Pseudo-peptides based on methyl cysteine or methionine inspired from mets motifs found in the copper transporter ctr1

Article abstract of DOI:10.1021/ic502962d

Most proteins involved in Cu homeostasis bind to intracellular Cu(I) in stable Cu(S-Cys)_x environments, thanks to well-conserved cysteine-rich sequences. Similarly, the Cu(I) transport protein Ctr1, responsible for copper acquisition, binds Cu(I) in Cu(S-Met)₃ environments in conserved methionine-rich MXMXXM sequences, referred as Mets motifs. Pseudo-peptides based on a nitrilotriacetic acid scaffold and functionalized with three amino acids bearing thioether side chains, either methyl cysteine in T¹ or methionine in T², were synthesized as mimics of the Mets sequences found in Ctr1. These two ligands were obtained with good overall yields from commercial amino acids and demonstrate efficient chelating ability for Cu(I). Only one species, the mononuclear [CuT^1,2]⁺ complex, was evidenced by electrospray ionization-mass spectroscopy (ESI-MS) and the circular dichroism signature obtained for the most constrained CuT¹ complex having the shortest side chains showed reorganization of the pseudo-peptide scaffold upon Cu(I) complexation. Considering that thioether functions are neutral sulfur donors, the stability constants measured by competition with ferrozine are quite large: log K ≈ 10.2-10.3. The CuT^1,2 complexes are significantly more stable that those formed with linear peptides, mimicking isolated Mets motifs MXMXXM of the Cu transport protein Ctr1 (log K ≈ 5-6). This may be attributed to the preorganized pseudo-peptide scaffold, which arranges the three neutral sulfur donors toward the metal center. Such moderate affinity Cu(I) chelators are interesting for applications in chelation therapy, for instance, to induce minimum disturbance of Cu homeostasis in Wilson's disease treatments.

Full text of DOI:10.1021/ic502962d

Article
pubs.acs.org/IC
Pseudo-peptides Based on Methyl Cysteine or Methionine Inspired
from Mets Motifs Found in the Copper Transporter Ctr1
Anne-Solene Jullien,†,‡ Christelle Gateau,†,‡ Colette Lebrun,†,‡ and Pascale Delangle*,†,‡
̀
†Univ. Grenoble Alpes, INAC-SCIB, F-38000 Grenoble, France
‡CEA, INAC-SCIB, Reconnaissance Ionique et Chimie de Coordination, F-38000 Grenoble, France
S
* Supporting Information
ABSTRACT: Most proteins involved in Cu homeostasis bind to intracellular
Cu(I) in stable Cu(S-Cys)x environments, thanks to well-conserved cysteine-rich
sequences. Similarly, the Cu(I) transport protein Ctr1, responsible for copper
acquisition, binds Cu(I) in Cu(S-Met)3 environments in conserved methionine-
rich MXMXXM sequences, referred as Mets motifs. Pseudo-peptides based on a
nitrilotriacetic acid scaffold and functionalized with three amino acids bearing
thioether side chains, either methyl cysteine in T1 or methionine in T2, were
synthesized as mimics of the Mets sequences found in Ctr1. These two ligands
were obtained with good overall yields from commercial amino acids and
demonstrate efficient chelating ability for Cu(I). Only one species, the
mononuclear [CuT1,2]+ complex, was evidenced by electrospray ionization−mass spectroscopy (ESI-MS) and the circular
dichroism signature obtained for the most constrained CuT1 complex having the shortest side chains showed reorganization of
the pseudo-peptide scaffold upon Cu(I) complexation. Considering that thioether functions are neutral sulfur donors, the
stability constants measured by competition with ferrozine are quite large: log K ≈ 10.2−10.3. The CuT1,2 complexes are
significantly more stable that those formed with linear peptides, mimicking isolated Mets motifs MXMXXM of the Cu transport
protein Ctr1 (log K ≈ 5−6). This may be attributed to the preorganized pseudo-peptide scaffold, which arranges the three
neutral sulfur donors toward the metal center. Such moderate affinity Cu(I) chelators are interesting for applications in chelation
therapy, for instance, to induce minimum disturbance of Cu homeostasis in Wilson’s disease treatments.
