Structure-Based Design of Melanocortin 4 Receptor Ligands Based on the SHU-9119-hMC4R Cocrystal Structure ?

Article abstract of DOI:10.1021/acs.jmedchem.0c01620

The melanocortin receptors (MC1R-MC5R) belong to class A G-protein-coupled receptors (GPCRs) and are known to have receptor-specific roles in normal and diseased states. Selectivity for MC4R is of particular interest due to its involvement in various metabolic disorders, including obesity, feeding regulation, and sexual dysfunctions. To further improve the potency and selectivity of MC4R (ant)agonist peptide ligands, we designed and synthesized a series of cyclic peptides based on the recent crystal structure of MC4R in complex with the well-characterized antagonist SHU-9119 (Ac-Nle4-c[Asp5-His6-DNal(2′)7-Arg8-Trp9-Lys10]-NH2). These analogues were pharmacologically characterized in vitro, giving key insights into exploiting binding site subpockets to deliver more selective ligands. More specifically, the side chains of the Nle4, DNal(2′)7, and Trp9 residues in SHU-9119, as well as the amide linkage between the Asp5 and Lys10 side chains, were found to represent structural features engaging a hMC4R/hMC3R selectivity switch.

Full text of DOI:10.1021/acs.jmedchem.0c01620

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Article
Structure-Based Design of Melanocortin 4 Receptor Ligands Based
on the SHU-9119-hMC4R Cocrystal Structure†
Charlotte Martin,¶ Luis E. Gimenez,¶ Savannah Y. Williams, Yu Jing, Yiran Wu, Charlie Hollanders,
Olivier Van der Poorten, Simon Gonzalez, Kevin Van holsbeeck, Santo Previti, Arthur Lamouroux,
́
Suwen Zhao, Dirk Tourwe, Raymond C. Stevens,* Roger D. Cone,* and Steven Ballet*
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ABSTRACT: The melanocortin receptors (MC1R−MC5R) be-
long to class A G-protein-coupled receptors (GPCRs) and are
known to have receptor-specific roles in normal and diseased states.
Selectivity for MC4R is of particular interest due to its involvement
in various metabolic disorders, including obesity, feeding regulation,
and sexual dysfunctions. To further improve the potency and
selectivity of MC4R (ant)agonist peptide ligands, we designed and
synthesized a series of cyclic peptides based on the recent crystal
structure of MC4R in complex with the well-characterized
antagonist SHU-9119 (Ac-Nle4-c[Asp5-His6-DNal(2′)7-Arg8-Trp9-
Lys10]-NH2). These analogues were pharmacologically characterized
in vitro, giving key insights into exploiting binding site subpockets to
deliver more selective ligands. More specifically, the side chains of
the Nle4, DNal(2′)7, and Trp9 residues in SHU-9119, as well as the amide linkage between the Asp5 and Lys10 side chains, were
found to represent structural features engaging a hMC4R/hMC3R selectivity switch.
Administration (FDA) for the treatment of erythropoietic
protoporphyria.8 Additionally, the cyclic nonselective agonist
Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-OH (bremelanotide,
the C-terminal carboxylic acid analogue of melanotan II,
MT-II) was recently approved to treat the hypoactive sexual
disorder.9 The former mediates its therapeutic effects via the
MC1R, and the latter via the MC4R. The development of
MC4R-selective ligands for the treatment of common dietary
obesity has so far not resulted in an approved drug, although
the nonselective MC4R agonist setmelanotide is currently
under priority review at the FDA for certain forms of
syndromic obesity.10,11
Despite intense research efforts in both academia and
industry, there remains a need for potent, selective, and stable
(ant)agonist ligands for the melanocortin receptors. So far,
efforts have focused on variations of known ligands, but the
structural requirements for designing receptor-selective ligands
are still not straightforward to unravel.2,12−14 Rising to this
challenge, structure-based design is a powerful technique that
INTRODUCTION
■
The melanocortin receptor (MCR) family is composed of five
members (MC1R−MC5R) and belongs to the rhodopsin
superfamily of G-protein-coupled receptors (GPCRs).1 All of
these receptors are activated by the endogenous peptide
agonists α-, β-, γ-melanocyte stimulating hormone (MSH) and
adrenocorticotropic hormone (ACTH), derived from the
proopiomelanocortin gene. These endogenous ligands, such
as α-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-
Lys-Pro-Val-NH2), contain the core tetrapeptide sequence
His-Phe-Arg-Trp as the essential pharmacophore.2 MCRs have
been shown to play receptor-specific roles in a variety of
physiological processes.3,4 Specifically, the MC4R has attracted
considerable attention as a therapeutic target owing to its
demonstrated involvement in obesity, energy homeostasis,
feeding behavior, and sexual dysfunction but also because of
potential roles in the regulation of emotional states, such as
anxiety and depression, and neuropathic pain and drug
addiction.5,6 As a result of intensive structure−activity studies,
a large number of peptide and nonpeptide agonists and
antagonists with varying selectivities have been developed, and
several have advanced into clinical trials.7 Unfortunately, the
approval of some candidate therapeutics has been hampered by
the occurrence of undesirable side effects. Only recently, the
linear nonselective agonist [Nle4, DPhe7]α-MSH (afamelano-
tide) has been approved by the EU and the Food and Drug
Received: September 16, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01620
© XXXX American Chemical Society
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A

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Figure 1. Top left: binding pose of the antagonist SHU-9119 in the MC4R (ribbon receptor backbone in shades of coral, and the SHU-9119
ligand in yellow; the model is based on the coordinates from PDB ID 6W25) with an indication of the nature of observed interactions (bottom
left). Right: chemical structures of the amino acids inserted (in black) per position in the SHU-9119 sequence (in green).
can provide a more efficient rational design and optimization
of lead candidates. However, its application to membrane-
bound GPCRs has been limited due to the extreme difficulties
in obtaining crystals that represent active or inactive states. In
recent years, breakthroughs in obtaining suitable crystals and in
diffraction techniques have resulted in the determination of
GPCR structures, allowing the observation of bound ligands
and providing a structural basis of signal transduction
mechanisms. These structures include GPCRs complexed
with peptide agonists or antagonists such as apelin
(APLNR),15 angiotensin (AT2),16 neurotensin (NTS1),17
endothelin (ETB),18 opioid (MOR and DOR),19−21 chemo-
kine (US28 and CXCR4),22−24 and component peptide
(C5a1)25 receptors. Recently, the crystal structure of MC4R
in complex with the MC3R/MC4R nonselective antagonist
SHU-9119 (Ac-Nle4-c[Asp5-His6-DNal(2′)7-Arg8-Trp9-Lys10]-
NH2) was described.26 Here, we report our first results to
design novel MC ligands by exploring the binding pockets for
critical residues in the SHU-9119 structure.
designed and synthesized, and their pharmacological profiles
were evaluated at both MC3R and MC4R. This revealed that
the Nle4 side chain (α-MSH numbering) is oriented toward a
large hydrophobic subpocket, delineated by receptor residues
Phe51, Phe117, and Ile121, suggesting that larger side chains
would fill this pocket more effectively and possibly engage
additional hydrophobic or π−π interactions. This led to the
design of Cha4 (SBL-MC-33), Phe4 (SBL-MC-34), and
Nal(2′)4 (SBL-MC-35) analogues of SHU-9119 (Figure 1,
right). This binding subpocket also has a proximal Glu100
residue (ca. 4.1 Å), enabling a potential interaction with a basic
side chain at position 4 of the cyclic peptides. Therefore,
[Orn4]-SHU-9119 (SBL-MC-36) and [DOrn4]-SHU-9119
(SBL-MC-37) were prepared.
