Design of MC1R Selective γ-MSH Analogues with Canonical Amino Acids Leads to Potency and Pigmentation

Article abstract of DOI:10.1021/acs.jmedchem.7b01295

Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The use of noncanonical amino acids in previous MC1R ligand development raises safety concerns. Here we report the development of the first potent and selective hMC1R agonist with only canonical amino acids. Using γ-MSH as a template, we developed a peptide, [Leu³, Leu⁷, Phe⁸]-γ-MSH-NH₂ (compound 5), which is 16-fold selective for the hMC1R (EC₅₀ = 4.5 nM) versus other melanocortin receptors. Conformational studies revealed a constrained conformation for this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration. [Leu³, Leu⁷, Phe⁸]-γ-MSH-NH₂ is ideal for inducing short-term skin pigmentation without sun for melanoma prevention.

Full text of DOI:10.1021/acs.jmedchem.7b01295

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Article
Design of MC1R selective #-MSH analogues with
canonical amino acids leads to potency and pigmentation
Yang Zhou, Saghar Mowlazadeh Haghighi, Ioanna Zoi,
Jonathon Randall Sawyer, Victor J. Hruby, and Minying Cai
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01295 • Publication Date (Web): 02 Nov 2017
Downloaded from http://pubs.acs.org on November 3, 2017
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Design of MC1R selective γꢀMSH analogues with canonical amino acids leads
to potency and pigmentation
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Yang Zhou,* Saghar Mowlazadeh Haghighi,* Ioanna Zoi, Jonathon R. Sawyer, Victor J. Hruby,
Minying Cai**
1Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721,
United States
ABSTRACT:
Melanoma is a lethal form of skin cancer. Skin pigmentation, which is regulated by the
melanocortin 1 receptor (MC1R), is an effective protection against melanoma. However, the
endogenous MC1R agonists lack selectivity for the MC1R and thus can have side effects. The
use of nonꢀcanonical amino acids in previous MC1R ligand development raises safety concerns.
Here we report the development of the first potent and selective hMC1R agonist with only
canonical amino acids. Using γꢀMSH as a template, we developed a peptide, [Leu3, Leu7, Phe8]ꢀ
γꢀMSHꢀNH2 (Compound 5), which is 16ꢀfold selective for the hMC1R (EC50= 4.5 nM) verse
other melanocortin receptors. Conformational studies revealed a constrained conformation for
this linear peptide. Molecular docking demonstrated a hydrophobic binding pocket for the
melanocortin 1 receptor. In vivo pigmentation study shows high potency and short duration.
[Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 is ideal for inducing short term skin pigmentation without sun
for melanoma prevention.
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INTRODUCTION
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Melanoma is the most dangerous form of skin cancer. In 2016, there is an estimated 73,870
new cases of melanoma, with an estimated of 10,130 deaths in the U. S. alone1. Exposure to UV
radiation is the primary risk factor for melanoma2. Skin pigmentation, on the other hand, is an
effective protection against melanoma. Melanocytes can produce melanin, which is able to
dissipate over 99.9% of UV radiation3. The strong correlation between skin pigmentation and
melanoma risk is also evidenced by the fact that in the U.S., Caucasians have 20ꢀ30 times higher
chances of getting melanoma than Asians and Blacks4. Current efforts seek to prevent UV
damage to human skin, which in many cases leads to melanoma and other skin cancers. As a
result, inducing skin pigmentation without UV exposure is considered an effective way to
prevent UV induced melanoma.
Skin pigmentation is regulated by the melanocortin 1 receptor (MC1R) on melanocytes5.
Activation of MC1R by melanocyte stimulating hormones (MSH) leads to cyclic adenosine
monophosphate (cAMP) production, which eventually leads to transcription of multiple pigment
synthesis genes and melanin production6.7ꢀ22. The natural MSHs consist of three peptides: αꢀ
melanocyteꢀstimulating hormone (αꢀMSH), βꢀmelanocyteꢀstimulating hormone (βꢀMSH) and γꢀ
melanocyteꢀstimulating hormone (γꢀMSH). All MSHs share the same pharmacophore of Hisꢀ
PheꢀArgꢀTrp. The MSHs not only activate MC1R, but they are also able to activate the
melanocortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R) and melanocortin 5 receptor
(MC5R), which regulate distinct physiological processes such as feeding behavior, energy
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homeostasis, sexual function, immune responses and sebaceous gland secretion23. Thus,
improving selectivity to MC1R is of critical importance for MSHs to trigger skin pigmentation
for melanoma prevention without interfering with other physiological functions.
