Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors

Article abstract of DOI:10.1016/j.bmcl.2010.03.007

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low μM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH₂, exhibited a K_d for hMC4R of 9.1 ± 1.4 μM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-α-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects.

Full text of DOI:10.1016/j.bmcl.2010.03.007

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2489–2492
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for
evaluation of binding of multivalent molecules to melanocortin receptors
Liping Xu a, Josef Vagner b, Ramesh Alleti c, Venkataramanarao Rao c, Bhumasamudram Jagadish c,
David L. Morse a, Victor J. Hruby b,c, Robert J. Gillies a, Eugene A. Mash c,
*
a H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL 33612, USA
b The Bio5 Institute, University of Arizona, Tucson, AZ 85721-0240, USA
c Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721-0041, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R)
Received 20 January 2010
Revised 26 February 2010
Accepted 1 March 2010
Available online 4 March 2010
with low
labeled ligand, Eu-DTPA-PEGO-His-
imately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a
competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent
MSH(4) constructs derived from squalene. The results were compared with results from a similar assay
that employed a more potent labeled ligand, Eu-DTPA-NDP-a-MSH. While results from the latter assay
reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity,
and/or cooperative binding effects.
l
M affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The
D
Phe-Arg-Trp-NH2, exhibited a Kd for hMC4R of 9.1 1.4 M, approx-
l
Keywords:
Multimeric ligands
Human melanocortin 4 receptor
NDP-a-MSH
Ó 2010 Elsevier Ltd. All rights reserved.
MSH(4)
Early detection of many human cancers would be facilitated by
of known potency. For example, labeled forms of NDP-a-MSH, a
the availability of imaging agents that selectively bind to cancer
cells and report their existence and location by noninvasive tech-
niques.1–4 Development of such imaging agents involves covalently
linking reporter moieties and multiple copies of ligands to produce
‘multivalent molecules’ that will cooperatively bind to receptors
expressed on the surface of cancer cells.5–11 Multivalent molecules
constructed from weakly binding ligands should display avidity for
such cells when compared with the affinity of a single copy of the
parental ligand.5–16
superpotent ligand that binds to human melanocortin recep-
tors,17,18 have been used for this purpose.19,20 In such assays one
often assumes thermodynamic control, that is, that the respective
on-rates and off-rates of the competing ligands are similar. If this is
not the case, details of how the assay is carried out (order and tim-
ing of reagent addition, timing of measurements taken) can affect
the outcome. Determination of on-rates and off-rates for binding
of multivalent molecules to living cells is difficult since labeled
probes may be taken up by the cells and receptors may aggregate
and cycle to and from the cell surface. In the absence of knowledge
of ligand on-rates and off-rates, a close match between the affinity
of the ligands used in construction of the multivalent molecule and
the labeled ligand used as the probe in the competitive binding as-
say would seem prudent.
As we are currently investigating the behavior of multivalent
molecules derived from the weakly binding ligand MSH(4),21–23
we wished to prepare and test a probe based on this ligand for use
in competitive binding assays. We report herein the synthesis of
the Eu-DTPA labeled derivative 1; studies of the binding affinity of
1 with the human melanocortin 4 receptor (hMC4R); and the use
of 1 in a competitive binding assay involving previously reported
monovalent and divalent MSH(4) constructs derived from
squalene.8
The affinity of a molecule for binding to a receptor is often
quantified by a competitive binding assay against a labeled ligand
Abbreviations: Boc, tert-butoxycarbonyl; BSA, bovine serum albumin; DCM,
dichloromethane; DIC, diisopropyl carbodiimide; DIEA, diisopropylethyl amine;
DMEM, Dulbecco’s Modified Eagle Medium; DMF, N,N-dimethylformamide; DMSO,
dimethyl sulfoxide; DTPA, diethylenetriaminepentaacetic acid; EC50, effective
concentration, 50%; FBS, fetal bovine serum; Fmoc, 9-fluorenylmethyoxycarbonyl;
FT-ICR MS, Fourier transform ion cyclotron resonance mass spectrometry; HBTU, 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; hMC4R,
human melanocortin 4 receptor; HOBt, 1-hydroxybenzotriazole; MSH(4), His-
DPhe-
Arg-Trp; NDP- -MSH, Ser-Tyr-Ser-Nle-Glu-His- Phe-Arg-Trp-Gly-Lys-Pro-Val; Pbf,
a
D
2,2,4,6,7-pentamethyldihydro-benzofuran-5-ylsulfonyl; PEGO, 19-amino-5-oxo-
3,10,13,16-tetraoxa-6-azanonadecan-1-oic acid; t-Bu, tert-butyl; TEAA, triethylam-
monium acetate; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TRF, time-resolved
fluorescence; Trt, triphenylmethyl.
