Development of melanoma-targeted polymer micelles by conjugation of a melanocortin 1 receptor (MC1R) specific ligand

Article abstract of DOI:10.1021/jm201226w

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH₂) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity.

Full text of DOI:10.1021/jm201226w

Article
pubs.acs.org/jmc
Development of Melanoma-Targeted Polymer Micelles by
Conjugation of a Melanocortin 1 Receptor (MC1R) Specific Ligand
Natalie M. Barkey,† Narges K. Tafreshi,† Jatinder S. Josan,‡ Channa R. De Silva,§ Kevin N. Sill,∥
Victor J. Hruby,‡,⊥ Robert J. Gillies,† David L. Morse,*,† and Josef Vagner*,‡
†Department of Imaging, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, Tampa, Florida, 33612, United
States
‡The BIO5 Research Institute, University of Arizona, 1657 E Helen Street, Tucson, Arizona 85721, United States
§Department of Chemistry and Physics, Western Carolina University, Cullowhee, North Carolina 28723, United States
∥Intezyne Technologies, Inc., 3720 Spectrum Boulevard, Suite 104, Tampa, Florida 33612, United States
⊥Department of Chemistry and Biochemistry, University of Arizona, 1306 E. University Boulevard, Tucson, Arizona 85721, United
States
S
* Supporting Information
ABSTRACT: The incidence of malignant melanoma is rising faster than that of any
other cancer in the United States. Because of its high expression on the surface of
melanomas, MC1R has been investigated as a target for selective imaging and therapeutic
agents against melanoma. Eight ligands were screened against cell lines engineered to
overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-
DPhe-Arg-Trp-NH2) exhibited high (0.2 nM) binding affinity for MC1R and low (high
nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-
terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect
the binding affinity. Finally, formation of the targeted polymer, as well as the targeted
micelle formulation, also resulted in constructs with low nanomolar binding affinity.
to design selective ligands that are highly specific for receptor
subtypes.
INTRODUCTION
■
The incidence of malignant melanoma is rising faster than that
of any other cancer in the United States, with diagnoses having
doubled from 1986 to 2001.1 Melanoma progression is
associated with altered expression of cell surface proteins,
including adhesion proteins and receptors.2 Over 80% of
malignant melanomas express high levels of isoform 1 of the
melanocyte stimulating hormone (αMSH) receptor (melano-
cortin 1 receptor, or MC1R).3 Thus, MC1R has been
investigated as a target for selective imaging and therapeutic
agents. MC1R belongs to a family of five G-protein-coupled
melanocortin receptors (MC1R−MC5R). Melanocortin recep-
tors have been discovered in a wide range of tissues and organs
throughout the body, ranging from the hair/skin (MC1R),4
kidneys (MC5R),5 adrenal glands (MC2R),6 and hypothalamus
(MC3R/MC4R)7 and are known to play a role in skin
pigmentation, hair coloration, obesity, metabolism, diabetes,
sexual behavior, erectile dysfunction, stress response, and
mood.4−8 Endogenously, agonists for the melanocortins are
the α-, β-, γ-melanocyte stimulating hormones (MSH) and
adrenocorticotropic hormone (ACTH, MC2R specific), all of
which contain the same central sequence of His-Phe-Arg-Trp.9
This high degree of pharmacophore homology makes it difficult
Because of its high expression on the surface of melanomas,
MC1R has been investigated as a target for selective imaging
and therapeutic agents, and a number of selective ligands have
been developed.10 The most well-known of these, [Nle4,D-
Phe7]-α-MSH (NDP-α-MSH),11 has been investigated exten-
sively by Chen who showed that 99αTc-CGCG labeled NDP-α-
MSH bound to melanomas with very high avidity (6.5% ID/
g).12 However, it is not selective, as NDP-α-MSH has strong
nanomolar binding affinities to MC3R, MC4R, and MC5R as
well.13 Such off-target binding is undesirable given the presence
of these receptors in sensitive organs such as the kidney and
brain. A co-injection of lysine has been reported to diminish
off-target binding in the kidneys,12,14 and presumably most
agents will not be able to cross the blood−brain barrier.
Nonetheless, the need for the development of highly specific
and selective ligands against MC1R for melanomas is of critical
importance.
Received: July 11, 2011
Published: October 19, 2011
© 2011 American Chemical Society
8078
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084

Journal of Medicinal Chemistry
Article
The development of ligands that can be attached to micelles
and/or liposomes to target cancer cells relative to healthy
organs is a major hurdle in current research. Stabilized micelles
and liposomes are emerging as important platforms for delivery
of lipophilic therapies to target tissues. Many such attempts fail
from (1) a loss of affinity resulting from the attachment of small
peptides to large micelles or liposomes, (2) an inherent
instability that results in collapse before entering the vicinity of
the tumor, or (3) a nanoparticle size that is too large to escape
the vasculature. In order to effectively design targeted
nanoparticles, each of these issues must be addressed.
