Binding analyses between Human PPARc–LBD and ligandsSurface plasmon resonance biosensor assay correlating with circular dichroic spectroscopy determination and molecular docking Changyin
Trang 1Binding analyses between Human PPARc–LBD and ligands
Surface plasmon resonance biosensor assay correlating with circular dichroic
spectroscopy determination and molecular docking
Changying Yu1,2, Lili Chen1, Haibing Luo1, Jing Chen1, Feng Cheng1, Chunshan Gui1, Ruihao Zhang1, Jianhua Shen1, Kaixian Chen1, Hualiang Jiang1and Xu Shen1
1 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 2 College of Marine Life Sciences,
Ocean University of China, Qingdao, China
The binding characteristics of a series of PPARc ligands
(GW9662, GI 262570, cis-parinaric acid, 15-deoxy-D12,14
-prostaglandin J2, LY171883, indomethacin, linoleic acid,
palmitic acid and troglitazone) to human PPARc ligand
binding domain have been investigated for the first time by
using surface plasmon resonance biosensor technology, CD
spectroscopy and molecular docking simulation The surface
plasmon resonance biosensor determined equilibrium
dis-sociation constants (KDvalues) are in agreement with the
results reported in the literature measured by other methods,
indicating that the surface plasmon resonance biosensor can
assume a direct assay method in screening new PPARc
agonists or antagonists Conformational changes of PPARc
caused by the ligand binding were detected by CD
deter-mination It is interesting that the thermal stability of the
receptor, reflected by the increase of the transition
tem-perature (Tm), was enhanced by the binding of the ligands
The increment of the transition temperature (DTm) of
PPARc owing to ligand binding correlated well with the
binding affinity This finding implies that CD could possibly
be a complementary technology with which to determine the binding affinities of ligands to PPARc Molecular docking simulation provided reasonable and reliable binding models
of the ligands to PPARc at the atomic level, which gave a good explanation of the structure-binding affinity relation-shipfor the ligands interacting with PPARc Moreover, the predicted binding free energies for the ligands correlated well with the binding constants measured by the surface plasmon resonance biosensor, indicating that the docking paradigm used in this study could possibly be employed in virtual screening to discover new PPARc ligands, although the docking program cannot accurately predict the absolute ligand-PPARc binding affinity
Keywords: PPARc; receptor binding; surface plasmon resonance biosensor; circular dichroism spectroscopy; molecular docking
The peroxisome proliferator-activated receptor (PPAR)
belongs to the nuclear receptor superfamily [1] that plays
an important role in the regulation of the storage and
catabolism of dietary fats [2] PPAR contains three
subtypes, PPARa, PPARb (also termed PPARd) and PPARc PPARc is a ligand-dependent transcription factor influencing the adipocyte differentiation and glucose homeostasis [3] Binding of ligands to PPARc causes conformational change in the receptor Upon binding of
an agonist to PPARc, a-helices H12, H3, H4, and H5 of the receptor form a charge clamp and a hydrophobic pocket, which are essential for the recruitment of coactivator– receptor complexing and the transcriptional activation of the PPARc target genes [4,5] It has been demonstrated that PPARc is the receptor of the thiazolidinedione (TZD) class
of ligands [6] Among the TZD type of anti-diabetic drugs, rosiglitazone and troglitazone are potent adipocyte differ-entiating agents, which activate ap2 gene expression in a PPARc-dependent manner [7] As PPARc ligands may regulate the adipogenesis, they can be designed and modified for the treatment of cardiovascular and diabetes diseases [2] Therefore, PPARc is an attractive target for new drug discovery
Ligand binding to PPARc is responsible for controlling the biological functions, and discovering new ligands that may modulate PPARc’s function is a major focus in the pharmaceutical industry Accordingly, using new technol-ogy to measure ligand–PPARc binding is significant for
Correspondence to H Jiang, Drug Discovery and Design Center,
State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, China.
Fax: + 86 21 50806918, Tel.: + 86 21 50807188,
E-mail: jiang@iris3.simm.ac.cn and X Shen, address as above.
Fax: + 86 21 50807088, Tel.: + 86 21 50806600 ext 2112;
E-mail: xshen@mail.shcnc.ac.cn.
Abbreviations: 15-d-PGJ 2 , 15-deoxy-D12,14-prostaglandin J 2 ; CPA,
cis-parinaric acid; GW9662, 2-chloro-5-nitrobenzanilide;
indometha-cin, 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid;
LBD, ligand binding domain; LBP, ligand binding pocket; LY171883,
1-{2-hydroxy-3-propyl-4-[(1H-tetrazol-5-yl)butoxyl]phenyl} ethanone;
PPARc, peroxisome proliferator-activated receptor c; RU, resonance
unit; SPR, surface plasmon resonance; TZD, thiazolidinedione.