Additionally, the His6 residue in SHU-9119 is interacting
with the Ile104 side chain of the receptor, in a relatively large
pocket. Because the literature indicated that a Pro residue was
tolerated at that position,12 the His6 residue was replaced by
Pro (SBL-MC-20) and several Pro derivatives, such as the
phenylated proline derivatives (4S-Ph)Pro6 and (4R-Ph)Pro6
(SBL-MC-42 and SBL-MC-43). Compounds SBL-MC-21
and SBL-MC-22, containing Hyp and Oic residues,
respectively, have already been reported in the literature27,28
but were included as reference compounds in this study.
RESULTS AND DISCUSSION
■
Design and Chemical Synthesis. Based on the binding
pose of the antagonist SHU-9119 in the MC4R (Figure 1, top
left panel), a series of SHU-9119 analogues were rationally
B
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Figure 2. (A) cAMP responses were determined in real time both in the presence of agonists and antagonists in the same experimental session. “B”
indicates the basal response measurement window (2 min) in the absence of the agonist, control antagonist (i.e., SHU-9119), or test peptide.
Window 1 (11 min) was used to determine the agonist activity of test peptides, or an ECMAX-equivalent concentration of the melanocortin
endogenous agonist α-MSH as maximum activity control (shown as a 1 μM α-MSH add). Window 2 (11 min) was used to measure the antagonist
activity of test peptides in the presence of an EC90-equivalent concentration of α-MSH (shown as a 0.1 μM agonist peptide add). (B) Substitution
of Nle4 by DOrn (SBL-MC-37) conferred a 103-fold increase in selectivity to MC4R while maintaining antagonist activity for cAMP response in
live cell assays. Shown are the concentration−response curves for the indicated compounds in the presence of an EC90-equivalent concentration of
α-MSH for cAMP production in cells stably expressing human MC3R or MC4R. Data points represent the mean standard error from one of two
independent experiments performed in triplicate. (C) Inhibition of 80 pM [125I]-[Nle4, DPhe7]-α-MSH specific binding by SBL-MC-37 at hMC3R
and hMC4R. (D) Binding pose of SBL-MC-37 docked to MC3R (ribbon receptor backbone in light blue, docked SBL-MC-37 in turquoise) or
MC4R (ribbon receptor backbone in coral, docked SBL-MC-37 in yellow). Both models were based on PDB ID 6W25.
The DNal(2′)7 side chain of SHU-9119 is located in a
receptor pocket delineated by hydrophobic (Ile185, Ile129)
and aromatic (Phe262, Phe284) residues. This pocket is,
however, sufficiently wide to accommodate larger alternative
aromatic residues. DPhe(pPh) (SBL-MC-44) and DPhe(mPh)
(SBL-MC-47) were introduced into the SHU-9119 sequence,
as well as DPhe residues with 1′-naphthyl (Np(1′)) and 2′-
naphthyl (Np(2′)) groups in para- or meta-position, which
lead to SBL-MC-49/46 or SBL-MC-48/45. This approach
was undertaken due to the large synthetic efforts required to
obtain the halogenated precursors of DNal(2′) and, contrast-
ingly, the commercial availability of different halogenated
DPhe isomers to perform cross couplings.
and 7-F-Trp (SBL-MC-55 to SBL-MC-57) were proposed and
synthesized.
In addition to the above point mutations in the SHU-9119
sequence, the variation of the amide bond in the cyclization
motif of the cyclic peptide was evaluated. Since the cycle amide
bond is in the cis configuration in the SHU-9119-MC4R crystal
structure, we replaced it with 1,5-substituted 1,2,3-triazole,
which mimics and locks the cis amide isomer, and is accessible
via Ru-catalyzed azide−alkyne cycloaddition (RuAAC) of
commercially available azidolysine (Azk) and propargylglycine
(Pra).29 For comparison, we also prepared the 1,4-substituted
1,2,3-triazole analogue using the CuAAC reaction. This isomer
is considered a trans amide bond mimic. Although the
cyclization tether is not directly interacting with the receptor,
the stereochemistry of the amide bond could influence the
conformation of the peptide macrocycle backbone and,
consequently, the positioning within the receptor and the
interaction with said receptor.
The peptides reported herein were manually synthesized
following the classical solid-phase peptide synthesis (SPPS)
using the standard 9-fluorenylmethyloxycarbonyl (Fmoc)
strategy with the appropriate orthogonal protecting groups.
All sequences were prepared on a Rink amide resin, and
couplings were carried out using N,N,N′,N′-tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU)
in the presence of diisopropylethylamine (DIPEA) as a base.
The sequences were acetylated on a solid support prior to
In the next position of the SHU-9119 sequence, the Arg8
side chain engages in electrostatic interactions with the
receptor’s Asp122 and Asp126 residues and provides an
additional interaction with Asn123. To potentially strengthen
these interactions, it was hypothesized that elongation of this
side chain by one methylene, through the insertion of hArg
(SBL-MC-50), or, alternatively, replacing it with an elongated/
rigid analogue, para-guanidinophenylalanine (Phe(pGnd)) (to
give SBL-MC-51), could be beneficial.
