Fluorescent CXCR4 chemokine receptor antagonists: Metal activated binding

Article abstract of DOI:10.1039/b614557d

The copper(ii) complex of a novel rhodamine-azamacrocycle conjugate binds to the CXCR4 chemokine receptor and competes effectively against anti-CXCR4 monoclonal antibodies. The copper macrocycle unit adopts a trans-II configuration in the solid state. The

Full text of DOI:10.1039/b614557d

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Fluorescent CXCR4 chemokine receptor antagonists: metal activated
binding{
Abid Khan,a Jon D. Silversides,a Leigh Madden,b John Greenmanb and Stephen J. Archibald*a
Received (in Cambridge, UK) 6th October 2006, Accepted 15th November 2006
First published as an Advance Article on the web 4th December 2006
DOI: 10.1039/b614557d
mono-N-benzylated cyclam macrocycle with an additional ethy-
lene bridge between adjacent nitrogen positions.16 The 4-nitro-
benzyl derivative was produced and could selectively be reduced to
the amine in high yields with no cleavage of the benzylic unit using
H2 and poisoned Pd/CaCO3. Reaction of the benzylamine
macrocycle, 4, with rhodamine isothiocyanate (RITC) gave 5 in
high yield. Copper complexation was carried out in methanol to
give the metallated form of the antagonist, Cu5Cl2. AMD3100
contains two macrocyclic rings which both contribute to the
binding interaction. Our new compound is mono-macrocyclic, but
we aim to sufficiently optimise binding through configurational
restriction and inclusion of a metal ion. There are other examples
of mono-macrocyclic CXCR4 antagonists.17
The copper(II) complex of a novel rhodamine-azamacrocycle
conjugate binds to the CXCR4 chemokine receptor and
competes effectively against anti-CXCR4 monoclonal antibo-
dies. The copper macrocycle unit adopts a trans-II configura-
tion in the solid state.
Research into immunotherapeutic drugs based around cytokines,
chemokines and growth factors is a rapidly developing and
expanding area.1–4 The chemokine and chemokine receptor family
is generally accepted to have recently reached completion with the
discovery of 18 chemokine receptors and 46 chemokines.2 Now the
challenge is to use this information to develop novel therapeutic
drugs and diagnostic applications.
The CXCR4 chemokine receptor is a seven transmembrane
G-protein coupled receptor with multiple critical functions in both
the normal and pathological states,5,6 having a sole natural ligand
(CXCL12). Synthetic small molecule antagonists exist, including a
xylyl bridged bicyclam AMD3100,7 which have been shown to
have both a high binding specificity and an effective inhibitory
action against a number of disease states including arthritis, cancer
and HIV infection.8–10 AMD3100 is currently in clinical trials for
stem cell mobilisation (Mozobil).11
For this type of molecule, binding to the chemokine receptor is
thought to occur via aspartate residues on the extracellular loops
and outer surface.18 The receptor has an overall surface charge of
92 and may interact with the protonated free macrocycle via
H-bonding. The copper(II) complex has an overall charge of 2+
and the additional potential for a coordination interaction between
the copper ion and a carboxylate from an aspartate residue (on
exchange of the bound chloride). Aspartate residues 171 and 262
have been shown to interact with AMD3100 using site-directed
mutagenesis studies.18 Previous data suggest that the presence of
the metal ion will significantly enhance binding to the CXCR4
receptor over the free macrocycle.19
In this work we aim to produce a fluorescently labeled small
molecule that selectively binds to a chemokine receptor. Such
compounds could be used for in vitro diagnostic assays and
competition binding studies in drug development. Our approach
has been to design configurationally restricted cyclam-based metal
complexes as CXCR4 antagonists.12 This locks the configuration
of the complex and optimises the metal coordination environment
for interaction with an aspartate residue on receptor binding. We
present the synthesis of a novel ethylene bridged cyclam-based bi-
functional chelator, the X-ray structure of a related copper
complex and the binding assays of the rhodamine conjugates in
competition with anti-CXCR4 monoclonal antibodies.
