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Introduction
Apoptosis is essential for normal development and
aging in multicellular organisms, and abnormal regulation of
apoptosis can result in multiple human diseases.
Mitochondria release apoptogenic proteins such as cytochrome C and
apoptosis-inducing factor (AIF) into the cytosol, which are
involved in the signaling pathway of caspases and induce
cell apoptosis[1每3]. One major pathway of the release of the
apoptogenic factors to the cytosol is via the rupture of the
outer mitochondrial membrane due to mitochondrial
permeability transition (MPT) pore
opening[3]. It is suggested that MPT pores play a potent role in cell
aging[4,5], and opening of the MPT pores may cause changes in mitochondrial shape
and function, such as the massive swelling of mitochondria,
rupture of the outer membrane and release of inter-membrane
components that induce apoptosis. It has been reported
that the agents that inhibit MPT may have therapeutic
potential for the treatment of human diseases such as
ischemia-reperfusion injury in peripheral organs, trauma and
neurodegenerative diseases[5每7].
Recent studies have shown that the MPT pore is
composed of three major proteins: the voltage-dependent anion
channel (VDAC) in the outer membrane that forms a large
H2O-filled pore with a diameter of 2.5每3.0 nm, the adenine
nucleotide translocator (ANT) that mediates the ADP-ATP
exchange in the inner membrane, and cyclophilin D
(CypD)[8]. CypD belongs to the family of highly homologous peptidyl
prolyl cis-trans isomerases (PPIases) that are thought to be
important for protein folding, and can bind to the
immuno-suppressor cyclosporin A
(CSA)[9,10]. It is known that CypD is a mitochondrial-targeted PPIase, even though its specific
physiological role is largely
obscure[11,12]. CypD has been confirmed to play a decisive role in MPT pore regulation,
and PPIase activity of CypD might be a necessary step in
MPT pore opening[8,13]. A model was recently proposed
concerning the mechanism of permeability transition-related
cytochrome c release, whereby the Ca2+ requirement for the
induction of the MPT pore opening might be due to the
Ca2+-dependent interaction between CypD and
ANT[14每16]. It has been reported that CypD inhibitor CSA and its analogues
may block MPT pore opening[17,18], which thereby makes
discovering the CypD inhibitor an appealing project. However,
to our knowledge, investigating the small molecular
CypD-specific inhibitor for allowing brain penetration is still a
challenge.
In this paper, we report 7 novel quinoxaline derivatives
(Scheme 1 and Figure 1) that inhibit the PPIase activity of
CypD. By using surface plasmon resonance (SPR) and fluo
rescence titration techniques, the kinetics of the
CypD-inhibitor interaction was investigated. The compounds＊
inhibition effects against rat liver
Ca2+-depedent mitochondrial swelling and
Ca2+ uptake/release were also determined. The
binding selectivity of CypD over CypA for the tested
compounds was analyzed, and explained based on the molecular
docking technique. We hope that this research will provide
a useful approach for the discovery of cyclophilin D inhibitors,
and thus help to develop promising compounds using CypD
as a drug target for the inhibition of MPT pore opening.
Materials and methods
All solvents and reagents were purchased commercially
and were used without further purification.
1H nuclear magnetic resonance (NMR) spectra (400 MHz) were recorded on
a Varian (Palo Alto, California, USA) Mercury-400
spectro-meter. Plasmid extraction was performed using the GenElute
Plasmid Miniprep Kit (Sigma-Aldrich, St Louis, Missouri,
USA).
The compound 6-amino-2,3-di(furan-2-yl)quinoxaline
was synthesized according to the patented
method[19]. (R)-ethyl nipecotate and
(S)-ethyl nipecotate were prepared according to a previously published
method[20].
General preparation procedure of compounds GW1每7
The chemical structures of the seven tested compounds are
shown in Figure 1, and the general synthetic procedure is
shown in Scheme 1. Briefly, the compounds were prepared
from 2,4-dinitroaniline in five steps.