(Received 31 July 2003, revised 10 November 2003,
accepted 20 November 2003)
Eur J Biochem 271, 386–397 (2004) FEBS 2003 doi:10.1046/j.1432-1033.2003.03937.x
Trang 2both the function study of the receptor and ligand discovery.
Numerous technologies, such as competition radioreceptor
assay [8–10], protease protection assay [11],
coactivator-dependent receptor ligand assay (CARLA) [12] and
scintil-lation proximity assay (SPA) [13], have been used to
measure the binding constants for ligand–PPARc
inter-actions and in screening of ligands By employing these
technologies, some important parameters evaluating the
binding affinity or activity for many ligands to PPARc, such
as Ki, KD, EC50 and IC50, have been obtained However,
these technologies either need specific radioligands for
labeling or the reporter gene has to be transfected in the cell
to be detected, both of which limit the screening speed for
finding new ligands, especially at the primary screening step
Recently, the surface plasmon resonance (SPR)
biosen-sor technology has been recognized as a powerful tool
in monitoring receptor–ligand interactions with
advan-tages of no labeling, real-time and noninvasive
measure-ments [14] This advanced technology will become a
potential secondary screening tool in drug screening It
has been successfully used to measure the binding
interactions of small molecules to the ligand-binding
domain (LBD) of human estrogen receptor [15] To the
best of our knowledge, there is to date no report
concerning the ligand–PPAR binding assay by using
SPR biosensor technology Promoted by the discovery of
new PPAR agonists with the eventual aim of developing
new drugs for the treatment of type II diabetes, we are
trying to construct screening modes and corresponding
assay methods SPR biosensor technology was used to
determine the binding affinities of PPARc ligand binding
domain (PPARc–LBD) with nine typical ligands, viz
GI262570 [16], troglitazone [16], linoleic acid [17,18],
GW9662 [19], cis-parinaric acid [20], 15-d-PGJ2 [17],
indomethacin [21], Palmitic acid, and LY171883 [22]
(Fig 1) It can be demonstrated that SPR biosensor
technology can quantitatively detect the binding affinities
of the tested ligands to PPARc, and the dissociation
constants (KDs) measured by SPR biosensor are in
agreement with data reported in the literature
Upon binding of ligands, great conformational changes
take place for PPARc [23] To address the ligand binding
effect to thermal stability of PPARc, circular dichroism
(CD) spectroscopic technology was used to investigate the
conformational changes of PPARc–LBD resulted from
the ligand binding In addition, the thermally induced
unfolding process of both apo-PPARc–LBD and its
ligand-bound complexes were also studied using CD
spectroscopy, and the transition temperature (Tm) for
each complex was estimated from the CD responses To
our knowledge, this is the first report of use of CD to
detect the conformational change and to monitor the Tm
of PPARc unfolding The result indicated that the thermal
stability of PPARc–LBD enhanced by the ligand binding,
the transition temperature increments (DTm) of PPARc–
LBD caused by ligand binding have a good correlation
with the binding affinities This finding suggests that CD
can also be used in studying ligand–PPARc binding and
in screening new ligands
To address the structure–binding affinity relationship,
molecular docking method was used to construct the
binding models of the tested ligands with PPARc–LBD
The 3D models provided a good explanation for the differences of the binding affinities from a structural viewpoint The predicted binding free energies of the ligands
to PPARc correlate well with the binding affinities derived from the SPR biosensor determination, indicating that the docking paradigm used in this study may be involved in the cycle of discovering new PPARc agonists or antagonists
as virtual screening tool
Experimental procedures
Preparation of ligand samples The structures of the ligands used in this study are shown in Fig 1 Indomethacin, cis-parinaric acid and palmitic acid were purchased from Calbiochem, 15-deoxy-D12,14 -prota-glandin J2(15-d-PGJ2) and linoleic acid were from Biomol and GW9662, LY171883 and troglitazone were from CAYMAN Chem Co (Ann Arbor, MI, USA) All the other reagents were purchased from Sigma in AR grade GI262570 was synthesized in our laboratory by using methods modified from Henke et al [16] and Collins et al [24].1H NMR (400 MHz, CDCl3): d(p.p.m.) 8.82 (s, 1H), 8.03 (m, 2H), 7.60–7.37 (m, 10H), 7.22 (d, 2H, J¼ 8.60Hz), 4.37 (m, 1H), 4.16 (t, 2H, J¼ 6.22 Hz), 3.20 (m, 2H), 3.00 (t, 2H, J¼ 6.22Hz), 2.36 (s, 3H); LRESI-MS: m/e 546(M-H)-; anal C30H30N2O5, found: C, 74.79; H, 5.51; N, 5.07; required: C, 74.71; H, 5.53; N, 5.12
Fig 1 Structures of the PPARc ligands used in this study.
