARR-LBD and the azetidinone derivative PPARR/γ dual
agonist BSM631707 (PDB code 2REW).24 This ligand forces
the side chain of F273, equivalent to F282 in PPARγ, to a folded
conformation that allows the accommodation of its terminal end.
In this regard, we solved the crystal structure of the complex
of PPARγ-LBD with the previously reported16 PPARR/γ dual
agonist (S)-2, structurally related to (S)-1, only differing in the
ethyl group replacing the distal aromatic ring of (S)-1 (see Figure
8).

PPARγ Partial Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7771
Figure 3. 2Fo - Fc electron density maps calculated around (A) (S)-1 and (B) (R)-1. All the maps are contoured at 1σ.
Binding of (R)-1 to PPARγ-LBD. Figure 3B represents the
R-enantiomer fitted into the 2Fo - Fc electron density map.
Figure 5B shows a CR superposition of the complexes PPARγ/
(S)-1 and PPARγ/(R)-1. Inside the cavity, (R)-1 occupies a
different region with respect to (S)-1, between H3 and the
ꢀ-sheet. In this way the polar head of (R)-1 cannot form the
usual network of H-bonds with H323, H449, and Y473, and it
does not interact at all with H11 and H12. The carboxylate group
of (R)-1 forms one H-bond with S342 NH of the ꢀ-sheet and
engages the charged R288 residue of H3 in an electrostatic
interaction. Obviously, the positioning of (R)-1 in a different
region of the cavity leaves the F282 side chain in its usual
conformation. The position of (R)-1 is similar to that of the
partial agonists nTZDpa and BVT.13 in the complexes with
PPARγ (PDB codes 2Q5S and 2Q6S);25,26 also in that case these
compounds formed a H-bond with the NH of S342.
Figure 4. Hydrogen bond network of (S)-1 in complex with PPARγ.
Molecular Mechanism of Partial Agonism. (S)-1 is a
PPARR/γ dual agonist that shows full agonism on PPARR
(Figure 2A and Table 2) and partial agonism on PPARγ (see
Figure 2C and Table 2). To explain this different subtype
behavior, we have docked (S)-1 into the PPARR-LBD and we
have superimposed the putative complex with the crystal
complex PPARγ/(S)-1, as shown in Figure 9A. The polar head
of the ligand in the complex with PPARR is shifted of about
1.5 Å toward H3 with respect to the position adopted in the
crystal complex of PPARγ. This shift is caused by the longer
protrusion of the Y314 side chain of PPARR, equivalent to H323
of PPARγ. As a consequence, the diphenyl group adopts a slope
different from that observed in the crystal complex of PPARγ,
approaching H11 more closely and partially losing interactions
with H3. The favorable van der Waals contacts between the
diphenyl group and residues belonging to H11 of PPARR are
the following: 3.6 and 2.9 Å, respectively, with CR and CO of
V444, 3.4 Å with the side chain of I447, and 3.4 Å with CR of
K448 (see Figure 9B). These contacts, together with that realized
with the loop 11/12, better stabilize the contiguous H12, with
a consequent improvement of the efficacy of the ligand toward
PPARR. On the other hand, the partial agonism of the
S-enantiomer toward PPARγ can be attributed to a lower
stabilization of H11 and increased stabilization of H3, as before
discussed.
Also, in this case the conformational change of the F282 side
chain induced by the ligand (see Supporting Information Figure
2a,b) has been observed. It could be argued that even the
presence of small substituents in the para position of the phenoxy
group of (S)-1 could provoke the rearrangement of F282 side
chain.
Since (S)-1 occupies only the first branch of the new L-shaped
region, we decided to start the preparation of some analogues
characterized from the presence on the diphenyl system of
groups that could allow the complete occupation of the entire
cavity with the aim of evaluating the effects due to a further
stabilization of H11, H12, and the loop 11/12 in terms of
potency, efficacy, and subtype selectivity. At the present, only
the (S)-3 and (S)-4 analogues reported in Figure 8 have been
synthesized and evaluated for their PPARR/γ activity. These
compounds were prepared similarly to (S)-1 (all synthetic details
will be reported elsewhere) and are characterized from an
increased flexibility due to the presence of one or two methylenic
groups between the phenyl rings of (S)-1 which in our mind
could allow the accommodation of the distal aromatic ring inside
the second branch of the L-shaped region. However, the results
do not show any significant modification of the activity on
PPARγ (EC50 ) 0.57 ( 0.01 µM, efficacy ) 40% ( 4% for
(S)-3; EC50 ) 0.40 ( 0.20 µM, efficacy ) 33% ( 5% for (S)-
4) and only an increased potency of PPARR activation for (S)-3
(EC50 ) 0.011 ( 0.002 µM, efficacy ) 107% ( 5% for (S)-3;
EC50 ) 0.15 ( 0.04 µM, efficacy ) 83% ( 10% for (S)-4).
