Heterobivalent dual-target probe for targeting GRP and Y1 receptors on tumor cells

Article abstract of DOI:10.1016/j.bmcl.2012.11.110

Receptor targeting ligands for imaging and/or therapy of cancer are limited by heterogeneity of receptor expression by tumor cells, both inter-patient and intra-patient. It is often more important for imaging agents to identify local and distant spread of disease than it is to identify a specific receptor presence. Two natural hormone peptide receptors, GRPR and Y1, are specifically interesting because expression of GRPR, Y1 or both is up-regulated in most breast cancers. We describe here the design and development of a new heterobivalent peptide ligand, truncated bombesin (t-BBN)/BVD15-DO3A, for dual-targeting of GRPR and Y1, and validation of its dual binding capability. Such a probe should be useful in imaging cells, tissues and tumors that are GRPR and/or Y1 positive and should target radioisotopes, for example, ⁶⁸Ga and/or ¹⁷⁷Lu, to more tumors cells than single GRPR or Y1 targeted probes. A GRP targeting ligand, J-G-Abz4-QWAVGHLM-NH₂ (J-G-Abz4-t-BBN), and an Y1 targeting ligand, INP-K[ε-J-(α-DO3A- ε-DGa)-K]-YRLRY-NH₂([ε-J-(α-DO3A-ε-DGa)-K]-BVD- 15), were synthesized and coupled to produce the heterobivalent ligand, t-BBN/BVD15-DO3A. Competitive displacement binding assays using t-BBN/BVD15-DO3A against ¹²⁵I-Tyr⁴-BBN yielded an IC₅₀ value of 18 ± 0.7 nM for GRPR in T-47D cells, a human breast cancer cell line. A similar assay using t-BBN/BVD15-DO3A against porcine ¹²⁵I-NPY showed IC₅₀ values of 80 ± 11 nM for Y1 receptor in MCF7 cells, another human breast cancer cell line. In conclusion, it is possible to construct a single DO3A chelate containing probe that can target both GRPR and Y1 on human tumor cells.

Full text of DOI:10.1016/j.bmcl.2012.11.110

Bioorganic & Medicinal Chemistry Letters 23 (2013) 687–692
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Heterobivalent dual-target probe for targeting GRP and Y1 receptors
on tumor cells
Ajay Shrivastava , Shu-Huei Wang , Natarajan Raju ,à, Izabela Gierach §, Haiming Ding,
⇑
Michael F. Tweedle
Department of Radiology, The Ohio State University, Columbus, OH 43210, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Receptor targeting ligands for imaging and/or therapy of cancer are limited by heterogeneity of receptor
expression by tumor cells, both inter-patient and intra-patient. It is often more important for imaging
agents to identify local and distant spread of disease than it is to identify a specific receptor presence.
Two natural hormone peptide receptors, GRPR and Y1, are specifically interesting because expression
of GRPR, Y1 or both is up-regulated in most breast cancers. We describe here the design and development
of a new heterobivalent peptide ligand, truncated bombesin (t-BBN)/BVD15-DO3A, for dual-targeting of
GRPR and Y1, and validation of its dual binding capability. Such a probe should be useful in imaging cells,
tissues and tumors that are GRPR and/or Y1 positive and should target radioisotopes, for example, 68Ga
and/or 177Lu, to more tumors cells than single GRPR or Y1 targeted probes. A GRP targeting ligand, J-G-
Received 17 October 2012
Revised 20 November 2012
Accepted 26 November 2012
Available online 5 December 2012
Keywords:
Heterobivalent dual-target probe
Gastrin-releasing peptide receptor
Y1 receptor
Neuropeptide Y
Imaging probe
DO3A
AMBA
BVD15
Hormone peptide
Bombesin
Neuromedin B
Breast cancer
Abz4-QWAVGHLM-NH2 (J-G-Abz4-t-BBN), and an Y1 targeting ligand, INP-K[
e-J-(a-DO3A-e-DGa)-K]-
YRLRY-NH2([ -J-( -DO3A- -DGa)-K]-BVD-15), were synthesized and coupled to produce the heterobiva-
e
a
e
lent ligand, t-BBN/BVD15-DO3A. Competitive displacement binding assays using t-BBN/BVD15-DO3A
against 125I-Tyr4-BBN yielded an IC50 value of 18 0.7 nM for GRPR in T-47D cells, a human breast cancer
cell line. A similar assay using t-BBN/BVD15-DO3A against porcine 125I-NPY showed IC50 values of
80 11 nM for Y1 receptor in MCF7 cells, another human breast cancer cell line. In conclusion, it is pos-
sible to construct a single DO3A chelate containing probe that can target both GRPR and Y1 on human
tumor cells.
