Multiple N -methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: Pharmacological and conformational studies

Article abstract of DOI:10.1021/ja101428m

Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation that resembles other α-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.

Full text of DOI:10.1021/ja101428m

Published on Web 05/24/2010
Multiple N-Methylation of MT-II Backbone Amide Bonds Leads
to Melanocortin Receptor Subtype hMC1R Selectivity:
Pharmacological and Conformational Studies
†
,‡
†,‡
‡,§
†,‡
Lucas Doedens, Florian Opperer, Minying Cai, Johannes G. Beck,
§
§
,§
,†
Matt Dedek, Erin Palmer, Victor J. Hruby,* and Horst Kessler*
Institute for AdVanced Study and Center for Integrated Protein Science at the Technische
UniVersit a¨ t M u¨ nchen, Lichtenbergstrasse 4, 85747 Garching, Germany, and Department of
Chemistry and Biochemistry, UniVersity of Arizona, Tucson, Arizona, 85721
Received February 18, 2010; E-mail: kessler@ch.tum.de; hruby@email.arizona.edu
Abstract: Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase
receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective
cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized
and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that
selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective
and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of
five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone
conformation that resembles other R-MSH analogs but possesses much less flexibility and in addition distinct
differences in the spatial arrangement of individual amino acid side chains.
Introduction
1
-3
N-Methylation of peptide bonds is long known
and has
often been used to modify biological properties of bioactive
4
peptides. However, it has become evident only recently that
multiple N-methylation is a novel technology to improve the
5
pharmacological properties of peptides and in extreme cases
6
even achieve oral bioavailability such as found for the antibiotic
7
,8
Cyclosporin. Here we present a complete N-methylation
library of the cyclic analogue of R-MSH, Ac-Nle-c[Asp-His-
2
D-Phe-Arg-Trp-Lys]-NH (MT-II), a biologically important and
potent but nonselective agonist for the melanocortin receptor
9
,10
subtype family.
The goal of our present study was to obtain
new peptidic compounds with melanocortin receptor activity
†
Institute for Advanced Study and Center for Integrated Protein Science
at the Technische Universit a¨ t M u¨ nchen.
‡
These authors contributed equally to this manuscript.
University of Arizona.
§
(
(
(
1) Lindenberg, H. J. Prakt. Chem. 1875, 12, 244–259.
2) Fischer, E.; Bergmann, M. Liebigs Ann. Chem. 1913, 398, 96–124.
3) Fischer, E.; Lipschitz, W. Ber. Dtsch. Chem. Ges. 1915, 48, 360–
5
6
7
8
9
3
78.
Figure 1. MT-II (Ac-Nle-cyclo(5ꢀf10ꢀ)(Asp -His -D-Phe -Arg -Trp -
10
(
4) Gilon, C.; Dechantsreiter, M. A.; Burkhart, F.; Frieder, A.; Kessler,
H. In Houben-Weyl Methods of Organic Chemistry; Georg Theime
Verlag: Stuttgart, 2003; Vol. E22c, pp 215-271.
2
Lys )-NH ) with sites of N-methylation indicated by arrows.
(
5) Chatterjee, J.; Gilon, C.; Hoffman, A.; Kessler, H. Acc. Chem. Res.
and improved selectivity, possessing pharmacological properties
superior to those of MT-II. Therefore, we exchanged in a
systematic manner His, D-Phe, Arg, Trp and Lys by their N -
methylated analogs and studied all of the 31 (2 - 1 ) 31)
possible MT-II-derivatives with one or more methylated back-
2
008, 41, 1331–1342.
(
6) Biron, E.; Chatterjee, J.; Ovadia, O.; Langenegger, D.; Brueggen,
J.; Hoyer, D.; Schmid, H. A.; Jelinek, R.; Gilon, C.; Hoffman, A.;
Kessler, H. Angew. Chem., Int. Ed. 2008, 47, 2595–2599.
R
5
(
(
(
7) Smith, J. M.; Hows, J. M.; Gordon-Smith, E. C. J. Clin. Pathol. 1983,
3
6, 41–43.
8) Sangalli, L.; Bortolotti, A.; Jiritano, L.; Bonati, M. Drug. Metab.
Dispos. 1988, 16, 749–753.
bone nitrogens (Figure 1).
9) Al-Obeidi, F.; Hadley, M. E.; Pettitt, B. M.; Hruby, V. J. J. Am.
Chem. Soc. 1989, 111, 3413–3416.
The melanocortin receptors are members of the 7 transmem-
brane (TM) spanning G-protein coupled receptor (GPCR)
superfamily. Five melanocortin receptor subtypes (MCR1-5)
(
10) Al-Obeidi, F.; Castrucci, A. M.; Hadley, M. E.; Hruby, V. J. J. Med.
Chem. 1989, 32, 2555–2561.
10.1021/ja101428m  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 8115–8128 9 8115

A R T I C L E S
Doedens et al.
8
,11-17
4
5
7
10
have been discovered thus far.
They all intracellularly
Met with Nle, Glu with Asp, Phe with D-Phe and Gly with
4
mediate their effects by activating pathways that are cyclic
adenosine monophosphate (cAMP) dependent. The diverse
MCRs are distributed widely in mammalian tissues, and regulate
numerous functions in the body including skin and hair
Lys, acetylation of the Nle -R-nitrogen, amidation of the
C-terminal carboxyl function, and lactam cyclization resulted
in the lactam bridged compound MT-II, a super potent but
nonselective agonist at 4 melanocortin receptors MC1R, MC3R,
1
8-20
21
22
coloration,
inflammation and immunomodulation, ste-
9,10,41
MC4R, MC5R
(not at the MC2R which has ACTH as its
2
3,24
25,26
roid production and release,
cardiovascular functions,
27
28-30
ligand, and where R-MSH and its analogues are not active).
energy homeostasis, feeding behavior,
penile erection and
3
1-33
sexual behavior
and many other functions. These very
diverse biological activities generally are associated with only
or 2 of the melanocortin receptors that are expressed in
different parts of the body. Thus, the search for highly selective
and potent agonist and antagonist analogues is critical.
A major focus in synthesizing new melanotropins is gaining
selectivity and improved pharmacological properties that are
suitable for medical applications of these compounds. When
creating peptidic ligands, one possible way to obtain these
properties is N-methylation of the amide bonds,
substitution of amide protons with methyl groups can result in
receptor subtype selectivity,
properties can be improved, such as metabolic stability
lipophilicity,
1
4–6,42-44
since
The natural endogenous agonistic ligands R-melanocyte
1
2
3
4
5
stimulating hormone (R-MSH, Ac-Ser -Tyr -Ser -Met -Glu -
45-48
but also other pharmacological
6
7
8
9
10
11
12
13
His -Phe -Arg -Trp -Gly -Lys -Pro -Val -NH ), ꢀ-MSH,
2
6
,49-51
γ-MSH and adrenocorticotropin (ACTH) all are derived from
the same precursor protein, pro-opiomelanocortin (POMC).
Structure-function examinations of R-MSH and of R-MSH
fragments have led to the identification of His-Phe-Arg-Trp as
the critical pharmacophore for pigmentary activity and other
functions of the melanotropin peptides.
the R-MSH sequence, keeping amino acids 4-10, exchange of
4
9–51
52-55
6,56,57
potency,
bioavailability
and con-
(
36) Medzihradszky, K. Recent DeVelopments in the Chemistry of Natural
19,20,34-41
Carbon Compounds;, Hungarian Academy of Science: Budapest,
976; pp 207-250.
Truncation of
1
(37) Schwyzer, R.; Eberle, A. Frontiers of Hormone Research; Karger:
Basel, 1977; Vol. 4, pp 18-25.
38) Hruby, V. J.; Wilkes, B. C.; Hadley, M. E.; Al-Obeidi, F.; Sawyer,
T. K.; Staples, D. J.; de Vaux, A. E.; Dym, O.; Castrucci, A. M.;
Hintz, M. F.; Riehm, J. P.; Rao, K. R. J. Med. Chem. 1987, 30, 2126–
2130.
(39) Castrucci, A. M.; Hadley, M. E.; Sawyer, T. K.; Wilkes, B. C.; Al-
Obeidi, F.; Staples, D. J.; de Vaux, A. E.; Dym, O.; Hintz, M. F.;
Riehm, J. P.; Rao, K. R.; Hruby, V. J. Gen. Comp. Endrocrinol.
1989, 73, 157–163.
(40) Hruby, V. J.; Cai, M.; Grieco, P.; Han, G.; Kavarana, M.; Trivedi,
D. Ann. N.Y. Acad. Sci. 2003, 994, 12–20.
(
(
11) The Melanotropic Peptides. Ann. N.Y. Acad. Sci. 1993, 680, 1-
87.
6
(
(
12) Chhajlani, V.; Wikberg, J. E. FEBS Lett. 1992, 309, 417–420.
13) Chhajlani, V.; Muceniece, R.; Wikberg, J. E. Biochem. Biophys. Res.
Commun. 1993, 195, 866–873.
(
14) Gantz, I.; Konda, Y.; Tashiro, T.; Shimoto, Y.; Miwa, H.; Munzert,
G.; Watson, S. J.; DelValle, J.; Yamada, T. J. Biol. Chem. 1993,
2
68, 8246–8250.
(
15) Gantz, I.; Miwa, H.; Konda, Y.; Shimoto, Y.; Tashiro, T.; Watson,
S. J.; DelValle, J.; Yamada, T. J. Biol. Chem. 1993, 268, 15174–
(41) Hruby, V. J.; Cai, M.; Cain, J. P.; Mayorov, A. V.; Dedek, M. M.;
Trivedi, D. Curr. Top. Med. Chem. 2007, 7, 1107–1119.
(42) Rajeswaran, W. G.; Hocart, S. J.; Murphy, W. A.; Taylor, J. E.; Coy,
D. H. J. Med. Chem. 2001, 44, 1416–1421.
1
5179.
(
(
(
16) Gantz, I.; Shimoto, Y.; Konda, Y.; Miwa, H.; Dickinson, C. J.;
Yamada, T. Biochem. Biophys. Res. Commun. 1994, 200, 1214–1220.
17) Labbe, O.; Desarnaud, F.; Eggerickx, D.; Vassart, G.; Parmentier,
M. Biochemistry 1994, 33, 4543–4549.
(43) Laufer, R.; Gilon, C.; Chorev, M.; Selinger, Z. J. Biol. Chem. 1986,
261, 10257–10263.
18) The Melanocortin System. Ann. N.Y. Acad. Sci. 2003, 994, 1-367,
provides the proceedings of a recent symposium discussing the latest
developments in this area.
(44) Laufer, R.; Wormser, U.; Friedman, Z. Y.; Gilon, C.; Chorev, M.;
Selinger, Z. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 7444–7448.
(45) Kawasaki, A. M.; Knapp, R.; Wire, W.; Kramer, T.; Yamamura, H. I.;
Burks, T. F.; Hruby, V. J. Peptides: Chemistry, Structure and Biology,
Proceedings of the EleVenth American Peptide Symposium 1990,
337-338.
(
19) Hadley, M. E. in The Melanotropin Peptides; CRC Press: Boca Raton,
FL, 1989.
(
20) Eberle, A. N. The Melanotropins: Chemistry, Physiology and
Mechanisms of Action; Karger: Basel, 1988.
21) Manna, S. K.; Aggarwal, B. B. J. Immunol. 1998, 161, 2873–2880.
22) Maaser, C.; Kannengiesser, K.; Specht, C.; Lugering, A.; Brzoska,
T.; Luger, T. A.; Domschke, W.; Kucharzik, T. Gut 2006, 55, 1415–
(46) Hruby, V. J.; Fang, S. A.; Knapp, R.; Kazmierski, W.; Lui, G. K.;
Yamamura, H. I. Int. J. Pept. Protein Res. 1990, 35, 566–573.
(47) Knapp, R. J.; Vaughn, L. K.; Fang, S. N.; Bogert, C. L.; Yamamura,
M. S.; Hruby, V. J.; Yamamura, H. I. J. Pharmacol. Exp. Ther. 1990,
255, 1278–1286.
(
(
1
422.
(
(
23) Xia, Y.; Wikberg, J. E. Cell Tissue Res. 1996, 286, 63–68.
(48) Chatterjee, J.; Ovadia, O.; Zahn, G.; Marinelli, L.; Hoffman, A.;
Gilon, C.; Kessler, H. J. Med. Chem. 2007, 50, 5878–5881.
(49) Cody, W. L.; He, J. X.; Reily, M. D.; Haleen, S. J.; Walker, D. M.;
Reyner, E. L.; Stewart, B. H.; Doherty, A. M. J. Med. Chem. 1997,
40, 2228–2240.
24) Buckley, D. I.; Ramachandran, J. Proc. Natl. Acad. Sci. U.S.A. 1981,
7
8, 7431–7435.
(
(
25) Low, M. J. J. Endocrinol. InVest. 2004, 27, 95–100.
26) Li, S. J.; Varga, K.; Archer, P.; Hruby, V. J.; Sharma, S. D.;
Kesterson, R. A.; Cone, R. D.; Kunos, G. J. Neurosci. 1996, 16,
(50) Haviv, F.; Fitzpatrick, T. D.; Swenson, R. E.; Nichols, C. J.; Mort,
N. A.; Bush, E. N.; Diaz, G.; Bammert, G.; Nguyen, A.; Rhutasel,
N. S.; Nellans, H. N.; Hoffman, D. J.; Johnson, E. S.; Greer, J. J. Med.
Chem. 1993, 36, 363–369.
5
182–5188.
(
(
(
(
(
(
(
27) Gantz, I.; Fong, T. M. Am. J. Physiol. Endocrinol. Metab. 2003, 284,
E468–474.
28) Fan, W.; Boston, B. A.; Kesterson, R. A.; Hruby, V. J.; Cone, R. D.
Nature 1997, 385, 165–168.
(51) Fairlie, D. P.; Abbenante, G.; March, D. R. Curr. Med. Chem. 1995,
2, 654–686.
29) Farooqi, I. S.; Keogh, J. M.; Yeo, G. S.; Lank, E. J.; Cheetham, T.;
O’Rahilly, S. N. Engl. J. Med. 2003, 348, 1085–1095.
30) Branson, R.; Potoczna, N.; Kral, J. G.; Lentes, K. U.; Hoehe, M. R.;
Horber, F. F. N. Engl. J. Med. 2003, 348, 1096–1103.
31) Wessells, H.; Gralnek, D.; Dorr, R.; Hruby, V. J.; Hadley, M. E.;
Levine, N. Urology 2000, 56, 641–646.
(52) Tonelli, A. E. Biopolymers 1976, 15, 1615–1622.
(53) Manavalan, P.; Momany, F. A. Biopolymers 1980, 19, 1943–1973.
(54) Ron, D.; Gilon, C.; Hanani, M.; Vromen, A.; Selinger, Z.; Chorev,
M. J. Med. Chem. 1992, 35, 2806–2811.
(55) Dechantsreiter, M. A.; Planker, E.; Math a¨ , B.; Lohof, E.; H o¨ lzemann,
G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. J. Med. Chem. 1999,
42, 3033–3040.
32) Wessells, H.; Levine, N.; Hadley, M. E.; Dorr, R.; Hruby, V. Int. J.
Impot. Res. 2000, 12 (4), S74–79.
(56) Ali, F. E.; Bennett, D. B.; Calvo, R. R.; Elliott, J. D.; Hwang, S. M.;
Ku, T. W.; Lago, M. A.; Nichols, A. J.; Romoff, T. T.; Shah, D. H.;
Vasko, J. A.; Wong, A. S.; Yellin, T. O.; Yuan, C.-K.; Samanen,
J. M. J. Med. Chem. 1994, 37, 769–780.
33) King, S. H.; Mayorov, A. V.; Balse-Srinivasan, P.; Hruby, V. J.;
Vanderah, T. W.; Wessells, H. Curr. Top. Med. Chem. 2007, 7, 1098–
1
106.
(
(
34) Eberle, A.; H u¨ bscher, W. HelV. Chim. Acta 1979, 62, 2460–2483.
35) Eberle, A.; Schwyzer, R. HelV. Chim. Acta 1979, 62, 2452–2459.
(57) Mazur, R. H.; James, P. A.; Tyner, D. A.; Hallinan, E. A.; Sanner,
J. H.; Schulze, R. J. Med. Chem. 1980, 23, 758–763.
8
116 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010