Trang 3All the test compounds were dissolved in DMSO as
20 mMstock solutions for the Biacore and CD experiments
Expression and purification of human PPARc ligand
binding domain (PPARc–LBD) protein
pET15b-hPPARc–LBD plasmid was kindly provided by
J Uppenberg (Department of Structural Chemistry,
Phar-macia and Upjohn, Stockholm, Sweden) The expression
and purification of the recombinant human PPARc–LBD
in Escherichia coli were carried out by using a method
slightly modified from Uppenberg et al [3] E coli
BL21(DE3) cells transformed with the plasmid were grown
in LB medium containing 50 lgÆmL)1 of ampicillin at
37C The expression of PPARc–LBD was induced by the
addition of 0.2 mM of isopropyl b-D-thiogalactoside
(IPTG) After induction for 5 h at 20C, the cells were
harvested and disrupted by sonication against NaCl/Pi
buffer The supernatant was applied to a Ni-nitrilotriacetic
acid column (1 mL resin), and the column was washed with
30 column volumes of loading buffer A (NaCl/Picontaining
10 mMimidazole, pH 8.8) followed by 10 column volumes
of loading buffer B (NaCl/Picontaining 25 mMimidazole,
p H 8.8) The PPARc–LBD protein was then eluted with
elution buffer C (NaCl/Pi containing 500 mM imidazole,
pH 8.8) For the Biacore experiments, imidazole in
PPARc–LBD protein was removed by dialysis against
HBS-EP buffer (10 mM Hepes, 150 mM NaCl, 3.4 mM
EDTA, 0.005% (v/v) surfactant P20, pH 7.4), while for
the CD experiment, imidazole in PPARc–LBD protein was
eradicated by dialysis against CD buffer (20 mM sodium
phosphate, pH 7.4) The PPARc–LBD protein sample was
concentrated by using Centriprep and Centricon
concen-trators Any insoluble materials in the protein were removed
by filtration The concentration of protein was
deter-mined from its molar extinction coefficient of e280¼
12 045M )1Æcm)1
Purification of PPARc–LBD/ligand complexes
To purify the PPARc–LBD/ligand complex, 20 lM
PPARc–LBD in 1.5 mL of CD buffer was incubated with
15 lL of the ligand stock solution [20 mM in dimethyl
sulfoxide (DMSO)] at 4C for 12 h, the excessive DMSO
and the ligand compound were then removed by use of
a HiTrapTM Desalting column (Amersham Pharmacia
Biotech AB) with CD buffer The PPARc–LBD/ligand
complex with desired concentration was concentrated
through a Centricon concentrator on demand
Surface plasmon resonance (SPR) analyses
The interaction analyses between immobilized PPARc–
LBD and its ligands were performed using the dual flow cell
Biacore 3000 instrument (Biacore AB, Uppsala, Sweden)
Immobilization of the protein to the hydrophilic
carboxy-methylated dextran matrix of the sensor chipCM5 (Biacore)
was carried out by the standard primary amine coupling
reaction The protein to be covalently bound to the matrix
was diluted in 10 mMsodium acetate buffer (pH 4.3) to a
final concentration of 0.35 mgÆmL)1 Equilibration of the
baseline was completed by a continuous flow of HBS-EP
buffer through the chipfor 1–2 h All the Biacore data were collected at 25C with HBS-EP as running buffer at a constant flow of 20 lLÆmin)1 All the sensorgrams were processed by using automatic correction for nonspecific bulk refractive index effects All the equilibrium constants (KDs) evaluating the protein–ligand binding affinity were determined by the steady state affinity fitting analysis of the results from Biacore data As the binding process for 15-d-PGJ2is slow, its kinetic analysis of the binding to PPARc– LBD regarding the association (kon) and dissociation (koff) rate constants were investigated based on the 1 : 1 (Lang-muir) binding fitting mode
CD spectral analyses
CD spectra of PPARc–LBD and its complexes at different temperatures were obtained by use of a JASCO 715 spectropolarimeter equipped with a Neslab water bath The CD spectra scans of the molar ellipticity were recorded using an optical cell with a 0.1 cm path-length for the
far-UV region Averages of six scans were collated The mean residue ellipticity of the protein was calculated using molar concentration multiplied by the number of residues The ellipticities at 222 nm for PPARc–LBD and its complexes were accumulated for analysis by ORIGIN 7.0 (http:// www.OriginLab.com), a program that combines numerical integration and nonlinear global fitting routines
