Structure-based design and synthesis of fluorescent PPARα/δ co-agonist and its application as a probe for fluorescent polarization assay of PPARδ ligands

Article abstract of DOI:10.1248/cpb.56.1357

Based on the result of X-ray crystallographic analysis of our peroxisome proliferator-activated receptor alpha and delta (PPARα/δ) co-agonist complexed with human PPAR ligand binding domain (LBD), we designed and synthesized an optically active fluorescent PPARα/δ co-agonist, which has a pyrene unit incorporated directly at the hydrophobic tail part of the structure as a fluorophore. This fluorescent co-agonist was applied in a homogeneous fluorescent polarization assay format for the identification of PPARδ ligands.

Full text of DOI:10.1248/cpb.56.1357

September 2008
Notes
Chem. Pharm. Bull. 56(9) 1357—1359 (2008)
1357
a d
Structure-Based Design and Synthesis of Fluorescent PPAR /
Co-agonist and Its Application as a Probe for Fluorescent Polarization
d
Assay of PPAR Ligands
Yo ko A RAYA,a Jun-ichi KASUGA,a Kenji TOYOTA,b Yuko HIRAKAWA,b Takuji OYAMA,b
Makoto MAKISHIMA, Kosuke MORIKAWA, Yuichi HASHIMOTO, and Hiroyuki MIYACHI
c
b
a
,a
*
a Institute of Molecular and Cellular Biosciences, University of Tokyo; Yayoi, Bunkyo-ku, Tokyo 113–0032, Japan: b The
Takara-Bio Endowed Division, Institute for Protein Research, Osaka University; 6–2–3 Furuedai, Suita, Osaka 565–0874,
Japan: and c School of Medicine, Nihon University; 30–1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173–8610, Japan.
Received May 20, 2008; accepted June 18, 2008
Based on the result of X-ray crystallographic analysis of our peroxisome proliferator-activated receptor
alpha and delta (PPARa/d) co-agonist complexed with human PPAR ligand binding domain (LBD), we designed
and synthesized an optically active fluorescent PPARa/d co-agonist, which has a pyrene unit incorporated di-
rectly at the hydrophobic tail part of the structure as a fluorophore. This fluorescent co-agonist was applied in a
homogeneous fluorescent polarization assay format for the identification of PPARd ligands.
Key words fluorescent ligand; phenylpropanoic acid; fluorescent polarization; peroxisome proliferator-activated receptor; fluo-
rescent peroxisome proliferator-activated receptor a/d co-agonist
Peroxisome proliferator-activated receptors (PPARs) are
As a part of our continuing research directed toward the
nuclear receptors with pleiotropic biological functions that structural development of characteristically subtype-selective
include regulatory roles in lipid, lipoprotein and glucose PPAR agonists, we required a rapid and simple method to de-
homeostasis. PPARs are activated by endogenous saturated termine PPARd binding activity without using isotope-la-
and unsaturated fatty acids and their metabolites and syn- beled material(s). Here, we report the design and the synthe-
thetic ligands.1) Three subtypes have been isolated to date: sis of a fluorescent PPARa/d co-agonist, suitable for use in a
PPARa (NR1C1), PPARd (NR1C2) and PPARg (NR1C3). homogeneous fluorescent polarization assay format for the
PPARa is mostly expressed in tissues involved in lipid oxi- identification of both endogenous and exogenous PPARd lig-
dation, such as liver, kidney, skeletal, cardiac muscle and ands. Perterson and co-worker have designed and synthesized
adrenal glands.2) PPARg is expressed in adipose tissue, a fluorescent PPARg agonist, based on GlaxoSmithKline
macrophages and vascular smooth muscles.3) In contrast to (GSK)’s potent PPARg agonist farglitazar, for high-through-
the specific distributions of PPARa and PPARg, PPARd is put screening of PPARg ligands.5) However, their ligand
ubiquitously expressed.4)
might not be suitable for the screening of other PPAR sub-
Two of these subtypes are the molecular targets of drugs types, such as PPARd, because the PPARg-selectivity of the
used for the treatment of dyslipidemia and type II diabetes mother compound, farglitazar, is extremely high (more than
mellitus, i.e., the dyslipidemic fibrate class of compounds are 1000-fold selectivity for PPARg over PPARa or PPARd).
agonists of PPARa, while the insulin-sensitising glitazones
Previously, we designed and synthesized a potent and
are agonists of PPARg. On the other hand, PPARd is the human PPARa/d co-agonist, TIPP-401 (2), based on KCL
least well investigated subtype. However, the availability of (1), a PPARa-selective agonist, as a lead compound (Fig.