The interpretation of these results by X-ray structure analysis
of the complexes of these new ligands with PPARγ-LBD is in
progress.
In order to check the interaction pattern of (S)-1 with H3
and H11 of both PPARR and PPARγ, we carried out MD
simulations in water solution at 300 K. In this regard, we
considered as an interacting couple, two noncovalently bonded
atoms experiencing a contact shorter than 3.9 Å with a
probability not lower than 0.37, corresponding to a negative
free energy of formation at 300 K. The result, schematically

7772 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Montanari et al.
Figure 5. (A) Hydrophobic interactions of (S)-1 in the PPARγ internal cavity; (B) CR superposition of the complexes of PPARγ with (S)-1
(yellow) and (R)-1 (cyan). H-bonds and electrostatic interactions of (R)-1 are shown by dashed lines.
Figure 8. Chemical structures of the (S)-1 analogues, (S)-2, (S)-3, and
(S)-4.
0.2 Å) characterizes H11 of both apo and complexed forms (see
Supporting Information Figure 3a,b).
Moreover, the analysis of the average distances between the
carboxylate oxygens of the ligand and the Nε2 of H449, the
Nε2 of of H323, the OH of Y473, and the OH of S289 (Nε2 of
H440, OH of Y314, Y464, and S280, respectively, in PPARR)
shows that in the MD simulation of the PPARγ complex only
the H-bond with Y473 is maintained, whereas in the PPARR
complex all the H-bonds are maintained during the simulation
(see Supporting Information Figures 4-11). Thus, the partial
agonism of (S)-1 could also be ascribed to the weakening of
significant H-bonds involving the carboxylate of the ligand with
some conserved residues of the protein, particularly that with
H449 of H11.
Figure 6. CR superposition of the complexes of PPARγ with (S)-1
(yellow) and farglitazar (pink). For clarity only the cartoon of PPARγ/
(S)-1 is shown (green). The orientation of F282 in the two complexes
is also shown (colored as the correspondent ligand).
Recently, H/D exchange kinetics and X-ray experiments have
been performed for six complexes of PPARγ with full and
partial agonists.25 On the basis of the structures of their
complexes, ligands were broadly grouped into those that occupy
the portion of LBD spanning from H11 and H12 beyond H3 to
those that occupy the region between H3 and the ꢀ-sheet.
Among the compounds of the first class, stronger transcriptional
efficacy was achieved with an increased stabilization of H12
and a corresponding decrease of stabilization of H3. The
compounds of this class with weaker transactivation profiles
(like the S-enantiomer here described) preferentially stabilize
H3 through closer hydrophobic contacts or H-bonds made with
residues of this helix. This relationship is in agreement with
our previous findings regarding two enantiomeric ureidofibrate
derivatives complexed with PPARγ, showing partial and full
agonism, respectively, toward this nuclear receptor.27 Even in
that case, while the full agonism of one enantiomer could be
related to stronger interactions with H11, H12, and the loop
11/12, the partial agonism of the other enantiomer could be
ascribed to closer contacts with a residue (Q286) of H3.
Compound (R)-1 belongs to the second class of PPARγ ligands;
compounds belonging to this class differentially stabilize other
Figure 7. New cavity calculated by VOIDOO (blue-colored mesh).
(S)-1 is shown in yellow.
reported in Figure 10, confirms that (S)-1 interacts with both
H3 and H11 of PPARR and only with H3 of PPARγ.
