Potent inhibition of protein tyrosine phosphatase 1B by copper complexes: Implications for copper toxicity in biological systems

Article abstract of DOI:10.1039/b925603b

A new dinuclear copper complex and several Cu-amino acid complexes inhibit protein tyrosine phosphatase 1B competitively at submicromolar levels, suggesting that copper complexes may interfere with cellular signaling pathways by inhibiting protein tyrosine phosphatases.

Full text of DOI:10.1039/b925603b

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Potent inhibition of protein tyrosine phosphatase 1B by copper
complexes: implications for copper toxicity in biological systemsw
Qingming Wang,a Liping Lu,*a Caixia Yuan,a Kai Pei,a Zhiwei Liu,b
Maolin Guo*b and Miaoli Zhu*ac
Received 8th December 2009, Accepted 17th March 2010
First published as an Advance Article on the web 9th April 2010
DOI: 10.1039/b925603b
A new dinuclear copper complex and several Cu-amino acid
complexes inhibit protein tyrosine phosphatase 1B competitively
at submicromolar levels, suggesting that copper complexes may
interfere with cellular signaling pathways by inhibiting protein
tyrosine phosphatases.
a dinuclear copper complex 1 [bis(diethyl(propane-1,3-diylbis-
(azanediyl))bis(phenylmethylene) diphosphonate)dicopper].zy
The copper complexes inhibit PTP1B4 competitively at sub-
micromolar level. The potent PTP1B4 inhibition activity of
copper complexes suggests that they may interfere with cellular
signaling pathways via inhibiting protein tyrosine phosphatases.
The crystal structure (Fig. 1)z revealed that 1 is a neutral
dinuclear molecule with a symmetrical center, each Cu(II) ion
adopting a square-pyramidal geometry with two N and two O
atoms from one ligand at the basal positions and an O atom
occupying the apical position from the other ligand. The apical
Cu–O is obviously longer than the other Cu–O bonds, due to
Jahn–Teller effects. In agreement with the crystal structure,
electrospray mass spectra in a 1 : 1 water–methanol solution
(Fig. S1, ESIw) showed that the species of [1]2ꢀ (m/z, 562.08)
existed as the major peak in negative ion mode, as well as a
smaller peak at m/z = 1124.92, assignable to [1]ꢀ species.
While in positive ion mode, [1 + Na]+ (m/z, 1149.00) and a
few solvent adducts were observed. UV-vis studies indicated
that 1 was stable in MOPS4 buffer, pH 7.2 (Fig. S2, ESIw).
EPR studies (Fig. S3, ESIw) confirmed the Cu(II) oxidation
state in 1 in the solid state as well as in the MOPS buffer in the
absence or presence of 2 mM GSH4 under aerobic conditions;
however, 1 was reduced by GSH4 to a monovalent copper
complex under anaerobic conditions.
Copper is an essential element for living organisms, being
required for enzymes involved in several key cellular activities
such as respiration, radical defense, neuronal myelination and
angiogenesis. However, copper is quite toxic to cells at high
levels.1,2 It is generally believed that the toxicity of copper is
due to the redox active metal facilitating oxidative damage
through generation of hydroxyl radicals (i.e., via the Fenton
reaction).2,3 Recent studies have suggested that the mechanism
of the cytotoxic effects of copper is complicated. In addition to
reactive oxygen species (ROS)4 generation, inhibition of
proteasome has been demonstrated.5 The 8-hydroxyquinoline
copper complex [Cu(8-OHQ)2] exhibited a complete proteasome
inhibition at 10 mM, resulting in a loss of viability of Jurkat
T cells.6 Furthermore, interference with protein kinases/
phosphatases and their related cell signaling cascades by copper
ions has been demonstrated,7 providing new opportunities to
investigate the mechanism of the cytotoxicity of copper and to
develop novel Cu-based therapeutic agents.
Protein tyrosine phosphatases (PTPs) are a large family of
enzymes which are critical in signal transduction and regu-
lation of cellular processes such as growth, differentiation and
proliferation.8 PTPs4 contain a highly active thiolate in the
conserved active site motif: HCX5R(S/T).8 Copper is expected
to have high affinity for this active site due to its coordination
property. Copper binding may inhibit the phosphatase
activities of PTPs and thus be cytotoxic.
The inhibition of PTP1B by 1 was investigated using
p-nitrophenol phosphate (pNPP4) as the substrate, using a
similar procedure reported previously.9 Compound 1 exhibited
In this communication, we report the potent inhibition of
human PTP1B4 by a few copper-amino acid complexes as well as
a Institute of Molecular Science, Key Laboratory of Chemical Biology
and Molecular Engineering of the Education Ministry,
Shanxi University, Taiyuan, Shanxi 030006, P. R. China.
