Azepinone-containing tetrapeptide analogues of melanotropin lead to selective h MC4R agonists and h MC5R antagonist

Article abstract of DOI:10.1021/ml500436s

To address the need for highly potent, metabolically stable, and selective agonists, antagonists, and inverse agonists at the melanocortin receptor subtypes, conformationally constrained indolo- and benzazepinone residues were inserted into the α-MSH phar

Full text of DOI:10.1021/ml500436s

Letter
pubs.acs.org/acsmedchemlett
Azepinone-Containing Tetrapeptide Analogues of Melanotropin
Lead to Selective hMC4R Agonists and hMC5R Antagonist
Olivier Van der Poorten,†,∥ Krisztina Feher,‡,∥ Koen Buysse,† Debby Feytens,† Ioanna Zoi,§
́
Steven D. Schwartz,§ Jose C. Martins,‡ Dirk Tourwe,† Minying Cai,§ Victor J. Hruby,§ and Steven Ballet*,†
́
́
†Research Group of Organic Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
‡Department of Organic Chemistry, University of Ghent, Krijgslaan 281 S4, 9000 Ghent, Belgium
§Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, United States
S
* Supporting Information
ABSTRACT: To address the need for highly potent, metabolically stable, and selective agonists, antagonists, and inverse
agonists at the melanocortin receptor subtypes, conformationally constrained indolo- and benzazepinone residues were inserted
into the α-MSH pharmacophore, His6-Phe7-Arg8-Trp9-domain. Replacement of His6 by an aminoindoloazepinone (Aia) or
aminobenzazepinone (Aba) moiety led to hMC4R and hMC5R selective agonist and antagonist ligands, respectively
(tetrapeptides 1 to 3 and 4, respectively). In peptides 1 to 3 and depending on the para-substituent of the D-Phe residue in
position 2, the activity goes from allosteric partial agonism (1, R = H) to allosteric full agonism (2, R = F) and finally allosteric
partial agonism (3, R = Br).
KEYWORDS: Melanocortin ligands, constrained aminoazepinones, hMC4R and hMC5R molecular modeling and ligand docking,
fluorine peptidomimetics, allosteric hMC4R ligands
he endogenous melanocortin peptides include the α-, β-,
and γ-melanocyte stimulating hormones (MSHs). These
focused on modifications at the level of this “message”
domain.6,7
T
peptide hormones derive from posttranslational processing of
the pro-opiomelanocortin (POMC) prehormone.1−4 They play
a role in a wide range of biological and physiological responses
such as feeding and learning behavior, sexual function, and
energy homeostasis. The melanocortin peptides operate
through interactions with the G protein-coupled melanocortin
receptors in both the peripheral and central nervous system and
activate the adenylate cyclase second messenger signal
transduction cascade.1−4
To date five melanocortin receptor subtypes have been
identified.1−4 It has been observed that the primary native
ligand for MC1R, MC3R, MC4R, and MC5R is α-melanocyte
stimulating hormone (α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-
Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2). The native ligands possess
neither a high selectivity for any of the above-mentioned MC
receptors5 nor a long half-life time as they are rapidly
hydrolyzed by proteases. In order to establish the biological
functions of the individual MC receptors, the need for highly
potent, metabolically stable, and selective agonists, antagonists,
and inverse agonists for the four receptors MC1R, MC3R,
MC4R, and MC5R became apparent.5 Since all melanocortin
peptides possess a central and conserved “His-Phe-Arg-Trp”
pharmacophore tetrapeptide sequence, most studies have
Initially, α-MSH was modified through the replacement of
Met4 by the pseudoisosteric amino acid norleucine (Nle) to
prevent oxidation of its side chain, and the configuration of
Phe7 was inverted to D-Phe7 to enhance stability against
proteases. The resulting peptide [Nle4,D-Phe7]-α-MSH (MT-I)
revealed not only to be more potent than α-MSH but also to
have a higher in vivo stability and improved pharmacokinetics.8
In addition, molecular modeling and NMR studies showed that
the bioactive conformation of several cyclic ligands involved a
β-turn structure at the level of the tetrapeptide pharmacophore.