In order to also evaluate the mechanical stabilization caused
by the above interactions, we focused our attention on the
change of the root mean square fluctuation (RMSF) of these
helices, upon complexation with PPARR and PPARγ. As a
result, we observed very low RMSF of both helices in the apo
form of PPARR, the result being practically unaffected upon
complexation. For PPARγ we registered a limited but clear
reduction of the RMSF, from 1.0 ( 0.2 Å (apo form) to 0.7 (
0.2 Å (complexed form) for H3. A rather low RMSF (0.3 (

PPARγ Partial Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7773
Figure 9. (A) CR superposition of PPARγ and PPARR LBD. (S)-1 is respectively complexed with PPARγ (yellow) and docked in the PPARR
LBD (green). (B) Binding pose of (S)-1 (green) into the PPARR LBD as calculated by Gold software.35 The more favorable hydrophobic interactions
are also shown.
Figure 10. Interaction pattern of (S)-1 with H3 and H11 (A) in complex with PPARγ and (B) in complex with PPARR. Residues of H3 and H11
interacting with the ligand at distances shorter than 3.9 Å are colored red. (S)-1, respectively complexed with PPARγ (yellow) and docked in the
PPARR LBD (green), is also shown.
regions of LBD rather than H12, for example, the ꢀ-sheet and
H3, with modest transactivation profiles (efficacy of <50%). It
has also been proposed that this differential stabilization could
suggest a distinct coactivator-binding surface, consistent with
the findings that regions outside the LxxLL motifs contribute
to receptor binding.28 Finally, the potency of (R)-1 is lower than
that of (S)-1 on the same receptor. This behavior can be easily
interpreted at the molecular level by the bindings found in the
relative complexes here reported. While (S)-1 forms direct
H-bonding interactions with H12, (R)-1 does not interact at all
with the active helix.
results obtained with two (S)-1 analogues endowed with
increased flexibility do not show remarkable change of PPARγ
activity; however, more structural modifications are needed for
an appropriate evaluation of this matter. For this purpose, the
preparation of new derivatives characterized by the presence
on the diphenyl system of groups with different stereoelectronic
properties is under way. A molecular mechanism not only
considering the stabilization of H12 but including even the
increased stabilization of H3 and the ꢀ-sheet has been elucidated
for the coactivator recruitment to the receptor in response to
ligands showing graded transcriptional responses. MD simula-
tions in water solution also showed that the possible weakening
of significant H-bonds realized by the ligand could be respon-
sible for its different subtype behavior.
Conclusions
New drug design strategies should be considered when
docking ligands on PPARs in light of the data here reported. In
fact, the F282 side chain conformation in PPARγ can be
changed, rendering accessible a new region never explored
before. Moreover, since (S)-1 occupies only the first branch of
this new region, it is evident that additional space is available
for bulkier substituents protruding toward H11 and H12. The
complete occupation of the entire cavity would confer a further
stabilization of H11, H12, and the loop 11/12 and could affect
potency, efficacy, and PPARR/PPARγ selectivity. This is an
important point, as the design of new molecules occupying this
pocket may provide the opportunity to modulate the receptor
activity via the selective recruitment of coactivators that confer
beneficial effects but avoid at the same time the typical side
effects observed with known PPARγ ligands. The preliminary
Experimental Section
Chemical Methods. Column chromatography was performed on
ICN silica gel 60 Å (63-200 µm) as the stationary phase. Melting
points were determined in open capillaries on a Gallenkamp
electrothermal apparatus and are uncorrected. Mass spectra were
recorded with a HP GC/MS 6890-5973 MSD spectrometer, electron
impact 70 eV, equipped with an HP chemstation. 1H NMR spectra
were recorded in CDCl3 on a Varian-Mercury 300 (300 MHz)
spectrometer. Chemical shifts are expressed as parts per million
(δ). Microanalyses of solid compounds were carried out with an
Eurovector Euro EA 3000 model analyzer; the analytical results
are within (0.4% of theoretical values. Optical rotations were
measured with a Perkin-Elmer 341 polarimeter at room temperature
(20 °C): concentrations are expressed as g/(100 mL). The enan-

7774 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Montanari et al.
tiomeric excesses of acids were determined by HPLC analysis of
their methyl esters, obtained by reaction with an ethereal solution
of diazomethane, on a Chiralcel OD column (4.6 mm i.d. × 250
mm, Daicel Chemical Industries, Ltd., Tokio, Japan). Analytical
liquid chromatography was performed on a PE chromatograph
equipped with a Rheodyne 7725i model injector, a 785A model
UV/vis detector, a series 200 model pump, and an NCI 900 model
interface. Chemicals were from Aldrich and were used without any
further purification.
GraphPad Prism, version 5.0a, for Macintosh (GraphPad Software,
San Diego, CA, www.graphpad.com).