MetXMet motifs containing 3−5 methionine residues per Mets
motif. The yeast Ctr1 (yCtr1) has 8 Mets motifs, with a total of
30 methionine residues in the 140-residues extracellular
domain, whereas the human Ctr1 (hCtr1) has 2 Mets motifs
in a 66-residue extracellular region. The Mets motifs have been
assumed to be involved in extracellular Cu acquisition ever
since Ctr1 was identified as a copper transporter.5,6,11 Other
proteins involved in copper transport use methionines for
Cu(I) binding such as Pco,12 Cop,13 and CusF,14 which are
three proteins found in copper-resistant bacteria. In all of the
Cu(I) transport proteins, the Mets motifs seem to be suitable
for both copper acquisition and copper internalization, since
these peptide sequences are able to stabilize reduced Cu(I),
which is more kinetically labile than Cu(II), in the extracellular
oxidizing environment.
Mets motifs have been extensively studied by Franz et
al.,15−18 who have focused on the thioether-only binding sites
found in these motifs, without any histidine residue in the
sequence.15,16 These methionine-only binding sites have been
found to bind Cu(I) with three methionine units in Cu(S-
Met)3 environments. The corresponding binding constant
values have been measured by both an ascorbic assay at pH 4.5
and mass spectrometry (MS). Binding constant values (log K)
INTRODUCTION
■
The molecular mechanisms of copper (Cu) trafficking are now
quite well elucidated, especially in the hepatocytes. Copper in
the +2 oxidation state (Cu(II)) in the oxidizing extracellular
environment is first reduced to Cu(I) via a reductase inserted in
the membrane.1 Then, a decisive step occurs: Cu(I) entry in
the hepatocytes by crossing the plasma membrane, taking
advantage of a favorable concentration gradient via the Cu(I)-
specific transporter Ctr1.2 The Cu(I) transport proteins (Ctr1)
are integral membrane proteins that are widely conserved from
yeast to humans.3,4 Genetic studies have confirmed that Ctr1 is
essential for copper acquisition, as yeast mutants lacking Ctr1
show clear signs of Cu deficiency5,6 and mice lacking mCtr1 die
in utero.7 Such studies have clearly established the essential role
of Ctr1 in Cu acquisition, which is of great importance for
mammalian development.
Ctr1 contains three transmembrane regions, with the amino
terminus in the extracellular space and the carboxyl terminus
facing the cytosol.3,6 The protein is made of a homotrimer,4−6,8
the center of which allows Cu(I) transport as evidenced by the
tryptophan scanning analysis performed by De Feo et al.9
Conserved cysteine and histidine residues located at the
cytosolic terminus have been shown to bind copper.2,10 The
extracellular domain of yeast Ctr1 (yCtr1), which is responsible
for Cu entrance in the cells, is mainly made of methionine-rich
domains (Mets motifs) arranged in both MetXXMet and
Received: December 11, 2014
© XXXX American Chemical Society
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Article
between 5 and 6 have been determined. These values are quite
low and indicate that the complexes formed with Mets motifs
are significantly less stable, with respect to Cu(I) species
formed with cysteine-rich proteins such as Cu chaperones or
metallothioneins (log K ≈ 16−19).2,19−23 This is in accordance
with the function of the Mets Motifs dedicated to the transport
of Cu(I) from the extracellular medium to the cytosol.
In this article, we report the design and synthesis of the two
new pseudo-peptides, T1 and T2, which bear three neutral soft
sulfur donors, as well as their Cu(I) complexation properties.
Interestingly these tripodal thioether chelating agents demon-
strate lower affinity for Cu(I) than the corresponding thiolate
compounds derived from cysteine and D-penicillamine but
significantly higher affinities than linear peptides, mimicking
isolated Mets motifs found in the Ctr1 extracellular domain.