Table 2. Affinity and Selectivity of Ligands Assayed in This
Publication
Ki (nM)
ligand
MC1R
MC4R
MC5R
1R/4R
1R/5R
1
2
3
4
5
6
7
8
0.17
1.8
160
988
27
58
950
560
160
33
a
a
a
NB
NB
NB
0.24
2.0
2.6
5.6
4.2
26
254
0.75
1.7
46
1058
0.38
0.67
0.13
1.0
192
0.76
1.4
0.38
0.57
0.13
0.93
0.77
4.4
0.71
3.9
In the current work, we screened eight putatively MC1R-
specific compounds (Table 1) against cell lines that were
a
a
4-targeted polymer
4-targeted micelles
NDP-α-MSH
NB
NB
a
a
2.9
1.8
NB
NB
Table 1. Structures of Ligands Screened for MC1R
Selectivity
19
9.9
10
5.5
a
NB: nonbinding.
compd
structure
1
2
3
4
5
6
4-phenylbutyryl-His-DPhe-Arg-Trp-NH2
stabilized micelles as controls (Figure 1, Table 2). The 4-
targeted micelle exhibits an increased binding avidity to the
hMC1R receptor compared to the targeted polymer, and both
are less avid than the native ligand (Ki of 2.9, 25.7, and 0.24 nM
for the micelle, polymer, and ligand, respectively). No binding
is observed with the untargeted polymer or untargeted micelles,
indicating that the triblock polymer does not interact
nonspecifically with the cell surface. Most notably, there is no
measurable interaction (≤10−5) between the 4-targeted micelle
and hMC4R or hMC5R, indicating that the conjugation of the
ligand to a micelle results in an increased selectivity.
Ac-homophenylalanine-His-DPhe-Arg-Trp-NH2
4-hydroxycinnamoyl-His-DPhe-Arg-Trp-NH2
4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH2
H-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH2
H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-
Arg-Phe-Gly-NH2
7
8
H-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH2
H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-
Arg-Phe-Gly-NH2
engineered to overexpress MC1R, MC4R, or MC5R,
respectively. Compounds were tested for their ability to
compete with Eu-NDP-α-MSH using a readout of time-
resolved fluorescence (TRF).13b,15 Of these, compound 1
exhibited high binding affinity for MC1R and low affinities for
MC4R and MC5R. Two analogues (compounds 2 and 3) have
been synthesized to allow attachment via their N-termini;
however, they exhibited a reduced (2) or complete loss (3) of
binding affinity. Therefore, 1 was modified at the C-terminus
(compound 4) to allow attachment to the micelle, and this
modification was shown to not affect the binding affinity.
Attachment to the stabilized polymer-based micelles was
accomplished with Cu-catalyzed click chemistry, and the
resulting construct was also shown to retain low nanomolar
binding affinity.
Micelle Physical Properties. Dynamic light scattering
(DLS) and zeta potential measurements showed the size and
surface charge of the 4-targeted micelles to be 91 ± 2 nm and
−10.6 ± 0.9 mV, respectively.
DISCUSSION
■
Historically, ligands that are known to interact with the hMC1R
receptor also demonstrate cross-reactivity with other melano-
cortin receptors, including hMC4R and hMC5R. While MC1R
is known to be expressed almost exclusively in melanoma cells
and melanocytes, hMC4R and hMC5R have high expression
levels in normal tissues, including kidney and brain; thus,
nonspecific ligand binding is not ideal. To combat this problem
and minimize off-target effects, we chose several ligands from
the literature that have previously been reported to possess
nanomolar binding affinities for hMC1R.
RESULTS
■
Ligand 1 was reported to have a high affinity and selectivity
for MC1R (1R/4R selectivity ratio of 1200)10c,16 and was
consequently chosen as a template for the design of the novel
ligands 2 and 3, which contain the same parent amino acid
sequence, as well as 4, which possesses a terminal alkyne.
Ligands 2 and 3 were initially designed for potential attachment
to a micelle through the N-terminus. Compound 2 represents
the α-amino analogue of 1, and compound 3 possesses a more
rigid C2 (cinnamate) aromatic linker compared to the 4-
phenylbutyryl, 1, which has free rotation about the C3 chain.
The 4-hydroxy group in compound 3 was intended as an
attachment point to the micelles via O-alkylation. As
mentioned, the rigid C2 linker in 3 reduces the free rotation
of compound 1 and thus we reasoned might potentially result
in improved interaction with the hydrophobic cleft of the
MC1R. Unfortunately, compound 3 exhibited no affinity to the
MC1R receptor, and compound 2 exhibited 10 times lower
binding affinity compared to 1. Therefore, we chose to proceed
Competitive Binding Assays. Competitive binding assays
were performed using HCT116/hMC1R, HEK293/hMC4R,
and HEK293/hMC5R cells with ligands 1−8. Seven of these
were observed to bind to MC1R with low nanomolar affinity.
Of these, only three related compounds (1, 2, and 4) were
̀
found to bind selectively to the hMC1R receptor, vis-a-vis
MC4R or MC5R (Table 2). Compound 1 displayed slightly
higher affinities for hMC1R compared to 2 (Ki is 0.17 and 1.77
nM for 1 and 2, respectively), as well as higher selectivity,
especially compared to MC5R (selectivity ratios of 160 and 33,
respectively). Ligands 5−8 demonstrated a high affinity for
hMC1R as well; however, they were also shown to have an
even stronger affinity for hMC4R and hMC5R (Table 2).
Ligand 3 demonstrated no affinity for the hMC1R.