2,3-di(furan-2-yl)-6-ethoxycarbonylamino quinoxaline
(GW1) To a solution of
6-amino-2,3-di(furan-2-yl)quinoxaline (83.1 mg, 0.30 mmol) and triethylamine (100
µL, 0.72 mmol) in dichloromethane (10 mL) we added triphosgene (30 mg, 0.10
mmol) while stirring. The mixture was stirred at room
temperature for 1 h, then ethanol (100 µL, 1.7 mmol) was added.
After another 1 h of stirring, the solvent was evaporated in a
vacuum to give the crude product, which was further
purified by flash column chromatography on a silica gel using
petro-ether/ethyl acetate (3:1) as the eluent. The obtained
compound GW1 (45.2 mg, 43% yield) was a yellowish
amorphous solid. 1HNMR
(CDCl3, 400 MHz). d: 8.10 (d, 1H, J=2.4
Hz), 8.05 (d, 1H, J=9.1Hz), 7.87 (dd, 1H, J=2.2 Hz, 9.1 Hz), 7.60
(m, 2H), 7.17 (s, 1H), 6.62 (m, 2H), 5.54 (m, 2H), 4.28 (q, 2H,
J=7.0 Hz), 1.32 (t, 3H, J=7.0 Hz); IR (KBr): 3419, 3246, 2982,
2928, 1732, 1622, 1572, 1539, 1495, 1261, 1226 per cm;
High-resolution mass spectra(electron impact) calculated value
[HRMS(EI) Calcd] for
C19H15N3O4
349.1063; Found 349.1068.
2,3-di(furan-2-yl)-6-N-(N
＊,N＊-diethylcarbamoyl)amino
quinoxaline (GW2) GW2 was synthesized by using a method
similar to that described for the preparation of GW1, except
that diethylamine was used instead of ethanol. GW2 is a
brown amorphous solid (63.0 mg, 56% yield).
1H NMR (CDCl3, 400 MHz). d: 8.03 (d, 2H, J=1.8 Hz), 7.96 (d, 1H, J=1.0 Hz),
7.59 (dd, 2H, J=0.7 Hz, 1.0 Hz), 6.78 (s, 1H), 6.61 (ddd, 2H, J=
0.7 Hz, 3.5 Hz, 12.2 Hz), 6.54 (m, 2H), 3.42 (q, 4H, J=7.2 Hz),
1.25 (t, 6H, J=7.2 Hz); IR (KBr): 3440, 3114, 2972, 2929, 1649,
1524, 1491, 1429, 1265 per cm; HRMS (EI) Calcd for
C21H20N4O3
376.1535; Found 376.1528.
2,3-di(furan-2-yl)-6-((R)-3-ethoxycarbonyl-piperidino)
carbonylamino quinoxaline (GW3) GW3 was prepared by
using a method similar to that described for the preparation
of GW1, except that (R)-ethyl nipecotate was used instead of
ethanol. GW3 is a yellow amorphous solid (66.4 mg, 48%
yield). 1H NMR (CDCl3, 400 MHz).
d: 8.13 (s, 1H), 8.02 (m, 1H), 7.95 (m, 2H), 7.59 (m, 2H), 6.59 (m, 2H), 6.53 (m, 2H), 4.22
(m, 2H), 3.99 (m, 2H), 3.48 (dd, 1H, J=3.3, 14.2 Hz), 3.10 (m,
1H), 2.70 (m, 1H), 2.20 (m, 1H), 1.45每1.95 (m, 3H), 1.29 (t, 3H,
J=6.1 Hz); IR (KBr): 3404, 2937, 2860, 1728, 1649, 1570, 1527,
1473, 1431, 1256 cm-1; HRMS(EI) Calcd for
C25H24N4O5
460.1747; Found 460.1725.
[a]D20 = -52º (c=0.83,
CH3OH).