E-mail: luliping@sxu.edu.cn, miaoli@sxu.edu.cn
b Department of Chemistry and Biochemistry,
University of Massachusetts Dartmouth, North Dartmouth,
MA 02747-2300, USA. E-mail: mguo@umassd.edu
c State Key Laboratory of Coordination Chemistry,
Nanjing University, Nanjing 210093, P. R. China
w Electronic supplementary information (ESI) available: Experimental
details, electrospray mass spectrometry, elemental analysis results,
UV–vis and EPR spectra of 1, PTP1B inhibition by 1 at different
times, PTP1B inhibition by four Cu-amino acid complexes, and so on.
CCDC 740931. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/b925603b
Fig. 1 X-Ray crystal structure of 1 with 50% thermal ellipsoids.
Hydrogen atoms, part disorder atoms and solvent water are omitted
for clarity, and the relative bond around metal ions (A) are presented:
Cu1–O1, 1.953(4); Cu1–O5, 1.970(4); Cu1–N2, 2.025(4); Cu1–N1,
2.026(5); Cu1–O2, 2.198(4). Symmetry code: (i) ꢀx, ꢀy, 1 ꢀ z.
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potent inhibition against PTP1B4 with IC50 at B0.156 mM.
Under the same conditions, the free ligand did not inhibit
PTP1B4 even at 10 mM. Previous studies indicate that the
active site cysteine residue (Cys215) in PTP1B4 may be
oxidized and inactivated by reactive oxygen species.10 Since
copper complexes may promote reactive oxygen species
production via the Fenton reaction,11 we investigated the
effects of O2 and GSH4 on the inhibition of PTP1B4 by 1 to
examine whether the inhibition involves a reactive oxygen
species produced by the Fenton reaction. In the presence of
O2, the IC50 value differs little in the presence or absence of
GSH (i.e. 0.156 and 0.134 mM in the assay buffer containing
2 mM GSH4 and no GSH, respectively), suggesting that GSH
has little effect on the inhibition under aerobic conditions.
However, under anaerobic conditions, 2 mM GSH decreased
the inhibitory effect about 14-fold (IC50 B2.183 mM). Further-
more, when both GSH4 and O2 were absent, 1 inhibited
PTP1B4 (IC50 B0.171 mM) with almost the same potency as
the combination of GSH4 and O2. These results suggest that
the inhibition may not involve ROS4 but may be related to the
oxidation state of copper. Under anaerobic conditions, 1 was
reduced by GSH4 to a monovalent copper complex which
decreased the inhibitory ability; but when O2 is present,
GSH4 could not reduce 1 to the monovalent copper complex
(Fig. S3, ESIw), and therefore 1 maintained its inhibitory
activity. Kinetic studies under aerobic conditions suggest that
the inhibition is time dependent (Fig. S4, ESIw) and about
40 min incubation is needed to reach the maximum inhibition.
To investigate the inhibition mode of 1 on PTP1B,4 detailed
kinetic analysis on the inhibition was conducted. The Michaelis–
Menten constant (Km) and maximum velocity (Vmax) of PTP1B4
were determined by Lineweaver–Burk reciprocal plots. As
shown in Fig. 2, the double-reciprocal plots in the presence
of various concentrations of 1 indicate a typical competitive
inhibition. The apparent inhibition constant (Ki) is estimated to
be B0.26 mM (Fig. 2B), indicating a strong affinity.
Since intracellular copper may exist as complexes of amino
acids (AA) at toxic levels of copper, we checked the inhibitory
effects of several copper–amino acid complexes on PTP1B.4
Due to the high affinities of Cu(II) with amino acids,13 the
copper complexes of four amino acids (glycine, histidine,
glutamate and arginine) were prepared by mixing CuCl2
with a 20-fold excess of the individual amino acid without
separating the Cu-amino acid complexes from the excess
amino acids (see S4, ESIw). The formation of the Cu(AA)2
complexes was confirmed by UV-vis titrations (see Fig. S5,
ESIw). The assay results (Fig. S6, ESIw) show that the amino
acid–copper complexes also inhibit PTP1B4 potently with
IC50 about 0.1–0.3 mM. The amino acids alone do not inhibit
PTP1B even at 10 mM (data not shown). The Cu-glutamic
acid complex also displayed competitive inhibition with
Ki B0.38 mM (Fig. S7, ESIw).
Fig. 2 (A) Lineweaver–Burk plots of 1/rate (min mMꢀ1) vs the
reciprocal of the pNPP concentration (mMꢀ1) at five fixed concentra-
tions of the complex 1 (nM); (B) Ki determination for complex 1. The
value of ꢀKi was determined from the x intercept of a plot of the slope
of the line from the double-reciprocal plot as a function of inhibitor
concentration (nM).
binding between 1 and the enzyme. The stoichiometry of 1
binding to PTP1B4 was found to be 1 : 1 and the binding constants
(K) were calculated to be 2.67 ꢂ 106 and 1.62 ꢂ 106 Mꢀ1 at
303 and 310 K, respectively (S5 and Fig. S9, ESIw). The binding
constants are comparable to the value (3.90 ꢂ 106 Mꢀ1) obtained
from Ki (Fig. 2B).