Competitive binding assays were also performed using 4-
targeted triblock polymers, 4-targeted stabilized triblock
polymer micelles, as well as untargeted polymer and untargeted,
8079
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084

Journal of Medicinal Chemistry
Article
Figure 1. Representative competitive binding assays for (A) 4-targeted polymer, (B) untargeted polymer, (C) 4-targeted micelles, (D) untargeted
micelles, and (E) 4 against HCT116/hMC1R cells. X-Axis concentrations for (A), (C), and (E) were normalized to the targeting ligand.
Concentrations for the ligand in (A), (C), and (E) and polymer and micelle in (B) and (D) were deliberately chosen to be the same for all assays.
with a C-terminal modification (i.e., compound 4), as the
binding affinity was not changed compared to the parent
compound 1. Additionally, ligands 5 and 7 were reported to
have moderate hMC1R selectivities over hMC4R and hMC5R
(1R/4R of 20.00 and 12.50 nM for 5 and 7, respectively; 1R/
5R of 11.67 and 2.06 for 5 and 7, respectively);13a thus, each
was functionalized with a terminal alkyne for attachment to a
nanoparticle scaffold via click chemistry and screened for
retained selectivity to MC1R.
The most specific of these ligands, 1, was determined by us
to have an hMC1R/hMC4R selectivity of 950, which is in good
agreement with the reported selectivity of 1200. Likewise,
ligand 2, based on the same parent amino acid sequence as 1,
was also found to have high 1R/4R selectivity; however, its 1R/
5R selectivity was substantially lower. Unfortunately, ligands 3
and 5−8 were found to be not at all selective for MC1R, with
ligand 3 possessing no affinity for any of the receptors tested.
Our results for ligands 5 and 7 deviate from that which has
been previously reported; however, this discrepancy may be
due to differences in the binding assays used to derive the
affinity constants. As detailed in the Experimental Section, our
lab derived Ki values based on europium time-resolved
fluorescence assays; however, previously determined EC50
values for these ligands were derived via 125I-labeled
competitive binding assays.
As 1 was determined to be the ligand with the highest
hMC1R affinity and selectivity, it was chosen for modification
with a terminal alkyne for attachment of a triblock polymer
micelle. Compound 4 did not demonstrate a loss of affinity of
MC1R following alkyne functionalization.
As predicted, 1 and 2 have similar binding profiles given the
similarity in their structures; however, it was surprising to see a
complete loss of affinity in 3. The differences in affinity among
these three ligands arise from the structural differences at the
N-terminal end of the peptide, given that they all share the
same R-HfRW-NH2 parent scaffold. However, whereas 1 and 2
contain Ph-(CH2)3-CO- and Ac-Hpe groups, both of which are
nonpolar, at the N-terminus, 3 contains a 4-hydroxy-Ph-CH
CH-CO-, which is more polar because of the incorporation of
the hydroxyl. Conversely, several analogues of 3 reported in the
literature possess low nanomolar affinities against MC1R with
varying selectivities. Thus, it seems reasonable to conclude that
the loss in affinity experienced by 3 results from the
incorporation of the alkene rather than the increased polarity
that arises from the addition of the hydroxyl group. While the
exact reasons behind the affinity of 3, or lack thereof, remain
unclear, it is plausible that incorporation of the alkene in this
ligand causes the peptide to adopt too rigid a structure, thereby
reducing its ability to conform to the receptor binding pocket.
8080
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084

Journal of Medicinal Chemistry
Article
Ligands 5−8 are about twice as large and display binding
affinities 1−2 orders of magnitude higher with hMC1R
compared to those described above. Ligands 6 and 8 were
synthesized as analogues of ligands 5 and 7, respectively, to be
used for potential attachment to nanoparticles. The similarity in
binding affinities of 5 versus 6 and 7 versus 8 further
demonstrates that the C-terminal end of these peptides is a
suitable location for the placement of an attachment of a
scaffold, as it does not seem to impact the binding ability of the
ligand.
A targeted, stabilized triblock polymer micelle was prepared
by combining 10% 4-targeted polymer with 90% untargeted
polymer (Scheme 2). As a control, competitive binding assays
were performed with targeted and untargeted polymer, as well
as untargeted stabilized micelles. For consistency, all binding
assays were normalized to the concentration of the ligand. As
previously stated, the 4-targeted micelle exhibits an increased
binding avidity to the hMC1R receptor compared to the
targeted polymer and a slightly weaker avidity than the native
ligand. The increase in binding avidity for the targeted micelles
compared to the targeted polymer is noteworthy in that (1) it
demonstrates the in vitro stability of this micelle system and (2)
it indicates that the binding avidity of the 4-targeted micelles
may be benefiting from multivalent interactions. Additionally,
the 4-targeted polymer and 4-targeted micelle demonstrated no
measurable interactions with either MC4R or MC5R, thereby
indicating that the targeted micelle is itself more specific than
the ligand alone.
The decrease in binding affinity of the targeted polymer
relative to the native ligand can be explained by the conjugation
of a large, flexible PEG group to a relatively small ligand. In
addition to adding to the entropy of the ligand system through
increased flexibility and size, PEG chains are known to have at
least moderate interaction with nonpolar hydrophobic
groups.18 Consequently, it is possible that the PEG moiety
on the end of the triblock polymer is weakly interacting with
the hydrophobic amino acids of the targeting group, thereby
decreasing its affinity for MC1R.