Molecular modeling The 3D structures of the ligands were constructed using standard geometric parameters of molecular modeling software packageSYBYL6.8 (http://www.tripos.com) The geometries of the ligands were subsequently optimized by using the Power method encoded inSYBYL6.8 to a root-mean-squared (rms) energy gradient of 0.05 kcalÆmol)1ÆA˚)1 Tripos force field [25] with Gasteiger–Hu¨ckel charges [26,27] was employed during the ligand minimization The protein models were constructed according to the crystal struc-ture of PPARc–LBD–thiazolidinedione (TZD) complex retrieved from the Brookhaven Protein Data Bank (PDB) [28,29], entry 2PRG [5] The ligand-binding pocket (LBP) of the receptor was defined as the collection of the amino acids enclosed within a sphere of 6.5 A˚ radius around the bound ligand (TZD) The binding models of the ligands to the receptor were constructed by docking the ligands into the LBP of PPARc–LBD employing the flexible docking program FLEXX [30] During the docking simulations, standard parameters of theFLEXX implemented in SYBYL
6.8 were used The global lowest-energy binding configur-ation of a ligand to the protein was identified by optimizing the rotation and translation of the ligand within the binding pocket NormallyFLEXXprovides more than 10 candidate configurations; configuration corresponding to the lowest interaction energy was selected as the final structure for further analysis The binding free energies of the ligands with the receptor were predicted by using the scoring function of AUTODOCK 3.0 [31] The scoring function of
AUTODOCKwas empirically calibrated at the level of binding free energy based on the traditional molecular force field terms, in which not only the restriction of internal rotors depending on the number of torsion angles of the ligand, but
388 C Yu et al (Eur J Biochem 271) FEBS 2003
Trang 4also on the desolvation upon binding and the hydrophobic
effect (solvent entropy changes at solute–solvent interfaces)
were calculated Thus, this scoring function can reflect the
ligand–protein binding free energies more accurately
All molecular modeling and docking simulations were
performed on a Silicon Graphics Origin3200 workstation
(with four CPUs)
Results
SPR determination of binding affinity
Immobilization of PPARc–LBD typically resulted in a
resonance signal at about 2000–2100 resonance units (RUs)
The binding responses in RUs were continuously recorded
and presented graphically as a function of time The
association could be described in a simple equilibrium
(A, analyte; B, ligand; AB, comp lex)
Aþ B Ð AB
To determine the equilibrium dissociation constant for the
interaction, the equilibrium response (Req) data were fit to
an independent-binding-site model [32]:
Req¼X
i
Rmax;i C Kon;i
1þ C Kon;i
ð1Þ
where, Rmax stands for the maximal response, C is the concentration of a ligand, and Kon is the equilibrium association constant For a single-site interaction, i¼ 1, for
a two-site binding, i¼ 2, and so on The Biacore biosensor determination results for the binding of the ligands with immobilized PPARc–LBD in the CM5 chipare shown in Fig 2 The response data indicate that, in reaching the equilibrium, both the association and dissociation of 15-d-PGJ2towards the immobilized PPARc–LBD are slow (Fig 2A) However, the association and dissociation phases
of the other compounds were transitory, the responses reach equilibrium towards PPARc–LBD quickly, within 2 s, and the compounds dissociated from the protein chip surface comp letely after 5 s as shown in Fig 2B
Two fitting methods are generally used in the data analyses for slow and fast response modes, respectively The first fitting method is the 1 : 1 (Langmuir) binding fitting model, in which the association rate constant (kon) and dissociation rate constant (koff) are fitted simultaneously by rate Equation 2,
Fig 2 Specificity of ligands binding to PPARc–LBDmeasured by SPR (Biacore 3000) Representative sensorgrams obtained from injections for 15-d-PGJ 2 at concentrations of 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 l M (A); for troglitazone at concentrations of 0.00977, 0.0195, 0.0391, 0.0781, 0.156, 0.625, 5.0, and 20.0 l M (B); for LY171883 at concentrations of 0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 l M (C) and for GW9662 at concentrations of 0.00977, 0.039, 0.156, 0.625, 2.5, 5.0, and 20.0 l M (D); over PPARc–LBD immobilized on the CM5 chip The ligands were injected for 120 s, and dissociation was monitored for more than 150 s.