Configuration of the cyclam rings in metal complexes
AMD3100 is of key importance to the binding interaction.20,21
To confirm the configuration of the macrocycle in our bridged
analogue, the structure of an analogous copper(II) complex,
[Cu6Cl](CuCl2) with the benzyl-armed bridged macrocycle, was
determined by X-ray crystallography (Fig. 1).{ Upon complexa-
tion to a copper(II) ion, the macrocycle adopts a trans-II
configuration with the piperazine ring in the boat conformation.22
The copper complex has a distorted square-based pyramidal
geometry with the four nitrogen donors from the macrocyclic
ligand and an axially coordinated chloride ion. The Cu–N bond
The piperazine-containing macrocycle was synthesized from a
bis-aminal unit (Scheme 1).{ The use of glyoxal bridged cyclam
allows for a high yielding mono-functionalisation reaction with a
benzyl halide due to the selective reactivity of the nitrogen
nucleophiles.13–15 The bis-aminal can then be reduced to give a
˚
distances are in the range 1.996(3)–2.072(4) A, and the Cu–Cl bond
2
length is 2.491(11) A. The counter anion is [CuCl2] , formed by
˚
reduction of the copper salt CuCl2?2H2O during the complexation
of the metal centre to the macrocycle.
aDepartment of Chemistry and Institute of Clinical Biosciences, The
University of Hull, Cottingham Road, Hull, UK HU6 7RX.
E-mail: s.j.archibald@hull.ac.uk; Fax: +44(0)1482 466410;
Tel: +44(0)1482 465488
Paramagnetic metal ions affect the fluorescence properties of
their ligands23 and fluorescence quenching has been used to detect
copper(II) ions.24 The magnitude of the effect is expected to be
reduced by an increased distance between the fluorophore and the
chelated metal ion. Significant quenching of the fluorescence
(approx. 75%) was observed on formation of Cu5Cl2; however, the
fluorescence emission was still sufficient to detect the compound in
bMedical Research Laboratory and Institute of Clinical Biosciences, The
University of Hull, Cottingham Road, Hull, UK HU6 7RX
{ Electronic supplementary information (ESI) available: Experimental
procedures (microbiology and chemical synthesis), fluorescence emission
spectra and X-ray crystallographic data. See DOI: 10.1039/b614557d
416 | Chem. Commun., 2007, 416–418
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Scheme 1 Synthesis of the mono-functionalised piperazino macrocycle and rhodamine attachment to the 4-amino benzyl derivative.
Fig. 1 Ball and stick representation of the X-ray crystal structure of the
copper(II) complex formed with the benzyl derivative of the piperazine
tetra-aza macrocycle. H-atoms and the [CuCl2]2 counter anion have been
omitted for clarity.
biological systems.{ Complex stability is also important. The
copper(II) complex of a related piperazino cyclam macrocycle has
been studied by Fabbrizzi and co-workers and shown to have a
demetallation half life of .20 days in 1 M HClO4.25
Cellular binding assays were carried out to determine whether
the mono-macrocyclic fluorescent compounds interact with the
CXCR4 chemokine receptor. A T-lymphocyte cell line (Jurkat)
that expresses high levels of the CXCR4 receptor was used.
Compounds 5 and Cu5Cl2 were analysed for binding to the Jurkat
cells by flow cytometry in competition with specific anti-CXCR4
monoclonal antibodies.
Two CXCR4-specific, monoclonal antibodies were used (mAbs
44716.111 and 44717.111), both of which bind to similar regions of
the extracellular loop.18 The binding assay was carried out by
saturation of the receptors with a high concentration of compound
(ca. 25 mM) followed by washing and introduction of the specific
antibodies. The amount of antibody present on the cell surface was
then quantified by addition of a secondary anti-mouse IgG
fluoresceinated reagent by flow cytometry. The free macrocycle
conjugate showed no shift from the positive control, suggesting
that either no binding occurs or 5 is completely displaced by the
antibodies [Fig. 2(A)]. The metal complex Cu5Cl2 binds and
competes successfully with both antibodies showing a significant
shift from the positive control [Fig. 2(B)].
Fig. 2 Flow cytometric plot for the binding of mAbs 44716.111 (green)
and 44717.111 (pink) in competition with compounds 5 (A) and Cu5Cl2
(B). (Negative) control (purple), (positive) controls 44716.111 (red) and
44717.111 (blue) are shown.
uptake can occur through passive diffusion and endocytosis.26 The
properties of the conjugates may be dominated by the rhodamine
component. Both a CXCR4 negative control cell line (Daudi) and
Jurkat gave a positive fluorescent response by flow cell cytometry.