However tempting it may be to ascribe the modest increase
in avidity of the targeted micelles relative to targeted polymer
solely to multivalent binding, such interactions are of high
complexity and can be attributed to the sum of multiple factors.
These variables include the enthalpies of individual binding
events, the entropic consequences experienced by the micelle
polymer upon binding, the topography of ligand positioning on
the micelle, the statistical proximity effect, and differences in
rates of cellular uptake.
positioned on one side of the micelle could prohibit binding of
ligands positioned on the opposite side. Since total ligand
concentration was considered when calculating avidities, having
a large fraction of ligands inaccessible to receptor at any given
time would effectively reduce the calculated avidity. However,
even if multivalent binding is completely inhibited in this
manner, increased binding avidity can be still observed because
of the statistical effect where proximal ligands can readily bind
following release of an existing interaction.21
Lastly, the rate of cellular uptake is likely different for the
targeted micelle relative to the targeted polymer or free ligand.
Constructs with faster rates of uptake can be calculated to have
higher avidities due to the loss of “off-rate” once internalized.
The sum of these variables and others not mentioned may
contribute to the observed binding avidity of these supra-
molecular systems.
CONCLUSION
■
An hMC1R ligand was modified for attachment to a stabilized
triblock polymer micelle. Functionalization and subsequent
attachment of the ligand to a ∼100 nm polymer micelle
resulted in a slight decrease in affinity, but increase in
specificity, to MC1R. Presumably, this decrease results from
the thermodynamic hurdles encountered in appending a small
peptide to a large nanoparticle, as well as an inherent handicap
in the assay design. As mentioned in the Introduction, three
hurdles must be overcome in the design of an effective targeted
nanoparticle delivery system: (1) it must be ensured that there
is no loss of ligand affinity resulting from the attachment of a
small peptide to a large nanoparticles, or any such loss in
affinity must be compensated by multivalent binding
interactions; (2) nanoparticles must be inherently stable; (3)
nanoparticles must be sufficiently small to escape the
vasculature and enter the tumor. We believe the current
publication addresses the first two of these concerns. We have
shown that our ligand remains selective after attachment and
the increased binding affinity observed between the 4-targeted
polymer and 4-targeted stabilized micelle has demonstrated the
in vitro stability of the system. On the basis of our DLS data, we
are confident that our micelles are of sufficient size to escape
the vasculature, and in vivo studies to evaluate the selectivity
and stability of this targeted micellar system in mice are
currently underway.
EXPERIMENTAL SECTION
■
Ligand Synthesis. N-α-Fmoc-protected amino acids, HBTU,
HCTU, HOCt, and HOBt were purchased from Anaspec (San Jose,
CA) or Novabiochem (San Diego, CA). Rink amide Tentagel S and R
resins were acquired from Rapp Polymere (Tubingen, Germany). Rink
amide 1% DVB PS resin was acquired from Novabiochem (San Diego,
CA). For the N-α-Fmoc-protected amino acids, the following side
chain protecting groups were used: Arg(Ng-Pbf); Asp(O-tBu);
His(Nim-Trt); Trp(Ni-Boc); Tyr(tBu); Lys(Nε-Aloc). Reagent grade
solvents, reagents, and acetonitrile (ACN) for HPLC were acquired
from VWR (West Chester, PA) or Aldrich-Sigma (Milwaukee, WI)
and were used without further purification unless otherwise noted. N-
Terminal heterocyclic acids, NMI, and scavengers were obtained from
Sigma-Aldrich or TCI. The solid-phase synthesis was performed in
fritted syringes using a Domino manual synthesizer obtained from
Torviq (Niles, MI). The C-18 Sep-Pak RC cartdridges for solid phase
extraction were purchased from Waters (Milford, MA).
Improved avidity through multivalent interactions is more
readily observed with ligands with relatively low affinity.19 In
the case of 4, it is possible that multivalent interactions would
not greatly enhance binding avidity given the high affinity of the
targeting ligand for binding the MC1R receptor. Also,
assembled polymer micelles have decreased entropy relative
to free polymer, via the hydrophobic effect, thereby potentially
leading to slower ligand−receptor off rates.20 Additionally,
ligand proximity and steric repulsions between ligands and
polymer chains are known to be important factors that
influence the degree, if any, to which multivalency is
experienced in micellar systems.19b
The targeted micelles herein are calculated to have
approximately 10 targeting groups each. This may seem to be
a large number; however, since these stabilized micelles are
relatively inflexible, ligands binding to the surface of a cell
Ligands 1−8 were prepared as previously published by solid-phase
synthesis as summarized in Scheme 1 on Rink amide Tentagel resin
(0.23 mmol/g) using a Fmoc/tBu synthetic strategy and standard
8081
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084

Journal of Medicinal Chemistry
Article
hMC5R as previously described, with slight modifications.13b
HCT116/hMC1R cells were plated in black PerkinElmer 96-well
plates, and HEK293/hMC4R and HEK293/hMC5R cells were plated
on SigmaScreen poly-D-lysine coated plates (Sigma-Aldrich), all at a
density of 10000−30,000 cells/well. Poly-D-lysine coated plates
contain a PDL polymer coating that creates a uniform positive charge
at the surface of the plastic, thereby facilitating cell attachment, growth,
and differentiation. Both PerkinElmer and SigmaScreen plates were
evaluated for nonspecific binding. Cells were grown in the 96-well
plates for 2−3 days. On the day of the experiment, the medium was
aspirated and 50 μL of nonlabeled competing ligand was added to each
well in a series of decreasing concentrations (ranging from ∼1 μM to
0.1 nM), followed by 50 μL of Eu-NDP-α-MSH at 10 nM. Both
labeled and nonlabeled ligands were diluted in binding medium
(DMEM, 1 mM 1,10-phenanthroline, 200 mg/L bacitracin, 0.5 mg/L
leupeptin, 0.3% BSA). In the case of the triblock polymer micelle
solutions, micelles were allowed to equilibrate in solution for 30 min
prior to cell addition. Cells were incubated with labeled and
nonlabeled ligands for 1 h at 37 °C. Following incubation, cells
were washed three times with wash buffer (50 mM Tris-HCl, 0.2%
BSA, 30 mM NaCl), and 100 μL of enhancement solution
(PerkinElmer) was added to each well. Cells were incubated for an
additional 30 min at 37 °C prior to reading. The plates were read on a
PerkinElmer VICTORx4 2030 multilabel reader using the standard Eu
time-resolved fluorescence (TRF) measurement (340 nm excitation,
400 μs delay, and emission collection for 400 μs at 615 nm).