Ó 2012 Elsevier Ltd. All rights reserved.
Prostate cancer
The use of Positron Emission Tomography (PET) for tumor
imaging and management has become routine for imaging hy-
per-metabolic tumors due to the development of hybrid PET/CT
machines, 18F-FDG (fluoro-deoxyglucose) distribution centers,
and improved reimbursement of imaging procedures.1 Although
18F-FDG is an effective tumor imaging agent, it has had limited use-
fulness in breast cancer2 and prostate cancer.3 In breast cancer, it
does not differentiate tumors from acute and chronic inflamma-
tion, physiological lactation, and benign focal breast masses,
including silicon granuloma, fat necrosis, fibroadenoma, and post-
surgical changes.2 In prostate cancer, 18F-FDG is accumulated by
normal, inflamed and infected prostate, and lacks the ability to dif-
ferentiate benign prostatic hyperplasia from severe forms of can-
cer.3 Other metabolic imaging agents like 18F- or 11C-acetate, and
18F or 11C-choline have similar limitations in imaging prostate can-
cers.3 There is thus a need for receptor targeted and more specific
PET imaging agent(s) that can differentiate breast and prostate tu-
mor tissues from non-tumor tissues.
An effective approach to breast and prostate tumor imaging is
to target hormone peptide receptors on tumor cells using radio-la-
beled peptides4,5 based upon the native agonists. Compared to
antibodies, the strengths of this approach derive from the small
size (<5 kDa) of the molecules that results in short circulation
times and rapid tumor permeation. Other advantages include high
binding affinity for the receptor, and the ability to use chemical
synthesis to tune the small molecules chemically6 to modulate
the weaknesses, such as greater renal retention, limited bioavail-
ability and undesirable normal tissue agonistic activity. A useful
attribute is the option of using exactly the same probe for imaging
and radiotherapy by changing the radio-metal (e.g., 68Ga/90Y,
Abbreviations: GRP, gastrin-releasing peptide; GRPR, gastrin-releasing peptide
receptor; NPY, Neuropeptide Y; Y1, Neuropeptide Y1 receptor; DGa, diglycolic acid;
J, 8-amino-3,6-dioxaoctanoic acid; Abz4, 4-aminobenzoic acid; DO3A, 1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid; ivDde, 1-(4,4-dimethyl-2,6-dioxocyclo-
hex-1-ylidene)-3-methylbutyl.
⇑
Corresponding author. Tel.: +1 44 614 247 4427.
E-mail address: Michael.Tweedle@osumc.edu (M.F. Tweedle).
These authors contributed equally to this work.
Current address: Enzo Life Sciences, Inc., 10 Executive Blvd, Farmingdale, NY
à
11735, United States.
§
Current address: Chemical Abstracts Services, 2540 Olentangy River Rd, Colum-
bus, OH 43210, United States.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.110

688
A. Shrivastava et al. / Bioorg. Med. Chem. Lett. 23 (2013) 687–692
68Ga/177Lu) labels. There are multiple receptors that are being indi-
vidually targeted using this approach including somatostatin
(sst1–sst5), gastrin-releasing peptide (BB2/GRPR), cholecystokinin
Section). AMBA analog (MW estimated 1545.8, MW found
1545.76), BVD15-DO3A (MW estimated 1607.9, MW found
1607.92), and t-BBN/BVD15-DO3A (MW estimated 3239.7, MW
found 3239.39) were 98%, 96%, and 98% pure.