The understanding of Cu homeostasis is of great interest for
inorganic medicinal chemists, since proteins involved in Cu
transport or sequestration are sources of inspiration for the
design of metal chelators. Our laboratory has been involved for
years in the design of bioinspired intracellular Cu(I) chelators
to treat copper overload in the orphan Wilson’s disease.24 In
particular, pseudo-peptides built up on a nitrilotriacetic acid
(NTA) template and extended with three cysteines or D-
penicillamines have been designed and extensively described in
previous publications.24−28 It has been demonstrated that these
tripodal architectures tightly bind Cu(I) in well-defined CuS3
cores either in mononuclear Cu(I) complexes or in Cu4S6 or
Cu6S9 metal−thiolate clusters. The affinities range from log K
≈ 16 to log K ≈ 19, and they are therefore competitive with
those of metallothioneins and copper chaperones found in cells.
Hence, tripodal architectures derived from NTA are particularly
efficient in binding Cu(I) and are therefore promising chelators
for the detoxification of Cu in Wilson’s disease.
Therefore, these efficient pseudo-peptide scaffolds have been
exploited to design Cu(I) chelators inspired from Mets motifs
found in Ctr1. The tripodal architectures T1 and T2 based on
the NTA template and extended by three amino acids bearing
thioether side chains (see Figure 1), either methyl cysteine
ethyl ester (T1) or methionine ethyl ester (T2) have been
synthesized. These new pseudo-peptides are expected to
complex Cu(I) in solution with properties close to Mets
motifs found in Cu transporters.
EXPERIMENTAL SECTION
■
General Procedure for Sample Preparation. The
procedures used for the syntheses of the chelators T1,2 are
described in the Supporting Information. Since Cu(I) is
sensitive to air oxidation, samples used for physicochemical
studies were all prepared in a glovebox under argon atmosphere
and sealed if used outside the glovebox. All the solutions used
for the sample preparation were deoxygenated and stored in the
glovebox. The chelators T1,2 were weighted and introduced in
the glovebox via a sealed flask under an argon atmosphere. All
the solutions were prepared in the glovebox. The samples were
dispersed in mixtures containing different proportions of
Millipore water buffered or not. At least 10% of acetonitrile
was added to each sample to prevent Cu(I) disproportionation
in solution.29 The Cu(I) solutions were prepared by dissolving
the appropriate amount of Cu(CH3CN)4 PF6 in acetonitrile.
The final concentration was determined by adding an excess of
sodium bathocuproine disulfonate (BCS) and measuring the
absorbance of Cu(BCS)2 (λmax = 483 nm, ε = 13 300 M−1
3−
cm−1).2
Electrospray Ionization−Mass Spectroscopy (ESI-MS)
Titrations with Cu(I). Mass spectra were acquired on a LXQ-
linear ion trap (THERMO Scientific, San Jose, USA) equipped
with an electrospray source. Electrospray full scan spectra in the
range m/z = 50−2000 amu or 2000−3000 amu were obtained
by infusion through a fused silica tubing at 2−10 μL/min. The
solutions were analyzed both in positive and negative modes.
The LXQ calibration (m/z = 50−2000) was achieved according
to the standard calibration procedure from the manufacturer
(mixture of caffeine, MRFA and Ultramark 1621). An ES-
Tuning Mix solution (Agilent) was used to calibrate the
spectrometer between 2000 amu and 3000 amu. The
temperature of the heated capillary for the LXQ was set in a
range of 200−250 °C, the ion-spray voltage was in the range of
3−6 kV and the injection time was 5−200 ms. Samples (∼100
μM) were prepared in MeCN/ammonium acetate buffer (10
mM, pH 7) or MeCN/water (1/9, v/v) and aliquots of the
metal solution were added.
Circular Dichroism (CD) Titrations with Cu(I). The
circular dichroism (CD) spectra were acquired with an Applied
Photo-Physics Chirascan spectrometer outside the glovebox. A
2-mL ligand solution (∼10−35 μM) in Millipore water/
CH3CN (9/1, v/v) was transferred in a sealed UV cell (1 cm
path) and the metal solution was prepared in a sealed flask.
Both samples were then stirred under argon atmosphere
outside the glovebox. Aliquots corresponding to 0.25 equiv of
the metal solution were successively added. The solution was
stirred during 10 min before each measurement to ensure the
complete formation of the metallic species.