Trang 5dt ¼ kon C ðRmax RÞ koff R ð2Þ
where, R represents the response unit, C is the
concentra-tion of the ligand This fitting model is normally used in the
determination of slow binding For the fast binding
ligands, steady state affinity fitting model has to be
employed in calculating the binding constants
Accord-ingly, the binding kinetic constants of 15-d-PGJ2 to
PPARc–LBD were calculated by using Equation 2 The
results are shown in Table 1 The binding constants, in
terms of KD, of other compounds to PPARc–LBD were
obtained employing steady state fitting methods; the steady
state plots against the concentrations of troglitazone are
shown in Fig 3A
For ligand LY171883, upto 20 lM, the response only
reached two units, as shown in Fig 2C, and its biosensor
RU was independent of the analyte concentration
Therefore, it can be tentatively concluded that
LY171883 did not bind or showed very weak affinity
to PPARc–LBD, at least in the present experimental
conditions For ligand GW9662, at concentrations ranging from 9.77 nM to 20 lM, the responses at equilibrium increased from approximate 0.3–17RUs (Fig 2D) Esti-mated from the steady state plot against the concentration (Fig 3B), the KDvalue of GW9662 binding to PPARc– LBD is about 1.59 lM Similar to GW9662, the KDvalues
of the remaining ligands binding to PPARc–LBD were evaluated employing the steady state-fitting model, which are listed in Table 2
CD determination Large conformational change occurs for the PPARc–LBD when binding with ligands, especially for helix 12 (H12) [23]
To investigate the thermal properties associated with the conformational changes caused by ligand binding and to identify the relationshipbetween the binding affinity and the thermal parameter, CD spectroscopic analyses were per-formed to both the apo-PPARc–LBD and its ligand complexes The CD spectroscopic data were collected at the temperatures ranging from 4 to 90C Because all the ligands do not exhibit CD spectroscopic reflection within far-ultraviolet wavelength (data are not shown), the CD responses may assign to conformational change of the protein
As an example, the CD spectra of PPARc–LBD in the absence and presence of Troglitazone and GI262570 at 4,
20, 40, 60, 90C, and 4 C again (cooled down to 4 C from
90C) are shown in Fig 4 Similar profiles were observed for the remaining ligands (data are not shown) Comparing the CD features of the apo- and ligand bound PPARc– LBDs, we can see that ligand binding indeed induced a secondary structure change for PPARc–LBD This is in agreement with the X-ray crystallographic results [33,34], which clearly demonstrated apo- and ligand bound PPARc– LBDs adopted different conformational arrangements When comparing the CD spectra at 4C with those at
4C cooled down from 90 C, a major difference of the CD features is observed, suggesting that the unfolding processes for either PPARc–LBD or its ligand complexes are irreversible (Fig 4) Corresponding to the thermally induced unfolding processes, transition temperatures exist between 40 and 60C (Fig 4) Thermal unfolding profiles
of apo-PPARc–LBD and its complexes with the tested ligands were obtained by monitoring the 222-nm ellipticities (h) as functions of temperature Dh is defined as the ellipticity determined at a given temperature subtracting that determined at the lowest experimental temperature (4C in this study); and Dhmaxis defined as the Dh at the highest experimental temperature (90C in this study) The profiles of Dh/Dhmaxfor apo-PPARc–LBD and its ligand-bound complexes plotted against temperature are shown
in Fig 5 The transition temperature (Tm) values were obtained by fitting Dh/Dh data inORIGIN7.0 The result
Table 1 The kinetic constants of 15-deoxy-D12,14-protaglandin J 2 (15-d-PGJ 2 ) binding to PPARc–LBD R max , maximum analyte binding capacity;
k on , association rate constant; k off : dissociation rate constant; K D , equilibrium dissociation constant K D ¼ k off /k on ; v 2 statistical value in Biacore.
Fig 3 Equilibrium data analysis of ligands binding to PPARc–LBD.
The data for the SPR sensorgrams (Fig 2) were fitted to a single-site
interaction model The plots of steady state RU vs the concentrations
of troglitazone (A) and GW9662 (B), respectively, were obtained by
using a steady-state fitting model.