One of these potent cyclic analogues was a lactam (Ac-Nle-
c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2, MT-II), which proved to
be an extremely potent, but nonselective, agonist of human
MC1R, MC3R, MC4R, and MC5R.9,10
In order to design potent and more selective peptide
melanocortin ligands, several interesting structural modifica-
tions of MT-II were reported.11 The replacement of His6 by
Pro in MT-II by Grieco et al. resulted in retention of agonist
Received: October 26, 2014
Accepted: December 3, 2014
Published: December 3, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
potency for most MC receptors.12 This was the first indication
that His6 was not crucial for agonism. Additionally, MT-II-
based MCR antagonists, such as SHU-9119 (Ac-Nle4-c[Asp5-
His6-D-Nal(2′)7-Arg8-Trp9-Lys10]-NH2) were developed.11,13
Upon substitution of His6 in SHU-9119 by conformationally
restricted amino acids, selective antagonists for the hMC3R and
hMC4R were discovered.14,15 Hence, the importance of
position 6 with regard to activity and MCR selectivity was
demonstrated.14,15
co-workers modified the melanocortin tetrapeptide at the para-
position of the D-Phe7 side chain. Halogenation at this position
was used to unravel important side chain determinants for
differentiation of the agonist versus antagonist activity of the
mMC3R.23 By insertion of a pF-D-Phe or pCl-D-Phe residue,
they achieved good to moderate activation of mMC1R and
mMC3R and excellent activation of mMC4R and mMC5R.23
The study presented herein employs ligand−receptor
structure−activity relationship (SAR) studies of locally con-
strained pharmacophore tetrapeptides 1−5 (Figure 2) to
Previously, we replaced the His6-D-Phe7 dipeptide segment in
MT-II by an Aba-Xxx motif (with Aba = 4-amino-1,2,4,5-
tetrahydro-2-benzazepin-3-one, Figure 1).16 Such azepinones
Figure 2. Locally constrained melanocortin tetrapeptides 1−5,
containing the Aia (1−3), Aba (4), and Ata (5) scaffolds.
Figure 1. [Aba]MT-II analogues Aba-2−4 and the linear equivalent
Aba-1.16
investigate the requirements for receptor selectivity and for
differentiation of agonist versus antagonist activity at the human
melanocortin receptors MC1R, MC3R, MC4R, and MC5R.
Since the MC2R is only activated by ACTH, it has been
excluded from this study. Aiming at compact and constrained
tetrapeptide analogues, we decided to replace the His6 residue
in the tetrapeptide pharmacophore with Aia, Aba, and Ata
scaffolds (Figure 2) and to evaluate their influence on receptor
selectivity and agonist versus antagonist activity (Table 1).
The affinity for tetrapeptide ligands 1−5 was evaluated by
competition binding experiments that were carried out using
HEK293 cells, which stably expressed the human MC1, MC3,
MC4, and MC5 receptors.15 The activity at these receptors was
evaluated in the same cells using a cAMP assay.15 The binding
affinities, expressed as IC50 values, and the cAMP accumulation
data are compared to MT-II and presented in Table 1.
Introduction of the Aia-D-Phe dipeptidomimetic in the linear
pharmacophoric sequence (tetrapeptide 1) resulted in weak
micromolar antagonist affinity for the hMC1R, nanomolar
partial agonism at hMC3R (EC50 = 52 nM), excellent partial
agonism at hMC4R (EC50 = 0.3 nM), and moderate
antagonism at the hMC5R. The fact that 1 was unable to
displace the radioligand at the hMC3R (and with low potency
at the hMC4R) is an indication of allosteric binding at these
receptors. Fluorination of the para-position of D-Phe7 led to
enhanced activity at the hMCRs,23 as low nanomolar selective
agonist activity (IC50 = 6.5 nM; EC50 = 13 nM) was indeed
observed at hMC4R for tetrapeptide 2. In addition, this
substitution gave moderate nanomolar binding as well as weak
partial agonism at hMC1R and hMC5R. In contrast to the pF-
D-Phe7 tetrapeptide 2, which resulted in a selective hMC4R
are frequently used in our group and by others for the
improvement of peptide stability, selectivity, and potency.16−21
Aba can be viewed as a topographically constrained Phe
building block. Molecular modeling indicated a good backbone
overlap of all Aba-containing analogues with the proposed
conformation of MT-II.16 On the basis of the analysis of
Cα(i)−Cα(i + 3) distances and the distances between the CO
group of Asp5 and the NH group of Arg8, the β-turn
conformation was, however, not adopted by these analogues.