* Corresponding author. Tel.: +1 520 621 6321; fax: +1 520 621 8407.
E-mail address: emash@u.arizona.edu (E.A. Mash).
Synthesis of Eu-DTPA-PEGO-MSH(4) (1). The synthesis of 1 is de-
picted in Scheme 1. MSH(4) was synthesized manually using an
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.007

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L. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2489–2492
O
(10 equiv) and HOBt (30 equiv) were dissolved in dry DMSO
(1 mL) at 50 °C and then stirred for 20 min at room temperature.
This mixture was injected into the syringe reactor which was sha-
ken overnight, then the resin washed with DMSO, THF, 20% aque-
ous THF, THF, 5% DIEA in THF (5 min), THF, DMF, THF, and DCM. A
cleavage mixture (10 mL/g of resin) consisting of trifluoroacetic
acid (91%), water (3%), 1,2-ethanedithiol (3%), and thioanisole
(3%) was injected into the syringe reactor containing the resin
and the mixture shaken for 4 h at room temperature. The solution
was then filtered off, and the resin was washed with TFA
(2 Â 3 min). Filtrates were collected and concentrated under a
stream of nitrogen, and the product was precipitated by addition
of cold ether to the residue. The peptide pellet was washed three
times with cold ether, dried, dissolved in 1.0 M acetic acid, and
lyophilized. The lyophilized DTPA-PEGO-MSH(4) construct was
purified by preparative HPLC and characterized by analytical HPLC
and FT-ICR MS.25
O
N
H
N
H
N
N
O
O
MSH(4)-NH2
HO
O
O
N
Eu
O
O
O
O
O
O
O
O
O
H
H
1
O
O
N
O
H
N
N
HO
N
NDP-α-MSH-NH2
Eu
O
O
O
O
O
O
O
H
H
2
OH
OH
OH
CH3
CH3
CH3
CH3
H3C
CH3
CH3
CH3
O
OH
OH
N
N
N
O
3
The metal-free precursor to 1 was dissolved in 0.1 M ammo-
nium acetate, the pH was adjusted to 8 with aqueous 0.1 M
NH4OH, and 3 equiv of EuCl3Á6H2O in water were added. The reac-
tion mixture was stirred at room temperature overnight. The ex-
cess EuCl3 and ammonium salts were removed using a SEPAC C-
18 reverse-phase column with repetitive washing (20 mL of HPLC
grade water). The final product was eluted using 50% aqueous ace-
tonitrile (4 mL), concentrated, lyophilized, and characterized by
analytical HPLC and FT-ICR MS. Data appear in Table 1.