390 C Yu et al (Eur J Biochem 271) FEBS 2003
Trang 6is listed in Table 2 The Tmvalue of apo-PPARc–LBD is
46.14 C, while for the ligand-bound complexes, the Tm
temperatures increased with the values of 46.91–53.06C
Binding models
For the tested ligands, only the co-crystal structure of
GI262570 with PPARc-LBP was reported [35], PDB entry
1FM9 Therefore, we obtained the binding models of the
tested ligands with PPARc-LBP employing the docking
program, FLEXX [30] The binding conformations of the
ligands to PPARc-LBP derived by docking are
schemati-cally presented in Fig 6 The corresponding hydrogen
bonds and hydrophobic interactions were, respectively,
calculated by usingHBPLUS[36] andLIGPLOT[37] program,
which are shown in Fig 7 The binding fashions of these
ligands with PPARc-LBP are in general analogous to that
of TZD class agonists: the polar head interacts with the
hydrophilic portion of the LBD, and the hydrophobic tail
stretches down into the large hydrophobic pocket of
PPARc forming strong hydrophobic contacts with several
lipophilic residues such as Cys285, Leu330, Ile341, Met348
and Met364 (Fig 6) The polar heads of the ligands can be
divided into three sorts: TZD, carboxylic acid,
o-hydroxyl-acetophenone Ligands with a TZD polar head (2:
troglitazone) form five hydrogen bonds with Gln286,
His449, Tyr473, His323 and Ser289 (Fig 7B); ligands with
a carboxylic acid polar head (5: cis-parinaric acid) form
four hydrogen bonds with His449, Tyr473, His323 and
Ser289 (Fig 7C); the polar head of LY171883 (9) forms
only three hydrogen bonds with Tyr327, Ser289 and
His323 (Fig 7D) As far as the hydrophobic interactions
are concerned, the a-substituted groups of carboxyl group
of GI262570 (1) form several hydrophobic contacts
(Fig 7A) with PPARc, besides the four highly conserved
hydrogen bonds
Based on the binding models derived by FLEXX, the
binding free energies of the ligands with PPARc-LBP were
predicted by usingAUTODOCKprogram [31] The predicted
data are listed in Table 3 As will be discussed later, the
AUTODOCK predicted binding free energies are in well
agreement with the KD values of Biacore (Table 3),
indicating again the reasonability of the binding models
for these ligands to PPARc-LBP
Discussion
Binding affinity derived from the SPR assay
In the present study, for the first time, SPR biosensor technology was used to directly measure the binding interactions of small ligands to PPARc–LBD The KD values of the tested ligands to PPARc–LBD derived from the SPR determinations are in general agreement with those measured by other methods (Table 2) Upon 15-d-PGJ2 binding to PPARc–LBD, the association rate constant (kon) and dissociation rate constant (koff) were estimated to be
257 ± 9.86M )1Æs)1and 3.90 ± 0.074· 10)3Æs)1(Table 1); these two rate constants have not been reported elsewhere From the rate constants, the KDof 15-d-PGJ2binding to the receptor was measured as 15.1 ± 1.05 lM, which is close
to the value of 11.6 lM produced from the radioligand competition-binding assay [17] (Table 2) Also, the SPR measured KDvalues of GI262570, troglitazone, linoleic acid, and indomethacin are in agreement with those determined
by other methods [16,17]
However, disagreement is observed between the Biacore-determined KDvalues and the data reported in the literature for GW9662 and cis-parinaric acid (CPA; Table 2) CPA is
a naturally existing polyunsaturated fatty acid, it is fluor-escent in a hydrophobic environment The binding affinity
of CPA to PPARc–LBD produced from Biacore assay (7.80 lM) is 10-fold larger than that (0.669 lM) obtained from fluorescent assay by Palmer and Wolf [20] This inconsistency may result from the fact that CPA is easily photochemically dimerized During the Biacore assay, the CPA solution could barely escape from the light and air, allowing the monitored concentration of CPA to be lower than expected Therefore, the higher KDvalue was measured
GW9662 has been reported as an irreversible ligand of PPARc–LBD with a very high binding affinity (IC50¼ 3.3 nM) [19] GW9662 may react with Cys285 of PPARc–LBD establishing as the site of covalent modifica-tion by releasing HCl molecule (Cl atom is from the structure of GW9662) [19] However, in the Biacore assay, such an irreversible binding was not observed Upon the response of GW9662 in Biacore measurement, after equi-librium phase for 120 s, the response returned to the
Table 2 The equilibrium constant and T m for the PPARc–LBDand the compounds complex The equilibrium constants (K D s) and T m values were obtained by Biacore and CD measurements, respectively K¢ D values are the equilibrium constants from the references (numbers in the parentheses).
a Ligand bound to GST–PPARc–LBD b IC 50 value.