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The major challenge is that sunscreen lotion is viewed as both a cosmetic and a drug in the
United States, and thus must follow regulations from the US Food and Drug Administration
(FDA)24. Since 2014, the αꢀMSH analogue [Nle4, ꢀPhe7] αꢀMSH (NDPꢀαꢀMSH, brand name
D
Scenesse) has been shown to effectively induce skin pigmentation25 and has been approved in
Europe for treating erythropoietic protoporphyria (EPP). However, the FDA approval for its use
in the U. S. is still pending. Two major aspects on NDPꢀαꢀMSH can be improved to pose less
health concerns and better fit FDA regulations. One is that NDPꢀαꢀMSH is a universal agonist
for all melanocortin receptors. The other is that it uses nonꢀcanonical amino acids that may not
be metabolized the same way as canonical amino acids. Our goal is to develop MC1R selective
peptides with canonical amino acids so that it will be more safe and easy degraded into natural
building block amino acids in the body.
Design of selective MC1R γꢀMSH analogues
Our earlier research led to a potent drug NDPꢀαꢀMSH (MTꢀI) or “Afamelanotide” for the
treatment of congenital erythropoietic porphyria in Europe and skin color disorders in Australia.
However, MTꢀI does not have high selectivity for any one of the melanocortin receptor subtypes,
which can lead to unexpected side effects such as headache and nausea25. One of our previous
studies of a γꢀMSH analogue (compound 1, Table 1) had improved potency and selectivity for
the MC1R. Efforts have been made to move compound 1 to be a melanoma prevention agent, but
the process of drug development has been very slow due to the unꢀnatural amino acids that are
involved.
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Table 1. Sequence of γꢀMSH analogues
no.
peptide sequences
9
1. Nle3. DNal26, DTrp8ꢀγꢀMSHꢀNH2
2. Leu3, Pro5, γꢀMSHꢀNH2
3. Leu3, Pro5, Phe8 γꢀMSHꢀNH2
4. Leu3, Leu7, γꢀMSHꢀNH2
5. Leu3, Leu7, Phe8 γꢀMSHꢀNH2
6. Leu3, Pro5, Le u7 γꢀMSHꢀNH2
7. Leu3, Pro5, Leu7, Phe8 γꢀMSHꢀNH2
8. Pro5 γꢀMSHꢀNH2
HꢀTyrꢀValꢀNleꢀGlyꢀHisꢀDꢀNal(2)ꢀArgꢀDꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀNH2
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HꢀTyrꢀValꢀLeuꢀGlyꢀProꢀPheꢀArgꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀLeuꢀGlyꢀProꢀPheꢀArgꢀPheꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀLeuꢀGlyꢀHisꢀPheꢀLeuꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀLeuꢀGlyꢀHisꢀPheꢀLeuꢀPheꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀLeuꢀGlyꢀProꢀPheꢀLeuꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀLeuꢀGlyꢀProꢀPheꢀLeuꢀPheꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀMetꢀGlyꢀProꢀPheꢀArgꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyrꢀValꢀLeuꢀGlyꢀHisꢀPheꢀArgꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀOH
HꢀTyrꢀValꢀLeuꢀGlyꢀHisꢀPheꢀArgꢀTrpꢀAspꢀArgꢀPheꢀGlyꢀ NH2
HꢀTyr1ꢀVal2ꢀMet3ꢀGly4ꢀHis5ꢀPhe6ꢀArg7ꢀTrp8ꢀAsp9ꢀArg10ꢀPhe11ꢀGly12ꢀ OH
HꢀTyr1ꢀVal2ꢀMet3ꢀGly4ꢀHis5ꢀPhe6ꢀArg7ꢀTrp8ꢀAsp9ꢀArg10ꢀPhe11ꢀGly12ꢀ NH2
9. Leu3, γꢀMSH
10. Leu3, γꢀMSHꢀNH2
11. γꢀMSH
12. γꢀMSHꢀNH2
13. AcꢀNDPꢀ γꢀMSHꢀNH2
14. αꢀMSH
AcꢀTyr1ꢀVal2ꢀNle3ꢀGly4ꢀHis5ꢀ
AcꢀSerꢀTyrꢀSerꢀMetꢀGluꢀHisꢀPheꢀArgꢀTrpꢀGlyꢀLysꢀProꢀValꢀ NH2
AcꢀSerꢀTyrꢀSerꢀNleꢀGluꢀHisꢀ ꢀPheꢀArgꢀTrpꢀGlyꢀLysꢀProꢀValꢀNH2
D
ꢀPhe6ꢀArg7ꢀTrp8ꢀAsp9ꢀArg10ꢀPhe11ꢀGly12ꢀ NH2
15. NDPꢀ αꢀMSH
D
This motivated us to design a selective MC1R ligand with only a canonical amino acid
starting from this γꢀMSH analogue. We introduced modifications on 5 different sites of γꢀMSH:
1. Introducing the Cꢀterminal amide group to γꢀMSH and its analogues was shown to increase
their binding affinities to all melanocortin receptor subtypes but MC3R26. 2. To keep the