2,3-di(furan-2-yl)-6-((S)-3-ethoxycarbonyl-piperidino)
carbonylamino quinoxaline (GW4) GW4 was prepared by
using a method similar to that described for GW1 except that
(S)-ethyl nipecotate was used instead of ethanol. GW4 was
a yellow amorphous solid (68.7 mg, 50%
yield). 1H NMR (CDCl3, 400 MHz).
d: 8.13(s, 1H), 8.02 (m, 1H), 7.95 (m, 2H),
7.59 (m, 2H), 6.59 (m, 2H), 6.53 (m, 2H), 4.22 (m, 2H), 3.99 (m,
2H), 3.48 (dd, 1H, J=3.3, 14.2 Hz), 3.10 (m, 1H), 2.70 (m, 1H),
2.20 (m, 1H), 1.45每1.95 (m, 3H), 1.29 (t, 3H, J=6.1 Hz); IR (KBr):
3404, 2937, 2860, 1728, 1649, 1570, 1529, 1475, 1431, 1254
cm-1; HRMS(EI) Calcd for
C25H24N4O5
460.1747; Found
460.1738. [a]D20 = +55º (c=1.78,
CH3OH).
2,3-di(furan-2-yl) -6-(pyrrolidin-1-yl)carbonylamino
quinoxaline (GW5) GW5 was prepared by using a method
similar to that described for the preparation of GW1 except
that pyrrolidine was used instead of ethanol. GW5 is a brown
amorphous solid (106.9 mg, 95% yield).
1H NMR (CDCl3, 400 MHz). d: 8.18 (dd, 1H, J=2.3, 9.2 Hz), 8.02 (m, 2H), 7.60 (m, 2H),
6.68 (s, 1H), 6.65 (d, 1H, J=3.4 Hz), 6.60 (dd, 1H, J=0.7,
3.4 Hz), 6.55 (m, 2H), 3.53 (t, 4H, J=6.6 Hz), 2.00 (t, 4H, J=6.6
Hz); IR (KBr): 3404, 2970, 2877, 1672, 1568, 1525, 1502, 1429,
1382, 1340, 1203 per cm; HRMS(EI) Calcd for
C21H18N4O3
374.1379; Found 374.1360.
2,3-di(furan-2-yl)-6-morpholinocarbonylamino
quinoxa-line (GW6) GW6 was prepared by using a method similar to
that described for the preparation of GW1 except that
morpholine was used instead of ethanol. GW6 was a yellow
amorphous solid (101.4 mg, 87% yield).
1H NMR (CDCl3, 400
MHz). d: 7.96每8.06 (m, 3H), 7.60 (m, 2H), 6.99 (s, 1H), 6.66 (dd,
1H, J=0.8, 3.5 Hz), 6.63 (dd, 1H, J=0.8, 3.5 Hz),
6.56 (m, 2H), 3.77 (t, 2H, J=4.9 Hz), 3.56 (t, 2H, J=4.9 Hz), 1.80
(b, 4H); IR (KBr): 3423, 2920, 2852, 1653, 1529, 1475, 1429,
1333, 1254 per cm; HRMS(EI) Calcd for
C21H18N4O4
390.1328; Found 390.1309.
2,3-di(furan-2-yl)
-6-N-(N＊,N
＊-diisopropylcarbamoyl)amino quinoxaline (GW7)
GW7 was prepared by using a method similar to that described for the preparation of GW1
except that diisopropylamine was used instead of ethanol.
GW7 is a brown amorphous solid (43.9 mg, 36%
yield). 1H NMR (CDCl3, 400 MHz).
d: 7.99每8.06 (m, 2H), 7.90 (d, 1H, J=
2.3 Hz), 7.57 (m, 2H), 6.76 (s, 1H), 6.59 (m, 2H), 6.51 (m, 2H),
3.99 (m, 2H), 1.32 (d, 12H, J=6.9 Hz); IR (KBr): 3427, 2968,
2928, 1647, 1566, 1520, 1495, 1433, 1375, 1205 per cm;
HRMS(EI) Calcd for
C23H24N4O3
404.1848; Found 404.1857.