Finally, the Trp9 side chain interacts with the Ile194 residue,
and sufficient space was clearly available to increase the size of
the indole substituent. Therefore, the halogenated analogues
5-, 6-, and 7-Cl-Trp (SBL-MC-52 to SBL-MC-54) and 5-, 6-,
C
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Table 1. Potency and Receptor Selectivity of SHU-9119 Analogues with Antagonist Action on Intracellular cAMP Levels after
a
α-MSH Stimulation
compound
code
MC3R
pIC50 SEM (IC50 nM)
MC4R
pIC50 SEM (IC50 nM)
selectivity (IC50MC3R/
IC50MC4R)
b
b
sequence
SHU-9119
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
7.08 0.31 (83)
6.53 0.17 (298)
6.26 0.28 (545)
6.01 0.00 (1349)
6.00 0.14 (1012)
5.29 0.05 (5140)
7.32 0.11 (48)
7.36 0.17 (43)
6.43 0.08 (374)
6.49 0.10 (322)
6.69 0.11 (206)
6.98 0.16 (105)
5.39 0.29 (4055)
6.12 0.34 (751)
7.11 0.17 (78)
7.09 0.18 (81)
6.46 0.10 (345)
7.12 0.01 (76)
6.57 0.18 (271)
7.36 0.23 (44)
6.23 0.24 (586)
7.42 0.20 (38)
6.91 0.27 (122)
7.01 0.50 (97)
6.28 0.26 (528)
7.32 0.34 (48)
7.30 0.32 (50)
7.75 0.01 (18)
7.69 0.02 (20)
6.77 0.26 (172)
7.18 0.01 (67)
7.11 0.15 (77)
7.39 0.31 (41)
6.40 0.59 (398)
7.11 0.21 (78)
7.19 0.21 (65)
7.43 0.01 (37)
7.28 0.12 (53)
7.45 0.10 (36)
7.41 0.15 (39)
7.77 0.08 (17)
7.48 0.16 (33)
2.18
2.44
5.60
1.84
21.26
103.04
2.69
2.14
2.18
4.83
2.66
2.56
10.20
9.59
1.20
2.17
6.56
2.14
7.00
2.56
17.60
SBL-MC-33 Ac-Cha-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-34 Ac-Phe-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-35 Ac-Nal(2′)-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-36 Ac-Orn-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-37 Ac-DOrn-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-20 Ac-Nle-c[Asp-Pro-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-21 Ac-Nle-c[Asp-Hyp-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-22 Ac-Nle-c[Asp-Oic-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-42 Ac-Nle-c[Asp-(4S-Ph)Pro-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-43 Ac-Nle-c[Asp-(4R-Ph)Pro-DNal(2′)-Arg-Trp-Lys]-NH2
SBL-MC-50 Ac-Nle-c[Asp-His-DNal(2′)-hArg-Trp-Lys]-NH2
SBL-MC-51 Ac-Nle-c[Asp-His-DNal(2′)-Phe(pGnd)-Trp-Lys]-NH2
SBL-MC-52 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(5-Cl)-Lys]-NH2
SBL-MC-53 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(6-Cl)-Lys]-NH2
SBL-MC-54 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(7-Cl)-Lys]-NH2
SBL-MC-55 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(5-F)-Lys]-NH2
SBL-MC-56 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(6-F)-Lys]-NH2
SBL-MC-57 Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(7-F)-Lys]-NH2
SBL-MC-38 Ac-Nle-click1,5[Pra-His-DNal(2′)-Arg-Trp-Azk]-NH2
SBL-MC-58 Ac-Nle-click1,4[Pra-His-DNal(2′)-Arg-Trp-Azk]-NH2
a
b
HEK 293 cells stably expressing the GScAMP22F cAMP reporter and either human MC3R or MC4R were used. Data were fit by nonlinear
regression to a logistic, four-parameter, variable-slope, sigmoid inhibition model. Data represent the mean standard error of the mean (SEM) of
ten (SHU-9119) or two (test peptides) independent experiments performed in triplicate.
cyclization. Peptide lactam cyclization was performed on resin
after selective Allyl/Alloc deprotection under neutral con-
ditions by means of catalytic amounts of tetrakis-
(triphenylphosphine)palladium(0) in the presence of phenyl-
silane. Formation of the cyclic 1,5-substituted 1,2,3-triazole
was carried out using on-resin RuAAC, while the cyclic peptide
containing the 1,4-substituted 1,2,3-triazole was prepared by
means of solution-phase CuAAC of the corresponding pure
linear compound. Importantly, for peptides containing the
Asp5-His6 motif, which are prone to aspartimide formation
during the Fmoc deprotection step, the Allyl/Alloc depro-
tection and cyclization steps were performed directly after Asp
coupling. Once cyclized, the sequences were subsequently
completed on resin before final cleavage. Final side chain
deprotection and cleavage of the peptides from the resin were
performed using a cocktail consisting of trifluoroacetic acid
(TFA)/triisopropylsilane (TIS)/H2O (95:2.5:2.5). After sol-
vent evaporation, precipitation, and subsequent lyophilization,
crude peptides were obtained. Purification was accomplished
by reverse-phase high-performance liquid chromatography
(RP-HPLC), and the pure compounds (>96%) were obtained
as TFA salts after lyophilization.
Pharmacological EvaluationIn Vitro Activity and
Affinity at hMC3R and hMC4R. We used a genetically
encoded cyclic adenosine monophosphate (cAMP) split-
luciferase reporter30,31 stably expressed in HEK 293 cells
together with human MC3R or MC4R to measure the cAMP
levels in live cells after stimulation. The investigated
compounds were evaluated in two measuring modes. In the
first measurement window (Figure 2A), test wells received the
test peptides in the absence of any other ligand to determine
the intrinsic agonist activity. The endogenous melanocortin
agonist α-MSH was added at various concentrations to control
wells to determine the maximum response for a given plate.
This response was used to normalize the signal from test wells
during analysis. In the second measurement window, an EC90-
equivalent dose of α-MSH (0.1 μM) was added to the test
wells to determine the inhibitory potency of the investigated
peptide analogues (Figure 2A). Figure 2A shows the typical
response from a control well that received a saturating
concentration (1 μM) of α-MSH during the agonist-mode
measurement window (measurement window 1). Also shown
are a control well that received vehicle during measurement
window 1 and 0.1 μM α-MSH during measurement window 2
and a test well to which 1.33 μM SHU-9119 was added during
measurement window 1 and the EC90-equivalent concentration
of the endogenous agonist during measurement window 2.
Relative test peptide potencies were derived from concen-
tration−response experiments after fitting to a four-parameter
sigmoid model.
Based on their antagonist or agonist potency and their
selectivity (Tables 1 and 2), a number of SHU-9119 analogues
were selected to be tested for their affinity at hMC3R or
hMC4R (Table 3). To do so, we performed competition-
binding experiments in the presence of a close-to-KD
concentration of the radioligand [125I]-[Nle4, DPhe7]-α-
MSH, the indicated test peptides, and crude membrane
preparations from cells stably expressing human MC3R and
MC4R. KD values were experimentally determined by
performing saturation binding experiments from the same
membrane extracts (Figure S1). In all instances, ligand binding
was determined at equilibrium after 150 min incubation at
room temperature. Affinity (Ki) values were derived using the
Cheng and Prusoff32 transformation after nonlinear regression
analysis fitting the data to a three-parameter sigmoid model.
The binding data are summarized in Table 3.
D
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Nle4 Substitution. While prior structure−activity relation-
ship (SAR) studies at position 4 revealed the preference for
lipophilic residues such as Nle or acidic residues, the insertion
of bulkier lipophilic amino acids (Cha, Phe, Nal(2′)) or basic
residues (Orn, DOrn) all maintained the antagonist character
(Table 1) of SHU-9119. Except for the Nal(2′) analogue SBL-
MC-35, moderately potent antagonists were obtained with
varying selectivity for hMC4R over hMC3R (Table 1). In
particular, the DOrn analogue SBL-MC-37 was highly
selective, being 103 times more potent at hMC4R than at
hMC3R, with a 1.3-fold lower antagonist potency at hMC4R
than SHU-9119 (Figure 2B and Table 1). This selectivity was
also confirmed during the determination of affinity in
competition binding experiments (Figure 2C and Table 3).
The DOrn4 analogue SBL-MC-37, which showed the highest
antagonist selectivity, was also the most selective analogue in
the binding assays. Its affinity is 1.45 times lower than that of
SHU-9119, but its selectivity is 20 times higher (at MC4R).
To understand this selectivity, molecular modeling (Prime
̈
module from the Schrodinger software platform) was used to
validate the hypothesis that this result might emerge due to a
potential interaction between the basic side chain of the DOrn4
of the cyclic peptide and the Asp113 residue in MC4R ECL1
(Figure 2D). The side chain of Asp113 was found to be
oriented toward the pocket, while Phe108 takes this position
according to the homology model of MC3R, leaving no acidic
residue in ECL1 pointing into the pocket (Figure 2D). We
positioned the structure of SBL-MC-37 based on SHU-9119
and refined its interactions with MC4R. Modeling confirmed
the salt bridge between DOrn4 and Asp113, thus suggesting
that this might be the key factor for the observed selectivity of
SBL-MC-37 for MC4R.