B/gastrin (CCK2/CCK-B), glucagon-like peptide-1 (GLP-1),
integrin, melanocortin 1 (MC1R), neurotensin (NTR1), Neuropep-
tide receptor (Y1), luteinizing hormone-releasing hormone
avb3-
The T-47D cells are known to overexpress human GRPR39–41
and were used for GRPR competition assays. The T-47D cells do
not express very much human Y1 (tested but data not shown).
Y
(LHRH-R), neurokinin 1 (NK-1) and chemokine (CXCR4) receptors.4
There are two peptide receptors, GRPR and Y1, that are specifically
interesting because the expression of GRPR, Y1 or both was up-reg-
ulated in most breast cancers.7 In 77 frozen primary breast tumor
tissues tested, 50 (65%) were GRPR positive, 45 (58%) were Y1
receptor positive and 63 (82%) were positive for either GRPR, Y1,
or both receptors. Apart from breast cancers, Y1 is also present in
prostate cancer cells,4,8,9 ovarian tumors, renal cell carcinoma
and gastrointestinal stromal tumors (GIST) among others.10 Simi-
larly, GRPR are present in prostate, breast, pancreas, gastric, small
cell lung cancer, colorectal cancer4,5,11–13 and in the neovasculature
of ovarian and bladder cancers.14,15 These data strongly suggest
that there may be advantages to developing a dual-target target
probe that can bind both GRPR and Y1. A further consideration is
the economics of the drug development process, now costing
>$100 million for an imaging drug, that favors a dual-target probe
over two separate single target probe development projects.16,17
Popularity of dual-target probes is increasing, as several heterova-
lent ligands targeting two different domains of one receptor18–22 or
two different receptors have recently appeared.23–35
The T-47D cells were pre-blocked with 1 lM NPY to prevent any
Y1 influence on GRPR binding. Bombesin and AMBA analogs
(Table 1) were used as controls to compare the affinity of hetero-
bivalent dual-target probe, t-BBN/BVD15-DO3A (Table1), with
the natural GRP ligand, BBN, and AMBA (based upon a truncated
BBN, t-BBN) that binds cancer specific GRPR subtypes, NMB and
BB2. The IC50 values of BBN and its AMBA analog were 2 0.1 nM
and 3.6 0.3 nM. The IC50 value of t-BBN/BVD15-DO3A was
18 0.7 nM (Fig. 3) in the same assay. The MCF7 cells express Y1
receptor37 and were used for Y1 receptor competition assays. The
MCF7 cells do not express very much human GRPR (tested but data
not shown). The MCF7 cells were pre-blocked with 1 lM BBN to
prevent any GRPR influence on Y1 binding. NPY and BVD15-
DO3A (Table 1) were used as controls to compare the affinity of
t-BBN/BVD15-DO3A, with the natural NPY ligand (binds all Y1–
Y5 receptors) and its monomeric BVD15 component (preferentially
binds cancer specific Y1). The IC50 value of NPY and BVD15-DO3A
were 2.6 1.2 and 9.8 1.2 nM (Fig. 4). In the same assay, the IC50
value of t-BBN/BVD15-DO3A was 80 11 nM (Fig. 4). The experi-
A heterobivalent dual-target probe, t-BBN/BVD15-DO3A, to rec-
ognize both GRPR and Y1 is described herein. The probe combines
AMBA, a ligand for two cancer specific GRPR subtypes36 and
BVD15, a ligand for Y1 receptor,37 a cancer-specific subtype of the
Neuropeptide Y receptor family. Bombesin is a 14 amino acids
(aa) long natural ligand of GRP receptors.38 AMBA comprises amino
acids 7–14 of bombesin (t-BBN), a 4-aminobenzoyl linker (Abz4),
and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) for
radio-labeling,36 and binds to two GRPR receptor subtypes, neurom-
edin B (NMB) and BB2, expressed in cancers preferentially to the
GRP receptor subtype, BB3, expressed in a few normal tissues.36
BVD15 is a ligand for Y1 that can be modified at the fourth lysine po-
sition with DO3A for radio-labeling.37 BVD15 is known to have some
binding to other members of Y1 receptor family, Y2 and Y4, but
[Lys4(DOTA)]BVD15 is shown to be Y1 selective.37 The heterobiva-
lent probe, t-BBN/BVD15-DO3A, is capable of competing with the
natural ligands, 125I-BBN and 125I-NPY, with reasonable affinities
and can recognize both GRPR and Y1 on human breast tumor cells.