Preparation of His-tagged human CypA
protein All cloning techniques including polymerase chain reaction (PCR),
restriction, ligation, E coli transformation, and plasmid DNA
preparation were carried out according to standard
methods[21]. The His-tagged CypA protein was expressed and purified
from the plasmid pQE30-CypA according to the published
procedure[22].
Preparation of rat CypD protein The plasmid
pcDNA3.1(+)/Zeo-CypD was kindly provided by Dr James
D LECHLEITER (University of Texas Health Science Center,
U3SA). By using the forward primer
5＊-ATAGAATTCATGCT-AGCTCTGCGCTGCG-3＊ (containing an
EcoRI site) and the reverse primer
5＊-ATATCTCGAGGCTCAACTGGCCACA-GTC-3＊ (containing an
XhoI site), the PCR product was sub-cloned into the vector pGEX-4T-1 between the
EcoRI and XhoI sites to obtain the expression plasmid pGEX-4T-1-CypD.
Sequencing was carried out to confirm the insertion.
E coli strains were prepared in Luria-Bertani medium
containing 100 mg/mL ampicillin. BL21 (DE3) bacteria
transformed with pGEX-4T-1-CypD were grown until the
OD600 reached 0.8, and
isopropylthio-b-D-galactoside (IPTG) was
added to a final concentration of 0.2 mmol/L to induce
GST-CypD expression at 25 C overnight.
Bacteria were harvested and lysed by sonication in a
sonication buffer [1℅phosphate-buffered saline (PBS), 1
mmol/L phenylmethylsulfonyl fluoride (PMSF), pH 7.3, 1
mmol/L ethylenediamine tetraacetic acid (EDTA), 1% Triton
X-100]. The bacterial lysate was centrifuged and the
supernatant was collected. GST-CypD protein was purified by
using a glutathione Sepharose 4B column (Amersham
Biosciences, Uppsala, Sweden), and the purified CypD
protein was obtained by the on-column cleavage of GST-CypD
using thrombin according to the instructions given by the
manufacturers of the glutathione Sepharose 4B column
(Amersham Biosciences). The purity of the obtained CypD
protein was verified by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis as a single band.
Surface plasmon resonance technology-based Biacore
3000 analyses The interactions between compounds
GW1每7 and CypD (A) were performed using the dual flow cell
Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). All
the experiments were carried out using HBS-EP (10 mmol/L
N-2-hydroxyethylpiperazine-N＊-2-ethanesulfonic acid
[HEPES], 150 mmol/L NaCl, 3.4 mmol/L EDTA and 0.005%
surfactant P20 at pH 7.4) as a running buffer at a constant
flow rate of 20 µL/min at 25 ∼C. The protein was immobilized
directly and covalently on the hydrophilic carboxymethylated
dextran matrix of the CM5 sensor chip (BIAcore) by using
the standard primary amine coupling reaction. The protein
to be bound to the sensor chip was diluted in 10 mmol/L
sodium acetate buffer (pH 6.5) to a concentration of 17
µmol/L. The concentrations of the compounds dissolved in the
running buffer varied from 1.18 to 10 µmol/L. All the data
analyses were carried out using BIAevaluation software, and the
sensorgrams were processed by automatic correction for
nonspecific bulk refractive index effects. The kinetic
analyses of the ligand binding to the protein were performed based
on the 1:1 Langmuir binding fit model according to the
procedures described in the software manual.
Fluorescence titration assay Fluorescence
measurements were performed on a Hitachi(Tokyo, Japan) F-2500
fluorescence spectrophotometer equipped with a thermal
controller. The change in the intrinsic tryptophan
fluorescence when the compound bound to the protein (CypA or
CypD) was monitored using a procedure similar to that
described in the literature[23,24]. The experiments were carried
out at 25 ∼C in PBS (pH 7.3) with the protein concentration
set at 13 µmol/L and the compound concentrations varied
from 0 to 40 µmol/L. The compounds were prepared in
dimethylsulfoxide as a stock solution of 10 mmol/L. The
fluorescent absorption was recorded with excitation at 280 nm and
emission at 340 nm.