His6 Substitution. Previous structure−activity relationship
studies demonstrated that position 6 is an important “key” to
identifying potent and selective ligands at MCRs when
conformationally constrained amino acids are inserted.28,33
As expected, all of the investigated analogues maintained the
antagonist character and were essentially nonselective.
Analogues SBL-MC-20 to SBL-MC-22 have already been
reported by Grieco et al. as antagonists at both hMC3R and
hMC4R with pA2 values between 8.2 and 9.8, showing no
selective affinity for one receptor.27 The substitution of the
His6 residue by the (4R-Ph)Pro isomer (SBL-MC-43)
maintained antagonist activity at hMC4R compared to SHU-
9119, while the (4S-Ph)Pro analogue SBL-MC-42 enhanced
the hMC4R selectivity by twofold.
DNal7 Substitution. Since substitutions of DNal7 were
performed using substituted DPhe analogues, these com-
pounds were closer analogues of the nonselective superagonist
MT-II. Indeed, SHU-9119 and MT-II only differ in the
seventh position compared to SHU-9119 bearing a DNal7
residue and MT-II bearing a DPhe7. In agreement, analogues
SBL-MC-44 to 49 behave as partial to full agonists at both
hMC3R and hMC4R (Table 2 and Figure 3). An interesting
trend of the substituents at the para position engaging in a
selectivity switch toward hMC3R is observed in terms of
agonist efficacy. As can be noticed, the pPh analogue SBL-MC-
44 is two times more selective for hMC3R and only evokes a
partial agonist response at hMC4R (Table 2 and Figure 3A),
the pNp(1′) analogue (SBL-MC-46) is five times more
hMC3R selective and similarly acts as a partial agonist at
hMC4R (Table 2 and Figure 3B), and the pNp(2′) analogue
(SBL-MC-45) is three times more hMC3R-selective (Table 2
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Table 3. Affinity and Receptor Selectivity As Determined by Radioligand Competition Binding Experiments in the Presence of
a
125I-NDP-MSH
compound
code
−log
−log
selectivity
(Ki MC3R/Ki MC4R)
b
b
sequence
Ki SEM(Ki in pM)−MC3R
Ki SEM(Ki in pM)−MC4R
SHU-9119
MT-II
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2
9.89 0.23 (129)
8.40 0.28 (3978)
8.43 0.13 (3754)
7.82 0.06 (15 080)
9.42 0.15 (378)
7.50 0.23 (31 410)
8.49 (3251)
10.55 0.27 (29)
9.34 0.32 (456)
10.38 0.02 (42)
9.34 0.05 (459)
10.56 0.07 (28)
9.26 0.01 (551)
9.11 (769)
4.5
8.7
SBL-MC-37
SBL-MC-47
SBL-MC-44
SBL-MC-49
SBL-MC-46
SBL-MC-48
SBL-MC-45
SBL-MC-52
SBL-MC-53
SBL-MC-54
SBL-MC-55
SBL-MC-56
SBL-MC-57
SBL-MC-38
SBL-MC-58
Ac-DOrn-c[Asp-His-DNal(2′)-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe(mPh)-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe(pPh)-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe(mNp(1′))-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe(pNp(1′))-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe(mNp(2′))-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DPhe(pNp(2′))-Arg-Trp-Lys]-NH2
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(5-Cl)-Lys]-NH2
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(6-Cl)-Lys]-NH2
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(7-Cl)-Lys]-NH2
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(5-F)-Lys]-NH2
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(6-F)-Lys]-NH2
Ac-Nle-c[Asp-His-DNal(2′)-Arg-Trp(7-F)-Lys]-NH2
Ac-Nle-click1,5[Pra-His-DNal(2′)-Arg-Trp-Azk]-NH2
Ac-Nle-click1,4[Pra-His-DNal(2′)-Arg-Trp-Azk]-NH2
90.1
32.9
13.7
57.0
4.2
8.00 0.06 (10 090)
8.05 0.01 (8872)
9.16 (690)
9.04 0.04 (915)
8.86 0.07 (1380)
10.46 (35)
11.0
6.4
19.9
1.1
10.66 (22)
10.71 (20)
10.51 0.05 (31)
9.45 (352)
10.78 0.28 (17)
10.59 (26)
1.9
13.7
2.1
10.49 0.06 (32)
9.48 (330)
10.82 0.35 (15)
10.67 (21)
15.4
8.4
9.87 0.27 (135)
9.41 0.07 (391)
10.80 0.01 (16)
10.68 0.03 (21)
18.5
a
b
Crude membrane extracts were prepared from HEK 293 cells stably expressing the human MC3R or MC4R. Data were fit by nonlinear
regression to a logistic, three-parameter, variable-slope, sigmoid competition model. Data represent the mean (when applicable) the standard
error of the mean.
Figure 3. (A−F) Extensions in the para- or meta-position of the DNal7 to DPhe7 substitution confer selectivity for MC3R (panels A−C) or MC4R
(panels D−F), respectively, in the context of agonist activity. Determinations of cAMP agonist responses are shown for the indicated test peptides
in cells stably expressing human MC3R or MC4R in live cells. (G−H) Inhibition of 80 pM [125I]-[Nle4, DPhe7]-α-MSH specific binding by MT-II,
SBL-MC-46, or SBL-MC-49 at hMC4R (G) or by SBL-MC-45 on hMC3R or hMC4R (H). Data points (A−H) represent the mean standard
error from one of two independent experiments performed in triplicate.
and Figure 3C). Among them, the pNp(2′) analogue SBL-
MC-45 is the most potent full agonist at hMC3R with an EC50
value of 41 nM. Interestingly, some of these analogues display
only partial agonism at the hMC4R, which opens a gateway
toward more selective hMC3R agonists.
On the contrary, the meta-substituted analogues show
selectivity for the hMC4R (SBL-MC-47 to 49, Figure 3D−
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Figure 4. Extensions in the para- or meta-position of the DNal7 to DPhe7 substitutions differentially fill a cavity in the orthosteric binding pocket of
MC3R and MC4R of an otherwise extensive network of π interactions. (A) Diagram depicting the amino acid residue interactions for SBL-MC-45
on MC3R and MC4R. (B) Extracellular view of the binding poses of MT-II (green), SBL-MC-46 (yellow), and SBL-MC-49 (purple) docked to
MC4R. The green dashed line indicates the approximate cutout plane for (C)−(E). (C−E) Side view cutouts of the MC4R binding pocket with
docked MT-II (C), SBL-MC-46 (D), or SBL-MC-49 (E). The MC4R model was based on PDB ID 6W25.
F). Molecular modeling suggests that SBL-MC-44 to 49 are
bulkier than SHU-9119 at the DNal7 position (and MT-II at
DPhe7) but further suggests as well that the para- and meta-
substituted analogues extend toward different directions: para-
substitution (SBL-MC-44 to 46) toward TM5/TM6, while
meta-substitution (SBL-MC-47 to 49) toward TM4/TM5.
DNal7 in SHU-9119 already reaches the bottom of the pocket;
thus, bulkier side chains may induce a conformational change
of the receptors in nearby space. However, the interacting
residues in MC3R and MC4R are identical (D121/126, C125/
130, L128/133, F179/184, S183/188, V188/193, and L192/
197).