Affinity for Cu(I). The apparent affinity constants at pH 7.4
of the Cu(I) complexes were measured by ultraviolet−visible
(UV-vis) titrations in the presence of ferrozine (Fz) as a
competitor.30,31 The spectra were recorded with a Varian
Cary50 spectrophotometer equipped with optical fibers
Figure 1. Structures of the thioether-based scaffolds T1 and T2
inspired from the Mets motifs found in the extracellular domain of
Ctr1.
B
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connected to an external cell holder in the glovebox. A solution
of the Cu(I) complex with Fz, Cu(Fz)2 (ca. 80 μM), was
ethyl bromide used as reagents in either pure acetonitrile
(MeCN) or in an acetonitrile/dimethylformamide mixture
(MeCN/DMF), depending on the solubility of the starting
materials. This reaction leads to 1a or 1b (86%), which are Boc-
deprotected to provide 2a and 2b quantitatively. The latters
were involved in coupling reactions with the activated ester
NTA(NHS)3 using diisopropylethylamine as a base to set the
pH at 7−8, leading to the pure products T1 (67%, 58% global
yield) and T2 (63%, 54% global yield).
Spectroscopic Characterization of the Cu(I) Com-
plexes. As expected, no characteristic signal of the binding of
the two ligands T1,2 to Cu(I) is detected in the UV spectra
acquired in the range of 200−800 nm. The coordination of the
neutral thioether groups to Cu(I) does not give rise to LMCT
bands, as in the case of the negatively charged thiolate
donors.25,28 Therefore, the complexation properties of the
chelators T1,2 have been investigated by circular dichroism
(CD). Aliquots of Cu(I) in acetonitrile have been added to a
solution containing the ligand from 0 to 2 equiv per ligand. CD
signals shifts or appearance of novel signals are expected due to
conformational changes of the ligand backbones caused by
Cu(I) binding. The titration recorded for the methyl cysteine
derivative T1 is represented in Figure 2 and shows relevant
3−
prepared in 100 mM Hepes buffer/MeCN (9/1, v/v), pH 7.4
in the UV cell (1 cm path). The mixture was stirred for 10 min
to ensure the formation of the complex. Aliquots of the pseudo-
peptide solution (∼2 mM in 100 mM Hepes buffer, pH 7.4)
3−
were then added to Cu(Fz)2 previously formed in the UV
cell. The spectra were then recorded and show the decrease of
the orange Cu(Fz)23− complex, which absorbs at 470 nm with a
molar extinction coefficient value ε = 4320 M−1 cm−1. The
stability of the absorbance at 470 nm was controlled before the
addition of any other aliquots of competitor. The stability
constants were then determined using the binding constant of
the Cu(Fz)2 complex (log β12 = 15.1)31 and the equations
3−
reported in the Boxed Text S1 in the Supporting Information.
Constants given in Table 1 are averaged values of significant
data points, i.e., points showing Cu displacements in the range
of 30%−70%.
Table 1. Conditional Stability Constants of the Cu(I)
Complexes with the Pseudo-peptides T1,2, Compared to Ctr1
Sequences Reported in the Literature
coordinating amino
a
b
ligand
acids
log K
method
reference
T1
M
M
10.3(1)
10.1(1)
5.0
Fz
Fz
this work
this work
ref 16
T2
yMets1
hMets1
hMets2
PcoC
M
AAA
M
5.0
AAA/ESI-MS ref 16
AAA/ESI-MS ref 16
AAA/ESI-MS ref 16
M
5.3
M
5.6
Ctr1-14
M/H
12.8
BCA
ref 17
a
b
Errors in the last figures are reported in parentheses. Legend: Fz =
ferrozine; AAA = ascorbic oxidation essay, 400 μM ascorbic acid
(H2Asc), pH 4.5; ESI-MS = 5 mM, H2Asc, pH 3.5 or 10 mM,
NH4OAc, 5 mM H2Asc, pH 7; BCA = Hepes buffer, pH 7.4, 0.5%
MeCN (where y denotes yeast, h denotes human, and M denotes
methionine or a methionine derivative); and H = histidine.