PPIase inhibition activity assay The PPIase activity
assay for the proteins CypA and CypD was performed based
on a published method[25] with some modifications. The
substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanlilide
(Suc-AAPF-pNA, S-7388) and a-chymotrypsin (C-7762) were
purchased from Sigma (St Louis, Missouri, USA).
Suc-AAPF-pNA was dissolved in tetrahydrofuran containing 400
mmol/L of LiCl, and the stock solution concentration was 10 mmol/L.
a-chymotrypsin was dissolved in 1 mmol/L HCl containing 2
mmol/L CaCl2, and the stock solution concentration was 80
mmol/L. The assay buffer (173 µL of 50 mmol/L HEPES, 100
mmol/L NaCl; pH 8.0 at 0 ∼C; final concentration 43 mmol/L
HEPES, 86 mmol/L NaCl), 15 µL of de-ionized water and CypD
(2 µL of a 2700 nmol/L stock solution) and the compounds
(final concentration ranging from 100 nmol/L to 50 µmol/L)
were pre-equilibrated for 3 h on ice. Immediately before the
assay was started, 7.5 µL of chymotrypsin solution was
added. Absorbance readings at 390 nm were recorded when
2.5 µL of the peptide substrate was added into the 1 cm path
length cuvette and the solution was mixed rapidly. The data
were collected on a Hitachi U2010 spectrophotometer.
Rat liver mitochondrial swelling and
Ca2+ uptake/release inhibition
assays The mitochondrial swelling and
Ca2+ uptake/release inhibition assays were carried out according
to published methods[26]. The mitochondria were isolated
by differential centrifugation from the livers of adult Wistar
rats (180每200 g) after overnight starvation treatment. The
rat livers were excised and washed with 0.25 mol/L sucrose.
The fat and connective tissue were removed, and the livers
were homogenized (1/10, w/v) using buffer A (250 mmol/L
mannitol, 0.5 mmol/L EDTA, 5 mmol/L HEPES, 0.1% bovine
serum albumin; pH 7.4) on ice. The homogenate was
centrifuged at 1000℅g for 10 min in a Biofuge Stratos centrifuge
(Hereus Company, Hanau, Germany). The sediment was
discarded and the supernatant was centrifuged at
1000℅g for 10 min twice. The collected supernatant was then further
centrifuged at 10 000℅g for 15 min. The pellet (mitochondrial
fraction) was resuspended in the test buffer (250 mmol/L
mannitol, 70 mmol/L sucrose, 5 mmol/L HEPES; pH 7.4). The
total mitochondrial protein was determined by using the
Lowry assay using bovine serum albumin as a standard. Rat
mitochondria were added to the test buffer to yield a final
concentration of 0.5 mg protein per
mL[27]. The tested compounds (100 µmol/L) were mixed with mitochondria for 1 h
before CaCl2 (200 µmol/L) was added. Mitochondrial
swelling was determined by monitoring absorbance at 540 nm
using a Hitachi U2010 spectrophotometer and the
mitochondrial Ca2+ uptake/release assay was monitored using a Hitachi
F-2500 fluorescence spectrophotometer as described
previously[26].