Confocal microscopy studies were carried out, Fig. 3. Images
The compounds were studied to see if any non-specific cellular
uptake occurred. Rhodamine is highly lipophilic and cellular
This journal is ß The Royal Society of Chemistry 2007
Chem. Commun., 2007, 416–418 | 417

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Support of this work by The Wellcome Trust (Grant 069719)
and the EPSRC (DTA for J. D. S.) is gratefully acknowledged. We
thank the EPSRC mass spectrometry service at Swansea.
Notes and references
{ Crystal data for [Cu6Cl](CuCl2): C19H32Cl3Cu2N4, T = 150(2) K,
˚
monoclinic, P21/c, a = 6.8348(7), b = 18.7560(14), c = 17.8251(18) A, b =
3
˚
91.838(8)u, V = 2283.9(4) A , Z = 4, F(000) = 1132, 21 632 reflections
measured of which 6350 were independent on F2 with Rint = 0.0544, final R
indices [I . 2s(I)]: R1 = 0.0429, wR2 = 0.1027, final R indices (all data):
R1 = 0.0719, wR2 = 0.1130. Structure was solved by direct methods
(SHELXS) and refined against F2 (SHELX97) using WinGX.28 CCDC
623207. For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b614557d.
1 L. Yan, G. M. Anderson, M. DeWitte and M. T. Nakada, Eur. J.
Cancer, 2006, 42, 793–802.
2 A. E. I. Proudfoot, Nat. Rev. Immunol., 2002, 2, 106–115.
3 J. J. Onuffer and R. Horuk, Trends Pharmacol. Sci., 2002, 23, 459–467.
4 S. Ribeiro and R. Horuk, Pharmacol. Ther., 2005, 107, 44–58.
5 C. Gerard and B. J. Rollins, Nat. Immunol., 2001, 2, 108–115.
6 C. Murdoch, Immunol. Rev., 2000, 177, 175–184.
7 S. Hatse, K. Princen, G. Bridger, E. De Clercq and D. Schols, FEBS
Lett., 2002, 527, 255–262.
8 T. Bourcier, T. Berbar, S. Paquet, N. Rondeau, F. Thomas, V. Borderie,
L. Laroche, W. Rostene, F. Haour and A. Lombet, Mol. Vision, 2003,
9, 96–102.
9 E. De Clercq, Biochim. Biophys. Acta, 2002, 1587, 258–275.
10 P. Matthys, S. Hatse, K. Vermeire, A. Wuyts, G. Bridger, G. W. Henson,
E. De Clercq, A. Billiau and D. Schols, J. Immunol., 2001, 167,
4686–4692.
Fig. 3 Confocal images of Jurkat cells after incubation with (A)
rhodamine, (B) compound 5, (C) compound Cu5Cl2 and (D) rhodamine
after cell surface blocking. The cell surface is outlined with a CD3 FITC
antibody conjugate. (The rhodamine conjugate shows up red and the
FITC conjugate green.)
revealed that the rhodamine macrocycle conjugates were present in
the cell cytoplasm. To determine whether the uptake of 5 and
Cu5Cl2 into the cell was via passive or active transport, cell surface
blocking studies were carried out. The conjugates were still taken
up into the Jurkat cells suggesting an active transport process.
Modifications are required to reduce non-specific uptake. No
significant cytotoxicity was observed in the course of these
experiments.
11 A. Larochelle, A. Krouse, M. Metzger, D. Orlic, R. E. Donahue,
S. Fricker, G. Bridger, C. E. Dunbar and P. Hematti, Blood, 2006, 107,
3772–3778.
12 G. C. Valks, G. McRobbie, E. A. Lewis, T. J. Hubin, T. M. Hunter,
P. J. Sadler, C. Pannecouque, E. De Clercq and S. J. Archibald, J. Med.
Chem., 2006, 49, 6162–6165.
13 E. A. Lewis, C. C. Allan, R. W. Boyle and S. J. Archibald, Tetrahedron
Lett., 2004, 45, 3059–3062.