Melanocortin Receptor Subtype hMC1R Selectivity
A R T I C L E S
4
8,51,58
formational rigidity.
It may also turn an agonist into an
observed as broad peaks or mixtures of two peaks with identical
mass, even after repeated synthesis. When the peaks were
separated and reinjected, similar chromatograms were observed.
This indicated the presence of conformational isomers rather
than diastereomers. To study the influence of N-methylation on
the conformation of the pharmacophore of MT-II, a library of
MT-II analogues with single and multiple N-methylation in the
core sequence of MT-II has been designed and synthesized
(Figure 1 and Table 1).
5
7
antagonist.
Application of the N-alkyl scan concept to the highly active
and selective cyclic pentapeptide cyclo(-RGDfV-) has led to
one of the most active (0.5 nM) and selective inhibitors for the
59
6
0
5
5,61
R
v
ꢀ
3
integrin,
the analog cyclo(-Arg-Gly-Asp-D-Phe-(Me)-
5
5,61
Val-], Cilengitide,
which is now in clinical phase III for
treatment of glioblastoma. N-Alkylation is also present in
naturally occurring peptides from plants, marine sources and
various microorganisms. Several of these compounds exhibit
biological activity like antibiotic,
immunosuppressor activity.
6
2-64
65-67
Results and Discussion
antitumor
and
6
8
Biological Data. Competitive binding assays with [125I]-
4
5
7
10
4
7
Peptide Design. Ac-Nle -c[Asp , D-Phe , Lys ]R-MSH(4-10)-
NH (MT-II) was chosen as a template for the design of a
2
[Nle ,D-Phe ]-R-MSH (NDP-R-MSH), and adenylate cyclase
assays were performed on HEK293 cells stably expressing the
hMC1R, hMC3R, hMC4R, and hMC5R (Table 1 and Table 2,
respectively).
combinatorial library to search for more selective melanotropin
peptides. Some N-methyl amino acids are commercially avail-
able, but most are expensive. However, several methods for
synthesizing N-methylated amino acids in solution and on solid
supports have been developed. We decided to do the N-
methylations on a solid support, which gives flexibility in library
design and facilitated coupling, because no coupling of N-
methylated amino acids to an N-methylated peptide is neces-
sary. We used the procedure originally described by Miller
and Scanlan which has been optimized by Biron et al. and
which is compatible with all commonly used amino acids.
A major advantage of the o-NBS protecting group coupled
to the free amine prior to N-methylation is that deprotection
with mercaptoethanol is selective for N-methylated derivatives
and does not occur when the protected amine is not alkylated.
Hence, it provides an inherent purification step during synthesis.
In the first group of peptides with the single N-methylation
screen (peptides 2-6), it is revealed that N-Me-D-Phe caused a
total loss of binding as well as adenylate cyclase activities
(Tables 1 and 2) at the hMC1R, hMC3R, hMC4R and hMC5R
for peptide 5. For the rest of the core sequence single
N-methylation does not cause this drastic loss of binding
activities (Table 1). The cAMP assay data (Table 2) show that
the N-methylated peptides 2, 3 and 6 still retain full agonist or
partial agonist activity toward all subtypes of melanocortin
receptors. This phenomenon parallels earlier studies that have
shown that D-Phe is the most critical amino acid for the binding
6
9
6
7
0
7
1
4
3,73,75
7
and cAMP activities to hMCRs.
The N-Me-D-Phe
71
substitution apparently led to a conformation which prevents
interaction of the aromatic ring with the third and the sixth
76
R
Couplings on N -methylamino acids are known to be more
transmembrane binding domains aromatic groups. The N-Me-
7
2
6
challenging than normal couplings. Hence, these couplings
were performed with HATU and HOAt instead of TBTU and
HOBt using DIEA as base in NMP yielding in complete
couplings after 3 h. Nevertheless, some peptides (3, 13, 25)
could not be purified as sharp peaks by HPLC and were
His analog reveals increased binding selectivity for the hMC1R
and hMC3R. This result parallels our earlier observation that
constraining the His residue can lead to potent antagonists
7
7
especially for the hMC3R.
The second group of peptides are dimethylated derivatives
in the core sequence of MT-II (peptides 7-16). In this group,
some exciting results are observed. It is first demonstrated that
(
(
(
(
58) Vitoux, B.; Aubry, A.; Cung, M. T.; Marraud, M. Int. J. Pept. Protein
7
any site of N-methylation combined with N-Me-D-Phe will lose
Res. 1986, 27, 617–632.
59) Sugano, H.; Higaki, K.; Miyoshi, M. Bull. Chem. Soc. Jpn. 1973,
the binding as well as cAMP activities at all hMCRs (peptides
4
6, 226–230.
9
, 12, 14, 16) confirming the results from monomethylation.
60) Aumailley, M.; Gurrath, M.; M u¨ ller, G.; Calvete, J.; Timpl, R.;
Kessler, H. FEBS Lett. 1991, 291, 50–54.
N-Methylation combined with N-Me-Lys reduces binding at the
hMC4R and hMC5R (peptide 7, 8, 10). The cAMP functional
assays show that except 7 at the hMC5R these peptides retain
partial agonist activities. N-Methylation combined with N-Me-
Trp increases the selective agonist activity at the hMC1R
(peptides 11, 13), and selective antagonist activity at hMC3R
61) Dechantsreiter, M. A.; Math a¨ , B.; Jonczyk, A.; Goodman, S. L.;
Kessler, H. Peptides 1996; Mayflower Scientific: Kingswinford, 1996;
p 329.
(
62) Shemyakin, M. M.; Ovchinnkov, Y. A.; Ivanov, V. T.; Kiryushkin,
A. A. Tetrahedron 1963, 19, 581–591.
(
63) Bevan, K.; Davies, J. S.; Hall, M. J.; Hassall, C. H.; Morton, R. B.;
Phillips, D. A.; Ogihara, Y.; Thomas, W. A. Experientia 1970, 26,
(peptide 13) and lead to large decreases in binding and functional
1
22–123.
activity at the hMC4R and hMC5R resulting in increased
selectivity of peptide 12 for the hMC3R. The combination of
N-Me-His and N-Me-Arg (peptide 15) reduces the binding at
(
64) Corbaz, R.; Ettlinger, L.; Gaumann, E.; Keller-Schierlein, W.;
Kradolfer, F.; Neipp, L.; Prelog, V. HelV. Chim. Acta 1957, 23, 199.
65) Jolad, S. D.; Hoffmann, J. J.; Torrance, S. J.; Wiedhopf, R. M.; Cole,
J. R.; Arora, S. K.; Bates, R. B.; Gargiulo, R. L.; Kriek, G. R. J. Am.
Chem. Soc. 1977, 99, 8040–8044.
(
(
66) Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald, C. L.;
Schmidt, J. M. J. Org. Chem. 1989, 54, 6005.
(73) Haskell-Luevano, C.; Miwa, H.; Dickinson, C.; Hruby, V. J.; Yamada,
T.; Gantz, I. Biochem. Biophys. Res. Commun. 1994, 204, 1137–
1142.
(
67) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner,
F. E.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.;
Bontems, R. J. J. Am. Chem. Soc. 1987, 109, 6883–6885.
(74) Haskell-Luevano, C.; Sawyer, T. K.; Hendrata, S.; North, C.;
Panahinia, L.; Stum, M.; Staples, D. J.; Castrucci, A. M.; Hadley,
M. F.; Hruby, V. J. Peptides 1996, 17, 995–1002.
(75) Sawyer, T. K.; Sanfilippo, P. J.; Hruby, V. J.; Engel, E. H.; Heward,
C. B.; Burnett, J. B.; Hadley, M. E. Proc. Natl. Acad. Sci. U.S.A.
1980, 77, 5754–5758.
(
68) Ruegger, A.; Kuhn, M.; Lichti, H.; Loosli, H.-R.; Huguenin, R.;
Quiquerez, C.; von Wartburg, A. HelV. Chim. Acta 1976, 59, 1075.
69) Biron, E.; Kessler, H. J. Org. Chem. 2005, 70, 5183–5189.
70) Miller, S. C.; Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301–
(
(
2
302.
71) Biron, E.; Chatterjee, J.; Kessler, H. J. Pept. Sci. 2006, 12, 213–
19.
72) Teixido, M.; Albericio, F.; Giralt, E. J. Pept. Res. 2005, 65, 153–
66.
(76) Chen, M.; Cai, M.; Aprahamian, C. J.; Georgeson, K. E.; Hruby,
V.; Harmon, C. M.; Yang, Y. J. Biol. Chem. 2007, 282, 21712–
21719.
(
(
2
(77) Grieco, P.; Cai, M.; Han, G.; Trivedi, D.; Campiglia, P.; Novellino,
E.; Hruby, V. J. Peptides 2007, 28, 1191–1196.
1
J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010 8117