The local constraint in the Aba-containing linear analogue of
MT-II (Aba-1, Figure 1) was not sufficient to induce efficient
binding affinity. Gratifyingly, the cyclic lactam analogue Aba-2
with the “Aba6-D-Phe7” motif proved to be a selective hMC3R
antagonist (IC50 of 50 nM at hMC3R). We concluded that the
Aba building block needed to be used in conjunction with a
global conformational constraint from these results since the
linear analogue Aba-1 did not bind at concentrations up to 10
μM.16
Haskell-Luevano and co-workers prepared a large series of
analogues of the linear tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2
in which the Phe7 residue was modified via the replacement of
other aromatic residues and various substituents at the para-
position of the benzyl side chain of D-Phe7. They obtained an
improved selectivity for the mouse (m)MC3R versus the
mMC4R as well as a differentiation of agonist versus antagonist
activity.22,23
This information could pave the way to the development of
low molecular weight peptidomimetics as the tetrapeptide
ligands are of intermediate molecular weight.22−24 Proneth and
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agonist, the pBr-D-Phe7 tetrapeptide 3 showed significant loss in
binding affinity to hMC4R (IC50 = 200 nM), but resulted in a
potent allosteric partial agonist (EC50 = 0.3 nM and 80 activity
%) at this receptor. In addition, tetrapeptide 3 is a weak agonist
at hMC1R, a partial agonist at hMC3R, and a moderate
antagonist at hMC5R (IC50 = 70 nM). These affinities reflect
the importance of the presence and nature (i.e., van der Waals
radius) of the halogen substituent, next to that of the
conformationally constrained Aia residue. Whereas the full
agonist activity observed in Aia tetrapeptide 2 against hMC4R
renders it a good candidate for further modifications,
tetrapeptide 1 might also serve as a good template for the
development of an allosteric hMC4R partial agonist. The
aminobenzazepinone Aba-D-Phe analogue 4 displayed very
weak agonist activity on hMC4R (IC50 = 220 nM). In contrast,
it resulted in a selective antagonist for hMC5R (IC50 = 37 nM),
which renders it an ideal template for the investigation of
hMC5R antagonism. In contrast to the Aia and Aba analogues,
the Ata-D-Phe analogue 5 showed no specific subtype receptor
selectivity. We did not, therefore, focus on this analogue in
subsequent docking studies (see below).
The preferential conformations of the most promising
peptidic ligands 2 and 4 were analyzed by 2D ROESY NMR
spectroscopy in DMSO solution at room temperature. The
3
resulting distance constraints and homonuclear JHN,HA
couplings (typically 7−9 Hz) were employed for restraining
main chain conformations in both cases. Unfortunately,
conformational analysis by NMR was unable to determine a
preferred topology for tetrapeptides 2 and 4 since the
topologies of the ten lowest energy conformers were all
substantially different. We concluded, therefore, that preorga-
nization in solution was absent or very limited. In contrast to
earlier studies,25 this result could be explained by the fact that
the conformational constraint was each time introduced as the
first amino acid of the tetrapeptide sequence (Figure 2).
Peptides 2 and 4 were docked into the active state model of
hMC4R proposed by Mosberg et al.,26 which was also used in
the docking study of MT-II and SHU-9119 by Grieco and co-
workers.27 Upon docking of the peptide ligands, multiple
binding poses were generated and clustered, yielding two
clusters for peptide 2 and one larger cluster for analogue 4. The
binding modes selected are high-ranking poses, which occurred
multiple times among the best scoring models. The binding
mode for both peptides positioned the conformationally
restricted Aia or Aba residues in a hydrophobic pocket at the
bottom of the extracellular binding site as shown in Figures 3
and 4b,c. The obtained docking poses of the peptides were
compared with the earlier proposed binding mode of the
pharmacophore of [Nle4,D-Phe7]-α-MSH, His6-D-Phe7-Arg8-
Trp9, also shown in Figures 3 and 4a.26 The residues of
[Nle4,D-Phe7]-α-MSH (dark brown) are buried slightly deeper
into the binding site than peptides 2 (magenta) and 4 (purple).