Protein Expression, Purification, and Crystallization. The
LBD of PPARγ was expressed as N-terminal His-tagged protein
using a pET28 vector and purified as previously described.27
Crystals of apo-PPARγ were obtained by vapor diffusion at 20 °C
using the sitting drop made by mixing 2 µL of protein solution (15
mg/mL in 20 mM Tris, DTT 5 mM, 0.5 mM EDTA, pH 8.0) with
2 µL of reservoir solution (0.8 M Na citrate, 0.15 mM Tris, pH
8.0). The crystals were soaked for 3 days in a storage solution (1.2
M Na citrate, 0.15 M Tris, pH 8.0) containing the ligands (0.5 mM).
The ligands dissolved in ethanol were diluted in the storage solution
so that the final ethanol concentration was 1%. The storage solution
with glycerol 20% (v/v) was used as cryoprotectant. Crystals of
PPARγ/(S)-1, PPARγ/(R)-1, and PPARγ/(S)-2 all belong to the
space group C2 with cell parameters shown in Table 1. The
asymmetric unit is formed by one homodimer (53.5% solvent for
all complexes).
Structure Determination. X-ray data were collected at 100 K
under a nitrogen stream using synchrotron radiation (beamline
XRD1 at Elettra, Trieste, Italy, for the complex PPARγ/(S)-1;
beamline ID14-1 at ESRF, Grenoble, France, for the other
complexes). The diffracted intensities were processed using the
programs DENZO and SCALEPACK29 for the complex PPARγ/
(S)-1 and using MOSFLM and SCALA30 for the other complexes.
Refinements were performed with CNS31 using the coordinates of
apo-PPARγ32 (PDB code 1PRG) as a starting model. All data
between 8 and 2.6 Å were included for PPARγ/(S)-1 (between 8
and 2.4 Å for PPARγ/(R)-1; between 8 and 2.4 Å for PPARγ/(S)-
2). The statistics of crystallographic data and refinement are
summarized in Table 1. The coordinates of PPARγ/(S)-1, PPARγ/
(R)-1, and PPARγ/(S)-2 complexes have been deposited in the
Brookhaven Protein Data Bank (PDB) with the codes 3B3K, 3D6D,
and 3CDS, respectively.
Preparation of (S)-Methyl 2-(4-Phenylphenoxy)-3-phenyl-
propanoate. To an ice-bath cooled suspension of triphenylphos-
phine-PS (DVB 2%, 3 mmol ·g-1, 0.460 g, 1.380 mmol) in
anhydrous toluene (5 mL) was added dropwise a solution of
diisopropylazodicarboxylate (DIAD, 0.273 g, 1.350 mmol) in
anhydrous toluene (5 mL). The resulting mixture was stirred for
0.5 h. A solution of (R)-methyl phenyllactate (0.201 g, 1.095 mmol)
in anhydrous toluene (5 mL) and 4-phenylphenol (0.173 g, 1.016
mmol) was added at room temperature. The reaction mixture was
stirred at room temperature overnight, the solid was filtered off,
and the filtrate was evaporated to dryness to give a white residue.
The title compound was purified by chromatography on silica gel
column (petroleum ether/ethyl acetate 9:1 as eluents), obtaining a
white solid (0.329 g, 0.991 mmol) in 97% yield. GC/MS, m/z: 332
(M+, 100). 1H NMR (CDCl3): δ 3.26-3.29 (m, 2H, CH2), 3.74 (s,
3H, CH3), 4.83-4.88 (dd, 1H, CH), 6.89-7.53 (m, 14H, aromatics).
The R-enantiomer was prepared following the same procedure,
starting from (S)-methyl phenyllactate, in 95% yield.
(-)-(S)-2-(4-Phenylphenoxy)-3-phenylpropanoic Acid. A solu-
tion of the methyl ester (0.301 g, 0.907 mmol) in THF (5 mL) and
1 N NaOH (5 mL) was stirred at room temperature for 4 h. The
organic layer was removed under reduced pressure, and the residue
was acidified with 6 N HCl and extracted with Et2O. The organic
layer was dried over Na2SO4 and evaporated to dryness, affording
the final acid as a white solid that was recrystallized from hexane
in 84% yield (0.242 g, 0.761 mmol). 1H NMR (CDCl3): δ
3.22-3.38 (m, 2H, CH2), 4.84-4.94 (m, 1H, CH), 5.70 (bs, 1H,
COOH, D2O exchanged), 6.88-7.60 (m, 14H, aromatics). Mp
149-50 °C; [R]D -1 (c 1.0, MeOH); 99% ee (methyl ester on
Chiralcel OD column, hexane/i-PrOH 98:2 as the mobile phase,
flow rate of 0.5 mL/min, detection at 230 nm).