Figure 2. Circular dichroism (CD) titrations of T1 (19 μM) in
MeCN/water (1/9, v/v) with Cu(I) (0−2 equiv): (red trace) free T1;
(blue trace) 0.25−0.75 equiv of Cu(I); and (black trace) 1−2 equiv of
Cu(I).
RESULTS
■
Syntheses of the Pseudo-peptides T1 and T2. The
synthetic strategies used to elaborate the ligands T1 and T2 are
depicted in Scheme 1. These syntheses are based on functional
transformations from either Boc-Cys(Me)−OH or Boc-Met-
OH and are widely inspired from our previous work on D-
penicillamine-based tripods.28 The first step consists in an
esterification reaction with cesium carbonate (Cs2CO3) and
changes evidencing Cu(I) binding. An optimal concentration of
T1 ≈ 20 μM allows to extract relevant signals from the noise
without saturation and to detect the small evolution of the
signals when the Cu(I) complex forms. Curves depicted in
Figure 2 show that Cu(I) binding induces conformational
changes of the ligand backbone, which are evidenced by both
a
Scheme 1. Syntheses of the Thioether-Based Scaffolds T1,2
a
Experimental conditions: (a) Cs2CO3, Et-Br in MeCN (1a) or DMF/MeCN (1b), RT, 24 h (1a, 86% ; 1b, 86%) ; (b) TES, DCM/TFA, RT, 1.5 h
(quantitative reactions); (c) 1 equiv of NTA(NHS)3 for 3 equiv of 2, DIEA, MeCN, pH 7−8, RT, 24 h (T1: 67%, 58% global yield ; T2: 63%, 54%
global yield).
C
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the negative band centered at 230 nm, the ellipcity of which
decreases with the additions of Cu(I), and the negative band
detected below 190 nm, the ellipcity of which increases with the
additions of Cu(I). Above 1 equiv of Cu(I) added, all the
curves are superimposed, which clearly ensures the formation of
CuT1 species in solution. These results are also in good
agreement with the observations made by Franz et al.,15 who
demonstrated that Cu(I) binding makes the high-energy
negative band (195 nm) of some Mets motifs sequences
(hMet2, Pco) shift to less-negative ellipcity values. These shifts
have also been attributed to conformational changes of the
peptide backbones caused by Cu(I) binding. More precisely, an
evolution from random coil states to β-type turn conformations
with the addition of Cu(I) has been found responsible for the
changes observed on the spectra.
The same experiments run with the methionine derivative T2
did not show any significant evolution of the CD spectra,
regardless of the stoichiometry and the concentrations used
(data not shown). The lack of sensitivity of the CD signature to
Cu(I) binding may be related to the longer, and therefore
more-flexible, arms of the tripodal ligand T2, in comparison to
T1. Indeed, the CD evolution seen for Cu(I) binding by T1 is
rather small. Since T2 is expected to be more flexible due to the
presence of one extra methylene group in each of the three
thioether arms, the effect of Cu(I) binding on the chirality of
the molecule is likely to escape from the detection by CD
spectroscopy.
Figure 3. (+) ESI-MS spectra of T2 (100 μM) with equimolar Cu(I)
(down) and 4 equiv (top) in water/MeCN (9/1, v/v). Inset shows
isotopic patterns (experimental (Exp.) and calculated (Theo.)).
Mass Spectrometry Signatures of the Cu(I) Com-
plexes. All the mass spectra have been performed on samples
prepared in MeCN/water (1/9, v/v) mixture with a chelator
concentration [T] ≈ 100 μM. Aliquots of Cu(I) in acetonitrile
have been added from 0 to 4 equiv per ligand T. The samples
were carefully maintained under argon until the injection in the
mass spectrometer to avoid any Cu(I) oxidation into Cu(II).