Competition curves were analyzed with GraphPad Prism software
using the sigmoidal dose−response (variable slope) classical equation
for nonlinear regression analysis.
a
Scheme 1. Synthetic Route for Compounds 1−8
Synthesis of Targeted Triblock Polymers. Triblock polymer,
azido-poly(ethylene glycol)-block-poly(aspartic acid)-block-poly-
(leucine-co-tyrosine), was obtained from Intezyne Inc. (Tampa, FL).
Alkyne functionalized ligand 4 was conjugated to the terminal azide of
the polymer by copper assisted click chemistry (CuAAC)24 (Scheme
a
(a) (i) Fmoc-AA-OH (3 equiv), HOCt or HOBt (3 equiv), and DIC
(3 equiv) in DMF/DCM (10 mL/1g of resin) for amino acid
couplings; (iii) piperidine/DMF (1:10, 2 + 20 min); (iv) 4-
phenylbutyric acid (6 equiv), and DIC (3 equiv) in DMF/DCM;
(b) (i) Pd(0) tetrakistriphenylphosphine (0.01 equiv), N,N′-
dimethylbarbituric acid (5 equiv) in degassed DCM (2 × 30 min);
(ii) 5-hexynoic acid (5 equiv) and DIC (3 equiv) in DMF/DCM for
compound 1; S-Trt-3-propanoic acid (5 equiv) and DIC (3 equiv) in
DMF/DCM for compound 2; (c) (i) TFA-scavenger cocktail (91%
TFA, 3% water, 3% thioanisole, 3% ethanedithiol); (ii) ether
extraction; (d) purification.
Scheme 2. Synthesis of Targeted, Stabilized Micelle System
activations.15,22 After final deprotection of the Fmoc group, the resin
was coupled with HOBt ester of 4-phenylbutyric acid (compounds 1
and 4), acetylated with acetic anhydride/pyridine (compound 2), or
left unreacted as a free amino group (compounds 5−8). 4-
Hydroxycinnamoyl-His-DPhe-Arg-Trp-NH-resin was treated with
50% piperidine in DMF to remove 4-hydroxycinnamoyl oligomers.
The ligands were cleaved off the resins with TFA-scavenger cocktail
(91% TFA, 3% water, 3% thioanisole, 3% ethanedithiol), extracted
with cold diethyl ether, then dissolved in 1.0 M aqueous acetic acid.
The crude ligands were purified by SEC and HPLC. All final
compounds were >95% pure by HPLC analysis. The pure compounds
were dissolved in deionized water or DMSO at approximately 1.0 mM,
and concentration was determined by Trp HPLC measurement.23
Ligand purification methods, mass spectra, and HPLC characterization
data are provided in Supporting Information
Cell Culture. HCT116 cells overexpressing hMC1R, and HEK293
cells overexpressing hMC4R13b or hMC5R were used in all studies.
The parental human colorectal carcinoma cell line HCT116
(American Type Culture Collection, CCL 247) was also used. Cells
were maintained under standard conditions (37 °C and 5% CO2) and
were grown in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% FBS and 5% penicillin/streptomycin. For
HCT116/hMC1R cells, Geneticin (G418S, 0.4 mg/mL) was added to
the medium to ensure proper selection.
2) using the following method. To a solution of 1:1 DMSO/H2O (10
mL) was added 4 (16 mmol, 1.2 equiv), triblock polymer (13.3 mmol,
1 equiv), sodium ascorbate (334.15 mmol, 25 equiv), (BimC4A)3
catalyst25 (13.42 mmol, 0.2 equiv), and CuSO4·5H2O (13.3 mmol, 1
equiv). The solution was heated to 50 °C and stirred for 2 days. The
mixture was then cooled and placed in a 3500 MW dialysis bag
(Spectra Por) and dialyzed against EDTA/H2O (×3) and H2O (×3).
Following purification by dialysis, the solution was lyophilized.