Computational Chemistry. Molecular modeling and graphics
manipulations were performed using the molecular operating
environment (MOE)33 running on a 2 CPU (PIV 2.0-3.0 GHz)
Linux workstation. Energy minimizations were realized by employ-
ing the AMBER 9 program,34 selecting the parm99 force field.35
Model building and geometry optimizations of (S)-1 enantiomer
were accomplished with the MMFF94X force field, available within
MOE. The carboxylate group was taken as dissociated. The
coordinates of PPARR in the complex with the R/γ dual agonist
BSM631707 (PDB code 2REW)24 were used in the docking
experiments. Bound ligand and water molecules were removed. A
correct atom assignment for N, Q, and H residues was done, and
hydrogen atoms were added using standard MOE geometries. Partial
atomic charges were computed by MOE using the AMBER99 force
field. All heavy atoms were then fixed, and hydrogen atoms were
minimized using the AMBER99 force field and a constant dielectric
(+)-(R)-2-(4-Phenylphenoxy)-3-phenylpropanoic Acid. Yield,
81%; mp 148-9 °C; [R]D +1 (c 1.0, MeOH); 98% ee (methyl ester
on Chiralcel OD column, hexane/i-PrOH 98:2 as the mobile phase,
flow rate of 0.5 mL/min, detection at 230 nm).
Cell-Based Transcription Assay. The expression vectors for
PPARR, PPARγ, and PPARꢀ/δ LBD fused in frame with the Gal4
DNA binding domain (pGal4-PPARRLBD, pGal4-PPARγLBD,
pGal4-PPARꢀ/δLBD, respectively) and the reporter vector contain-
ing five copies of the Gal4 upstream activating sequences
(pGal4UAS-luciferase) driving the transcription of the luciferase
reporter gene were kind gifts from Krister Bamberg (AstraZeneca,
Mo¨lndal, Sweden). pCMVꢀ, containing the E. coli ꢀ-galactosidase
gene driven by the cytomegalovirus early promoter/enhancer
(Clontech, Mountain View, CA) was used as an internal standard
to normalize for transfection efficiency across wells. All other
vectors expressing Gal4 fusion proteins with the ligand binding
domains of tested nuclear receptors have been described previ-
ously.27 Cell-based transcription assays were performed by trans-
fecting HepG2 cells with 100 ng of pGal4UAS-luciferase along
with 50 ng of receptor vector and 300 ng of pCMVꢀ with the
calcium phosphate coprecipitation technique for 4 h as described.16
After removal of the DNA coprecipitates, cells were washed with
phosphate-buffered saline and refed with serum-free medium
(Dulbecco’s modified Eagle medium/F-12 nutrient 1:1, Invitrogen,
Milano, Italy) containing 100 U of penicillin G/mL and 100 µg of
streptomycin sulfate/mL and the indicated concentrations of tested
ligands or vehicle (0.1% ethanol). After 20 h, cells were harvested
and luciferase and ꢀ-galactosidase activities were measured as
described.16 All transfection experiments were repeated at least three
times. EC50 values for all tested ligands were calculated by using
of 1, terminating at a gradient of 0.001 kcal mol-1 Å-1
.
Enantiomer (S)-1 was docked into the active site of PPARR using
the GOLD 3.2 program,36 computing interaction energies within a
sphere of a 18 Å radius centered on the OH atom of Y464 in the
PPARR structure. The poses obtained with the original Goldscore
function are rescored and reranked with the GOLD implementation
of the CHEMscore function. To perform a thorough and unbiased
search of the conformation space, each docking run was allowed
to produce 30 poses without the option of early termination, using
standard default settings. In this docking run, the 30 poses produced
by GOLD resulted in only one cluster on the basis of their
conformations. The top solution (CHEMscore fitness ) 41.2 kJ/
mol) obtained after reranking of the poses with CHEMscore was
selected to generate the PPARR/(S)-1 complex.