A R T I C L E S
Doedens et al.
8
118 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010

Melanocortin Receptor Subtype hMC1R Selectivity
A R T I C L E S
J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010 8119

A R T I C L E S
Doedens et al.
the hMC4R and the hMC5R, therefore increase the agonist
selectivity for the hMC1R and hMC3R.
More interesting results are found in the third group of
peptides (peptides 17-26), tri-N-methylated derivatives in the
core sequence of MT-II caused a loss in binding affinities
(
peptides 18, 20, 22, 23, 25, 26), especially, with any of the
combinations with N-Me-D-Phe. In this group, as long as D-Phe
is not N-methylated, most ligands will retain binding activity
at hMCRs (peptides 17, 19, 21, 24), but they all have reduced
cAMP activity (Table 2) except at the hMC1R (partial agonists).
Tri-N-methylations generally increase the agonist selectivity for
the hMC1R (peptides 17, 19, 21, 24). It can be seen from Table
1
that 24 has totally lost activity for all receptors but the hMC1R,
which still exhibits good binding affinity and >1000 fold
selectivity vs the hMC3, hMC4 and hMC5 receptors. The
compound has agonist activity as well (Table 2).
Selectivity, but also loss of activity, are further shown for
the five tetra-N-methylated MT-II derivatives (peptides 27-31).
Tetra-N-methylation caused a loss in binding affinities and
cAMP activities at all subtypes of hMCRs except for peptide
2
8, which has potent binding affinity (14 nM) at the hMC1R
and has full agonist activity. Thus, a highly potent and selective
agonist of hMC1R has been obtained. Peptides 29, 30 and
especially 32 are weak, but selective antagonists for the hMC5R.
Figure 2. Molecular structure of compound 28.
N
coupling constants, H temperature gradients) and on restrained
and unrestrained molecular dynamics a distinct and preferred
structure could be derived for the peptide backbone. The amide
R
In addition to 31 N -methylated analogs, one ε-N-methylated
lysine analogue (peptide 33) was investigated. It is interesting
to note that in the case of peptide 33, there was a loss in binding
to the hMC4R, while selective antagonist activity was seen for
the hMC5R in the nanomolar range (peptide 33 is still a full
agonist at the hMC1R and the hMC3R). Thus far there are few
cases of selective peptide antagonists at the hMC5R.
It has been demonstrated that the MC1R, which is activated
selectively by the 4-fold N-methylated peptide 28 is involved
in pain, in immune response, and in melanoma cancer. Thus,
5
10
bridged side chains of the residues Asp and N-Me-Lys that
are also part of the cyclic core were found to be flexible. The
resulting good agreement between measured and calculated
distances clearly indicates a preferred backbone conformation
of the structure obtained from the restrained MD simulation
8
3
(Table 4). In a very extended (30 ns) unrestrained MD
simulation in explicit water this structure proved to be stable
except for slight changes of four backbone dihedral angles in
the range of 20-40° (Table 5). Indicators for a reliable structure
can be seen in the predominantly parallel orientation of CO(i)
7
8
highly selective MC1R ligands have potential for treatment of
pigmentary disorders,
therapy,
1
8–20,79,80
78
treatment of pain, cancer
81,82
R
R
84
and inflammatory disorders of the skin. The hMC1R
to C H (i + 1) bond vectors and in an overall high dispersion
R
is just one subtype of hMCRs in the melanocortin system. It is
mainly distributed in the peripheral system in mammals, and
the other subtypes of hMCRs are distributed in both of the
peripheral and the central nervous system. Designing highly
constrained, lipophilic hMC1R selective N-methylated MT-II
derivatives will have widest application for specific targeting
of disease.
NMR Conformational Studies. The conformation of the
highly potent and selective compound 28 (Figure 2) was
investigated by NMR spectroscopy, restrained distance geometry
calculations (DG), restrained 150 ps MD simulation in explicit
water (rMD) and by unrestrained 30 ns MD simulation in
explicit water (MD). Based on NMR assignments (Table 3) and
other NMR data (ROEs, homo- and heteronuclear scalar
of backbone chemical shifts (H : 4.231 to 6.021 ppm (Figure
3), H : 1.559 to 3.115 ppm). In our hands a comparative
Me
attempt to discuss the spectral data of non-N-methylated MT-
II by one single preferred conformation failed. There was
considerable backbone dynamics as indicated by a high het-
erogeneity of the best 30 out of 50 MT-II conformers obtained
from DG calculations (rmsd of the backbone carbon and nitrogen
atoms of 1.26 Å). Moreover, a low dispersion of chemical shifts
1
8
R
N
(
H : 4.198 to 4.644 ppm (Figure 3), H : 7.871 to 8.541 ppm),
N
a lack of strong differentiation of all seven backbone H
temperature gradients (-8.61 to -5.24 ppb/K) and of amide
proton exchange rates as estimated by ROESY exchange peaks,
as well as a smaller preference of distinct side chain conforma-
tions was observed. Altogether, these indicators suggest a more
flexible peptide backbone of MT-II as compared to peptide 28.
(
78) Mogil, J. S.; Wilson, S. G.; Chesler, E. J.; Rankin, A. L.; Nemmani,
K. V. S.; Lariviere, W. R.; Groce, M. K.; Wallace, M. R.; Kaplan,
L.; Staud, R.; Ness, T. J.; Glover, T. L.; Stankova, M.; Mayorov,
A.; Hruby, V. J.; Grisel, J. E.; Fillingim, R. B. Proc. Natl. Acad.
Sci. U.S.A. 2003, 100, 4867–4872.
Structure of the Peptide Backbone of 28. The conformation
of compound 28 is shown in Figure 4. The ROE pattern
demonstrates that all peptide bonds are trans configurated. As
most of the amide bonds are N-methylated, turn-structures are
85
(
79) Dorr, R. T.; Dvorakova, K.; Brooks, C.; Lines, R.; Levine, N.;
Schram, K.; Miketova, P.; Hruby, V. J.; Alberts, D. S. Protochem.
Photobiol. 2000, 72, 526–532.
not only defined by intramolecular hydrogen bonds. We think
(
(
(
80) Hadley, M. E.; Hruby, V. J.; Sharma, S. D.; Dorr, R. T.; Levine, N.
Ann. N.Y. Acad. Sci. 1993, 680, 424–439.
(83) Kessler, H.; Griesinger, C.; Lautz, J.; M u¨ ller, A.; van Gunsteren,
W. F.; Berendsen, H. J. C. J. Am. Chem. Soc. 1988, 110, 3393–
3396.
81) Sharma, S. D.; Jiang, J.; Hadley, M. E.; Bentley, D. L.; Hruby, V. J.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13715–13720.
82) Jiang, J.; Sharma, S. D.; Fink, J. L.; Hadley, M. E.; Hruby, V. J.
Exp. Dermatol. 1996, 5, 325–333.
(84) Heller, M.; Sukopp, M.; Tsomaia, N.; John, M.; Mierke, D. F.; Reif,
B.; Kessler, H. J. Am. Chem. Soc. 2006, 128, 13806–13814.
(85) Matthews, B. W. Macromolecules 1972, 5, 818–819.
8
120 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010