Molecular modeling and docking The CypD sequence
from Rattus norvegicus was retrieved from GenBank (GenBank
protein ID U68544; http://www.ncbi.nlm.nih.gov). The
CLUSTAL W program was used to carry out sequence
alignment between the sequences of CypD from
Rattus norvegicus and human
CypA[28]. The sequence similarity identity between CypD and CypA was 63%, and positives
were 81%, making the Protein Data Bank (PDB) of human
CypA an ideal template for CypD 3-D model building. The 3-
D model of the TrpRS was generated based on PDB
templates 1AK4[29],
1AWT[30], and 1NMK[31] retrieved from the
Protein DataBase by using the MODELLER
program[32] encoded in Insight
II[33]. MODELLER uses a spatial restraint
method to build up 3-D protein models. The structure of
each template protein was used to derive spatial restraints
expressed as probability density functions for each of the
restrained features of the models. The structure with the
lowest violation score and lowest energy score was chosen
as the candidate. Refinements of the routine in the
Homology module of Insight II were used to adjust the positions of
the side chains. Finally, the structural models were
optimized using Amber force field[34] with the following
parameters: a distance-dependent dielectric constant of 4.0,
nonbonded cut-off 10 Å, and Kollman-all-atom
charges[34]. The structures were first minimized by steepest descent, then
by conjugating the gradient method to the energy gradient
root-mean-square <0.05 kcal﹞(mol﹞Å)-1. Several structural
analysis software packages were used to check the
structure quality. The Prostat module of Insight II was used to
analyze the bonds, angles and torsions. The Profile-3D
program[35] was used for checking the structure and sequence
compatibility. The 3-D structures of the compounds
GW1每7 were constructed from scratch by Sybyl
6.8[36], and optimized to energy convergence with the Tripos force field and
MMFF94 charges.
The major residues possibly comprising the binding site
of CypD were identified by sequence alignment with human
CypA, and the SiteID program encoded in Sybyl
6.8[36]. The surface structure of the binding pocket was constructed by
using the MOLCAD module of Sybyl 6.8.
The DOCK suite of programs is designed to find
possible orientations of a ligand in a ※receptor§
site[37]. The orientation of a ligand is evaluated with a shape-scoring
function and/or a function approximating the
ligand-receptor binding energy. The shape-scoring function is an
empirical function resembling the van der Waals＊ attractive
energy. The ligand-receptor binding energy is taken to be
approximately the sum of the van der Waals＊ and
electrostatic interaction energies. After the initial orientation and
scoring evaluation, a grid-based rigid body minimization is
carried out for the ligand to locate the nearest local energy
minimum within the receptor binding site. The position and
conformation of each docked molecule were optimized using
the single anchor search and torsion minimization method of
DOCK 4.0. Thirty conformations per ligand building a cycle
and 50 maximum anchor orientations were used in the
anchor-first docking algorithm. All docked configurations were
energy minimized using 100 maximum iterations and one mini
mization cycle.
Results and Discussion
Synthesis of the compounds Generally, the compounds
GW1每7 were synthesized as outlined in Scheme 1.
Kinetic analysis of CypD (A) binding to GW1每7 by
surface plasmon resonance In order to perform kinetic analyses
of the binding of GW1每7 to CypD and CypA, the Biacore
3000 instrument (based on surface plasmon resonance [SPR]
technology) was used. As a typical example, the Biacore
sensorgrams for the binding of GW2 to the immobilized CypD
are shown in Figure 2. The 1:1 Langmuir binding fit model
was used for determining the equilibrium dissociation
constant (KD), and the association
(kon) and dissociation
(koff) rate constants by using Equations (1) and (2).
where R represents the response unit,
C is the concentration of the analyte, and
The obtained results were evaluated by c2
analysis. All the kinetic parameters are listed in Table 1.
The Biacore results show that all the 7 tested compounds
exhibited strong binding affinities with CypD, with
KD values approximately 3每6 µmol/L. Due to the high structural
homology of CypD and CypA, the tested compounds had
high binding affinities with CypA, as indicated in Table 1.
However, compound GW5 exhibited higher binding
specificity with CypD than with CypA. This was further verified
by the intrinsic fluorescence titration analysis and cyclophilin
PPIase activity inhibition assay as shown in Tables 2 and 3.
Structurally, the R group of the compound (Scheme 1) might
play an important role in the ligand binding selectivity for
CypD over CypA.