Europium Binding Assays. Competitive binding assays were
performed using HCT116/hMC1R cells and HEK293/hMC4R or
8082
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084

Journal of Medicinal Chemistry
Article
Successful click coupling was verified through visualization of the
triazole-H in H NMR (8.02 ppm).
a protein with a molecular weight of 76,000. Cancer Res. 1987, 47 (3),
841−845. (b) Wang, R.; Kobayashi, R.; Bishop, J. M. Cellular
adherence elicits ligand-independent activation of the Met cell-surface
receptor. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (16), 8425−8430.
(c) Wang, Y.; Rao, U.; Mascari, R.; Richards, T. J.; Panson, A. J.;
Edington, H. D.; Shipe-Spotloe, J. M.; Donnelly, S. S.; Kirkwood, J.
M.; Becker, D. Molecular analysis of melanoma precursor lesions. Cell
Growth Differ. 1996, 7 (12), 1733−1740. (d) Wang, Z.; Margulies, L.;
Hicklin, D. J.; Ferrone, S. Molecular and functional phenotypes of
melanoma cells with abnormalities in HLA class I antigen expression.
Tissue Antigens 1996, 47 (5), 382−390. (e) Yang, P.; Farkas, D. L.;
Kirkwood, J. M.; Abernethy, J. L.; Edington, H. D.; Becker, D.
Macroscopic spectral imaging and gene expression analysis of the early
stages of melanoma. Mol. Med. 1999, 5 (12), 785−794. (f) Hsu, M.;
Andl, T.; Li, G.; Meinkoth, J. L.; Herlyn, M. Cadherin repertoire
determines partner-specific gap junctional communication during
melanoma progression. J. Cell Sci. 2000, 113 (Part 9), 1535−1542.
(3) Siegrist, W.; Solca, F.; Stutz, S.; Giuffre, L.; Carrel, S.; Girard, J.;
Eberle, A. N. Characterization of receptors for alpha-melanocyte-
stimulating hormone on human melanoma cells. Cancer Res. 1989, 49
(22), 6352−6358.
1
Micelle Formulation. Triblock polymers were dissolved at 20
mg/mL in 30% tert-butanol/H2O at room temperature, stirred for 4 h,
and then lyophilized (Scheme 2).26 Micelles were stabilized with an
Fe(III) cross-linking (Scheme 2) by dissolving the micelle (20 mg/
mL) in FeCl3 (1.35 mg/mL) in Tris buffer, adjusting to pH 8, and
stirring for 12 h. For the targeted micelle system, 10% targeted
polymer and 90% untargeted polymer were used in the formulation
mixture. Micelle size was determined by dynamic light scattering
(DLS, Wyatt Technology, DynaPro), and surface charge was
determined by ζ measurement (Malvern, Zetasizer).
ASSOCIATED CONTENT
* Supporting Information
■
S
Complete information on general methods and synthesis, solid
phase synthesis QC and purification, and mass spectrometry.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
(4) Garcia-Borron, J. C.; Sanchez-Laorden, B. L.; Jimenez-Cervantes,
C. Melanocortin-1 receptor structure and functional regulation. Pigm.
Cell Res. 2005, 18, 393−410.
*For D.L.M.: phone, 813-745-8948; fax, 813-745-8357; e-mail,
david.morse@moffitt.org. For J.V.: phone, 520-626-4179; fax,
520-626-4824; e-mail, vagner@email.arizona.edu.
(5) Rodrigues, A. R.; Pignatelli, D.; Almeida, H.; Gouveiaa, A. M.
Melanocortin 5 receptor activates ERK1/2 through a PI3K-regulated
signaling mechanism. Mol. Cell. Endocrinol. 2009, 303, 74−81.
(6) Webb, T. R.; Clark, A. J. L. Minireview: the melanocortin 2
receptor accessory proteins. Mol. Endocrinol. 2009, 24 (3), 475−484.
(7) (a) Ploeg, L. H. T. V. d.; Martin, W. J.; Howard, A. D.; Nargund,
R. P.; Austin, C. P.; Guan, X.; Drisko, J.; Cashen, D.; Sebhat, I.;
Patchett, A. A.; Figueroa, D. J.; DiLella, A. G.; Connolly, B. M.;
Weinberg, D. H.; Tan, C. P.; Palyha, O. C.; Pong, S.-S.; MacNeil, T.;
Rosenblum, C.; Vongs, A.; Tang, R.; Yu, H.; Sailer, A. W.; Fong, T. M.;
Huang, C.; Tota, M. R.; Chang, R. S.; Stearns, R.; Tamvakopoulos, C.;
Christ, G.; Drazen, D. L.; Spar, B. D.; Nelson, R. J.; MacIntyre, D. E. A
role for the melanocortin 4 receptor in sexual function. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99 (17), 11381−11386. (b) Begriche, K.;
Sutton, G. M.; Fang, J.; Butler, A. A. The role of melanocortin
neuronal pathways in circadian biology: a new homeostatic output
involving melanocortin-3 receptors? Obes. Rev. 2009, 10 (Suppl.2),
14−24.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
ACKNOWLEDGMENTS
■
The authors acknowledge Intezyne Inc. for support and
scientific contributions. Research was funded by the Bankhead
Coley Melanoma Pre-SPORE Program (Grant 02-15066-10-03
to D.L.M.), NIH/NCI (Grant R01 CA 097360 to R.J.G. and
D.L.M.), and Intezyne Inc. (Award 84-16301-01-01 to R.J.G.)
for a portion of N.M.B.’s salary and polymer materials.