Bombesin (BBN, #20665, >95% pure, MW 1620.9) and Neuro-
peptide Y (NPY, #22465, >95% pure, MW 4271.8) were obtained
from AnaSpec. Human 125I-Tyr4-BBN (#NEX258050UC) and por-
cine 125I-NPY (NEX222050UC) were obtained from Perkin–Elmer.
Other peptides were synthesized using solid phase peptide synthe-
sis methods.46 All the intermediates and final compound were
purified using reverse phase high-performance liquid chromatog-
raphy (HPLC) and characterized by NMR and mass spectrometry.
Sequences of all the compounds tested are shown in the Table 1
(details of experiments are described in the References and Notes
ment shown here was done with 1 lM BBN to avoid any conse-
quences of undetectable or low GRPR on NPY expression. In the
same experiment, we also obtained IC50 of NPY without pre-block-
ing with BBN (4.7 1.9 nM) to compare with BBN blocking and
found no significant effect (data not shown in Fig. 4). To confirm
Y1 binding of t-BBN/BVD15-DO3A, IC50 of t-BBN/BVD15-DO3A
was also done in SK-N-MC (human neuronal epithelioma) cells
(74 5 nM) which are known to express only Y1 receptor (data
not shown).42,43 Binding of t-BBN/BVD15-DO3A to Y2 and Y4
receptor was not explored because BVD15 modified at Lys-4
position, [Lys4(DOTA)]BVD15, has been shown to be Y1 selective.37
Finally, our data clearly shows that t-BBN/BVD15-DO3A can bind
both GRPR and Y1.
Imaging of breast and prostate cancers actually suffers from two
problems. First, the sensitivity of PET/CT for detection of lesions
<1 cm is poor. PET/CT has made significant advances over PET in
this regard, and MRI/PET is poised to further increase the sensitiv-
ity for detection of small lesions.1 The second problem is specificity
for malignancy, and is better addressed with a new probe. For
example, in early triage of breast cancer, FDG is not used because
its high false positive rate creates inefficiencies in time and costs
as false leads are followed up in a triage where time to treatment
is important. A reasonable goal for a new PET/CT probe will there-
fore be to perform at greater specificity for cancer versus normal
(e.g., inflamed) tissue. Dual-target binding of our bivalent probe
is so intended: the AMBA analog binds to GRPR subtypes, NMB
and BB2 with no or minimal BB3 binding,36 and the BVD15 analog
binds specifically to Y1.37
Table 1
Sequences of peptide compounds
Compound
Molecular weight
Sequence
Bombesin analog
AMBA analog
Human NPY
BVD15-DO3A
t-BBN/BVD15-DO3A
1620.9
1555.85
4271.8
1607.9
3240.7
Pyr-QRLGNQWAVGHLM-NH2
DO3A-Dab4-Abz4-QWAVGHLM-NH2
YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY-NH2
INPK(DO3A)YRLRY-NH2
INP-K[e-Aoa-K(a-DO3A-e-DGa-Aoa-G-Abz4-QWAVGHLM-NH2)]-YRLRY-NH2
Abbreviations used are Pyr (pyroglutamic acid), DO3A (1,4,7,10-tetraazadodecaundecane-1,4,7,10-tetraacetic acid), Dab4 (1,4-diamino-
acid), and DGa (diglycolic acid).
L-butyric acid), Abz4 (4-aminobenzoic

A. Shrivastava et al. / Bioorg. Med. Chem. Lett. 23 (2013) 687–692
689
Figure 1.
Figure 2.

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A. Shrivastava et al. / Bioorg. Med. Chem. Lett. 23 (2013) 687–692
heteromultivalent probes to simultaneously target two different
receptors on the same cell24,31–33,35 or two different receptors in
the same tumor environment.23,25–30,34 Heterobivalent ligands tar-
geting two different receptors on the same cell can simultaneously
bind both receptors generating higher affinity than corresponding
monovalent peptides for one of the receptors, and greater avidity
for cells expressing both receptors.24,31–33,35 Heterobivalent ligands
targeting two different receptors in the same tumor had similar or
lower affinity than corresponding monovalent peptides for two
targeted receptors and did not simultaneously bind both receptors,
but still showed higher tumor affinity in vivo.23,25–30,34 It was
hypothesized that increased tumor avidity is due to increased local
ligand concentration and the availability of two different receptors
for binding,34 although receptor interaction or pharmacokinetics
differences are possible contributing variables.