This could reflect the fact that [Nle4,D-Phe7]-α-MSH is more
potent than the presented peptides. The 2D ligand interaction
maps of [Nle4,D-Phe7]-α-MSH, peptides 2 and 4, together with
a detailed comparison of the interactions found in the binding
poses, are presented in the Supporting Information. Peptide 2
has a slightly higher number of hydrophobic and H-bonding
interactions. In particular, the Aia residue is in contact with six
hydrophobic residues (two of which are the conserved residues
Phe261 and His264), while the Aba residue in 4 is only in contact
with two hydrophobic residues. Furthermore, the fluorinated D-
Phe in peptide 2 is in contact with three residues: hydrophobic
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Figure 5. Combined docking poses for Aia peptide 2 (magenta,
calculated affinity: −8.0 kcal/mol) and Aba peptide 4 (purple,
calculated affinity: −9.4 kcal/mol) in hMC5R. Extracellular part of
TMH4 (green helix) and -5 (yellow helix) are hidden for better
visibility of the poses.
Figure 3. Combined docking poses for Aia peptide 2 (magenta,
calculated affinity: −10.8 kcal/mol), Aba peptide 4 (purple, calculated
affinity: −10.0 kcal/mol), and [Nle4,D-Phe7]-α-MSH (dark brown) in
hMC4R. Extracellular part of TMH4 (green helix) and -5 (yellow
helix) are hidden for better visibility of the poses.
that binding of peptide 2 to hMC5R is sterically hindered by
the bulkiness of the Aia residue, while the Aba residue in
peptide 4 fits into the binding pocket but is unable to activate
the receptor.
interactions to Met130 and Ile129 and one polar interaction to
the CO of Asp126, with Phe284, Phe261, and His264 also in the
vicinity. The nonfluorinated D-Phe in peptide 4, however,
features only three intermolecular contacts. Contacts to the
fluorine could further stabilize the receptor interaction with
peptide 2, and the larger van der Waals radius of the fluorinated
benzyl in pF-D-Phe might contribute to favorable conforma-
tional changes for the induction of agonism.
In order to understand the selective hMC5R antagonist
activity of peptidomimetic 4, we compared the binding of
peptides 4 and 2 to hMC5R as shown in Figure 5. The best
docking pose of Aba-containing peptide 4 in hMC5R is very
similar to the one in hMC4R: the Aba and D-Phe residues
position themselves at the bottom of the hydrophobic part of
the binding pocket and interact with conserved residues, such
as Phe254. The Aia peptide 2, however, either fails to dock into
the binding site or assumes a docking pose in which the Aia
residue is located outside the binding pocket, while Trp and D-
Phe residues occupy the hydrophobic interior. This suggests
In conclusion, the insertion of constrained aminobenzo- and
indoloazepinone-based residues into the core melanocortin
tetrapeptides has resulted in compact and selective human
melanocortin receptor ligands. The nature (steric bulk) and
substitution pattern (para-halogenation) are important struc-
tural features for the modulation of receptor affinity, selectivity,
and activity in these constrained peptides. Peptide 2 is a very
potent and selective agonist for hMC4R, rendering it a good
candidate for studies related to obesity and diabetes. Moreover,
the newly discovered antagonist activity of peptide 4 at hMC5R
can prove very useful to studies investigating anxiety and other
neurological disorders. As further modification of the azepinone
cores can improve and fine-tune the pharmacodynamics of
these ligands, they represent valuable tools toward the
unravelling of the individual role of each receptor subtype,
but they may also be used for the development of specific
Figure 4. Best docking pose for (A) [Nle4,D-Phe7]-α-MSH (dark brown), (B) Aia peptide 2 (magenta), and (C) Aba peptide 4 (purple) in hMC4R.
Extracellular part of TMH4 (green helix) and -5 (yellow helix) are hidden for better visibility of the poses.