ABBREVIATIONS USED
■
(8) (a) Hall, J. E.; Silva, A. A. d.; do Carmo, J. M.; Dubinion, J.;
Hamza, S.; Munusamy, S.; Smith, G.; Stec, D. E. Obesity-induced
hypertension: role of sympathetic nervous system, leptin, and
melanocortins. J. Biol. Chem 2010, 28 (23), 17271−17276. (b) Jun,
D.-J.; Na, K.-Y.; Kim, W.; Kwak, D.; Kwon, E.-J.; Yoon, J. H.; Yea, K.;
Lee, H.; Kim, J.; Suh, P.-G.; an, S. H. R.; Kim, K.-T. Melanocortins
induce interleukin 6 gene expression and secretion through
melanocortin receptors 2 and 5 in 3T3-L1 adipocytes. J. Mol.
Endocrinol. 2010, 44, 225−236. (c) Fridmanis, D.; Petrovskaa, R.;
Aloc, allyloxycarbonyl; (BimC4A)3, potassium 5,5′,5″-(2,2′,2″-
(nitrilotris(methylene))tris(1H-benzimidazole-2,1-diyl))-
tripentanoate; Boc, tert-butyloxycarbonyl; tBu, tert-butyl;
DMSO, dimethylsulfoxide; DVB, divinylbenzene; Fmoc, (9H-
fluoren-9-ylmethoxy)carbonyl; HBTU, 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt,
N-hydroxybenzotriazole; HOCt, 6-chloro-1H-hydroxybenzo-
triazole; NMI, N-methylimidazole; Pbf, 2,2,4,6,7-pentamethyl-
dihydrobenzofuran-5-sulfonyl; PS, polystyrene; RP-HPLC,
reverse-phase high performance liquid chromatography; TFA,
trifluoroacetic acid; Trt, triphenylmethyl (trityl)
Kalnina, I.; Peculisa, M. S.; Schioth, H. B.; Klovins, J. Identification of
̈
domains responsible for specific membrane transport and ligand
specificity of the ACTH receptor (MC2R). Mol. Cell. Endocrinol. 2010,
321, 175−183.
(9) Corander, M. P.; Fenech, M.; Coll, A. P. The science of self
preservation: how melanocortin action in the brain modulates body
weight, blood pressure and ischaemic damage. Circulation 2010, 120
(22), 2260−2268.
(10) (a) Cai, M.; Varga, E. V.; Stankova, M.; Mayorov, A.; Perry, J.
W.; Yamamura, H. I.; Trivedi, D.; Hruby, V. J. Cell signaling and
trafficking of human melanocortin receptors in real time using two-
photon fluorescence and confocal laser microscopy: differentiation of
agonists and antagonists. Chem. Biol. Drug Des. 2006, 68 (4), 183−193.
(b) Mayorov, A. V.; Han, S. Y.; Cai, M.; Hammer, M. R.; Trivedi, D.;
Hruby, V. J. Effects of macrocycle size and rigidity on melanocortin
receptor-1 and −5 selectivity in cyclic lactam alpha-melanocyte-
stimulating hormone analogs. Chem Biol Drug Des 2006, 67 (5), 329−
335. (c) Koikov, L. N.; Ebetino, F. H.; Solinsky, M. G.; Cross-Doersen,
ADDITIONAL NOTE
■
Abbreviations used for amino acids and designation of peptides
follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977−983.
REFERENCES
■
(1) Welch, H. G.; Woloshin, S.; Schwartz, L. M. Skin biopsy rates
and incidence of melanoma: population based ecological study. Br.
Med. J. 2005, 331, 481−489.
(2) (a) Lehmann, J. M.; Holzmann, B.; Breitbart, E. W.;
Schmiegelow, P.; Riethmuller, G.; Johnson, J. P. Discrimination
between benign and malignant cells of melanocytic lineage by two
novel antigens, a glycoprotein with a molecular weight of 113,000 and
8083
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084

Journal of Medicinal Chemistry
Article
D.; Knittel, J. J. Sub-nanomolar hMC1R agonists by end-capping of
the melanocortin tetrapeptide His-D-Phe-Arg-Trp-NH(2). Bioorg.
Med. Chem. Lett. 2003, 13 (16), 2647−2650.
(11) Sawyer, T. K.; Sanfilippo, P. J.; Hruby, V. J.; Engel, M. H.;
Heward, C. B.; Burnett, J. B.; Hadley, M. E. 4-Norleucine, 7-D-
phenylalanine-α-melanocyte-stimulating hormone: a highly potent α-
melanotropin with ultralong biological activity. Proc. Natl. Acad. Sci.
U.S.A. 1980, 77 (10), 5754−5758.
(25) Rodionov, V. O.; Presolski, S. I.; Gardinier, S.; Lim, Y.-H.; Finn,
M. G. Benzimidazole and related ligands for Cu-catalyzed azide−
alkyne cycloaddition. J. Am. Chem. Soc. 2007, 129 (42), 12696−12704.
(26) Fournier, E.; Dufresne, M.-H.; Smith, D. C.; Ranger, M.;
Leroux, J.-C. A novel one-step drug-loading procedure for water-
soluble amphiphilic nanocarriers. Pharm. Res. 2004, 21 (6), 962−968.