In addition, the KD values obtained from the Biacore
assay agreed with the apparent equilibrium dissociation
constants (KD＊) from the intrinsic fluorescence titration analysis
and the IC50 values in the cyclophilin PPIase activity
inhibition determination as shown in Tables 2 and 3. In agreement
with Huber et al[38], we suggest that Biacore is a powerful
and useful method for screening cyclophilin inhibitors.
Intrinsic fluorescence titration analysis of compounds
binding to CypD(A) Because both of the binding sites of
CypD and CypA have a tryptophan residue (Trp 124 for CypD
and Trp 121 for CypA), we investigated the binding
affinities of the tested compounds for CypD and CypA by using
an intrinsic fluorescence titration
technique[23]. During the assay, a 1:1 ratio of CypD(A) to binding compound was used
based on published information about CypA/CSA
interactions[39,40] and the results of our molecular docking analyses.
The apparent equilibrium dissociation constant
(KD＊) used for evaluating CypD(A) binding affinity to the tested com
pound was calculated according to the method in the
literature[23]. We assumed that a 50% occupancy of CypD (or
CypA) is set at a fractional fluorescence change of 0.5
(FC0.5), and at this point the concentration of the bound ligand
is equal to that of the bound protein, which is half of the total
concentration of protein. Accordingly,
KD＊ is equal to the total ligand concentration minus the concentration of the
bound protein at FC0.5[23].
Figure 3 shows the typical tryptophan fluorescence
quenching of CypD induced by titration of the tested
compounds with an increase in their concentration. Since none
of the compounds showed any intrinsic fluorescence
absorption, their possible effects on the experiments could
be discounted (data not shown). The
KD＊ values of GW1每7 are summarized in Table 2. Obviously, the binding
selectivity of GW5 for CypD over CypA could also be determined,
and the KD＊ values are very comparable to the
KD values determined by the Biacore assay.
CypD (A) PPIase activity inhibition assay
Both CypD and CypA belong to the PPIase family, and classic
spectrophotometric methods[41] can be used to determine the PPIase
inhibition activity of GW1每7 against CypD and CypA.
During the assay, the rate constants for the
cis-trans interconver-sion were evaluated by fitting the data to the integrated
first-order rate equation by nonlinear least-square
analysis[25,41].
As a typical example, Figure 4 shows the CypD PPIase
inhibition results with increases in the concentrations of the
compounds, and Table 3 shows the IC50 values of GW1每7
against CypD and CypA. The fact that the
IC50 values accord well with the Biacore and fluorescence titration results
(Tables 1, 2) confirms the reliability of these three detection
approaches.
Rat Ca2+-dependent mitochondrial swelling and
Ca2+ uptake/release inhibition assays
In general, MPT pores are open when mitochondria encounter abnormally high
concentrations of exogenous Ca2+ ions. These pores allow
solutes of <1500 Da in size across the inner mitochondrial
membrane, leading to mitochondrial swelling. Such swelling
can be detected by time scans of absorbance at 540 nm
(A540) and the extent of swelling is proportional to
A540[26].
Fluo-5N fluorescence is quite low without binding to
Ca2+ in controls, because the high mitochondrial membrane
potential prevents the release of endogenous mitochondrial
Ca2+. When exogenous Ca2+ was added, Fluo-5N
fluorescence increased immediately and decreased rapidly as
Ca2+ ions were taken up into the mitochondria. Subsequently,
the accumulation of cations in the mitochondria led to
mitochondrial swelling and depolarization.
Ca2+ ions were then released from mitochondria as a consequence of the onset
of MPT, as indicated by an increase in Fluo-5N fluorescence.
Ca2+ release was completely blocked by 1 µmol/L CSA.
Fluo-5N fluorescence also revealed that compounds GW1每7
inhibited the uptake/release of exogenously added
Ca2+ to a certain extent.