The goal of the current study was to create a heterobivalent
dual-target probe, t-BBN/BVD15-DO3A, for imaging and radiother-
apy of cells, tissues and tumors positively expressing receptors,
GRPR, Y1 or both. We conclusively showed that t-BBN/BVD15-
DO3A is capable of binding both GRPR and Y1 on two different
breast cancer cells, T-47D (ductal carcinoma) and MCF7 (adenocar-
cinoma). Therefore, t-BBN/BVD15-DO3A is a potentially useful can-
didate for imaging and radiotherapy of GRPR and Y1 receptor
positive tumors, a class including both breast and prostate cancers.
The IC50 values of t-BBN/BVD15-DO3A for GRPR and Y1 receptors
were significantly higher than the IC50 value of the monomeric
components, t-BBN and BVD15. A similar phenomenon was ob-
served previously with the RGD-BBN heterobivalent ligand but
that ligand still worked well in vivo.34 A similar phenomenon
might be expected in GRPR and Y1 positive tumors using t-BBN/
BVD15-DO3A.
Figure 3.
Our work provides a proof of concept for dual-target probe tar-
geting of GRPR and Y1 receptors. Further characterization of t-BBN/
BVD15-DO3A will require radio-labeling with 68Ga for imaging,
177Lu for radiotherapy, in vitro cell binding and in vivo tumor
animal studies with radio-labeled material, determining whether
t-BBN/BVD15-DO3A is an agonist or antagonist, and the kinetics
of its receptor binding.
Acknowledgment
The authors acknowledge Ms. Keisha Milum for maintaining the
cell cultures and reviewing the manuscript.
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deprotection (Fmoc) from
G residue under 20% piperidine and then was
subsequently coupled with Fmoc-J-OH, under HBTU conditions. Eventually the
peptide was released from the resin using a cocktail containing trifluoroacetic
39. Ma, L.; Yu, P.; Veerendra, B.; Rold, T. L.; Retzloff, L.; Prasanphanich, A.;
Sieckman, G.; Hoffman, T. J.; Volkert, W. A.; Smith, C. J. Mol. Imaging 2007, 6,
171.
acid, phenol, tris-isopropylsilane and water in a ratio of 95:2:2:1 and
precipitated into methyl-tert-butyl ether discussed previously. The
precipitate was filtered and the crude solid was purified on preparative HPLC
40. Parry, J. J.; Andrews, R.; Rogers, B. E. Breast Cancer Res. Treat. 2007, 101, 175.
41. Prasanphanich, A. F.; Retzloff, L.; Lane, S. R.; Nanda, P. K.; Sieckman, G. L.; Rold,
T. L.; Ma, L.; Figueroa, S. D.; Sublett, S. V.; Hoffman, T. J.; Smith, C. J. Nucl. Med.
Biol. 2009, 36, 171.
[Shimadzu preparatory purification unit (LC8A)] using C18 column (10 lm,
50 ꢀ 250 mm, 60 min runtime at 100 mL/min) with water (0.1% TFA): MeCN
(0.1% TFA)-10–100% solvent. Finally, the fully protected acid intermediate 8
(60.0 mg, 0.02 mmol) was dissolved in DMF (100 lL), and HATU (8.0 mg,
42. Chatenet, D.; Cescato, R.; Waser, B.; Erchegyi, J.; Rivier, J. E.; Reubi, J. C. EJNMMI
Res. 2011, 1, 21.
0.04 mmol) and NMM (8.0 mg, 0.08 mmol) were added with stirring; the
activation was continued for 30 min. The amine, J-Gly-Abz4-Gln-Trp-Ala-Val-
43. Wahlestedt, C.; Regunathan, S.; Reis, D. J. Pharmacol. Lett. 1992, 50, PL7.
44. Pochon, S.; Tardy, I.; Bussat, P.; Bettinger, T.; Brochot, J.; von Wronski, M.;
Passantino, L.; Schneider, M. Invest. Radiol. 2010, 45, 89.