PPARd knockout animals and selective ligands prompted us 1A).6) We have solved the X-ray crystallographic structure of
to examine the involvement of PPARd in fatty acid metabo- TIPP-401 complexed with human PPARd ligand binding do-
lism, insulin resistance, reverse cholesterol transport, and in- main at 2.7 A resolution (Fig. 1B) (The Protein Data Bank
flammation. Activation of PPARd was reported to improve code for the PPARd/TIPP-401 complex is 2ZNQ). The PPAR
glucose tolerance and insulin resistance in ob/ob mice. Con- ligand-binding pocket is reported to form a large Y-shaped
sequently, there is increasing interest in this receptor as a tar- cavity that extends from the C-terminal helix to the b-sheet
get for drug development.
lying between helices H3 and H6.7) We confirmed the pres-
Fig. 1. (A) Structures of KCL and TIPP-401, (B) an Overview of the Structure of TIPP-401 Complexed with hPPARd Ligand Binding Domain (LBD) and
(C) Zoomed View of the Hydrophobic Tail Part of TIPP-401
∗ To whom correspondence should be addressed. e-mail: miyachi@iam.u-tokyo.ac.jp
© 2008 Pharmaceutical Society of Japan

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Vol. 56, No. 9
Reagents and conditions; a) pyrene-1-carboxamide, triethylsilane, trifluoroacetic acid, toluene, reflux, 24 h, 70%; b) LiOH H2O, 30% H2O2, THF : H2Oꢂ4 : 1 (v/v), 0 °C, 85%.
Chart 1. Synthetic Route to Fluorescent PPARa/d Co-agonist
ence of the cavity in our structure, and found that the
hydrophobic tail part, the 2-fluoro-4-trifluoromethylphenyl
group, of TIPP-401, is located in the narrow bottom cavity of
the Y-shaped pocket composed of Trp264, Val281, Phe282,
Arg284, Leu339, Val341 and Val348 (Fig. 1C). The depth of
the bottom cavity is limited by a wall composed of the
bulkier side chains of Trp264 and Arg284. Based on these
insights, we anticipated that a structurally planar and smaller
fluorophore, pyrene, could be incorporated directly as a hy-
drophobic tail part of the molecule to afford a fluorescent
PPAR agonist without losing the potent PPARa/d co-agonist
nature.
Chemistry The benzaldehyde derivative 48) was amide-
alkylated9) with pyrene-1-carboxamide (5), followed by re-
moval of the chiral auxiliary to afford the desired (S)-config-
uration product, 3 (Chart 1).10) The antipodal (R) enantiomer
was prepared via similar procedures, using (S)-N-butyryl-4-
benzyloxazolidinone as the reagent.
Fig. 2. Fluorescence Polarization Changes upon Addition of hPPARa (A)
and hPPARd (B) to (S)-3 (100 nM) in PBS and (C) the Dose–Response Rela-
tionships of the Binding of Representative Compounds to PPARd
Results and Discussion
As expected, (S)-3 exhibits PPARa/d co-agonistic activity be more than 1000 nM (data not shown). Based on these ex-
as potent as that of TIPP-401, while the PPARg agonistic periments, the optimal PPARa and PPARd LBD concentra-
activity is weak. The EC50s (nM) towards PPARd/PPARa/ tion for subsequent competition binding experiments was de-
PPARg were 40/10/ꢀ10000 for (S)-3, 400/100/ꢀ10000 for termined to be 500 nM in both cases.
(R)-3, and 24/10/2200 for TIPP-401 in our assay system (the
Thus, we have successfully constructed a homogeneous
EC50 values were derived by curve-fitting using Origin® soft- fluorescent polarization assay method for measuring the
ware (Origin Lab)). Clear enantio-dependency of the transac- binding of test compounds with PPARd. Using this method,
tivation activity towards the PPAR subtypes was also found, we reevaluated our synthetic PPARd ligands (JKPL-5, JKPL-
and (S)-3 exhibited more potent PPARa/d transactivation ac- 22, and JKPL-67). The PPARd agonistic activities of these
tivity than the antipodal R isomer, (R)-3. Thus, we obtained a compounds had previously been evaluated by means of a
fluorescent PPARa/d co-agonist by introducing a pyrene functional biochemical method of transactivation assay, but
group as a fluorophore directly in place of the 2-fluoro-4-tri- the binding abilities remained uncertain. As shown in Fig.
fluoromethylphenyl group of TIPP-401 (2).