Figure 5A shows the results of the rat mitochondrial
swelling inhibition assay for GW1每7 (100 µmol/L) with CSA (1
µmol/L) as a control. Figure 5B gives the results of the
mitochondrial Ca2+ uptake/release inhibition assay for GW1每7
(100 µmol/L). The results show that the inhibition abilities of
compounds GW1每7 against Ca2+ uptake/release are in good
agreement with their inhibition abilities against
mitochondrial swelling. We found that compounds GW2, 5, 6, and 7
had a strong ability, whereas GW1, 3, and 4 did not have any
inhibition activity, which could be because of the R group.
Compared with GW2, 5, 6, and 7, the tails of GW1, 3, and 4
were ethoxycarbonyl, which might prevent them from
transferring into mitochondria through the membranes or cause
the loss of inhibition ability for other (unclear) reasons. The
behavior of CypD in mitochondria is much more complicated
than that of the purified CypD protein, so the different R
groups might cause different results. CSA had the highest
inhibition activity, and the relative general inhibition
abilities of the other compounds were: GW5>GW7>GW6>GW2.
Such a sequence seems to be consistent with the CypD
PPIase inhibition ability of the compounds (Table 3). This
result thus confirms the fact that CypD inhibitors may
possess possible inhibition activity against
Ca2+-dependent MPT pore opening.
Molecular docking analyses To gain further insight into
the CypD(A)/inhibitor interaction model at the atomic level,
docking analysis based on molecular modeling was carried
out without the published rat CypD crystal data. Our rat
CypD model tallies very well with the human CypD
crystal[PDB ID: 2bit] structure shown in Figure 6A. The weighted
root mean square distance is 0.6040 and the identity score is
95.7%. Because of the similar structures of the compounds,
they share the same precursor, with some overlapping
structural elements in common (Figure 6B). Similar to CypA, the
binding pocket of CypD is also fairly large and shallow, and
is composed of residues Arg58, Ile60, Phe63, Met64, Glu66,
Gly75, Thr76, Gly77, Ala104, Asn105, Ala106, Gln114, Phe116,
Thr122, Trp124, Leu125, Lys128, and His129. GW1每7 and
CSA bind to the same binding site of CypD. Unlike the case
of full occupation by CSA, GW1每7 occupied only part of the
binding pocket and might swing in the pocket. Helekar and
Patrick even demonstrated that Arg55 of CypA was a key
determinant against PPIase activity[42]. Compounds GW1每7
showed their hydrophobic contact with Arg58 of CypD
(Arg55 of CypA). Therefore, the PPIase activity of CypD
could be inhibited by hydrophobic interactions with the
inhibitors. In addition, GW1每7 formed stacking interactions
with Trp124 of CypD (Trp121 of CypA) and the only
tryptophan residues of CypD and CypA that contribute to the
change in fluorescence intensity (data not shown ).
Conclusion In this work, we reported on 7 small
quinoxa-line derivatives as novel CypD inhibitors.
In vitro assays indicated that compounds GW2, 5, 6, and 7 inhibit
Ca2+-dependent rat liver mitochondrial swelling and
Ca2+ uptake/release. By using SPR and fluorescence titration techniques,
kinetic analysis of CypD/inhibitor interactions were
quantitatively performed. The measured
IC50 values for the tested compounds are all in good agreement with the SPR and
fluorescence titration results, which suggests that these are
powerful methods for identifying CypD
inhibitors[38]. Further studies indicated that GW5 has binding selectivity for CypD
over CypA.
In summary, in this present work we used an appropriate
and powerful approach for identifying CypD inhibitors, and
developed a small compound that shows specific
ligand-binding ability for CypD, which could be used in the
inhibition of MPT pore opening.
Acknowledgements
We thank Dr James D LECHLEITER for providing the rat
CypD gene and Research Collaboratory for Structural
Bioinformatics Protein Data Bank (RCSB PDB) for providing
human cyclophilin D structure information.
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