Melanocortin Receptor Subtype hMC1R Selectivity
A R T I C L E S
a
Table 3. Resonance Assignment of Peptide 28 (Sodium Acetate-d4 Buffer (50 mM), pH 4.5, 298 K)
N
NMe
Hr
Hꢀ
Hγ
Hδ
Hε
Hꢁ
Hη
H
(H
)
others
Nle4
8.166
8.328
4.231
1.653
.701
1.281
1.320
0.893
-
-
HAcetyl
2.062
:
1
Asp5
5.183
5.392
4.668
5.164
6.021
5.238
proR: 2.271
proS: 2.521
proR: 3.272
proS: 3.063
proR: 2.901
proS: 2.661
proR: 1.245
proS: 1.518
proR: 3.287
proS: 3.216
1.914
-
-
-
-
-
1: 8.563
7.327
-
-
-
-
-
-
-
-
N-Me-His6
D-Phe7
(3.115)
7.657
2: 7.084
7.152
-
7.320
-
-
N-Me-Arg8
N-Me-Trp9
N-Me-Lys10
(1.559)
(2.695)
(2.774)
1.168
1.255
-
3.084
7.144
-
1: 7.249
1: 10.180
3: 7.639
3.046
2: 7. 535
3: 7.150
8.174
2: 7.321
-
1.146
1.291
1.470
1.594
HAmide: 7.145
7.575
1
.914
3.530
a
1
Chemical shifts are referenced on sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 ( H at 0.000 ppm).
1
0
R
that steric effects and dipole orientation such as the above-
N-Me-Lys H ) were violated during the unrestrained MD
simulation. This can be traced back directly to the aforemen-
tioned changes in backbone dihedral angles. As illustrated in
more detail in the sections describing the side chain dynamics
and structure calculations, we think that the changes in the
backbone dihedral angles were caused by artificial strains in
R
R
mentioned parallel orientation of CO(i) to C H (i + 1) bond
84
vectors contribute most strongly to the conformation of smaller
N-methylated cyclic peptides.
6
According to the N-Me-His backbone dihedral angles (Φ )
-
98°, Ψ ) 78°) the structure obtained from restrained MD
5
10
85
the amide linked Asp and N-Me-Lys side chains. These were
introduced within the DG calculation as our structure calculation
protocol did not take conformational averaging explicitly into
account. Accordingly, the conformer obtained from restrained
MD seems to be the best structural model for the peptide
backbone and we focused the analysis of side chain conforma-
tion on this structure.
In consideration of the high binding affinity to hMC1R (IC50
) 14 nm) and the strong restriction that cyclization and 4-fold
N-methylation pose on conformational changes within the
backbone of peptide 28, we think that its backbone conformation
in aqueous solution is very close to the conformation present
in the receptor bound state.
The conformation of the peptide backbone also offers an
explanation for the strong interference of D-Phe N-methylation
with hMCR affinity, as N-methylation goes in hand with an
increased spatial requirement and increased hydrophobicity in
comparison to the replaced amide proton. If D-Phe is N-
methylated, these hydrophobic and steric effects would prohibit
the formation of the backbone conformation present in peptide
simulation possesses an inverted γ-turn centered at N-Me-
6
5
His . A distance of 3.4 Å between the Asp carbonyl oxygen
7
and the D-Phe amide nitrogen, a hydrogen bond angle of 141°
and a moderately negative temperature gradient of D-Phe H
7
N
(
-5.92 ppb/K in the aqueous buffer, -5.55 ppb/K in DMSO)
indicate that the hydrogen bond within this inverted γ-turn is
rather weak and protection form the solvent is incomplete.
8
6
According to the minimal requirement for ꢀ-turns, which
R
and CRi+3
,
i
consists in a distance of less than 7 Å between C
the inverted γ-turn is located within a ꢀ-turn ranging from Asp
5
8
R
R
to N-Me-Arg (C -C distance: 6.4 Å). A distance of 7.6 Å
6
9
between the R-carbon atoms of N-Me-His and N-Me-Trp
almost fulfills the criterion for a second overlapping ꢀ-turn,
7
7
which is close to type II′ ꢀ-turn geometry, as D-Phe Φ, D-Phe
8
8
7
Ψ, N-Me-Arg Φ, and N-Me-Arg Ψ possess dihedral angles
of 96°, -126°, -135° and 80°, respectively. The overlapping
turns result in a virtually complete helical twist (R-turn) that
5
9
7
extends from residues Asp to N-Me-Trp . Hydrophobic cluster-
9
5
ꢀ
ing of the N-Me-Trp N-methyl group with the Asp H , N-Me-
8
R
10
γ
6
Arg H , N-Me-Lys H atoms and the N-Me-His N-methyl
5
group (indicated by the presence of ROESY cross peaks between
28, as close proximity between the Asp carbonyl oxygen and
9
5
ꢀ
8
R
7
the N-Me-Trp methyl protons and the Asp H , N-Me-Arg H ,
the D-Phe N-methyl group is disfavored. Hence, the N-Me-D-
10
γ
6
7
N-Me-Lys H , N-Me-His N-methyl protons) seems to stabilize
this helical twist. Within the unrestrained 30 ns MD simulation
starting from the structure of the restrained MD, slight changes
occurred in a few backbone dihedral angles as compared to the
Phe substitution would not simply displace a hydrogen bond
donor, but also lead to an altered conformation of the peptide
backbone, which would affect the presentation of the pharma-
cophore and prevent interaction of the aromatic ring with the
third and the sixth transmembrane binding domains aromatic
5
average structure from the restrained MD simulation. Asp Ψ,
6
7
9
76
N-Me-His Ψ, D-Phe Φ and N-Me-Trp Ψ were most affected
and changed from 144° to 113°, 78° to 117°, 96° to 75° and
groups.
Side Chain Structure and Dynamics. Investigation of side
chain conformation about the ꢁ angle requires a careful analysis
1
of homo- and heteronuclear J-couplings as well as the consid-
eration of NOE distances in stereospecifically assigned ꢀ
protons. Extended MD simulations of a solution structure in
explicit solvent can further clarify which structural flips of side
chain dihedral angles are correlated.
The sums of the JHR-Hꢀ coupling constants of the individual
amino acid side chains are in the order of 15 Hz, which excludes
6
3° to 96°, respectively (Table 5). The rmsd between the atoms
5 10
of the peptide backbone from Asp to N-Me-Lys of structures
obtained from the unrestrained and restrained MD simulation
6
8
7
is 1.0 Å. According to the N-Me-His backbone dihedral angles
(
Φ ) -102°, Ψ ) 117°) the inverted γ-turn is less pronounced
in the structure obtained from unrestrained MD simulation.
Upper bounds of some distance restraints within the cyclic core
structure (Asp H -D-Phe H ; Asp H -N-Me-His H ; D-Phe H -
3
5
ꢀ
7
N
5
ꢀ
6
Me
7
N
8
R
8
R
10 Me
9
Me
N-Me-Arg H ; N-Me-Arg H -N-Me-Lys H ; N-Me-Trp H -
(
87) Kessler, H.; Griesinger, C.; Wagner, K. J. Am. Chem. Soc. 1987,
109, 6927–6933.
(
86) Venkatachalam, C. M. Biopolymers 1968, 6, 1425–1436.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010 8121