We speculate that the subtype selectivity of para- and meta-
substitutes is caused by the subtle difference of conformational
flexibility of MC3R and MC4R in these regions. Docking
analysis revealed that the receptor residues that might interact
with the DNal7 surrogates are identical for hMC3R and
hMC4R (Figure 4A). When the DPhe7 aromatic ring is
extended in the meta- or para-position by the insertion of an
additional phenyl, 1′- or 2′-naphthyl ring, the receptor
subpocket in the hMC4R and modeled hMC3R (not shown)
is nicely filled, as shown in Figures 4D,E, for SBL-MC-46 and
SBL-MC-49, respectively, contrasting with MT-II (Figure
4C). DPhe7 participates in a π network involving many
aromatic residues (Phe201, Phe202, Phe254, Trp258, Phe261,
Phe262, His264, and Phe284 in hMC4R). These interactions
are very sensitive to the positions of aromatic rings; thus,
subtle conformational differences between MC4R and MC3R
can lead to distinct subtype selectivity of the para- and meta-
substituted ligands (Figure 4).
In the agonist series of compounds, the MC4R selectivity of
the meta-substituted DPhe7 analogues was also confirmed in
terms of binding data (Table 3). However, whereas the para-
substituted DPhe7 analogues showed MC3R selectivity in the
cAMP agonist assay, this selectivity was not confirmed in the
binding assay, thereby highlighting the limitations of radio-
ligand-binding experiments in predicting agonist coupling.
Arg8 Substitution. The elongation of the Arg side chain by
incorporating homoArg (SBL-MC-50) or the more rigid p-
guanidino-Phe (SBL-MC-51) did not result in improved
antagonist potency, indicating that these modified side chains
are not able to engage in beneficial electrostatic interactions
with the Asp122, Asp126, or Asn123 residues of the hMC4R,
as initially hypothesized during the design of the respective
ligands. Although analogue SBL-MC-51 provides a tenfold
selectivity increase for MC4R, the activity is only in the
micromolar concentration range.
Trp9 Substitution. The position of the chloro- or fluoro-
substituent on the indole ring was found to have a clear effect
on receptor subtype selectivity. Although being modestly
potent (IC50 = 78 nM), the 5-chloro-Trp analogue (SBL-MC-
52) has significant selectivity for hMC4R (ca. tenfold, Figure
5A and Table 1), the 6-chloro isomer (SBL-MC-53) seems to
be a more balanced hMC3R/hMC4R ligand (Figure 5B and
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Figure 5. Halogenated Trp9 substitutions in the indole 5 through 7-position transition from MC4R to slight MC3R selectivity. (A−C)
Determinations of cAMP agonist responses for the indicated test peptides in cells stably expressing human MC3R or MC4R in live cells. (D−F)
Inhibition of 80 pM [125I]-[Nle4, DPhe7]-α-MSH specific binding by indicated test compounds on hMC3R or hMC4R. (G) Extracellular view of
the binding pose of SBL-MC-52 on hMC3R and hMC4R. MC3R (receptor backbone in blue, docked SBL-MC-52 in turquoise) or MC4R
(receptor backbone in coral, docked SBL-MC-52 in yellow). The MC3R and MC4R models were based on PDB ID 6W25. Data points (A−F)
represent the mean standard error from one of two independent experiments performed in triplicate.
Table 1). In the fluoro-series, the 7-fluoro-Trp analogue SBL-
MC-57 is the most hMC4R selective (i.e., sevenfold, Figure 5C
and Table 1).
of SHU-9119 with the docked structure of the 1,5-substituted
triazole isomer SBL-MC-38 revealed a very similar binding
mode. Notable was that a small change in orientation was
observed for the His6 residue. The intramolecular hydrogen
bond between the imidazole and the cis-amide bond in SHU-
9119 is not present when the triazole is inserted. The relative
orientation of the triazole is responsible for the missing
hydrogen bond (Figure S2). Compared to SHU-9119,
analogue SBL-MC-38 is a more potent antagonist at the
hMC4R with a modest twofold selectivity over the hMC3R
(Figure 6A), which was also captured in the radioligand
binding experiments performed in the presence of NDP-MSH
(Figure 6C). The 1,4-substituted triazole analogue SBL-MC-
58, in which the disubstituted triazole mimics a trans amide
bond, is equipotent at the hMC4R but is about 18 times more
selective for hMC4R over hMC3R, unexpectedly providing
another hMC4R over hMC3R selectivity handle (Figure 6B,D
and Table 1). For this reason, this structural feature might be
combined in conjunction with amino acid substitutions in the
main chain of the peptide to form even more selective and
promising compounds.
Molecular modeling shows that the 5-position of Trp9 points
toward the aromatic ring of DNal7, meaning that a bulky
substitution might alter the ligand’s conformation. This can
explain the ninefold activity loss observed for the 5-chloro-Trp
analogue (SBL-MC-52) for MC3R. However, on MC4R, the
impact is much smaller (twofold). This difference between the
two receptor subtypes may be attributed to the involvement of
Tyr268 in MC4R, which could act as a “cap”, covering Trp9
and causing the ligand conformation to have reduced impact,
due to hampered ligand dissociation. The equivalent position
on MC3R (Ile265) does not form such a cap and possibly does
not impede ligand dissociation, as shown in Figure 5G, for
SBL-MC-52. Of note, the compound with the highest MC4R
affinity is the DTrp(6-F)9-bearing analogue SBL-MC-56
(Table 3), with an impressive binding affinity of 15 pM,
compared to 29 pM for SHU-9119 (i.e., a twofold improve-
ment).
Asp-Lys Amide Bond in the Cyclization Bridge. In the
receptor-bound conformation of SHU-9119, the amide bond
in the Asp-Lys cyclization bridge is in the cis configuration.
This is rather unusual since secondary amide bonds (in
unconstrained macrocycles) have a strong preference for the
trans configuration. An overlap of the receptor-bound structure
CONCLUSIONS
■
A visual inspection of orthosteric binding site subpockets was
made possible by the recently published SHU-9119-hMC4R
co-crystal structure,26 enabling the rational design of our
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plicities were described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), broad (br), or a combination thereof. The
corresponding coupling constants (J values) were reported in hertz
(Hz). Analytical RP-HPLC was performed on a VWR-Hitachi
Chromaster HPLC with a Chromolith HighResolution RP-18C
column from Merck (150 mm × 4.6 mm, 1.1 μm, 150 Å). The flow
rate was 3 mL/min, and ultraviolet (UV) detection occurred at 214
nm. The solvent system used consisted of 0.1% TFA in ultrapure
water (A) and 0.1% TFA in acetonitrile (B) with a gradient from 3 to
100% B over a 6 min runtime. For liquid chromatography−mass
spectrometry (LC/MS) analysis, the HPLC unit used was a Waters
600 system combined with a Waters 2487 UV detector at 215 nm and
as a stationary phase an EC 150/2 NUCLEODUR 300-5 C18 column
(150 mm × 2.1 mm, 3 μm, 300 Å). The solvent system used was 0.1%
formic acid in water (A) and 0.1% formic acid in acetonitrile (B) with
a gradient going from 3 to 100% B over 20 min with a flow rate of 0.3
mL/min. The MS unit, coupled to the HPLC system, was a
Micromass QTOF-micro system. For high-resolution mass spectros-
copy, the same MS system was used with a reserpine (2.10−3 mg/
mL) solution in H2O/CH3CN (1:1) as a reference. Microwave
syntheses were performed using a Biotage Initiator+ SP Wave.