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ASSOCIATED CONTENT
* Supporting Information
Complete experimental details along with the characterization
of the synthesized tetrapeptides 1−5. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +32 2 6293292. E-mail: sballet@vub.ac.be.
Author Contributions
■
∥O.V.D.P and K.F contributed equally to this work. The
manuscript was written through contributions of all authors.
O.V.D.P., S.B., D.F., K.B., and D.T. were in charge of the
peptide synthesis; I.Z., M.C., and V.J.H. for the functional
bioactivities of tetrapeptides 1−5 at the human melanocortin
receptors; O.V.D.P., K.F., and J.C.M. for the conformational
NMR analysis of tetrapeptides 2 and 4; and K.F., I.Z., M.C., and
V.J.H. for the homology modeling of hMC4R and hMC5R and
ligand docking. All authors have given approval to the final
version of the manuscript.
Funding
The work of O.V.D.P., K.F., J.C.M., S.B., D.F., K.B.,and D.T.
was supported by the Research Foundation−Flanders (FWO
Vlaanderen) and the Agency for Innovation by Science and
Technology (IWT). The bioassay studies were supported in
part by grants from the U.S. Public Health Service, National
Institutes of Health, DK017420, GM 108040, and DA06284.
Notes
(13) Hruby, V. J.; Lu, D.; Sharma, S. D.; de Lauro Castrucci, A.;
Kesterson, R. A.; Al-Obeidi, F. A.; Hadley, M. E.; Cone, R. D. Cyclic
Lactam α-Melanotropin Analogues of Ac-Nle4-c[Asp5, D-Phe7,
Lys10]α-MSH(4−10)-NH2 with Bulky Aromatic Amino Acids at
Position 7 Show High Antagonist Potency and Selectivity at Specific
Melanocortin Receptors. J. Med. Chem. 1995, 38, 3454−3461.
(14) Grieco, P.; Lavecchia, A.; Cai, M.; Trivedi, D.; Weinberg, D.;
MacNeil, T.; Van der Ploeg, L. H. T.; Hruby, V. J. Structure-Activity
Studies of the Melanocortin Peptides: Discovery of Potent and
Selective Affinity Antagonists for the hMC3R and hMC4R Receptors.
J. Med. Chem. 2002, 45, 5287−5294.
The authors declare no competing financial interest.
ABBREVIATIONS
■
Aba, 4-amino-1,2,4,5-tetrahydro-3H-2-benzazepin-3-one;
ACTH, adrenocorticotropic hormone; Aia, 4-amino-1,4,5,10-
tetrahydroazepino[3,4-b]indol-3(2H)-one; Ata, 7-amino-7,8-
dihydro-4H-[1,2,3]triazolo[1,5-a][1,4]diazepin-6(5H)-one;
cAMP, cyclic(′-adenosine monophosphate); GPCR, G-protein
coupled receptor; MC1R, melanocortin-1 receptor; MC2R,
melanocortin-2 receptor; MC3R, melanocortin-3 receptor;
MC4R, melanocortin-4 receptor; MC5R, melanocortin-5
receptor; MCR, melanocortin receptor; MSH, melanocyte-
stimulating hormone; POMC, pro-opiomelanocortin; SAR,
structure−activity relationship; α-MSH, α-melanocyte stimulat-
ing hormone; β-MSH, β-melanocyte stimulating hormone; γ-
MSH, γ-melanocyte stimulating hormone
(15) Cai, M.; Cai, C.; Mayorov, A. V.; Xiong, C.; Cabello, C. M.;
Soloshonok, V. A.; Swift, J. R.; Trivedi, D.; Hruby, V. J. Biological and
Conformational Study of β-Substituted Prolines in MT-II Template:
Steric Effects Leading to Human MC5 Receptor Selectivity. J. Pept.
Res. 2004, 63, 116−131.
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́
B.; Palmer, E. S.; Van Rompaey, K.; Misicka, A.; Tourwe, D.; Hruby,
V. J. Novel Selective Human Melanocortin-3 Receptor Ligands: Use of
the 4-Amino-1,2,4,5-Tetrahydro-2-Benzazepin-3-One (Aba) Scaffold.
Bioorg. Med. Chem. Lett. 2007, 17, 2492−2498.
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