Trang 7baseline rapidly, followed by another binding in the next
cycle, suggesting that the binding of GW9662 to PPARc–
LBD is reversible rather than irreversible The binding
affinity produced from Biacore assay is only 1.59 lM
(Table 2) The reversible nature of GW9662 binding to
PPARc–LBD may be attributed to the fact that in the
Biacore experiment, the incubation time of GW9662 with
PPARc–LBD is not long enough for the ligand to react with
Cys285 In addition, reaction conditions such as pH value
and temperature might also affect the covalent modification
of PPARc by GW9662
Palmitic acid was reported as a natural ligand of PPARa
[38], but there is no quantitative binding affinity for this
ligand to PPARc as yet For the first time, we found that
palmitic acid was also a weak ligand of PPARc Biacore SPR biosensor determination revealed that the binding constant of this ligand to PPARc–LBD is 156 lM
(Table 2) LY171883 is an LTD4receptor antagonist, which was reported to be capable of activating PPARc by transactivation assay at micromolar concentrations [22] However, SPR determination did not detect the binding of LY171883 to PPARc, even at millimolar concentrations (Fig 2C)
SPR biosensor experiments require immobilization of a receptor or ligand on a surface and monitoring its binding to
a second component in solution [14] Without an appropri-ate method for immobilizing one reactant onto the detecting chip, SPR Biacore technology cannot be applied in binding assay and drug screening Omitting the ligands with uncertain KDvalues (GW9662 and cis-parinaric acid), the SPR Biacore values of KDhave a good correlation with those from reported binding affinities (K¢Din Table 2), the correlation relationshipbetween these two data sets is
KD¼ 1.062K¢D, the correlation coefficient R is as higher as 0.985 This demonstrates that SPR Biacore technology and the protein immobilizing method can be used to monitor the ligand–PPARc binding With the advantages of SPR Biacore technology in binding assay such as label-free and real time detections [14], the measurement methods esta-blished in this study can also be extended to drug screening for discovering new agonists or antagonists of PPARc Thermal stability correlates with the binding affinity X-ray crystal structures indicated that ligand-bound PPARc adopts different conformations with respect to the apo-PPARc [5,33–35] The CD spectra indeed reflect the conformational changes induced by the bound ligands (Fig 4) However, ligands studied in this paper with similar function (agonists) bind to a similar conformation of
Fig 5 Temperature dependence of ellipticity of apo-PPARc–LBDand its complexes at 222 nm Plots were obtained by fitting Dh/Dh max data with the temperature for apo-PPARc–LBD (¤), GI262570 (—), linoleic acid (j), cis-parinaric acid (m) 15-d-PGJ 2 (·), troglitazone (…).
Fig 4 Circular dichroism spectra of PPARc–LBD(A), troglitazone/
PPARc–LBD(B) and GI262570/PPARc–LBD(C) complexes Plots
were obtained at 4 C (—), 20 C (j), 40 C (m), 60 C (·), 90 C (¤),
and 4 C again cooled down from 90 C ( .).
392 C Yu et al (Eur J Biochem 271) FEBS 2003
Trang 8PPARc-LDB because different ligand-bound PPARc
pro-duced a similar CD spectral feature (Fig 4) This is also in
agreement with the crystal structures of ligand–PPARc–
LDB complexes [5,33–35] Nevertheless, the CD
determin-ation indicated that ligand binding increased the thermal
stability of PPARc To quantitatively analyze the
relation-shipbetween transition temperature and binding affinity, we
defined the transition temperature increment (DTm) as the
Tmof a ligand complex subtracting that of the apo-PPARc–
LBD The DTm data might reflect thermal stability of
PPARc–LBD caused by the ligand binding It is interesting
that the DTm values correlate linearly with the binding
affinities of the ligands except GW9662 (Fig 8) The
departure of GW9662 from the linear relationship is also
derived from the experimental condition (see Discussion in
the above section) Regression analysis without GW9662
resulted following the relationshipbetween DTmvalues and
binding affinities of the ligands to PPARc–LBD:
log KD¼ 2:52 þ 0:90 DTm
n¼ 8; SD¼ 0:293; R2¼ 0:952 ð3Þ
where, n is the number of tested ligands, SD is the standard
error, R2 is the correlation coefficient This correlation
implies the direct relationship between the ligand binding
affinity and the thermal stability Apparently, strong
binding of a ligand increases the thermal stability of
PPARc–LBD, which thereby increases the Tmof thermally
induced unfolding of PPARc–LBD This finding implies
that CD spectroscopic method can also be used in detecting
the binding affinity of ligands to PPARc and in screening new PPARc binders Those compounds exhibiting larger
Tmvalues using this paradigm would therefore be expected
to have potent binding affinity
Structure–affinity relationship
To explore the binding characteristics of the ligands to PPARc at the molecular level, molecular docking method was applied to construct the ligand–PPARc binding models and to predict the binding affinities Due to the uncertain binding affinity, GW9662 was not included in the docking analysis.AUTODOCKpredicted binding free energies of the eight tested ligands to PPARc to have a good correlation with the binding constants (Table 3 and Fig 9) The regression equation for SPR Biacore measured binding affinity (–logKD), which was obtained by using the predicted binding free energy (Table 3) as a unique descriptor By means of a simple linear regression analysis, the statistical results are presented in Eqn 4:
log KD¼ 2:93 0:34 DGbinding
n¼ 8; SD¼ 0:726; R2¼ 0:846 ð4Þ where, n is the number of tested ligands, SD is the standard error, and R2is the correlation coefficient This correlation between the predicted binding free energies and the Biacore-measured binding affinity demonstrates again that the binding models of the ligands to PPARc derived from docking simulation are, in a way, reliable However, Fig 9
Fig 6 The binding conformations of the test PPARc ligands The first image is the conformational superposition within the binding pocket of PPARc, showing that these ligands adopt a similar fashion to PPARc The yellow structure in the first image is the binding conformation of GI262570 retrieved from the crystal structure of the PPARc-GI262570 complex (PDB entry 1FM9).