Automated flash chromatography was performed using a Grace
Reveleris X2 system equipped with an evaporative light scattering
detector (ELSD) and a UV detector (254 or 280 nm). The normal
phase columns used for the systems were Grace Silica Flash
Cartridges of 40 g with a flow rate of 40 mL/min unless stated
otherwise. Semipreparative RP-HPLC-purifications were done using a
Gilson HPLC system with a Gilson 322 pump equipped with an
INTERCHIM Vydac 150HC C18 column (250 mm × 22 mm, 10
μm) and a Waters UV/vis-156 detector at 215 nm. The same solvent
system was used as applied for the analytical RP-HPLC with a flow
rate of 20 mL/min.
Figure 6. Cis to trans Asp-Lys amide cyclization bond manipulations
affect potency and selectivity for MC4R. (A−B) Inhibition of the α-
MSH EC90-elicited cAMP response by varying the concentrations of
the indicated test peptides in cells expressing MC3R or MC4R. (C−
D) Inhibition of 80 pM [125I]-[Nle4, DPhe7]-α-MSH specific binding
by SBL-MC-38 (C) and SBL-MC-58 (D) on hMC3R or hMC4R.
Data points for (A)−(D) represent the mean standard error from
one of two independent experiments performed in triplicate.
Fmoc Protection of Halo Tryptophane. To a round-bottomed
flask containing amino acid hydrochloric acid (1 equiv) and water
(0.176 M), a premade solution of Fmoc N-hydroxysuccinimide ester
(1.2 equiv) and Na2CO3 (1.3 equiv) in water/acetone (0.088 M, 1:3)
was slowly added. The pH was checked after 30 min and adjusted to
pH 8 with Na2CO3 if necessary. The mixture was stirred until
completion was observed by HPLC. The mixture was concentrated to
approximately half volume in vacuo and acidified with HCl (1 M).
The aqueous phase was extracted three times with dichloromethane
(DCM), and the combined organic phases were dried over MgSO4,
filtered, and concentrated in vacuo. The crude compound was
subsequently purified using semipreparative HPLC.
Pd-Catalyzed Suzuki−Miyaura Coupling. For accessing the
unnatural DPhe-based amino acids of peptides SBL-MC-44 to 49,
a microwave vial was charged with K2CO3 (2.5 equiv), N-Fmoc-
protected amino acid (1.1 equiv), PdCl2 (dppf) (5 mol %) and
boronic acid (1.2 equiv), dissolved in H2O/iPrOH (0.05 M, 1:1). The
vial was flushed with argon and then capped. The mixture was
vigorously stirred at 40 °C for 20 h. After cooling to room
temperature, the mixture was concentrated and acidified to pH 3 with
HCl (1 M). The aqueous phase was extracted with DCM (three
times), and the combined organic phases were dried over MgSO4,
filtered, and concentrated in vacuo. The crude product was
subsequently purified using normal-phase, automated flash chroma-
tography.
Peptide Synthesis. The syntheses were performed manually using
standard Fmoc-based solid-phase peptide synthesis (SPPS) on a Rink
amide AM resin. Fmoc amino acids (3 equiv) (1.5 equiv of unnatural
amino acids) were coupled using HBTU (3 equiv) and DIPEA (5
equiv) in N,N-dimethylformamide (DMF) for 40 min (1.5 h for
unnatural amino acids). Fmoc removal was performed using 20% 4-
methylpiperidine in the DMF solution twice (5 and 15 min each). N-
terminal acetylation was performed on resin using a solution of acetic
anhydride (10 equiv) and DIPEA (5 equiv) in DMF for 1 h. After
completion of the sequences, the peptides were deprotected and
cleaved from the resin by treatment with a cleavage cocktail
constituted of TFA/TIS/H2O (95:2.5:2.5) for 4 h at room
temperature. The resulting cleavage mixtures were evaporated, and
improved hMC4R ligands. Our results demonstrate the
importance of structure-based design in the search for high
affinity and subtype receptor-selective ligands. Through
replacement of all SHU-9119 residues by a limited number
of (un)common amino acid surrogates, ligands with improved
affinity for the hMC4R were discovered and, moreover, several
structural features leading to enhanced hMC4R over hMC3R
selectivity were revealed (e.g., DOrn4, Trp(5-Cl)9, 1,4-triazolyl
tether). Importantly, an extension of the DPhe7 residue by
inserting an additional phenyl/naphthyl ring in meta- and para-
position of this aromatic side chain gave way to full to partial
agonist ligands with a higher potency at hMC4R and hMC3R,
respectively. Altogether, these results represent significant
progress to improve upon the receptor subtype specificity of
agonist and antagonist ligands of the melanocortin receptors.
Our findings will contribute toward improved pharmacological
tools and drug leads and help to fully exploit the therapeutic
potential of these receptors.
EXPERIMENTAL SECTION
Materials and Methods for Chemistry. Unless stated
■
otherwise, all commercial chemicals were used without further
1
purification. H and 13C NMR spectra were recorded using a Bruker
Avance II 500 MHz at ambient temperature with the frequencies of
1H and 13C set, respectively, at 500.03 and 125.74 MHz. The chemical
shifts were reported in delta (δ) units in parts per million (ppm)
relative to the signal of the deuterated solvent. For CDCl3, the singlet
1
in H NMR was calibrated at 7.26 ppm and the central line of the
triplet in 13C NMR at 77.0 ppm. When recording in MeOD or
DMSO-d6, the calibration was performed, respectively, at 3.31 and
2.50 ppm for 1H NMR and 49.0 and 39.5 ppm for 13C NMR.
Assignments were made using one-dimensional (1D) 1H and 13C
spectra and two-dimensional (2D) heteronuclear single quantum
coherence (HSQC), heteronuclear multiple bond correlation
(HMBC), and correlation spectroscopy (COSY) spectra. Multi-
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the peptides precipitated with cold diethylether. After centrifugation,
the precipitated peptides were dissolved in a mixture of H2O/CH3CN
(1:1) and lyophilized. Finally, the crude peptides were purified by
preparative reversed-phase HPLC (RP-HPLC) and the pure fractions
were lyophilized. Peptides were obtained as a TFA salt and were
characterized by high-resolution mass spectroscopy (HRMS). The
purity of all peptides was found to be ≥96% according to HPLC
analysis.
Lactam Cyclization. After synthesis of the resin-bound linear
precursor peptides, the Allyl/Alloc side chain protecting groups were
selectively removed under neutral conditions by means of catalytic
amounts of tetrakis(triphenylphosphine)palladium (0) (0.2 equiv) in
the presence of phenylsilane (24 equiv) in DCM (two times for 30
min each). The resin was then washed with a solution of 11.6 mM
sodium diethyldithiocarbamate and 0.01% DIPEA solution in DMF,
followed by three washing steps with DCM. Subsequently, the on-
resin cyclization through lactamization was performed overnight using
HBTU (6 equiv), HOBt·H2O (6 equiv), and DIPEA (12 equiv) in
DMF. Peptides were cleaved and purified as outlined in the Peptide
Synthesis section.