45. Larhammar, D.; Blomqvist, A. G.; Yee, F.; Jazin, E.; Yoo, H.; Wahlestedt, C. J. Biol.
Chem. 1992, 267, 10935.
46. Notes: Peptide synthesis: monomeric peptides were synthesized using Fmoc
strategy. For each mmol of the amine on the resin, protected amino acid
4.0 mmol was activated with 4.0 mmol of the appropriate coupling agent like
HATU/HBTU and 8.0 mmol of DIEA for 5 min and the activated acid was
transferred to the amine on the solid phase and the vessel was shaken for
90.0 min. The final products and the protection groups were released from the
resin (process repeated twice, 10 mL) using a cocktail containing trifluoroacetic
Gly-His-Leu-Met-NH2 (25.0 mg, 0.02 mmol) in 100 lL of DMF (pH adjusted to
8.0 with NMM) was transferred to the acid and stirring was continued for 20 h.
The reaction mixture was diluted with t-butyl methyl ether, and the
precipitate was filtered, washed with water and air dried. Fully protected
peptide 9 (crude; 80.0 mg) was air dried and subjected to cleavage with
10.0 mL of cocktail reagent B for 3 h. All the volatiles were removed under
reduced pressure and the residue was triturated with dry ether; the clear ether
layer was decanted and discarded. This step was repeated two more times and
the residue was then dissolved in water and filtered. The filtrate was loaded on
to a preparative HPLC column and purified. Fractions with >90% purity were
pooled and checked for the product by MS. The fractions with the required
mass and purity >90% were pooled and freeze dried to yield the product as a
colorless fluffy solid, 16.0 mg (4% yield). The chemical structure of t-BBN/
BVD15-DO3A is shown in Figure 2. Cell culture: the GRPR competition assays
were done using T-47D human breast cancer cells (ductal carcinoma;
American Type Culture Collection, cat#HTB-133). T-47D cells were
maintained in DMEM high glucose supplemented with 10% fetal bovine
serum (Atlanta Biologicals) and 1% Pen Strep (Gibco, Life Technologies) and
sub-cultured using 0.25% trypsin once per week using standard cell culture
techniques. The Y1 receptor competition assays were done using MCF7,
another human breast cancer cell (adenocarcinoma; American Type Culture
Collection, cat#HTB-22). MCF7 cells were maintained in DMEM high glucose
containing additional 2 mM glutamine supplemented with 10% fetal bovine
serum (Atlanta Biologicals) and 1% Pen Strep (Gibco, Life Technologies) and
sub-cultured using 0.25% trypsin acid twice per week using standard cell
culture techniques. Cells were seeded in 96-well plate at 30,000 cells per well
acid, phenol, tris-isopropylsilane and water in
precipitated into methyl-tert-butyl ether. The heterobivalent dual-target
target probe synthesis was accomplished using multistep process.
a ratio of 95:2:2:1 and
a
A
scheme for synthesis of t-BBN/BVD15-DO3A is shown in Figure 1. As a first
step, Sieber amide resin (0.5 mmol/g) was loaded on to a reaction vessel,
placed on an ABI 433A automated peptide synthesizer (0.4 g, 0.2 mmol) and
the synthesis was carried out using the standard peptide synthesis protocol
until the last amino acid was added (intermediate 1). The resin was then
transferred to another reaction vessel and shaken with 10.0 mL of 5% hydrazine
in N,N-dimethylformamide or DMF (volume/volume or v/v) for 10.0 min. The
resin was drained and the above procedure was repeated two more times. The
resin was then washed with DMF (3 ꢀ v) (intermediate 2) and taken to the
next step. A solution of Fmoc-J (Fmoc: 9-fluorenylmethyloxycarbonyl, 0.385 g,
1.0 mmol, in 2.0 mL of DMF) was activated with O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluroniumhexafluorophosphate
or
HBTU
(0.378 g,
in 200 lL of cell culture medium for competition assays. GRPR competition
1.0 mmol) and N,N-diisopropylethylamine or DIEA (0.26 g, 2.0 mmol). The
solution was stirred for 5.0 min, transferred to the vessel containing the resin
from above (intermediate 2) and the remaining activated acid was transferred
to the vessel with 5.0 mL more of DMF. The coupling was carried out for
60.0 min by shaking on a mechanical shaker; resin was drained by positive
pressure of nitrogen and then thrice washed with DMF, to obtain intermediate
3. The resin in the vessel from intermediate 3 was de-protected using the
standard protocol and washed three times with DMF to obtain intermediate 4.