Trang 9shows several dots, especially those corresponding to
linoleic acid and LY171883, that depart from the regression
line This indicates that docking parameters would be
improved if Eqn 4 was used in predicting ligand-PPARc
binding affinity accurately
AUTODOCKpredicted that binding free energy (DGbinding)
contains three terms: intermolecular electrostatic interaction
(DG ), intermolecular atomic affinity (DG ) and
intra-molecular torsional free energy (DGtor), which, respectively, represent the contributions of the receptor–ligand electro-static interactions, non-electroelectro-static interactions (including hydrogen bonding and hydrophobic interaction), and the entropy effect from the loss of torsion degrees of freedom upon ligand binding (Table 3) The separated terms of the predicted binding free energies indicate that non-electro-static interactions dominate the binding of the ligands and
Fig 7 Schematic representations of hydrogen bonds and hydrophobic interactions of PPARc with GI262570 (A), troglitazone (B), cis-parinaric acid (C), and LY171883 (D) The corresponding hydrogen bonds and hydrophobic interactions were, respectively, calculated by using HBPLUS [36] and
LIGPLOT [37] programs Dashed lines represent hydrogen bonds and spiked residues form hydrophobic contacts with the ligands.
394 C Yu et al (Eur J Biochem 271) FEBS 2003
Trang 10receptor Moreover, the non-electrostatic interactions
cor-relate well with the total binding free energies, and the
correlation coefficient (R2) is as high as 0.92, while the other
two terms do not correlate with the total binding free
energy The result predicted by docking is in good
agreement with the structural properties of both receptor
and ligands The majority of binding sites of PPARc is
lipophilic, and the lipophilicity of the ligands is also very high, so a hydrophobic effect must play a key role in receptor–ligand binding
On the other hand, the polar head of each ligand forms strong hydrogen bonds with the polar pocket of PPARc Structurally, the polar heads of troglitazone, cis-parinaric acid and LY171883 form 5, 4 and 3 hydrogen bonds with PPARc, respectively, indicating that the ability of the polar heads in forming hydrogen bonds with PPARc is in a decreasing order of TZD > carboxylic acid group> o-hydroxylacetophenone (Fig 7) By considering the fact that the tails of the ligands are located in the same hydrophobic pocket of PPARc (Fig 6), the above order explains adequately why LY171883 is the weakest PPARc binder and troglitazone is much more active than the ligands containing a carboxylic acid polar head such as cis-parinaric acid, linoleic acid, 15-d-PGJ2and palmitic acid (Table 2) In comparison with other ligands, GI262570 forms several additional hydrophobic contacts with PPARc (Fig 7A), which enhances the binding affinity of GI262570 to PPARc
On the contrary, the hydrophobic tail of indomethacin is shorter than those of other ligands, which decreases the hydrophobic interactions with PPARc–LBD The flexible palmitic acid contains a bond with more rotational potential than cis-parinaric acid; binding with the receptor the former ligand lost more entropy than the later (Table 3) This is one
of the reasons that cis-parinaric acid binds to PPARc more tightly than does palmitic acid (Table 2)
In conclusion, we demonstrated that SPR biosensor technology can quantitatively measure the binding affinity for ligand–PPARc interaction, and thereby can be poten-tially extended in the compounds screening for discovering the new agonists or antagonists of PPARc CD spectros-copy detected the conformational changes of PPARc induced by ligand binding Ligand binding enhances the thermal stability of PPARc, which is reflected in the increase
of the transition temperature (Tm), and correlates well with the ligand binding affinity The binding models constructed
by using docking modeling for the ligands to PPARc provided a good explanation for the structure-binding affinity relationship, and provided an attractive way for predicting the overall binding affinity, although its separate components cannot be as accurately predicted This result indicated that the binding models, docking paradigm and scoring function might be extended to virtual screening for finding new hits of PPARc ligands from the available databases Accordingly, combining above three methods is
Fig 8 The correlation between binding affinity and thermal stability.
The negative logarithm of K D was plotted against the DT m The data
were analyzed by linear fitting method using ORIGIN 7.0.
Fig 9 The correlation between the SPR binding affinities and
Auto-Dock-predicted binding free energies.
Table 3 The binding free energies of the ligands binding to PPARc The binding free energies (kcalÆmol)1) of the protein–ligand complex were estimated by the scoring function of AUTODOCK 3.0.