MSH(4)-NH2
MSH(4)-NH2
O
N
N
N
OH
OH
CH3
CH3
CH3
CH3
O
H3C
O
CH3
CH3
CH3
OH
OH
N
N
N
O
4
Saturation binding of 1 to hMC4R. Hek293 cells overexpressing
hMC4R were used to assess ligand binding. Quantitative recep-
tor-binding assays were carried out following a previously de-
scribed method.19 Cells were grown in Dulbecco’s Modified Eagle
Medium (DMEM) supplemented with 10% FBS. Cells were seeded
in a 96-well plate at a density of 15,000 cells per well and were al-
lowed to reach 80–90% confluence. On the day of the experiment,
media was aspirated from all wells. Probe 1 diluted in binding buf-
fer (DMEM, 1 mM 1,10-phenanthroline, 200 mg/L bacitracin,
0.5 mg/L leupeptin, 0.3 BSA) was added to the wells to result in fi-
a
N -Fmoc/t-Bu solid-phase peptide synthesis strategy and standard
DIC/HOBt or HBTU/DIEA activations on Rink amide Tentagel re-
sin.24 Attachment of the PEGO linker was performed using DIC/
HOBt activation (3 equiv Fmoc-PEGO, 3 equiv of HOBt, and 3 equiv
of DIC). Next, the DTPA chelator was attached to the N-terminus of
the resin-bound PEGO-MSH(4) construct as follows. After Fmoc re-
moval, the resin was washed with DMSO. DTPA dianhydride
H-His(Nim-Trt)-DPhe-Arg(Nd-Pbf)-Trp(Ni-Boc)-NH
Fmoc-PEGO-OH
H
His(Nim-Trt)-DPhe-Arg(Nd-Pbf)-Trp(Ni-Boc)-NH
FmocNH
O
O
N
O
O
O
O
O
Fmoc deprotection
DTPA dianhydride/HOBt
TFA cleavage
O
HO
N
H
N
H
N
His-DPhe-Arg-Trp-NH2
HO
O
O
N
N
O
O
O
O
O
O
OH HO
O
EuCl3.6H2O (3eq)
0.1 M Ammonium acetate (pH=8)
O
O
H
H
N
N
His-DPhe-Arg-Trp-NH2
HO
O
O
N
N
O
N
O
O
O
Eu
O
O
O
O
O
O
O
H
1
H
Eu(III)-DTPA
Scheme 1. Synthesis of Eu-DTPA-PEGO-MSH(4) (1).
PEGO linker
MSH(4)

L. Xu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2489–2492
2491
Table 1
buffer, enhancement solution was added, and fluorescence mea-
sured as described above. Competitive binding data were analyzed
by nonlinear regression analysis using GraphPad Prism software
and fitted to a classic one site binding competition equation. Re-
sults are given in Table 3.
FT-ICR MS and HPLC characterization of compound 1
a
Formula
Calculated masses [ion] Masses found (error) tR
C60H85N16O19151Eu 743.27604
743.27683
(1.1 ppm)
744.27971
(3.4 ppm)
11.36
[M+2]2+
C
60H85N16O19153Eu 744.27718
[M+2]2+
Using probe 1, the Ki values for compounds
3 [scaf-
fold + 1 Â MSH(4)] and 4 [scaffold + 2 Â MSH(4)] were approxi-
mately sevenfold higher and threefold lower, respectively, than
the values for the parental MSH(4) ligand. These results indicate
that attachment of MSH(4) to the squalene-derived scaffold had
a modest detrimental effect on monovalent ligand binding, and
that some combination of statistical, proximity, and/or cooperative
effects resulted in enhancement of affinity in the divalent ligand,
despite attachment to the scaffold. The Ki for 4 relative to 3 showed
an 18-fold enhancement.
a
Analyzed on a 3 Â 150 mm 3.5 Å Waters C18 XBridge column, flow rate 0.3 mL/
min, linear gradient from 10% to 60% B in A over 30 min, where A is 0.1% TEAA in
water and B is 90% acetonitrile and 10% A, detection at 220 and 280 nm. Purity
>95%.
Table 2
Binding constants for Eu-DTPA-PEGO-MSH(4) (1) and Eu-DTPA-NDP-
hMC4R
a
-MSH (2) with
Using probe 2, the Ki values for compounds 3 and 4 were
approximately equal to and threefold lower than the values for
the parental MSH(4) ligand, respectively. The latter result is consis-
tent with the prior study8 and with statistical probability. How-
Compounds
Kd
9.1 1.4
18.8 1.7 nM
na
1
2
l
M
4
(Ref. 19)
a
ever, compound
3 exhibited significantly greater affinity for
The value given is the average of n independent binding experiments, each done
in quadruplicate.
hMC4R than was previously reported.8 The reason for this differ-
ence is unknown. The current results using the high affinity com-
peting probe 2 indicate that, to the extent that an MSH(4) ligand
can compete with 2 for hMC4R, ligand bound to scaffold is as po-
tent as the parental ligand.