Some of the mass spectra acquired in the positive mode for T2
are shown in Figure 3.
such as BCA (log β2 = 17.3)32 and BCS (log β2 = 19.8).2 Thus,
competition experiments performed using Fz allow for the
measurement of quite low affinity constants, with pCu+ in the
range of 9.8−13.8, with enough accuracy.31 Recently, Wedd et
al. emphasized that Fz may be sensitive to reducing agents
while BCA and BCS are not. Therefore, the competitive
experiments were performed without any reducing agent and in
the glovebox to avoid any trace of oxygen, which may lead to
Cu(II).
In the presence of equimolar Cu(I), the free form T2,
detected as the sodium adduct [T2+Na]+ at 691.4 and the
Cu(I) complex CuT2 detected as [T2+Cu]+ at 731.3 are both
observed, the latter being significantly more intense. This
equilibrium is displaced toward the CuT2 complex with the
addition of Cu(I), and above 2 equiv, the only signal of the
CuT2 complex is found to remain on the spectrum. The
examination of the Cu complex isotopic pattern confirms that
Cu is in the +1 oxidation state (Cu(I)). Very similar data are
observed for the Cu(I) complex of T1, which is detected as
[T1+Cu]+ at 689.3 in Figure S1 in the Supporting Information
and corroborate the CD experiment results with the formation
of a 1:1 Cu(I) complex.
3−
The complex Cu(Fz)2 was mixed with aliquots of the two
ligands T1 and T2 at pH 7.4 to induce Cu displacements of
30%−70%. The affinity constants (K) were obtained,
considering the formation of a single Cu(I) complex, as
evidenced by mass spectrometry and circular dichroism,
according to eq 1.
Cu(I) + T1,2 = CuT1,2
K
(1)
The equations used for the determination of the constants are
reported in Boxed Text S1 in the Supporting Information.
As seen in Table 1, affinity constants values of log K =
10.3(1) and 10.1(1) have been determined for Cu(I) binding
by the two pseudo-peptides, T1 and T2, respectively. These two
compounds bind Cu(I) with greater affinities than linear
peptides mimicking isolated Mets motifs found in yeast or
human Ctr1 (log K = 5−6).15 Although different methods have
been used to measure these binding constants, striking
differences are observed between the tripodal chelators T1,2
and peptides containing Mets-only sequences. The larger
affinity for Cu(I) demonstrated by the pseudo-peptides T1 and
T2 may be attributed to the rigid NTA chemical scaffold, which
provides more preorganized binding sites and contributes to
reinforce the Cu(I) binding, with respect to the more-flexible
Mets Motifs. As expected, the affinities of T1,2 for Cu(I) are
lower than those measured for peptide sequences that include
Hence, electrospray ionization−mass spectrometry (ESI-MS)
clearly indicates that the two tripodal pseudopeptides T1 and
T2 form monometallic CuT complexes of significant stabilities
in water. This was confirmed by measuring the conditional
stability constants of these two Cu(I)−thioether species.
Affinity for Cu(I). The affinities of the chelators T1,2 for
Cu(I) have been determined by competition experiments using
ferrozine (Fz) as a competitor.30,31 Fz is known to form a 1:2
3−
stoichiometry complex Cu(Fz)2 that absorbs at 470 nm
470 nm
(εCu(Fz)
= 4320 M−1 cm−1). The conditional stability
2
constant at neutral pH of the complex Cu(Fz)23−, log
pH=7
KCu(Fz)2
= 15.1 has been recently determined and is rather
small compared to those reported for other Cu(I) competitors
D
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Article
both methionine and histidine residues (log K ≈ 13), since
histidine is known to coordinate more efficiently Cu(I) than
methionine at physiological pH.17,18
entrance in cancer cells.36 Considering the affinity of T1 and T2
for Cu(I), they may be appropriate candidates to compete for
Cu(I) with Ctr1 and to enhance cisplatin incorporation and
efficacy. However, since Pt(II) is known to bind to sulfur
donors and in particular to methionine of Mets motifs,37 the
interaction of T1 and T2 with cisplatin should also be taken into
account.
The moderate affinity of the two methionine- or methyl
cysteine-based pseudo-peptides may also be of interest to
induce minimum disturbance of Cu homeostasis in chelation
therapy treatments such as in Wilson’s disease.