To eliminate any residual geometric strain, the obtained complex
was energy-minimized for 5000 steps using combined steepest
descent and conjugate gradient methods until a convergence value
of 0.001 kcal/(mol Å). Upon minimization, the protein backbone
atoms were held fixed. The geometry optimization was performed
using the SANDER module in the AMBER suite of programs,
employing the Cornell et al. force field to assign parameters for

PPARγ Partial Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7775
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D. Crystal structure of a mutant hERalpha ligand-binding domain
reveals key structural features for the mechanism of partial agonism.
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reveals the molecular basis of heterodimerization among nuclear
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13924.
the standard amino acids. General AMBER force field (GAFF)
parameters were assigned to ligands, while the partial charges were
calculated using the AM1-BCC method as implemented in the
ANTECHAMBER suite of AMBER.
Molecular Dynamics (MD) simulations of PPARγ/(S)-1 and
PPARR/(S)-1 were carried out in aqueous solution according to
the following protocol.
The complexes were put at the center of a box of 7.2 × 7.6 ×
7.6 nm3 volume. As starting structures, we considered the complex
formed by docking (S)-1 into the PPARR-LBD, and the crystal
complex of PPARγ/(S)-1. The box was then filled with 11 476
Single Point Charges37 water molecules to reproduce the typical
liquid density. The electrical neutrality of the systems was ensured
by adding the proper number of positive (Na+) counterions, i.e.,
seven for PPARγ/(S)-1 and two for PPARR/(S)-1. The same
conditions were applied for simulating apo-PPARγ and apo-
PPARR. All the simulations were initiated with a steepest descent
optimization and solvent relaxation. Subsequently, a slow heating
from 10 to 300 K was performed using short simulations of 50.0
ps. The simulations were then propagated for 13 ns in a NVT
ensemble using an integration step of 2.0 fs with the rototransla-
tional constraint applied to the solute.38 The temperature was kept
constant by the isokinetic temperature coupling,39 and all bond
lengths were constrained by adopting the LINCS algorithm.40 Long
range electrostatics was computed by the particle mesh Ewald
(PME) method,41 with 34 wave vectors in each dimension and a
fourth-order cubic interpolation. The Gromos force field42 was used
for the protein, at neutral pH, and for (S)-1. The point charges of
the latter were calculated with a standard fitting scheme43 carried
out on density functional theory calculations using the Becke3LYP
functional44 with 6-311+g(d) basis set using the Gaussian 03
package.45 The Gromacs software46 was employed for all the MD
runs.
(19) Shi, G. Q.; Dropinski, J. F.; McKeever, B. M.; Xu, S.; Becker, J. W.;
Berger, J. P.; MacNaul, K. L.; Elbrecht, A.; Zhou, G.; Doebber, T. W.;
Wang, P.; Chao, Y. S.; Forrest, M.; Heck, J. V.; Moller, D. E.; Jones,
A. B. Design and synthesis of alpha-aryloxyphenylacetic acid deriva-
tives: a novel class of PPARalpha/gamma dual agonists with potent
antihyperglycemic and lipid modulating activity. J. Med. Chem. 2005,
48 (13), 4457–4468.
Statistical evaluations on selected observables were calculated
by dividing the trajectory in two subparts and reporting the
semidispersion around the average value.
(20) Lu, I. L.; Huang, C. F.; Peng, Y. H.; Lin, Y. T.; Hsieh, H. P.; Chen,
C. T.; Lien, T. W.; Lee, H. J.; Mahindroo, N.; Prakash, E.; Yueh, A.;
Chen, H. Y.; Goparaju, C. M.; Chen, X.; Liao, C. C.; Chao, Y. S.;
Hsu, J. T.; Wu, S. Y. Structure-based drug design of a novel family
of PPARgamma partial agonists: virtual screening, X-ray crystal-
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(21) Kleywegt, G. J.; Jones, T. A. Acta Crystallogr. 1994, D50, 178–185.
(22) Nettles, K. W.; Sun, J.; Radek, J. T.; Sheng, S.; Rodriguez, A. L.;
Katzenellenbogen, J. A.; Katzenellenbogen, B. S.; Greene, G. L.
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and beta: implications for other nuclear receptors. Mol. Cell 2004, 13
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Acknowledgment. The authors acknowledge CASPUR
(Roma) for the use of Gaussian 03.
Supporting Information Available: X-ray and molecular
dynamics data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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