Table 3
Competitive binding of MSH(4), 3, and 4 to hMC4R
This study has demonstrated that compound 1 binds specifi-
cally to hMC4R and is useful as a fluorescent probe in assays of
competitive binding at that receptor. Probe 1 is easier and less
expensive to prepare than is probe 2. In addition, probe 1 appears
to be more sensitive than probe 2 to the binding of low affinity li-
gands to hMC4 receptors. Characterizations of the interactions of
other multivalent MSH(4) constructs with melanocortin receptors
using probes 1 and 2 are in progress.
Compounds
Probe 1 Kia
Probe 2 Kib
MSH(4)
3
4
0.76 0.03
5.0 0.95
0.27 0.02
lM (n = 4)
lM (n = 4)
lM (n = 4)
0.72 0.09
0.85 0.13
0.27 0.02
lM (n = 4)
lM (n = 5)
lM (n = 4)
a
The Ki was calculated using the equation Ki = EC50/(1 + ([ligand]/Kd)), where
[ligand] refers to the concentration of probe 1 used as the labeled competed ligand
(0.5 M).
Here the [ligand] refers to the concentration of probe 2 (10 nM).
l
b
Acknowledgments
nal concentrations ranging from 2 to 30
nonspecific binding, cells were incubated with various concentra-
tions of probe 1 in the presence of 100 M of unlabeled MSH(4)
at 37 °C for 1 h. The cells were washed with wash buffer, enhance-
ment solution was added, and fluorescence measured on a Wallac
VICTOR3 instrument using standard Eu TRF measurement condi-
lM. In wells used to test
The authors thank Renata Patek for laboratory assistance. This
work was supported by Grants R33 CA 95944, RO1 CA 97360,
RO1 CA 123547, and P30 CA 23074 from the National Cancer
Institute.
l
References and notes
tions (340 nm excitation, 400
ls delay, and emission collection
for 400 s at 615 nm). Saturation curves were analyzed with
GraphPad Prism software using the one site binding (hyperbola)
classical equation for nonlinear regression analysis.
l
1. Weissleder, R.; Mahmood, U. Radiology 2001, 219, 316.
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5. Handl, H. L.; Vagner, J.; Han, H.; Mash, E.; Hruby, V. J.; Gillies, R. J. Expert Opin.
Ther. Targets 2004, 8, 565.
As shown in Table 2, compound 1 possessed a lower binding
affinity (Kd = 9.1 1.4 lM) at hMC4R when compared with com-
pound 2 (Kd = 18.8 1.7 nM),19 well within the expected range in
a saturation binding assay for an MSH(4)-based fluorescent probe.
Binding assays using 1 and 2. Eu-DPTA-PEGO-MSH(4) (1) and Eu-
6. Vagner, J.; Handl, H. L.; Monguchi, Y.; Jana, U.; Begay, L. J.; Mash, E. A.; Hruby, V.
J.; Gillies, R. J. Bioconjugate Chem. 2006, 17, 1545.
7. Bowen, M. E.; Monguchi, Y.; Sankaranarayanan, R.; Vagner, J.; Begay, L. J.; Xu, L.;
Jagadish, B.; Hruby, V. J.; Gillies, R. J.; Mash, E. A. J. Org. Chem. 2007, 72, 1675.
8. Jagadish, B.; Sankaranarayanan, R.; Xu, L.; Richards, R.; Vagner, J.; Hruby, V. J.;
Gillies, R. J.; Mash, E. A. Bioorg. Med. Chem. Lett. 2007, 17, 3310.
9. Handl, H. L.; Sankaranarayanan, R.; Josan, J. S.; Vagner, J.; Mash, E. A.; Gillies, R.
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Hruby, V. J. Angew. Chem., Int. Ed. 2008, 47, 1685.
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Mash, E. A.; Hruby, V. J.; Gillies, R. J. Mol. Cancer Ther. 2009, 8, 2356.