A R T I C L E S
Doedens et al.
Table 4. Comparison between the Experimentally Derived
Distance Restraints (dlow), (dupp) and Calculated (dMD) Interproton
Distances of Compound 28 as obtained from Restrained MD
Table 5. Φ and Ψ Dihedral Angles of the Average Structure from
the Restrained MD (rMD) and from the Trajectory of the
Unrestrained MD (MD)
a
Calculation
Amino acid
residue
interproton distance
dlow [Å]
dupp [Å]
dMD [Å]
dViol [Å]
Φ
rMD [°]
ΦMD [°]
Ψ
rMD [°]
ΨMD [°]
5
ꢀ
5
R
R
Nle4
Asp
N-Me-His
D-Phe
N-Me-Arg
N-Me-Trp
N-Me-Lys
Asp H proR
Asp H
2.32
2.58
2.08
4.13
2.52
4.73
4.15
3.15
3.43
2.62
3.06
2.77
3.21
2.05
3.71
3.4
2.84
3.15
3.19
5.05
3.68
5.78
5.07
3.85
4.19
3.78
3.74
3.38
4.43
2.51
5.23
4.64
4.62
4.97
4.64
3.09
3.53
4.61
3.22
5.16
4.52
5.34
4.39
3.18
5.87
5.29
5.06
4.59
4.53
4.5
2.629
3.050
2.758
4.635
3.286
5.248
4.608
3.520
4.100
2.716
4.014 +0.27
3.054
3.913
2.390
4.326
4.334
4.500
3.689
3.660
3.053
3.187
4.748 +0.14
2.679
4.752
4.450
4.472
3.872
2.779
5.526
5.034
4.816
4.579
4.760 +0.23
4.184
4.359
4.123
3.806
4.327 +0.25
4.472
5.333 +0.14
3.905
2.996
4.889 +0.06
4.504
5.450
3.964
3.057
4.620
5.457
3.617
4.258 +0.42
3.447
2.532
5.053
5.731 +0.07
3.218
2.597
3.798
4.133
3.897
3.349
3.889
4.116
-101
71
-98
96
-135
-120
-114
-75.5 ( 39.6
-89.7 ( 32.7
-102.4 ( 15.7
75.0 ( 18.8
-122.9 ( 8.0
-136.6 ( 14.0
-120.7 ( 9.9
109
144
78
29.4 ( 82.5
112.7 ( 16.9
117.4 ( 19.2
-116.0 ( 10.1
83.2 ( 9.9
5
ꢀ
5
5
Asp H proS
Asp H
5
R
6
Me
R
6
Asp H
N-Me-His H
N-Me-His H
N-Me-His H
N-Me-His H
N-Me-His H
D-Phe H
D-Phe H
N-Me-Trp H
N-Me-Lys
N-Me-Lys
N-Me-His H
D-Phe H
D-Phe H
D-Phe H
D-Phe H
N-Me-Trp H
N-Me-Trp H
D-Phe H
D-Phe H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Trp H
N-Me-Arg H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
5
ꢀ
6
7
Asp H proR
-126
5
ꢀ
6
Me
R
8
Asp H proR
80
63
0
5
ꢀ
6
9
Asp H proS
96.0 ( 12.7
83.0 ( 63.4
5
R
6
R
10
Asp H
5
ꢀ
7
N
N
Asp H proR
5
ꢀ
7
Asp H proS
5
ꢀ
9
Me
Asp H proR
difference between the two 3JHR-Hꢀ coupling constants together
with a sum of both of about 15 Hz indicate however a preferred
5
R
10
ꢂ
ꢂ
Asp H
H
5
ꢀ
10
Asp H proR
H
5
3
6
Me
R
6
R
(ꢁ
) -60°) conformation for Asp ( JHR-HꢀproS ) 10.7 Hz) and
9 3 6
N-Me-His H
N-Me-His H
N-Me-His H
N-Me-His H
N-Me-His H
N-Me-His H
N-Me-His H
D-Phe H
D-Phe H
D-Phe H
D-Phe H
D-Phe H
D-Phe H
D-Phe H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Arg H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
1
6
7
N
N
N
R
N-Me-Trp ( JHR-HꢀproR ) 11.1 Hz), whereas N-Me-His and
N-Me-Arg with identical JHR-HꢀproR and JHR-HꢀproS coupling
6
ꢀ
7
8
3
3
6
Me
R
7
constants populate the (ꢁ
1
) -60°) and (ꢁ
1
) 180°) conforma-
tions. For D-Phe the difference of the JHR-Hꢀ coupling constants
is small indicating a less pronounced preference of the ꢁ
6
7
3.78
3.24
3.4
7
3
6
Me
Me
9
Me
6
9
δ1
)
1
7
N
ꢀ
7
R
10
3
2.53
2.32
3.77
2.11
3.08
2.65
4.37
3.17
2.08
4.23
3.98
3.34
3.75
3.3
60° over the ꢁ
coupling constants indicate populations of 70 to 30% for the ꢁ
-60° and ꢁ ) 180° rotamers or vice versa. It is not clear,
1
) 180° rotamer. For N-Me-Lys the JHR-Hꢀ
7
7
N
1
7
N
R
ꢀ
8
R
)
1
7
8
Me
ꢀ
7
8
which of both is higher populated as the chemical shifts of the
two ꢀ protons are degenerated. The N-Me-Lys10 H signal is
slightly broadened to a singlet of 18 Hz line width which has
weak shoulders (see Supporting Information) but lacks the
expected double duplet or triplet structure expected from the
coupling to the two δ-Protons when the NMR data is processed
for signal intensity. Optimization of the line width by Gaussian
multiplication of the FID prior to Fourier transformation led to
ꢂ
7
ꢀ
8
Me
7
R
9
ꢀ1
8
Me
8
R
8
R
9
Me
8
ꢀ
9
R
8
Me
Me
R
9
R
8
9
Me
R
8
9
8
Me
Me
Me
Me
Me
Me
R
9
δ1
ꢀ1
ꢀ3
η2
ꢂ2
ꢂ3
3
two similar JHꢀ-Hꢂ coupling constants of 6.5 to 7.0 Hz. The
similar JHR-Hꢀ coupling constants indicate an equilibrium of
8
9
3.27
3.63
3.6
3
8
9
4.9
4.86
4.7
5
8
9
different conformers within the amide bridged Asp and N-Me-
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Lys
N-Me-Lys
10
10
ꢂ
8
9
3.45
2.89
3.32
3.26
3.15
2.23
3.56
3.34
3.63
2.85
2.85
3.78
3.95
2.31
2.57
2.83
2.42
4.33
4.33
2.99
2.57
2.88
2.96
3.22
2.87
2.94
3.43
Lys side chains. The slightly broadened N-Me-Lys H signal
further suggests that the underlying conformers exchange on a
time scale slower than the ns regime what prohibits their
sampling by MD simulation in explicit water.
8
9
4.08
4.56
5.19
4.37
3.35
4.83
4.58
5.59
4.04
4.04
4.62
5.52
4.14
3.84
4.16
4.36
5.66
5.66
5.06
3.85
4.22
4.87
4.44
4.21
4.99
5.38
8
10
Me
H
8
ꢀ
10
Me
H
9
Me
R
9
R
N-Me-Trp H
9
10
Me
R
Dynamics of the side chains that are not involved in
cyclization were further investigated by 30 ns molecular
dynamics simulations in explicit water (Figure 5). For analyzing
the side chain dynamics based on the structure obtained from
the rMD calculation, position restraints were applied on the
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
H
9
Me
Me
Me
Me
Me
R
10
10
10
10
10
10
N
H
H
H
H
H
H
9
ꢂ
9
δ
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
N-Me-Trp H
9
γ1
γ2
R
9
5
9
carbon and nitrogen atoms of the peptide backbone from Asp
N-Me-Lys
Amide
9
R
11
R
10
R
N-Me-Trp H
H
C to N-Me-Lys C . Analysis of the MD simulation performed
with such position restraints revealed that all ꢁ populations
N-Me-Lys Hꢀ
10
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
N-Me-Lys
10
10
10
10
10
10
10
10
10
10
10
H
H
H
H
H
H
H
H
H
H
H
Me
R
1
N-Me-Lys1
0
0
0
0
0
0
0
0
0
0
0
0
H
H
H
H
H
H
H
H
H
H
H
H
H
δ
8
10
except of the ꢁ
1
populations of N-Me-Arg and of N-Me-Lys
N-Me-Lys1
δ
ꢂ
3
N-Me-Lys1
δ
ꢀ
were well consistent with the JHR-Hꢀ coupling constants (Table
6, Figure 5). The strong preference of ꢁ
5
1
δ1
δ2
ꢀ
Me
Me
γ
) -60° for Asp and
N-Me-Lys
1
N-Me-Lys1
N-Me-Lys1
N-Me-Lys1
for N-Me-Trp as well as the evenly populated ꢁ ) -60° and
9
1
4
6
ꢁ
J
1
) 180° rotamers for Nle and N-Me-His are indicated by
γ
R
3
HR-Hꢀ coupling constants and well reproduced by the MD
1
γ
ꢂ
N-Me-Lys
7
N-Me-Lys1
γ
Me
R
simulation. For D-Phe , the preference of the ꢁ
1
) 60° rotamer
1
Me
R
with respect to the ꢁ
) 180° rotamer, which is indicated by
N-Me-Lys
N-Me-Lys
1
N-Me-Lys1
Amide HN
11
3
the JHR-Hꢀ coupling constants, is not reflected by the MD
trajectory, which suggests similar populations of the two
rotamers.
1
ꢀ
11
11
N
N
N-Me-Lys
Amide
Amide
H
H
N-Me-Lys10
Me
a
As indicated by the MD simulation, by the ROEs and by the
upfield or downfield chemical shifts, hydrophobic clustering is
crucial for the different side chain conformations of peptide 28
in aqueous solution. Stacking of the N-Me-His and D-Phe side
chains, that was also reported in other structural investigations
of R-MSH analogs is well observed within the MD trajectory
Violations of upper bounds (positive sign) and of lower bounds
(
negative sign) are given in the last column (dViol).
6
7
higher populations of the ꢁ
1
) 60° conformation (gauche+)
) -60° conforma-
tion (gauche-) for the D-Phe residue (Table 6). A strong
for the L amino acid residues, and of the ꢁ
1
7
8
122 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010

Melanocortin Receptor Subtype hMC1R Selectivity
A R T I C L E S
R
Figure 3. H regions of 1H NMR spectra of peptide 28 (A and B) and MT-II (C and D). Spectra A and C were detected in 50 mM sodium acetate-d
4
D
2
O
R
buffer (pH 4.5), B and D were detected in DMSO-d
6
. Numbers refer to H atoms of the respective residues in the R-MSH sequence.
Table 6. 3
from E.COSY
J
HR-Hꢀ Coupling Constants as Experimentally Determined
a
3
JHR-Hꢀ [Hz]
p(ꢁ
1
) -60°) p(ꢁ
[%]
1
) 180°) p(ꢁ
[%]
1
) 60°)
[%]
HR-Hꢀ proR HR-Hꢀ proS
Nle4
Asp
N-Me-His
D-Phe
N-Me-Arg
N-Me-Trp
N-Me-Lys
8.2; 6.2
84
74
48
14
40
16
15
4
61
20
3
5
3.8
7.9
5.3
7.0
11.1
10.7
7.9
9.2
7.0
4.7
11
48
25
40
19
6
7
8
9
78
1
0
9.5, 6
100
0
a
1
The according ꢁ populations were derived by a linear combination
of 3JHR-Hꢀ(ap) ) 12 Hz, JHR-Hꢀ(ga) ) 3.5 Hz.
3
Me
indolyl protons as well as by a strongly upfield shifted H
6
resonance at 1.559 ppm. ROEs between the N-Me-His methyl
protons and the N-Me-Trp δ1 and ꢀ1 protons as well as between
D-Phe H and N-Me-Trp H are well consistent with the
9
7
R
9
ꢀ1
orientation of the indolyl ring given in Figure 4A and B. An
6
additional ROE between the N-Me-His methyl protons and the
9
N-Me-Trp ꢀ3 proton indicates another orientation of the indolyl
Figure 4. Stereoviews of the solution structure of peptide 28 as determined
by NMR spectroscopy and MD calculations. The conformers shown in A
and B possess the same cyclic core structure. The different side chain
conformations of N-Me-His , D-Phe , and N-Me-Arg in A and B,
respectively, reflect dynamics in ꢁ dihedral angles observed within the MD.
group that was not sampled during 30 ns unrestrained MD
simulation. In this orientation the indolyl group also seems to
stack on top of the N-Me-Arg N-methyl group as shown in
Figure 4 but with the six membered ring of the indolyl group
pointing to the left and the five membered ring pointing to the
right.
A dispersion of chemical shifts of peptide 28 in DMSO-d6
(H : 4.245 to 5.824 ppm (Figure 3), H : 1.893 to 3.058 ppm),
which is similar to the dispersion in the aqueous buffer (H :
4.231 to 6.021 ppm (Figure 3), H : 1.559 to 3.115 ppm),
indicates that hydrophobic interactions observed in aqueous
buffer are also present in the slightly more hydrophobic DMSO.
This suggests that such hydrophobic interactions might also be
present in hMC1R bound state.
In addition it is often found that stronger conformational
preference which is accompanied by stronger biological activity
6
7
8
8
and is also present in the exemplary conformation shown in
7
Figure 4A. Hydrophobic contacts between the D-Phe and N-Me-
8
R
Me
Arg side chains that agree with strong upfield shifts of the
8
R
N-Me-Arg ꢀ and γ protons are also observed within the MD
Me
trajectory and depicted in the conformation shown in Figure
3
4
B. Overall side chain dynamics as indicated by JHR-Hꢀ coupling
6
constants, is reflected by the rotamers shown for N-Me-His ,
D-Phe and N-Me-Arg in Figure 4A and B, respectively. The
clustering of the N-Me-Trp indolyl ring with the N-methyl
group of N-Me-Arg , which is observed in Figures 4A andB, is
7
8
9
8
confirmed by ROE contacts between methyl protons and all
J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010 8123