(12) Chen, J.; Giblin, M. F.; Wang, N.; Jurisson, S. S.; Quinn, T. P. In
vivo evaluation of 99mTc/188Re-labeled linear alpha-melanocyte
stimulating hormone analogs for specific melanoma targeting. Nucl.
Med. Biol. 1999, 26 (6), 687−693.
(13) (a) Cai, M.; Mayorov, A. V.; Cabello, C.; Stankova, M.; Trivedi,
D.; Hruby, V. J. Novel 3D pharmacophore of α-MSH/γ-MSH hybrids
leads to selective human MC1R and MC3R analogues. J. Med. Chem.
2005, 48 (6), 1839−1848. (b) Handl, H. L.; Vagner, J.; Yamamura, H.
I.; Hruby, V. J.; Gillies, R. J. Lanthanide-based time-resolved
fluorescence of in cyto ligand−receptor interactions. Anal. Biochem.
2004, 330 (2), 242−250. (c) Yang, Y.; Hruby, V. J.; Chen, M.; Crasto,
C.; Cai, M.; Harmon, C. M. Novel binding motif of ACTH analogues
at the melanocortin receptors. Biochemistry 2009, 48, 9775−9784.
(14) (a) Wei, L.; Butcher, C.; Miao, Y.; Gallazzi, F.; Quinn, T. P.;
Welch, M. J.; Lewis, J. S. Synthesis and biologic evaluation of 64Cu-
labeled rhenium-cyclized α-MSH peptide analog using a cross-bridged
cyclam chelator. J. Nucl. Med. 2007, 48 (1), 64−72. (b) Chen, J.;
Cheng, Z.; Hoffman, T. J.; Jurisson, S. S.; Quinn, T. P. Melanoma-
targeting properties of (99m)technetium-labeled cyclic alpha-melano-
cyte-stimulating hormone peptide analogues. Cancer Res. 2000, 60
(20), 5649−5658.
(15) Handl, H. L.; Sankaranarayanan, R.; Josan, J.; Vagner, J.; Mash,
E. A.; Gillies, R. J.; Hruby, V. J. Bioconjugate Chem. 2007, 18, 1101−
1109.
(16) Koikov, L. N.; Ebetino, F. H.; Solinsky, M. G.; Cross-Doersenb,
D.; Knittela, J. J. Analogs of sub-nanomolar hMC1R agonist LK-184
[Ph(CH2)3CO-His-D-Phe-Arg-Trp-NH2]. An additional binding site
within the human melanocortin receptor 1? Bioorg. Med. Chem. Lett.
2004, 14, 3997−4000.
(17) Koikov, L. N.; Ebetino, F. H.; Hayes, J. C.; Cross-Doersen, D.;
Knittel, J. J. End-capping of the modified melanocortin tetrapeptide (p-
Cl)Phe-D-Phe-Arg-Trp-NH2 as a route to hMC1R agonists. Bioorg.
Med. Chem. Lett. 2004, 14, 4389−4842.
(18) Sheth, S. R.; Efremova, N.; Leckband, D. E. Interactions of
poly(ethylene oxide) brushes with chemically selective surfaces. J. Phys.
Chem. B 2000, 104, 7652−7662.
(19) (a) Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.;
Kiessling, L. L. Selective tumor cell targeting using low-affinity,
multivalent interactions. ACS Chem. Biol. 2007, 2 (2), 119−127.
(b) Martin, A. L.; Li, B.; Gillies, E. R. Surface functionalization of
nanomaterials with dendritic groups: toward enhanced binding to
biological targets. J. Am. Chem. Soc. 2008, 131, 734−741.
(20) Tanford, C. The hydrophobic effect and the organization of
living matter. Science 1977, 200, 1012−1018.
(21) Handl, H.; Vagner, J.; Han, H.; Mash, E.; Hruby, V.; Gillies, R.
Hitting multiple targets with multimeric ligands. Expert Opin. Ther.
Targets 2004, 8 (6), 565−586.
(22) Vagner, J.; Handl, H. L.; Gillies, R. J.; Hruby, V. J. Bioorg. Med.
Chem. Lett. 2004, 14, 211−215.
(23) (a) Vagner, J.; Xu, L.; Handl, H. L.; Josan, J. S.; Morse, D. L.;
Mash, E. A.; Gillies, R. J.; Hruby, V. J. Heterobivalent ligands crosslink
multiple cell-surface receptors: the human melanocortin-4 and δ-
opioid receptors. Angew. Chem., Int. Ed 2008, 47, 1685−1688.
(b) Josan, J. S.; Vagner, J.; Handl, H. L.; Sankaranarayanan, R.;
Gillies, R. J.; Hruby, V. J. Solid-phase synthesis of heterobivalent
ligands targeted to melanocortin and cholecystokinin receptors. Int. J.
Pept. Res. Ther. 2008, 14, 293−300.
(24) Lewis, W. G.; Magallon, F. G.; Fokin, V. V.; Finn, M. G.
Discovery and characterization of catalysts for azide−alkyne cyclo-
addition by fluorescence quenching. J. Am. Chem. Soc. 2004, 126,
9152−9153.
8084
dx.doi.org/10.1021/jm201226w|J. Med. Chem. 2011, 54, 8078−8084