The above intermediate resin was coupled to Fmoc-K(ivDde)-OH (ivDde:1-
binding: cell culture medium was removed 24 h after plating and cells were
washed twice with binding buffer (RPMI 1640, 20 mM HEPES, 0.1% BSA w/v,
0.1 mg/mL bacitracin) at room temperature (rt). Cells were placed in a 4 °C
refrigerator for 30 min for slow cooling. All the subsequent steps used ice cold
solutions and incubations were performed at 4 °C. Cells were pre-blocked with
70 lL of 1 lM NPY (AnaSpec, cat#22465) in binding buffer for 30 min to reduce
any binding to Y1 receptors in T-47D cells. Peptide mixtures for the
competition assay were prepared using constant concentration of 125I-Tyr4-
BBN (0.22
l
Ci/mL), constant concentration of NPY (1
l
M) and varied (0.1 nM–
L of
(4,4-dimethyl-2,6-dioxocyclohexylidine)-3-methylbutyl)
standard coupling procedure as described for intermediate
intermediate 5. The intermediate
employing
a
1
lM) concentration of competing peptide. Cells were incubated with 70 l
3
to obtain
peptide mixtures in each well for 1 h. Each concentration of competing peptide
was tested in triplicate. After 1 h, cells were washed five times with ice cold
wash buffer (25 mM HEPES, 150 mM NaCl, pH 7.4). Cells were lysed by adding
5
was de-blocked to remove the Fmoc
protecting group and washed for subsequent coupling to DO3A-tri-t-butyl acid
under standard coupling conditions for 1 h, drained and washed with DMF to
obtain intermediate 6. The resin was shaken with 5% hydrazine in DMF for
10 min and then drained. The procedure was repeated two more times and
then washed thrice with DMF to yield the free amine on the resin as
intermediate 7. The resin was suspended in 10.0 mL of anhydrous pyridine,
diglycolic acid anhydride (0.464 g, 4.0 mmol) was added followed by
200 lL of 1 N NaOH kept at 37 °C twice to each well, solutions were transferred
to tubes and radioactivity was measured using an automatic gamma counter
(Perkin–Elmer Wizard II, Model 2480). Data were analyzed using GraphPad
Prism 5. Y1 Competition Binding: Cell culture medium was removed 24 h after
plating and cells were washed twice with binding buffer (25 mM Hepes,
2.5 mM CaCl2, 1 mM MgCl2, 1% BSA and 0.1 mg/mL bacitracin, pH 7.4) at rt.

692
A. Shrivastava et al. / Bioorg. Med. Chem. Lett. 23 (2013) 687–692
Cells were pre-blocked with 70
l
L of 1
l
M bombesin (AnaSpec, cat#20665) in
cells were washed five times with the binding buffer at rt. Cells were lysed by
adding 200 L of 1 N NaOH kept at 37 °C twice to each well, solutions were
transferred to tubes and radioactivity was measured using an automatic
gamma counter (Perkin–Elmer Wizard II, Model 2480). Data were analyzed
using GraphPad Prism 5. Porcine 125I-NPY was used instead of human because
it is commercially available and is equipotent with human NPY.45
binding buffer for 30 min at rt to reduce any binding to GRPR in MCF7 cells.
Peptide mixtures for the competition assay were prepared using a constant
l
concentration of 125I-NPY (porcine) (0.2229
l
Ci/mL), constant concentration of
M) concentration of competing peptide.
L of peptide mixtures in each well for 2 h at rt.
Each concentration of competing peptide was tested in triplicate. After 2 h,
BBN (1
lM) and varied (0.1 nM–1 l
Cells were incubated with 70
l