14 E. H. Wong, G. R. Weisman, D. C. Hill, D. P. Reed, M. E. Rogers,
J. S. Condon, M. A. Fagan, J. C. Calabrese, K. C. Lam, I. A. Guzei and
A. L. Rheingold, J. Am. Chem. Soc., 2000, 122, 10 561–10 572.
15 M. Le Baccon, F. Chuburu, L. Toupet, H. Handel, M. Soibinet,
I. Dechamps-Olivier, J. P. Barbiere and M. Aplincourt, New J. Chem.,
2001, 25, 1168–1174.
16 R. A. Kolinski, Pol. J. Chem., 1995, 69, 1039–1045.
17 S. Hatse, K. Princen, E. De Clercq, M. M. Rosenkilde, T. W. Schwartz,
P. E. Hernandez-Abad, R. T. Skerlj, G. J. Bridger and D. Schols,
Biochem. Pharmacol., 2005, 70, 752–761.
18 S. Hatse, K. Princen, K. Vermeire, L. O. Gerlach, M. M. Rosenkilde,
T. W. Schwartz, G. Bridger, E. De Clercq and D. Schols, FEBS Lett.,
2003, 546, 300–306.
19 L. O. Gerlach, J. S. Jakobsen, K. P. Jensen, M. R. Rosenkilde,
R. T. Skerlj, U. Ryde, G. J. Bridger and T. W. Schwartz, Biochemistry,
2003, 42, 710–717.
It is highly significant that the metal complex competes for
receptor binding more effectively than the free protonated
chelator, supporting the idea that a direct coordination interaction
with the receptor from the metal centre not only enhances but
activates binding. Configurational restriction via an ethylene
bridge offers an advantage for the metal complexes, in the
presentation of only one molecular shape.27 However, it also
reduces the potential for H-bonding for the free macrocycle
relative to the cyclam analogue. This could account for the
inability of 5 to compete with the anti-CXCR4 antibodies in the
competition binding assay.
20 X. Y. Liang, J. A. Parkinson, M. Weishaupl, R. O. Gould, S. J. Paisey,
H. S. Park, T. M. Hunter, C. A. Blindauer, S. Parsons and P. J. Sadler,
J. Am. Chem. Soc., 2002, 124, 9105–9112.
21 X. Y. Liang, M. Weishaupl, J. A. Parkinson, S. Parsons, P. A. McGregor
and P. J. Sadler, Chem.–Eur. J., 2003, 9, 4709–4717.
22 B. Bosnich, C. K. Poon and M. L. Tobe, Inorg. Chem., 1965, 4,
1102–1108.
In conclusion, we have produced a new fluorescent aza-
macrocyclic derivative that can bind copper(II) to form a complex
with the cyclam ring in the trans-II configuration. The high
yielding synthesis of these compounds exploits the selective
reactivity of rigid bis-aminal intermediates. The copper(II) complex
Cu5Cl2 effectively competes with both the antibodies used in a
CXCR4 binding assay. We have achieved the goal of binding a
fluorescent dye to the CXCR4 chemokine receptor via a small
molecule antagonist conjugate. Further research into using these
fluorescent antagonists in drug screening assays and manipulation
of cellular uptake properties is underway. A full investigation of
the biological stability, photophysical properties and the effects of
varying the metal ion will also be carried out.
23 L. Fabbrizzi, F. Foti, M. Licchelli and A. Poggi, Inorg. Chem., 2002, 41,
4612–4614.
24 S. Banthia and A. Samanta, New J. Chem., 2005, 29, 1007–1010.
25 M. Boiocchi, M. Bonizzoni, L. Fabbrizzi, F. Foti, M. Licchelli, A. Poggi,
A. Taglietti and M. Zema, Chem.–Eur. J., 2004, 10, 3209–3216.
26 M. S. Dordal, A. C. Ho, M. Jacksonstone, Y. F. Fu, C. L. Goolsby and
J. N. Winter, Cytometry, 1995, 20, 307–314.
27 T. M. Hunter, S. J. Paisey, H. S. Park, L. Cleghorn, A. Parkin,
S. Parsons and P. J. Sadler, J. Inorg. Biochem., 2004, 98, 713–719.
28 L. J. Farrugia, J. Appl. Crystallogr., 1999, 32, 837–838.
418 | Chem. Commun., 2007, 416–418
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