2C, the dose-dependent decrease in the relative fluorescent
In order to investigate the utility of (S)-3 as a fluorescent polarization values indicates that these compounds bind
probe, the dependence of the spectroscopic properties upon PPARd dose-dependently. The rank order of the binding ac-
the polarity of the solvent was examined. We found, that the tivity was JKPL-5ꢁJKPL-22ꢁJKPL-67, which is consistent
physicochemical properties were not greatly affected by the with the rank order of functional PPARd transactivation ac-
solvent polarity, i.e., the excitation maxima and the emission tivity. These results confirm the usefulness of the present
maxima of (S)-3 are 350 nm and 399 nm in phosphate buffer assay method.
saline (PBS) containing 0.1% DMSO and 354 nm and
394 nm in CH2Cl2, respectively.
Then, we applied this method to evaluate the binding abil-
ity of naturally occuring fatty acid derivatives. n-3 Polyunsat-
To examine the binding of PPARa and PPARd to this flu- urated fatty acids (PUFA), such as eicosapentaenoic acid
orescent probe, the human PPARa (or human PPARd) ligand (EPA) and docosahexaenoic acid (DHA), have been reported
binding domain (LBD) was added to PBS containing (S)-3 at to improve lipid profiles,11) reduce blood pressure,12) improve
the concentration of 100 nM (this concentration provides suf- insulin resistance,13) and so on. These beneficial pharmaco-
ficient fluorescence polarization). In both cases, saturation of logical effects of EPA and DHA are thought to be mediated
binding to the PPAR LBD was obtained (Figs. 2A, B), and through binding to and activating PPARs, especially PPARa
the affinity constants (Kd) were determined to be 97 nM and and PPARg. We hypothesize that PPARd binding and activa-
120 nM, respectively (The Kd values of the tested compounds tion also contribute to the effects of PUFA. The results are
were derived by curve-fitting using Origin® software (Origin summarized in Fig. 3. As expected, the PUFAs tested bind
Lab.)). On the other hand, the binding was not saturated in dose-dependently to PPARd. We found that the binding was
the case of human PPARg LBD, even when 1000-fold excess independent of the degree of unsaturation of the fatty acids,
of (S)-3 was used, and the affinity constant (Kd) appeared to although saturated fatty acids tested did not exhibit signifi-

September 2008
1359
v/v) to obtain 218 mg (74%) of the intermediate 5 as a colorless oil. The in-
termediate 5 (210 mg, 0.34 mmol) was dissolved in 20 ml of a mixed solvent
of tetrahydrofuran and water (4 : 1 v/v), under an argon atmosphere with
ice-cooling. To this solution was added 30% aqueous hydrogen peroxide
(0.17 ml, 1.50 mmol). Then lithium hydroxide monohydrate (21.0 mg,
0.50 mmol) was added, and the mixture was stirred overnight under ice-cool-
ing. Sodium hydrogen sulfite solution was added dropwise. The reaction
mixture was concentrated, poured into ice water, acidified with 5% HCl. The
whole was extracted with ethyl acetate, washed with water and brine, dried
over anhydrous magnesium sulfate and concentrated. The residue was
purified by silica gel column chromatography (eluant; n-hexane : ethyl
acetateꢂ2 : 3 v/v) to afford 104 mg (67%) of the title compound as a color-
1
less crystals; H-NMR (500 MHz, CDCl3) d 8.46 (d, 1H, Jꢂ9.4 Hz), 8.17
(m, 2H), 7.98 (m, 5H), 7.32 (s, 1H), 7.10 (d, 1H, Jꢂ5.6 Hz), 6.80 (d, 1H,
Jꢂ5.6 Hz), 6.63 (m, 1H), 4.70 (m, 2H), 3.82 (s, 3H), 2.87 (m, 1H), 2.74 (m,
1H), 2.56 (m, 1H), 1.50 (m, 2H), 0.94 (t, 3H, Jꢂ7.5 Hz); HR-MS; (MꢃH)ꢃ
Calcd for C30H27NO4, 466.2018, Found 466.2029; Anal. Calcd for
C30H27NO4: C, 77.40; H, 5.85; N, 3.01. Found: C, 77.24; H, 6.07; N, 3.00;
[a]D 24.6° (cꢂ0.20, MeCN).
Fluorescence Polarization Assays Assays employed compound (S)-3
(conc.ꢂ500 nM) in binding buffer (10 mM HEPES, MgCl2 2 mM, NaCl
150 mM, HEPES 10 mM, DTT 5 mM pH 7.3). The data shown in Figs. 2 and 3
were obtained with a JASCO spectrofluorometer FP-6500. The Kd values
were calculated by nonlinear least squares curve fitting using binding mod-
els from ORIGIN® (lightstone Corp., Japan).