RuAAC Cyclization. The resin-bound linear precursor peptide (0.1
mmol) was transferred to a microwave vial and dried under high
vacuum, after which 4 mL of DMF (degassed with argon for 30 min)
was added. Subsequently, argon was bubbled through the mixture for
30 min using a needle, followed by the addition of 8 mg (20 mol %
b a s e d o n i n i t i a l r e s i n s u b s t i t u t i o n ) o f c h l o r o -
(pentamethylcyclopentadienyl)(cyclooctadiene)ruthenium(II)
(Cp*RuCl(COD)). After bubbling argon gas through the mixture for
10 min, the vial was sealed and subjected to a microwave treatment
for 22 h at 60 °C. Subsequently, the resin was washed with three
washing steps each of, respectively, DMF, 11.6 mM sodium
diethyldithiocarbamate and 0.01% DIPEA in DMF, methanol,
acetonitrile, and DCM. SBL-MC-38 was obtained after deprotection,
cleavage, and purification as outlined in the Peptide Synthesis section.
CuAAC Cyclization. After deprotection, cleavage, and purification,
61 mg (42% yield, >98% according to HPLC analysis) of the linear
precursor peptide SBL-MC-58-linear was obtained, which was
dissolved in a mixture of methanol and a 0.1 M aqueous
NH4HCO3 solution (0.8 mM, 1:2). After bubbling argon through
the solution for 10 min, CuSO4·5H2O (16 equiv) and sodium
ascorbate (16 equiv) were added. After stirring the mixture for 1 h at
room temperature, methanol was removed under reduced pressure,
and the resulting suspension was lyophilized. SBL-MC-58 was
obtained after purification as outlined in the Peptide Synthesis
section.
serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL
amphotericin B, 200 μg/mL hygromycin B (for positive selection of
the GScAMP22f luciferase reporter), and Geneticin (G418) 700 μg/
mL (for MC3R or MC4R selection). Cell line identity is routinely
verified by qPCR and MC3 and MC4R-specific oligonucleotides.
The assay for the determination of the cAMP response in live cells
was described previously.26 Briefly, cells were seeded at a density of
20 000 cells per well using 384-well poly-D lysine-coated, clear
bottom, and black-wall assay plates (Corning Inc. Corning, NJ). Cells
were allowed to attach to the plates for 18−24 h after which growth
media was removed and 20 μL of 4% D-luciferin (Promega) in CO2-
independent medium (Thermo Fisher Scientific) was added to each
well. The luciferase substrate was allowed to permeate the cells for
120 min at 37 °C. Intracellular cAMP levels were measured using an
FDSS 7000EX Functional Drug Screening System (Hamamatsu
Photonics, Hamamatsu, Japan) at the Center for Chemical Genomics
at the Life Sciences Institute. This instrument allowed the in-line
addition of test peptides and receptor agonists while simultaneously
acquiring the luminescence signal from live cells. Assay read steps are
set as follows: baseline acquisition of 2 min, the addition of 10 μL of
varying 3× concentrations of test peptides or vehicle followed by an
11 min measurement (measurement window 1), and the addition of
10 μL of 4× concentration of the endogenous melanocortin agonist α-
MSH (Bachem, Bubendorf, Switzerland) followed by an additional 11
min response measurement (measurement window 2). The resulting
final concentration of α-MSH was close to the respective receptor
EC90 dose for each receptor. Intraplate concentration−response
curves for α-MSH and SHU-9119 (Phoenix Pharmaceuticals,
Burlingame, CA) were included as reference controls. A submaximal
forskolin (20 μM) concentration was also included to serve as a
normalization reference to account for cell number variations and
differences in the assay transducer efficiency between cell lines.
With this setup, it was possible to evaluate the direct agonist effect
of the test peptides during measurement window 1, while the
antagonist profile in the presence of EC90 α-MSH was determined on
measurement window 2 (Figure 2A). For data analysis, baseline
luminescence (i.e., the maximum luminescence signal from the initial
0−2 min window) was subtracted from the maximum luminescence
obtained during measurement window 1 (2−13 min) and measure-
ment window 2 (13−24 min) to yield the test-peptide-elicited
responses. EC50 or IC50 potency values were determined by nonlinear
regression by fitting the data to a sigmoid four-parameter variable
slope model using the GraphPad Prism version 8.4 software package
(San Diego, CA).
Radioligand Binding Experiments. The stable cell lines expressing
the human MC3R and MC4R used for radioligand binding
experiments are described in the previous section. Crude membranes
were prepared from these cell lines as follows: 5 × 106 cells were
seeded onto each of the ten cell culture-grade 150 mm culture dishes
per receptor type and grown up to 90−95% confluency. TrypLE
Express (1×) cell dissociation reagent (Thermo Fisher Scientific) was
used to detach the cells from the growth vessels after 5 min incubation
at 37 °C. Trypsin was neutralized with an equal volume of complete
growth media, and cells were harvested by centrifugation at 200g for 5
min. The cell pellets were washed twice with phosphate-buffered
saline (PBS) solution. Washed pellets were resuspended in a
“membrane extract buffer” consisting of 50 mM Tris-HCl (pH 7.4),
10 mM MgSO4, 1.5 mM CaCl2, 0.5 mM ethylenediaminetetraacetic
acid (EDTA), and complete EDTA-free protease inhibitor cocktail
(Roche) and homogenized using a T10 Ultra Turrax (IKA Works,
Wilmington, NC). Homogenates were pelleted at 35 000g for 20 min.
Homogenization and pelleting were repeated twice. The final pellet
was resuspended with 10× v/w of membrane preparation buffer
supplemented with 10% sucrose, and aliquots were stored at −80 °C.
The total protein content from the crude membrane extracts was
determined by the bicinchoninic acid assay34 using a commercially
available kit (Pierce BCA Protein Assay Kit, Thermo Fisher
Scientific).
Peptide Purification. All peptides were purified by preparative
reversed-phase high-performance liquid chromatography (HPLC) on
a Gilson HPLC system accommodated with Gilson 322 pumps over a
Supelco Discovery BIO Wide Pore C18 column (25 cm × 21.2 mm,
10 μm) using a UV/vis-156 detector at 215 nm. The solvent system
consisted of Milli-Q water (containing 0.1% TFA) and acetonitrile
(containing 0.1% TFA). Gradients were determined according to the
retention time observed on the HPLC chromatogram of the crude
product. Prior to injection, the crude peptides were dissolved in
DMSO in a ratio of approximately 100 mg of crude product per mL of
DMSO, depending on the solubility of the crude product. The
solution was then filtered using a CHROMAFIL syringe filter. The
collected pure fractions were combined and lyophilized to retrieve the
purified peptides as a white powder with a purity exceeding 96%
according to HPLC analysis.
Methodology for Pharmacological In Vitro Assays. Determi-
nation of Intracellular cAMP Levels in Live Cells. A genetically
encoded cAMP split-luciferase reporter stably-expressing cell line
(Promega, Madison, WI)30,31 was used as the basis for the generation
of stable clones expressing the human MC4R receptor (a gift from
Promega) or the human MC3R (generated in-house by monoclonal
selection). The stable cell lines were grown and maintained in
selection media consisting of Dulbecco’s modified Eagle’s media
(DMEM) with 4.5 g/L D-glucose and 4 mM L-glutamine (Thermo
Fisher Scientific, Waltham, MA), supplemented with 10% fetal bovine
Competition binding experiments at equilibrium were performed in
the presence of 0.4−4 μg of total protein from the respective crude
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membrane extracts, a close-to-KD concentration of [125I]-
[Nle4,DPhe7]-α-MSH, and varying concentrations of the indicated
test peptides in a final volume of 200 μL of binding assay buffer (25
mM HEPES pH 7.4, 1 mM MgSO4, 1 mM CaCl2, 0.2% bovine serum
albumin (BSA), and complete EDTA-free protease inhibitor cocktail).