In summary, we have synthesized a dinuclear copper(II)
complex which can bind strongly to PTP1B4 and inhibit its
phosphatase activity via a competitive mode (Ki B0.26 mM).
Similar potent inhibition was also observed for a few Cu(II)-amino
acid complexes. These data suggest that copper complexes may
interfere with cellular signaling pathways by inhibiting protein
tyrosine phosphatases, providing a new mechanism to explain the
cytotoxicity of copper. Moreover, as PTP1B4 is over-expressed in
many cancer cell lines and has emerged as an attractive target for
diabetes and cancer therapy,12 the potent PTP1B4 inhibition
property of copper complexes may be harnessed to develop
copper-based anticancer or antidiabetic agents.
The work was supported financially by the National
Natural Science Foundation of China (grant No: 20471033),
the Province Natural Foundation of Shanxi province of
China (grant No: 20051013), the Overseas Returned Scholar
Foundation of Shanxi Province in 2008, China Scholarship
Council and University of Massachusetts, Dartmouth, USA. We
are grateful to Prof. J. D. Smith (University of Massachusetts
Dartmouth) for careful editing on the manuscript.
To gain further insight into the interactions between 1 and
PTP1B,4 we studied their interactions by fluorescence spectro-
scopy. As shown in Fig. 3, with the addition of 1 into PTP1B4
solution at 310 K (or at 303 K, see Fig. S8, ESIw), there was a
decrease in the intensity of the fluorescence band of PTP1B4 at
335 nm accompanied by an increase in intensity at 420 nm. An
isosbestic point was located at 385 nm, suggesting a specific
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dissolved in 24 ml of H2O) were mixed and the mixture was kept
stirring for 4 h. The resulting blue solution was then kept at room
temperature for slow evaporation. Blue block crystals were obtained
after three days. Yield, 0.4156 g (24%). C42H60Cu2N4O16P4ꢃ4H2O
requires C, 42.04, H, 5.71, N, 4.67. Found: C, 42.07, H, 5.65, N,
4.67%. IR (cmꢀ1): 3421m, n(O–H, N–H); 2979m, n(C–H); 1167s,
n(PQO); 1074s, 1048s, n(P–O–C); 947s, n(P–C). UV-vis
(e/Mꢀ1 cmꢀ1(l/nm): e(242) = 7.33 ꢂ 103 , e(281) = 1.05 ꢂ 104;
e(684) = 104.6. Exact mass for 1 ꢀ 4H2O: 1126.15, ESI-MS (negative
mode) [1]2ꢀ (m/z, 562.08), [1]ꢀ (m/z, 1124.92); (positive mode)
[1 + Na]+ (m/z, 1149.00).
z Crystal data for 1: unit cell parameters were determined by least-
squares fit of 7275 reflections having 2y values in the range
5.798–43.9951. Crystal dimension: 0.30 ꢂ 0.20 ꢂ 0.20 mm; T =
ꢀ
298(2) K; triclinic, P1; a = 9.871(3), b = 11.180(4), c = 14.456(5) A,
a = 105.683(5), b = 101.778(6), g = 104.361(5)1, V = 1423.6(8) A3,
Z = 1, Dc = 1.400 g cmꢀ3, 2ymax = 441; 7266 reflections collected
of which 4922 were unique; Rint = 0.703, R1 = 0.063, wR2 = 0.152 for
I 4 2s(I); residual electron density: 0.955, ꢀ0.524 e Aꢀ3
.
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4 Abbreviations: ROS, reactive oxygen species; PTP, protein
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MOPS, 3-morpholinoropanesulfonic acid; AA, amino acid.
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Fig.
3 (A) Fluorescence emission spectra of the titration of
PTP1B with 1. Conditions: lex = 282 nm, 310 K, [PTP1B] = 2.0 ꢂ
10ꢀ7 mol Lꢀ1, [1]: (a)–(m), 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0,
2.25, 2.5, 2.75, 3.0 (10ꢀ7 mol Lꢀ1), respectively. (B) The fluorescence
intensity at 335 nm is plotted against the ratio of [1]/[PTP1B]. Inset:
Plot for the binding constant (K) and stoichiometry (n) determination
(for details see S5, ESIw).
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Notes and references
z Expression and purification of human PTP1B as well as PTP1B
inhibition assays have been described previously.9
y Synthesis of 1. The ligand (see S2 in ESIw) (3.0 mmol, 1.674 g,
dissolved in 48 ml of methanol) and CuCl2ꢃH2O (3.0 mmol, 0.5114 g,
13 C. W. Childs and D. D. Perrin, J. Chem. Soc. A, 1969, 1039.
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