Fig. 3. Dose–Response Relationships of the Fluorescence Polarization of
Fatty Acid Derivatives
cant binding at the concentration of 100 mM. The binding
ability of the PUFAs was lost upon methyl esterification, but
Acknowledgement The work described in this paper was partially sup-
ported by Grants-in-Aid for Scientific Research from The Ministry of Edu-
interestingly, acyl-CoA esterification restored the binding cation, Culture, Sports, Science and Technology of Japan.
ability. All the acyl-CoA ester derivatives of PUFAs tested
References
bound dose-dependently to PPARd independently of the de-
gree of unsaturation of the PUFAs. These results are con-
1) Alberti K. G., Zimmet P. Z., Diabet. Med., 15, 539—553 (1998).
2) Mukherjee R., Jow L., Noonan D., McDonnell D. P., J. Steroid
sistent with our hypothesis the beneficial pharmacological ef-
fects of PUFAs are mediated through binding to and activat-
ing all PPAR subtypes.
In summary, we have developed a fluorescent PPARa/d
co-agonist suitable for use in a homogeneous fluorescent po-
larization assay format for the screening of PPARd ligands.
The optimization of the assay format and its application to
find structurally new PPARd ligands among compounds of
natural or synthetic origin is in progress.
Biochem. Mol. Biol., 51, 157—166 (1994).
3) Okuno A., Tamemoto H., Tobe K., Ueki K., Mori Y., Iwamoto
K., Umesono K., Akanuma Y., Fujiwara T., Horikoshi H., Yazaki Y.,
Kadowaki T., J. Clin. Invest., 101, 1354—1361 (1998).
4) Willson T. M., Brown P. J., Sternbach D. D., J. Med. Chem., 43, 527—
550 (2000).
5) DeGrazia M. L., Thompson J., Vanden Heuvel J. P., Peterson B. R.,
Bioorg. Med. Chem., 11, 4325—4332 (2003).
6) Kasuga J., Makishima M., Hashimoto Y., Miyachi H., Bioorg. Med.
Chem. Lett., 16, 554—558 (2006).
7) Nolte R. T., Wisely G. B., Westin S., Cobb J. E., Lambert M. H.,
Kurokawa R., Rosenfeld M. G., Willson T. M., Glass C. K., Milburn
M. V., Nature (London), 395, 137—143 (1998).
Experimental
Melting points were determined by using a Yanagimoto hot-stage melting
point apparatus and are uncorrected. Elemental analyses were carried out in
the Microanalytical Laboratory, Faculty of Pharmaceutical Sciences, Univer-
sity of Tokyo, and were within plus or minus 0.3% of the theoretical values.
NMR spectra were recorded on a JEOL JNM-GX500 (500 MHz) spectrome-
ter. Chemical shifts are expressed in ppm relative to tetramethylsilane. Mass
spectra were recorded on a JEOL JMS-DX303 spectrometer.
Preparation of (S)-2-(4-Methoxy-3-((pyrene-1-carboxamido)methyl)-
benzyl)butanoic Acid A mixture of 4 (187 mg, 0.47 mmol), pyren-1-yl-
carboxamide (348 mg,1.42 mmol), triethylsilane (0.20 ml, 1.42 mmol), triflu-
oroacetic acid (0.10 ml, 1.42 mmol), and 30 ml of dehydrated toluene was re-
fluxed overnight. The mixture was evaporated, and the residue was purified
by silica gel column chromatography (eluant; n-hexane : ethyl acetateꢂ2 : 1
8) Kasuga J., Hashimoto Y., Miyachi H., Bioorg. Med. Chem. Lett., 16,
771—774 (2006).
9) Dube D., Scholte A. A., Tetrahedron Lett., 40, 2295—2298 (1999).
10) Kasuga J., Yamasaki D., Araya Y., Nakagawa A., Makishima M., Doi
T., Hashimoto Y., Miyachi H., Bioorg. Med. Chem., 14, 8405—8414
(2006).
11) Mori T. A., Burke V., Puddey I. B., Watts G. F., O’Neal D. N., Best J.
D., Beilin L. J., Am. J. Clin. Nutr., 71, 1085—1094 (2000).
12) Prisco D., Paniccia R., Bandinelli B., Filippini M., Francalanci I.,
Giusti B., Giurlani L., Gensini G. F., Abbate R., Neri Serneri G. G.,
Thromb. Res., 91, 105—112 (1998).
13) Holness M. J., Greenwood G., Smith N., Sugden M., Endocrinology,
144, 3958—3968 (2003).