A R T I C L E S
Doedens et al.
side chain of compound 28 is pointing down from the peptide
9
plane and the N-Me-Trp side chain of compound 28 is pointing
8
up. This provides further evidence that positively charged Arg
is critical for hMC3R and hMC4R binding and functional
7
6,89,90
activities both for the ligand and for the receptor.
Our
earlier chimeric and multiple receptor mutation work demon-
8
strated that the positive charged Arg plays an important role
1
22
in binding to the third transmembrane domain using the Asp
and Asp1 residues of the hMC3R and hMC4R to achieve
binding and functional activities. Multiple N-methylation in-
creases the hydrophobicity of the ligand and further reduces
26
1
22
the ability of the guanidyl group to interact with the Asp
,
Asp1 to activate the hMCRs, leading to a loss in the binding
and functional activity use. These strong structural differences
suggest that the side chain presentation required for MCR
activity varies strongly for the individual receptor subtypes.
26
76
8
8,89
Inherent MT-II flexibility,
seems to be high enough for the
adaptation to the binding regions of the individual receptor
subtypes, whereas 28 possesses a rigid peptide backbone that
hinders the pharmacophore of MT-II from interacting properly
with the binding pockets of the hMC3R, hMC4R and hMC5R
therefore leading to hMC1R selectivity.
Backbone dihedral angles of 96°, -126°, -135° and 80° for
D-Phe7 Φ, D-Phe7 Ψ, N-Me-Arg8 Φ, and N-Me-Arg Ψ,
respectively, resemble type II′ ꢀ-turn positions i+1 and i+2.
This directed our attention on obvious similarities between
8
91-93
peptide 28 and potent somatostatin analogs,
which possess
a type II′ ꢀ-turn with an aromatic D amino acid residue in
position i+1 and a positively charged L amino acid residue in
6
position i+2. Both bind selectively to distinct GPCR subtypes
of individual GPCR families (peptide 28 to hMC1R, soma-
tostatin to sst2 and sst5 of the SRIF receptors). Hydrophobic
interactions between aromatic side chains stabilize the turn
structure of somatostatin analogs, while hydrophobic interactions
9
5
10
of the N-Me-Trp methyl group with Asp and N-Me-Lys side
chain protons are present in peptide 28. These similarities in
peptide structure and receptor selectivity suggest further simi-
larities between R-MSH analogs and somatostatin analogs with
respect to their receptor subtype preference. For somatostatin
Figure 5. ꢁ dihedral angles as observed during the unrestrained 30 ns MD
simulation. The column number (from the left to the right) corresponds to
the ꢁ dihedral angle positions within the side chains.
γ
the empirical correlation of Lys H upfield shifts with the
potency of the ligands have been extensively used to predict
activities. Possibly, comparable correlations between Arg H
upfield shifts and potency could be found in R-MSH analogs
as well.
indicates a closer similarity to the bioactive conformation. To
our experience these effects seem to be stronger than calculated
from populations of conformations. Maybe this can be attributed
to the importance of entropic effects in the binding process.
Comparison with Structures of Other Peptidic GPCR
Effectors. According to our knowledge, no inverted γ-turn
93
γ
DG Calculations. DG calculations resulted in a very homo-
geneous ensemble of compound 28 conformers that were in
good agreement with the distance restraints and very similar to
the structure shown in Figure 4. The best 30 out of 50 calculated
structures possessed an rmsd of 0.27 ( 0.10 Å with respect to
6
centered at His has been described so far in structures of similar,
but not N-methylated R-MSH analogs, like MT-II. ꢀ-turns
5
8
ranging from Asp to Arg have more frequently been described
5
the backbone nitrogen and carbon atoms of residues Asp to
6
9
than ꢀ-turns ranging from His to Trp which agrees with the
structure of 28 to such a degree as the distance between the
10
3
N-Me-Lys . Although J coupling constants and broadening
10
ꢂ
of the N-Me-Lys H signal indicated dynamics in the amide
5
8
Asp and N-Me-Arg R carbon atoms (6.4 Å) is smaller than
linked side chains, the structures obtained from DG possessed
6
9
the distance between the N-Me-His to N-Me-Trp R carbon
atoms (7.6 Å). In spite of certain similarities with respect to
8
8
(
89) Fr a¨ ndberg, P.-A.; Xu, X.; Chhajlani, V. Biochem. Biophy. Res.
Commun. 1997, 236, 489–492.
the occurrence of ꢀ-turns, the presentation of the side chains of
8
9
residues N-Me-Arg and N-Me-Trp differs strongly from MT-
(90) Grieco, P.; Balse, P. M.; Weinberg, D.; MacNeil, T.; Hruby, V. J.
8
J. Med. Chem. 2000, 43, 4998–5002.
II. A structure investigation of MT-II suggested that the Arg
(
91) Veber, D. F.; Holly, F. W.; Paleveda, W. J.; Nutt, R. F.; Bergstrand,
S. J.; Torchiana, M.; Glitzer, M. S.; Saperstein, R.; Hirschmann, R.
Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 2636–2640.
side chain is pointing up from the peptide plane and the Trp9
side chain is below the peptide plane, when the backbone is
oriented as shown in Figure 4. In contrast, the N-Me-Arg8
8
8
(92) Sukopp, M.; Schwab, R.; Marinelli, L.; Biron, E.; Heller, M.;
Varkondi, E.; Pap, A.; Novellino, E.; Keri, G.; Kessler, H. J. Med.
Chem. 2005, 48, 2916–2926.
(
88) Ying, J.; Kover, K. E.; Gu, X.; Han, G.; Trivedi, D. B.; Kavarana,
(93) Arison, B. H.; Hirschmann, R.; Veber, D. F. Bioorg. Chem. 1978, 7,
447–451.
M. J.; Hruby, V. J. Biopolymers 2003, 71, 696–716.
8
124 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010

Melanocortin Receptor Subtype hMC1R Selectivity
A R T I C L E S
1
0
very similar homogeneous N-Me-Lys ꢁ
) -55.2° +/- 2.01 Å, ꢁ ) -109° +/- 3.73°). The
observation of only one preferred ꢁ dihedral angle did not
reflect the JHR-Hꢀ coupling constants (Table 6) and the only
populated eclipsed ꢁ rotamer was thermodynamically unfavor-
able. Apparently, conformation averaging in the N-Me-Lys
side chain led to a number of conformation averaged distance
1
and ꢁ
2
dihedral angles
in strong receptor subtype selectivity with retained or even
improved activity for an individual receptor subtype. This
(
ꢁ
1
2
94,95
1
approach, discussed by us already a quarter of a century ago
is not achievable only by cyclization but can be further increased
3
4
8
2
by multiple N-methylation. With an increasing extent of
introduction of N-methyl groups into its backbone, MT-II
derivatives lose their ability to bind to particular hMCRs out
of the total family of melanocortin receptors. This leads in two
cases (peptides 24 and 28) to highly selective and potent agonists
for the hMC1R, but also to antagonists and compounds with
both, agonist and antagonist activities. These compounds possess
multiple amide bonds that are N-methylated which was recently
10
1
0
restraints that forced the lactam bridged N-Me-Lys side chain
into conformations during DG calculations that could not be
highly populated at equilibrium.
Restrained Molecular Dynamics Calculations. The best DG
structure in respect of violations of experimental distance
restraints was subjected to refinement by restrained molecular
dynamics (rMD) calculation in explicit water. The average
structure of the rMD calculation was very similar to the DG
starting structure (rmsd ) 0.69 Å with respect to backbone
5
reported to be a possible key toward bioavailability and maybe
6
also enhanced oral bioavailability of peptides. Whereas the
effect of N-methylation on receptor subtype selectivity is
strongly supported by our results, the effect on pharmacological
5
10
4–6
heavy atoms from Asp to N-Me-Lys ). Within the cyclic core
structure significant changes occurred only in the N-Me-Lys10
side chain, that was strained in the starting conformer as side
properties that have been improved in other examples has to
be demonstrated for the N-methylated MT-II derivatives. These
investigations are in progress.
chain dihedral angles were eclipsed (ꢁ
137°). As expected, the force field applied during restrained
MD calculation rotated the dihedral angles within the N-Me-
2
) -111° and ꢁ
3
)
-
Experimental Section
R
Materials. N -Fmoc-amino acids, peptide coupling reagents,
1
0
Lys side chain to thermodynamically feasible values (ꢁ
169° and ꢁ ) 163°).
Unrestrained Molecular Dynamics Calculations. The average
2
)
Rink amide MBHA resin and solvents were reagent grade and used
without further purification unless otherwise noted. These chemicals
were obtained from Aldrich, Novabiochem, Iris Biotech GmbH,
Merck, NeoMPS, ORPEGEN Pharma and GLS. The following
amino acids were used: Fmoc-Lys(Alloc)-OH, Fmoc-Trp(Boc)-OH,
Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe-OH, Fmoc-His(Trt)-OH, Fmoc-
Asp(OAllyl)-OH and Fmoc-Nle-OH. The polypropylene reaction
vessels (syringes with frits) were purchased from B. Braun
Melsungen AG. The purity of the peptides was checked by
analytical reverse-phase HPLC using an Amersham Pharmacia
-
3
structure obtained from the rMD calculation was further refined
by extended (30 ns) unrestrained MD simulation in explicit
water. This lead to significant changes of some backbone
dihedral angles (Table 5) that were accompanied by further
conformational changes in the lactam bridged N-Me-Lys10 side
chain. The changes of backbone dihedral angles led to significant
3
violations of distance restraints and most
constants were not consistent with the ꢁ
JHR-Hꢀ coupling
¨
Biotech Akta Basic 10F with a ODS-A C18 (120 Å, 5 µm, 250
1
populations extracted
mm ×4.6 mm) column (Omnicrom YMC Europe GmbH) moni-
tored at 220 and 254 nm and by high resolution mass spectral
analysis.
3
from the MD trajectory. Consistency with JHR-Hꢀ coupling
constants and with distance restraints was much higher in a
trajectory of an MD simulation in which the conformation of
the peptide backbone obtained from rMD was conserved by
applying position restraints on all backbone carbon and nitrogen
Synthesis. The MT-II analogues were synthesized manually by
the Fmoc-SPPS using methods described in refs 96 and 97.
Rink amide MBHA resin (0.3 g, 4-(2′,4′-dimethoxyphenyl-Fmoc-
aminomethyl)-phenoxyacetamido-norleucyl-MBHA resin) in a 10
mL syringe with a frit on the bottom was swollen in NMP (3 mL)
for 10 min. The Fmoc protecting group on the Rink linker was
removed by 20% piperidine in NMP (1 × 8 min, 1 × 12 min).
5
R
10
R
atoms from Asp C to N-Me-Lys C .
We believe that the artificial strains in the lactam bridged
side chains, that were described above, induced the conforma-
tional changes that were observed in the peptide backbone
during 30 ns non position restrained MD simulation. The slight
variations in Φ and Ψ dihedral angles seemed to be energetically
less demanding than flips between individual staggered con-
formations within the N-Me-Lys1 side chain. Apparently, the
backbone compensated for the strains within the lactam bridged
side chains during the unrestrained MD simulation and position
restraining of the backbone during the unrestrained MD simula-
tion seems to be a legitimate way to avoid faulty changes of
backbone dihedral angles.
R
The resin was washed with NMP (5 × 3 mL). The first N -Fmoc-
R
amino acid was coupled using preactivated ester (3 equiv N -Fmoc-
amino acid, 3 equiv TBTU, 3 equiv HOBt and 8 equiv DIEA) in
NMP. The coupling mixture was transferred into the syringe with
the resin and shaken for 3 h. Afterward the resin was washed with
NMP (5 × 3 mL). Complete coupling was confirmed by HPLC
monitoring. Therefore the resin was washed with DCM (2 × 3
mL). A small amount of resin was taken out of the syringe and
three drops of a mixture of trifluoracetic acid/triethylsilane/water
0
(
95:2.5:2.5, v/v/v) was added. After 10 min, acetonitrile and water
were added, and after filtration, HPLC and ESI-MS showed
complete coupling. The peptide sequences were continued by
consecutively coupling of the particular amino acids. N-Methylation
was carried out after coupling of the corresponding amino acid.
Conclusions
Multiple N-methylation in MT-II led to several derivatives
in which the affinity and activity for hMC1R could be retained,
but simultaneously the binding affinity to the other 3 melano-
cortin receptors was completely lost. Hence, selectivity and
retained activity was achieved in this way. A careful analysis
of the conformation of the most active (13 nM) compound
reveals a strong preference of one backbone conformation,
which indicates a strongly reduced conformational space in
comparison to the nonmethylated MT-II. Hence, this work again
shows that restriction of peptide backbone conformation resulted
R
This procedure includes three steps. First, the N-terminal N -Fmoc-
deprotected amino acid was protected as an N -o-nitrobenzene
sulfonamide by reaction with 5 equiv of o-nitrobenzenesulfonyl
chloride (o-NBS) and 10 equiv of collidine in NMP (15 min). The
R
(94) Kessler, H. Angew. Chem., Int. Ed. 1982, 21, 512–523.
(
(
95) Hruby, V. J. Life Sci. 1982, 31, 189–199.
96) Mayorov, A. V.; Han, S. Y.; Cai, M.; Hammer, M. R.; Trivedi, D.;
Hruby, V. J. Chem. Biol. Drug. Des. 2006, 67, 329–335.
(97) Heckmann, D.; Kessler, H. Methods Enzymol. 2007, 426, 463–503.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010 8125