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2754.
DTPA-NDP-a-MSH (2) were each used as the probe in a previously
described competitive binding assay19 to characterize the interac-
tions of the monovalent and divalent constructs 3 and 4 with
hMC4R.
In brief, Hek293 cells overexpressing hMC4R (6 Â 105 receptors/
cell on the cell surface) were plated in a 96-well plate. On the day
of the assay, the cells had reached 80–90% confluence. Media was
aspirated from all wells, and 50 lL of the compounds to be tested
(dilutions ranging from 2 Â 10À4 to 1 Â 10À11 M) and 50
lL of Eu-
13. Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Angew. Chem., Int. Ed. 2006, 45,
2348.
14. Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.; Kiessling, L. L. ACS
Chem. Biol. 2007, 2, 119.
labeled ligand (probe 1, 0.5 lM or probe 2, 10 nM) were added to
each well. Cells were incubated in the presence of unlabeled and
labeled ligands at 37 °C for 1 h. The cells were washed with wash
15. Trouche, N.; Wieckowski, S.; Sun, W.; Chaloin, O.; Hoebeke, J.; Fournel, S.;
Guichard, G. J. Am. Chem. Soc. 2007, 129, 13480.
16. Diestler, D. J.; Knapp, E. W. Phys. Rev. Lett. 2008, 100, 178101(4).
17. For the ‘high-affinity’ ligand NDP-
a-MSH (Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-
Compounds 3 and 4 are mixtures of stereoisomers and regioisomers; the sites of
ligand attachment shown are arbitrary; see Ref. 8.
Trp-Gly-Lys-Pro-Val) see: Sawyer, T. K.; Sanfilippo, P. J.; Hruby, V. J.; Engel, M.

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HOBt in DMF, DMF, and the next N -Fmoc amino acid was coupled using
a
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18. Hadley, M. E.; Anderson, B.; Heward, C. B.; Sawyer, T. K.; Hruby, V. J. Science
of HOBt, and 3 equiv of DIC). The resin slurry was shaken for 2 h or until a
Kaiser test became negative. If the test failed, the resin was washed with DMF
and the amino acid was coupled again using the HBTU/DIEA procedure (0.3 M
1981, 213, 1025.
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solution of 3 equiv of N -Fmoc amino acid, 3 equiv of HBTU, and 6 equiv of
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21. For the ‘low-affinity’ ligand MSH(4) (His-DPhe-Arg-Trp) see: Hruby, V. J.;
DIEA in DMF) for 3 h. If the second coupling did not result in a negative Kaiser
test, the resin was washed with DMF and the remaining amino groups were
capped using 50% acetic anhydride in pyridine for 10 min. When the coupling
reaction was finished, the resin was washed with DMF, and the coupling
procedure repeated for the next amino acid until all the amino acids in the
sequence were attached.
Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.; Sawyer, T. K.; Staples, D. J.; de Vaux,
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V. J.; Dickinson, C.; Gantz, I. J. Med. Chem. 1997, 40, 2133.
25. DTPA-PEGO-MSH(4) was purified on
a
22 Â 250 mm 10–22 Å Vydac
218TP1022 column, flow rate 5 mL/min, linear gradient from 0% to 30% B in
A over 30 min, where A is 0.1% TFA in water and B is 0.08% TFA in acetonitrile,
detection at 230 nm, and analyzed on a 3 Â 150 mm 3.5 Å Waters C18 XBridge
column, flow rate 0.3 mL/min, linear gradient from 10% to 60% B in A over
30 min, where A is 0.1% TFA in water and B is 0.08% TFA in acetonitrile,
detection at 220 nm, tR = 12.06 min, purity >95%. ESI-MS: calculated [M+2]2+
669.3; found 669.2.
24. The Rink resin (0.23 mmol/g) was swollen in DMF for 1 h, washed with DMF,
a
and the N -Fmoc protecting groups were removed using 50% piperidine in
DMF (1 Â 2 min and 1 Â 20 min). The resin was washed again with DMF, 1.0 M