Test peptides and crude membranes were preincubated for 30 min at
room temperature. After the addition of the radioligand, assay tubes
were incubated for an additional 120 min. Bound [125I]-[Nle4,DPhe7]-
α-MSH was rapidly separated from free ligand by filtration through
GF/B glass fiber filters (Brandel, Gaithersburg, MD) using a cell
harvester (Brandel) manifold. During filtration, the filters were
washed five times in rapid succession with cold 25 mM Tris−HCl pH
7.4. Equilibrium dissociation constant (KD) values for human MC3R
or MC4R were determined ad hoc with [125I]-[Nle4,DPhe7]-α-MSH
saturation binding experiments (Figure S1). IC50 values were derived
from nonlinear regression fitting the data to a sigmoid function model.
Ki values were derived by applying the Cheng and Prusoff32
transformation.
Olivier Van der Poorten − Research Group of Organic
Chemistry, Vrije Universiteit Brussel, B-1050 Brussels,
Belgium
Simon Gonzalez − Research Group of Organic Chemistry,
Vrije Universiteit Brussel, B-1050 Brussels, Belgium
Kevin Van holsbeeck − Research Group of Organic
Chemistry, Vrije Universiteit Brussel, B-1050 Brussels,
Belgium
Santo Previti − Research Group of Organic Chemistry, Vrije
Universiteit Brussel, B-1050 Brussels, Belgium
Arthur Lamouroux − Research Group of Organic Chemistry,
Vrije Universiteit Brussel, B-1050 Brussels, Belgium
Suwen Zhao − iHuman Institute and School of Life Science
and Technology, ShanghaiTech University, Shanghai
201210, China; orcid.org/0000-0001-5609-434X
́
Dirk Tourwe − Research Group of Organic Chemistry, Vrije
Universiteit Brussel, B-1050 Brussels, Belgium
Methodology for Molecular Docking. Modeling was per-
̈
formed using the Maestro application of the Schrodinger software
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01620
platform version 2015-4. The homology model of hMC3R was
constructed based on the crystal structure of hMC4R using the
Advanced Homology Modeling tool. The new peptide ligand
structures were manually generated based on the published structure
of SHU-9119 cocrystalized with hMC4R26 and minimized by
Author Contributions
¶C.M. and L.E.G. contributed equally. The manuscript was
written through the contribution of all authors. All authors
have given approval to the final version of the manuscript.
OPLS3e forcefield. The complexes were refined using the Prime
35
̈
module (Schrodinger).
Funding
ASSOCIATED CONTENT
C.M., D.T., and S.B. thank the Spearhead (SRP) program of
the Vrije Universiteit Brussel for the financial support. K.V.h.
and S.B. acknowledge the Research Foundation Flanders
(FWO Vlaanderen) for providing a PhD fellowship to K.V.h.
S.Z. thanks the National Natural Science Foundation of China
grant 31971178. R.D.C. was supported by NIHRO1
DK070332.
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01620.
Peptide characterization including HPLC and HRMS
data for the described analogues as well as pharmaco-
logical data of the tested analogues including in vitro
activity and affinity at hMC3R and hMC4R (PDF)
Notes
The authors declare the following competing financial
interest(s): R.C.S. acknowledges that his primary academic
affiliation is the University of Southern California, he is an
adjunct professor at ShanghaiTech University, and he is the
founder of the biotech company ShouTi focused on GPCR
structure-based drug discovery. R.D.C., S.Y.W., and L.E.G.
have equity interests in Courage Therapeutics, Inc. and are
inventors of intellectual property optioned to the company.
R.D.C. is a founder, board member, and SAB Chair of Courage
Therapeutics, Inc.
Molecular formula strings (ZIP)
AUTHOR INFORMATION
■
Corresponding Authors
Raymond C. Stevens − iHuman Institute and School of Life
Science and Technology, ShanghaiTech University, Shanghai
201210, China; Email: stevens@stevensgrp.org
Roger D. Cone − Life Sciences Institute and Department of
Molecular and Integrative Physiology, University of Michigan,
Ann Arbor, Michigan 48109, United States; Email: rcone@
umich.edu
Steven Ballet − Research Group of Organic Chemistry, Vrije
Universiteit Brussel, B-1050 Brussels, Belgium; orcid.org/
0000-0003-4123-1641; Email: steven.ballet@vub.be
DEDICATION
■
†The authors want to dedicate this paper to the scientific
career of Prof. Em. Victor J. Hruby.
ABBREVIATIONS USED
■
Authors
ACN, acetonitrile; Azk, azidolysine; cAMP, cyclic adenosine
monophosphate; Cha, cyclohexylalanine; CuAAC, copper(I)-
catalyzed azide−alkyne cycloaddition; DCM, dichlorome-
thane; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s
modified Eagle’s medium; DMF, N,N-dimethylformamide;
ECL, extracellular loop; EDTA, ethylenediamine tetraacetic
acid; FDA, Food and Drug Administration; Fmoc, 9-
fluorenylmethyloxycarbonyl; Phe(pGnd), para-guanidinophe-
nylalanine; GPCR, G protein-coupled receptor; hArg, homo
arginine; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-
1-yl)uronium hexafluorophosphate; HEPES, 4-(2-hydroxyeth-
yl)-1-piperazineethanesulfonic acid; HOBt, 1-hydroxybenzo-
triazole; HPLC, high-performance liquid chromatography;
Charlotte Martin − Research Group of Organic Chemistry,
Vrije Universiteit Brussel, B-1050 Brussels, Belgium
Luis E. Gimenez − Life Sciences Institute, University of
Michigan, Ann Arbor, Michigan 48109, United States
Savannah Y. Williams − Life Sciences Institute, University of
Michigan, Ann Arbor, Michigan 48109, United States
Yu Jing − iHuman Institute, ShanghaiTech University,
Shanghai 201210, China
Yiran Wu − iHuman Institute, ShanghaiTech University,
Shanghai 201210, China
Charlie Hollanders − Research Group of Organic Chemistry,
Vrije Universiteit Brussel, B-1050 Brussels, Belgium
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01620
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Swaminath, G.; Zhao, S.; Stevens, R. C.; Hu, L. A.; Zhong, W.; Zhang,
M.; Xu, F. Structural basis for apelin control of the human apelin
receptor. Structure 2017, 25, 858−866.
HRMS, high-resolution mass spectroscopy; Hyp, hydroxypro-
line; LC−MS, liquid chromatography−mass spectrometry;
MC, melanocortin; MCR, melanocortin receptor; Nal(2′), 3-
(2-naphthyl)-L-alanine; Nle, norleucine; NMR, nuclear mag-
netic resonance; Oic, octahydroindole-2-carboxylic acid;
OPLS, optimized potentials for liquid simulation; Orn,
ornithine; RP-HPLC, reverse-phase high-performance liquid
chromatography; RuAAC, ruthenium-catalyzed azide−alkyne
cycloaddition; SAR, structure−activity relationship; SD, stand-
ard deviation; SEM, standard error of the mean; SPPS, solid-
phase peptide synthesis; TFA, trifluoroacetic acid; TIS,
triisopropylsilane; TM, transmembrane domain
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