A R T I C L E S
Doedens et al.
9
8
methylation was done under Mitsunobu conditions with 5 equiv
of triphenylphosphine, 10 equiv of methanol and 5 equiv of
diisopropyl azodicarboxylate (DIAD) in THF (10 min). Then the
o-NBS group was removed with 10 equiv of 2-mercaptoethanol
and 5 equiv of DBU in NMP (5 min). This deproctection procedure
was repeated once. For the coupling of the next amino acid, 3 equiv
HATU, 3 equiv HOAt and 8 equiv DIEA were used. After coupling
of the last amino acid Fmoc-Nle-OH and Fmoc deprotection as
described above, acetylation was achieved by treatment of the DCM
washed resin (3 × 3 mL) with acetic anhydride/DCM/DIEA (25:
200 mg/L bacitracin. The assay medium was subsequently
removed, the cells were washed once with basic medium, and
then lysed by the addition of 100 µL of 0.1 M NaOH and 100
µL of 1% Triton X-100. The lysed cells were transferred to 12
×
75 mm borosilicate glass tubes, and the radioactivity was
measured by a Wallac 1470 WIZARD Gamma Counter. The
results are shown in Table 1.
Adenylate Cyclase Assay. HEK 293 cells transfected with
1
03
human melanocortin receptors were grown to confluence in
MEM medium (GIBCO) containing 10% fetal bovine serum,
100 units/mL penicillin and streptomycin, and 1 mM sodium
pyruvate. The cells were seeded on 96-well plates 48 h before
assay (50 000 cells/well). For the assay, the cell culture medium
was removed and the cells were rinsed with 100 µL of MEM
buffer (GIBCO). An aliquot (100 µL) of the Earle’s balanced
salt solution with 5 nM isobutylmethylxanthine (IBMX) was
placed in each well along for 1 min at 37 °C. Next, aliquots
(25 µL) of melanotropin peptides of varying concentration were
added, and the cells were incubated for 3 min at 37 °C. The
reaction was stopped by aspirating the assay buffer and adding
60 µL ice-cold Tris/EDTA buffer to each well, then placing
the plates in a boiling water bath for 7 min. The cell lysates
were then centrifuged for 10 min at 2,300g. A 50 µL aliquot of
the supernatant was transferred to another 96-well plate and
7
5:2.5, v/v/v, 3 mL) for 30 min and washing of the resin with DCM
ε
(
5 × 3 mL). The next step was the removal of the N -Alloc group
9
6,97,99
of Lys and the ꢀ-allyl group of Asp.
For this, a solution of
3
PhSiH (24 equiv) in 1 mL DCM was added to the peptide resins.
Then a solution of Pd(PPh ) (0.25 equiv) in 2 mL DCM was added
3 4
and the reaction allowed to proceed for 30 min. The peptide resins
were washed with DCM (3 × 3 mL), NMP (2 × 3 mL), afterward
twice with 0.5% DIEA in DMF (v/v) and 0.5% sodium dieth-
yldithiocarbamate trihydrate (DEDTC) in DMF (w/w) to remove
1
00
any remaining traces of the Pd catalyst, followed by NMP (5 ×
3
mL). The macrocyclic lactam ring formation was then mediated
by addition of TBTU (3 equiv), HOBt (3 equiv) and DIEA (8 equiv)
for 3 h. The cyclized peptides were cleaved off solid support using
3
3
mL trifluoracetic acid/triethylsilane/water (95:2.5:2.5, v/v/v, 1 ×
h, 1 × 15 min), and the crude peptides were precipitated by
dropping the solution in chilled diethyl ether to give white to light
yellow precipitates. The resulting peptide suspensions were cen-
trifuged for 5 min at 4000 rpm, and the liquid was decanted. The
crude peptides were washed with chilled diethyl ether (2 × 30 mL),
and after the final centrifugation, dried under vacuum overnight.
Purification was performed by HPLC using a ODS-A C18 (120
Å´, 5 µm, 250 mm ×20 mm) column (Omnicrom YMC Europe
GmbH) eluting with a linear gradient of acetonitrile and water
containing 0.1% TFA. The products were obtained by lyophilization
of the appropriate fractions after removal of the acetonitrile by rotary
evaporation. Analysis by analytical HPLC and HPLC-MS (Sup-
porting Information) showed the peptides to be pure (>95%). The
purified peptides were isolated in 2-14% overall yields.
3
placed with 50 µL [ H] cAMP and 100 µL protein kinase A
(
PKA) buffer in an ice bath for 2-3 h. The PKA buffer
consisted of Tris/EDTA buffer with 60 µg/mL PKA and 0.1%
bovine serum albumin by weight. The incubation mixture was
filtered through 1.0 µm glass fiber filters in MultiScreen-FB 96-
3
well plates (Millipore, Billerica, MA). The total [ H] cAMP
was measured by a Wallac MicroBeta TriLux 1450 LSC and
Luminescence Counter (PerkinElmer Life Science, Boston,
MA). The cAMP accumulation data for each peptide analogue
was determined with the help of a cAMP standard curve
generated by the same method as described above. IC50 and
EC50 values represent the mean of two experiments performed
in triplicate. IC50 and EC50 estimates and their associated
standard errors were determined by fitting the data using a
nonlinear least-squares analysis, with the help of GraphPad
Prism 4 (GraphPad Software, San Diego, CA). The maximal
cAMP produced at 10 µM concentration of each ligand was
compared to the amount of cAMP produced at 10 µM
concentration of the standard agonist MT-II, and is expressed
in percent (as % max effect) in the Table 2. The antagonist
properties of the lead compounds were evaluated by their ability
to competitively displace the MT-II agonist in a dose-dependent
manner, at up to 10 µM.
Biological Activity Assays
Receptor Binding Assay. Competition binding experiments
were carried out using whole HEK293 cells stably expressing
human MC1, MC3, MC4, and MC5 receptors as described
1
01-103
101–103
before.
HEK293 cells transfected with hMCRs
were seeded on 96-well plates 48 h before assay (50,000 cells/
well). For the assay, the cell culture medium was aspirated and
the cells were washed once with a freshly prepared MEM buffer
containing 100% minimum essential medium with Earle’s salt
MEM, GIBCO), and 25 mM sodium bicarbonate. Next, the
cells were incubated for 40 min at 37 °C with different
concentrations of unlabeled peptide and labeled [ I]-[Nle ,D-
Phe ]-R-MSH (Perkin-Elmer Life Science, 20 000 cpm/well,
3.06 pM) diluted in a 125 µL of freshly prepared binding buffer
containing 100% MEM, 25 mM HEPES (pH 7.4), 0.2% bovine
serum albumin, 1 mM 1,10-phenanthrolone, 0.5 mg/L leupeptin,
(
1
25
4
Data Analysis. IC50 and EC50 values represent the mean of
two experiments performed in triplicate. IC50 and EC50 estimates
and their associated standard errors were determined by fitting
the data using a nonlinear least-squares analysis, with the help
of GraphPad Prism 4 (GraphPad Software, San Diego, CA).
NMR Spectroscopy. DQF-COSY, E.COSY, TOCSY, ROE-
SY, 13C-HMBC and C-HSQC spectra were recorded at 298
K on a Bruker DMX spectrometer operating at 600 MHz. A
7
3
13
(
(
98) Mitsunobu, O. Synthesis 1981, 1, 1–28.
99) Thieriet, N.; Alsina, J.; Giralt, E.; Guibe, F.; Albericio, F. Tetrahedron
Lett. 1997, 38, 7275–7278.
104
COLOC spectrum was recorded at 298 K on a Bruker Avance
(
100) Mayorov, A. V.; Cai, M.; Chandler, K. B.; Petrov, R. R.; VanScoy,
III spectrometer operating at 600 MHz. A phase sensitive
HMBC and a reference HSQC spectrum with offset and rf-
A. R.; Yu, Z.; Tanaka, D. K.; Trivedi, D.; Hruby, V. J. J. Med. Chem.
2
006, 49, 1946–1952.
(
(
(
101) Cai, M.; Mayorov, A. V.; Ying, J.; Stankova, M.; Trivedi, D.; Cabello,
105,106
107,108
amplitude-compensated BEBOP
and BIBOP pulses
C.; Hruby, V. J. Peptides 2005, 26, 1481–1485.
102) Cai, M.; Mayorov, A. V.; Cabello, C.; Stankova, M.; Trivedi, D.;
Hruby, V. J. J. Med. Chem. 2005, 48, 1839–1848.
(104) Kessler, H.; Griesinger, C.; Zarbock, J.; Loosli, H.-R. J. Magn. Reson.
1984, 57, 331–336.
103) Cai, M.; Cai, C.; Mayorov, A. V.; Xiong, C.; Cabello, C. M.;
Soloshonok, V. A.; Swift, J. R.; Trivedi, D.; Hruby, V. J. J. Pept.
Res. 2004, 63, 116–131.
(105) Skinner, T. E.; Reiss, T. O.; Luy, B.; Khaneja, N.; Glaser, S. J. J.
Magn. Reson. 2003, 163, 8–15.
8
126 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010