DISCUSSION
■
The pseudo-peptides T1 and T2 are inspired from Cu(S-Met)3
coordination spheres of the methionine-only binding sites
found in the Cu(I) transporter Ctr1 and many bacteria copper-
resistant proteins.12−16 These ligands are based on a tripodal
nitrilotriacetic acid (NTA) scaffold functionalized with three
converging thioether functions, either from three methionine
esters in T2 or from three more constrained methyl cysteine
esters in T1. The synthesis of these two neutral soft sulfur
donors were performed using synthetic strategies established in
previous work on cysteine and D-penicillamine deriva-
tives.25,26,28 T1 and T2 were obtained from commercial amino
acids with 58% and 54% global yields, respectively.
ASSOCIATED CONTENT
■
S
* Supporting Information
Syntheses and characterizations of chelators T1,2, supplemen-
tary MS figures, equations for the calculation of the stability
constants. This material is available free of charge via the
Internet at http://pubs.acs.org.
The data presented in this paper show the formation of
mononuclear Cu(I)T1,2 complexes. The affinity for Cu(I) was
measured by using ferrozine (Fz) as a competitor. Indeed, Fz is
a Cu(I) chelating agent with a moderate affinity and therefore
allows determination of the affinity constants for Cu(I) inferior
to 1013. The two pseudo-peptides T1 and T2 demonstrate
interesting affinity for Cu(I) with log K ≈ 10.1−10.3. The
constants are very similar, demonstrating that the modification
of the chemical architecture due to the substitution of methyl
cysteine arms in T1 with methionines in T2 does not affect the
Cu(I) chelation properties. Interestingly, these values are
significantly larger than those reported for linear peptides
mimicking isolated methionine-only binding sites found in Ctr1
sequences (log K ≈ 5−6).15,16 This may be assigned to the
preorganization of the tripodal ligands T1 and T2 that present
three converging sulfur donors to coordinate the Cu(I) ion.
Therefore, these pseudo-peptides are likely to compete with the
Mets motifs for Cu(I) in vivo.
AUTHOR INFORMATION
■
Corresponding Author
* E-mail: Pascale.delangle@cea.fr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the “Agence Nationale pour la
Recherche” (COPDETOX, No. ANR-11-EMMA-025), the
́
“Fondation pour la Recherche Medicale” (Grant No.
DCM20111223043) and the Labex ARCANE (Grant No.
ANR-11-LABX-0003-01).
REFERENCES
■
The two neutral sulfur donors T1 and T2 may also be
compared to the previously reported pseudo-peptides L1
(derived from cysteine25,26) and L4 (derived from D-penicill-
amine28), which present three negatively charged sulfur thiolate
donors and mimic [CuS3]2− coordination sites found in
metallothioneins. T1 and T2 have the same chemical
architecture than the thiolate derivatives L1 and L4 but present
three neutral sulfur donors of the soft thioether groups to
chelate Cu(I) in [CuS3]+ environments. So, as expected, they
show significantly lower affinity than the cysteine or D-
penicillamine derivatives, which chelate Cu(I) with extremely
large affinities, log K = 17−19.
Ligands based on thioethers as neutral sulfur donors could
contribute to the development of novel agents for chelation
therapy, which is a field that is under constant evolution.33
Indeed, in contrast to thiol derivatives, they are not sensitive to
extracellular oxidizing environments. Therefore, they are
interesting candidates for extracellular Cu(I) chelation, for
instance when Cu(I) is formed before cellular incorporation via
Ctr1. Such extracellular chelators are currently proposed to
promote cisplatin entrance in cisplatin-resistant cancer cells.
Indeed, cisplatin is known to interfere in Cu homeostasis and
interacts with Cu transporting proteins such as Atox1 or
Ctr1.34,35 In particular, its cellular uptake is proposed to be
mediated by the copper transporter Ctr1; therefore, extrac-
ellular Cu(I) competes with cisplatin for binding to Ctr1.35
Some Cu chelators such as tetrathiomolybdate (TTM) have
already been used to deplete Cu and to promote cisplatin
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(2) Xiao, Z.; Loughlin, F.; George, G. N.; Howlett, G. J.; Wedd, A. G.
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