Melanocortin Receptor Subtype hMC1R Selectivity
A R T I C L E S
were detected at 298K on a Bruker Avance III spectrometer
derived distance restraints were used for structure calculations,
1
09,110
5
operating at 750 MHz.
Samples were prepared in 50 mM
O, 0.05% NaN ) at
with restraints between the lactam bridged residues Asp and
N-Me-Lys10 counted as sequential.
Sodium acetate-d buffer (pH 4.5, 10% D
4
2
3
concentrations of 12-38 mM. This low pH was chosen to keep
hydrogen exchange induced line broadening small and to
enhance comparability with numerous earlier structural studies
on MCR effectors that were performed under the same condi-
3JHN-HR coupling constants were determined from 1D 1H NMR
3
spectra, JHR-Hꢀ coupling constants from E.COSY and hetero-
3
nuclear JC-H coupling constants from HMBC and reference
109,110,114,115
HSQC spectra.
1
tions. Sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 ( H at
Structure Calculations for 28. The structural NMR refinement
protocol included distance geometry (DG) calculations, energy
minimizations, and molecular dynamics (MD) simulations. The
DG program DISGEO was used to generate structures consistent
0.000 ppm) was used as internal standard. Data were processed
with Topspin 1.3 software from Bruker. The homo- and
heteronuclear experiments DQF-COSY, E.COSY, TOCSY,
13
ROESY, and magnitude mode C-HMBC were performed with
1
16,117
1
13
with the 63 distance restraints derived from the ROEs.
a spectral width of 11 ppm for H and 180 ppm for C.
1
3
The DG procedure started with the embedding of 50 structures
using random metrization. The structures obtained from DG
were evaluated according to the lowest total restraint violations
and additionally counterchecked for close contacts of protons
that did not show corresponding ROESY crosspeaks. Among
30 structures that were in best accordance with experimentally
derived restraints, all had identical peptide backbone conforma-
tions.
Individual HSQC spectra covering aliphatic ( C offset ) 35
1
3
ppm, spectral width ) 50 ppm) and aromatic C resonances
13
(
C offset ) 120 ppm, spectral width ) 30 ppm) were detected.
The phase sensitive HMBC spectrum and the reference HSQC
1
spectrum were detected with a spectral width of 9 ppm for H
and 190 ppm for 1 C. A COLOC spectrum was detected with
3
1
13
spectral widths of 9.5 ppm for H and 190 ppm for C. The
increments in t1 and t2 were adjusted to the information
extracted from the individual experiments, ranging from 384
to 2048 increments in t1 and from 4096 to 16 384 complex data
points in t2. Depending on the sample concentration and the
individual experiments, 16 to 48 transients were averaged for
each t1 value. A mixing time of 80 ms was used for TOCSY
Restrained MD calculations (rMD) were carried out employ-
ing the module DISCOVER of the INSIGHT II 2001 program
(Biosym/MSI Inc.) with the CVFF force field. The ROE
-
1
restraints were included with a force constant of 10 kcal mol
-2
Å . The calculations were done with the explicit-image model
of periodic boundary conditions. The best structure resulting
from the DG calculation was placed in a cubic box of length
30 Å and soaked with water. After energy minimization using
steepest descent and conjugate gradient, the system was heated
gradually starting from 10 K and increasing to 50, 100, 150,
(
spin lock field: 6 kHz; mixing sequence MLEV-17). Water
signal suppression was achieved by WATERGATE tech-
1
11
niques. The sequential assignment was obtained from het-
eronuclear J correlations that were extracted from HSQC and
HMBC spectra. A compensated ROESY experiment, which was
used for the extraction of inter proton distances, was performed
2
00, 250 and 300 K in 1 ps steps, each by direct scaling of
1
12
with 150 ms mixing time and a spin lock field of 4000 kHz.
velocities. The system was equilibrated for 50 ps with temper-
ature bath coupling. Coordinates were saved every 100 fs for
another 150 ps. The average structure of the 150 ps rMD
calculation was subjected to energy minimization and further
investigated by unrestrained MD simulations.
The volume integrals of the individually assigned cross-peaks
were compensated for offset effects and converted into distance
1
13
constraints using the isolated spin pair approximation.
To
compensate for watergate solvent signal suppression artifacts,
of any two cross peaks representing an internuclear distance,
the peak with the higher water resonance offset in the direct
dimension was preferably considered for distance calculations.
The ROESY cross-peak volumes were calibrated against the
The GROMACS 4.0 software package (www.gromacs.
118-120
org)
was used to perform unrestrained MD calculations.
Visualization of the simulation trajectories was performed using
121
122
the software packages VMD and SYBYL 8.0. The scripts
g_cluster, g_dist and g_angle, that were used for analysis of
the MD trajectory, were all packaged with GROMACS. The
10
ꢀ
distance (1.78 Å) between the prochiral N-Me-Lys H protons.
Upper and lower distance limits were set to plus and minus
1
0% of the calculated distances, respectively. For distance
5
3a6 united atom (CH, CH
single atom) forcefield, one of the GROMOS96 force fields,
was used for the molecular dynamic simulations. Rigid SPC
2 3
and CH groups represented as a
restraints referring to nonstereospecifically assigned methylene
protons, another 0.7 Å was added on upper bounds as pseudoa-
toms and multiplicity corrections. For distance restraints refer-
ring to methyl protons, subtraction of 10% for the lower bounds
was omitted and another 0.9 Å was added on upper bounds as
pseudoatoms and multiplicity corrections. 18 intraresidual, 36
sequential interresidual and 9 nonsequential interresidual ROE
1
23
124
water which was constrained using SETTLE served as water
model. Solute bonds were constrained by the SHAKE algo-
(114) Eberstadt, M.; Gemmecker, G.; Mierke, D. F.; Kessler, H. Angew.
Chem., Int. Ed. 1995, 34, 1671–1695.
(
115) Richardson, J. M.; Titman, J. J.; Keeler, J.; Neuhaus, D. J. Magn.
Reson. 1991, 93, 533–553.
(
(
(
(
106) Skinner, T. E.; Reiss, T. O.; Luy, B.; Khaneja, N.; Glaser, S. J. J.
Magn. Reson. 2004, 167, 68–74.
(116) Mierke, D. F.; Kessler, H. Biopolymers 1993, 33, 1003–1017.
(117) Havel, T. F. Prog. Biophys. Mol. Biol. 1991, 56, 43–78.
(118) Lindahl, E.; Hess, B.; van der Spoel, D. J. Mol. Model. 2001, 7,
306–317.
107) Kobzar, K.; Skinner, T. E.; Khaneja, N.; Glaser, S. J.; Luy, B. J.
Magn. Reson. 2004, 170, 236–243.
108) Luy, B.; Kobzar, K.; Skinner, T. E.; Khaneja, N.; Glaser, S. J. J.
Magn. Reson. 2005, 176, 179–186.
(119) van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A. E.;
Berendsen, H. J. C. J. Comput. Chem. 2005, 26, 1701–1718.
(120) van der Spoel, D.; Lindahl, E.; Hess, B.; van Buuren, A. R.; Apol,
P. J.; Meulenhoff, P. J.; Tieleman, D. P.; Sijbers, A. L. T. M.;
Feenstra, K. A.; van Drunen, R.; Berendsen, H. J. C. Gromacs User
Manual Version 4.0, 2005; http://www.gromacs.org.
109) Verdier, L.; Sakhaii, P.; Zweckstetter, M.; Griesinger, C. J. Magn.
Reson. 2003, 163, 353–359.
(
(
110) Kobzar, K.; Luy, B. J. Magn. Reson. 2007, 186, 131–141.
111) Piotto, M.; Saudek, V.; Sklen a´ r, V. J. Biomol. NMR 1992, 2, 661–
6
65.
(
(
112) Griesinger, C.; Ernst, R. J. Magn. Reson. 1987, 75, 261–271.
(121) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14
(33-38), 27–38.
113) Kumar, A.; Wagner, G.; Ernst, R.; Wuethrich, K. J. Am. Chem. Soc.
1
981, 103, 3654–3658.
(122) SYBYL 7.3; Tripos International: St. Louis, MO, 2006.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010 8127

A R T I C L E S
Doedens et al.
1
25
rithm and temperature and pressure control was executed by
at 298 K. The final 30 ns MD simulation was carried out at
298 K. Coordinates were saved every 10 ps. For the MD
simulation with strict conservation of the peptide backbone
obtained from the restrained MD simulation, position restraints
1
26
Berendsen coupling.
Periodic boundary conditions were
employed on a octahedral simulation box, which was built with
a distance of 1.4 nm for the solute. Cut off distances of 1.4 nm
for electrostatic and Lennard-Jones nonbonding interactions were
applied. Simulation time steps were set to 2 fs. Upon addition
of two acetate counterions and soaking of the box with water,
the system was equilibrated by an initial minimization and
subsequent 50 ps MD simulations at 50, 100, 150, 200, 250,
and 298 K using position restraints. Within the individual MD
steps, the temperature was gradually increased, while the force
constants of the position restraints were decreased exponentially
-
1
-2
of 250 000 KJmol nm were applied on all backbone carbon
5
R
10
R
and nitrogen atoms between Asp C and N-Me-Lys C .
Acknowledgment. These studies were supported in parts by
grants from the U.S. Public Health Service, National Institutes of
Health, DK017420 and DA06284. We also thank the Humboldt
Foundation for the support via the Max-Planck Award, the
Deutschen Forschungsgemeinschaft (DFG) for financial support and
Prof. Eberle in Basel for initial measurements of activities. J.G.B.
thanks Oliver Demmer for help with GROMACS and Dr. Burkhard
Luy for help with HMBC and reference HSQC spectra.
-1
-2
-1
-2
from 250 000 KJmol nm at 50 K to 25 KJmol nm at 250
K. At 298 K no position restraints were applied. For adjacent
pressure equilibration a 100 ps MD simulation was performed
(
123) van Gunsteren, W. F.; Billeter, S. R.; Eising, A. A.; Hunenberger,
P. H.; Krueger, P.; Mark, A. E.; Scott, W. R. P.; Tironi, I. G. in
Biomolecular simulation: the GROMOS96 manual and user guide,
Supporting Information Available: Table listing sequences
and physicochemical properties of the N-methylated MT-II
1
analogs, HPLC chromatograms, H NMR spectra, and f2 slice
1
st ed., Hochschulverlag AG an der ETH Z u¨ rich, Z u¨ rich, 1996.
124) Miyamoto, S.; Kollman, P. A. J. Comput. Chem. 1992, 13, 952–
62.
125) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys.
1
0
ꢂ
5
(
(
(
of the ROESY crosspeak between N-Me-Lys H and Asp
H
9
ꢀ
proS. This material is available free of charge via the Internet
1
977, 23, 327–341.
at http://pubs.acs.org.
126) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Dinola,
A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684–3690.
JA101428M
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128 J. AM. CHEM. SOC. 9 VOL. 132, NO. 23, 2010