similarity and stability of compound 1 we substitute Nle3 with Leu3 since natural γꢀMSH has
Met3 which is easily oxidized. 3. His5 was substituted to proline, which was previously shown to
increase potency and selectivity to MC1R26. 4. Trp8 was substituted to phenylalanine, which was
also shown to improve MC1R selectivity27. 5. Most importantly, our chimeric receptor studies
demonstrated that the electrostatic interaction, Arg(L)ꢀAsp(R), between the Arg8 of the NDPꢀαꢀ
MSH and the Asp122, Asp126 of the hMC4R is of critical importance to achieve binding and
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receptor activation, as Asp126Asn mutation on the MC4R caused more than a 400ꢀfold increase
in the EC50 value28. Similarly, a key interaction between the Arg8 of NDPꢀαꢀMSH and the
Asp154 as well as the Asp158 of the MC3R is necessary, as Asp158Ala mutation on MC3R
caused more than 350ꢀfold increase for the EC50 value29. In contrast, mutation of either Asp117
or Asp121 to alanine on MC1R only had around a 10 fold influence on the IC50 and EC50 of
NDPꢀαꢀMSH, suggesting a smaller role of ionic interactions between MC1R and its agonists30.
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Finally, our latest docking studies of hMC1R with selective hMC1R ligand revealed that there is
a very hydrophobic binding pocket for the hMC1R. This suggested that increasing
hydrophobicity of the hMC1R ligand can improve the selectivity (Data in the process of
publication). Therefore, our hypothesis is switching the arginine in the γꢀMSH analogue to a
neutrally charged amino acid, in particular Leucine should reduce binding towards the hMC3R
and the hMC4RWe envision that enhanced selectivity towards the MC1R can be reached with
reduced electrostatic interaction between the Arg (L)ꢀAsp(R) of the γꢀMSH analogues and the
respective aspartic acids on the MC3R and MC4R receptors. Herein, a series of Leu3, Pro5, Leu7,
Phe8 γꢀMSHꢀNH2 analogues are designed and synthesized to test these hypotheses. (Table 1).
RESULTS
Bioassay Results. Competitive binding assays with [125I]ꢀNDPꢀαꢀMSH and adenylate cyclase
assays were performed with HEK293 cells stably expressing human MC1R (hMC1R), human
MC3R (hMC3R), human MC4R (hMC4R) and human MC5R (hMC5R) on the γꢀMSH
analogues (Table 1, Table 2). γꢀMSH (Compound 11) is a universal agonist to melanocortin
receptors with 1.8ꢀfold selectivity to hMC1R (EC50=300 nM). By adding the Cꢀterminal amide
group to γꢀMSH, compound 12 loses its selectivity to hMC1R, as the potency on hMC1R
(EC50=70 nM) is in the same range as the potency on hMC4R (EC50=65 nM). However, the
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potency to hMC1R is enhanced 3ꢀfolds. Compound 9 and 10 with methionine substituted by
leucine both have enhanced potency and selectivity to the hMC1R. Compound 9 has a 10ꢀfold
increase in potency (EC50= 31 nM) to the hMC1R comparing to γꢀMSH with 13ꢀfold selectivity.
Compound 10 has better potency and selectivity with EC50 of 8 nM on the hMC1R and a
selectivity of over 22ꢀfold. The methionine to leucine substitution also caused compound 9 and
10 to have less binding efficiency and no activation on the hMC5R, which is consistent with the
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D
ꢀamino acid scan showing Met3 in γꢀMSH is important for hMC5R activation31. Thus, we
focused our further design using Leu3 γꢀMSHꢀNH2 (compound 10) as a template.
To improve selectivity on the hMC1R, we did modifications on the melanotropin
pharmacophore His5ꢀPhe6ꢀArg7ꢀTrp8. Peptides were designed with a combination of mutations
on His5, Arg7 and Trp8. As expected, compound 2 with the proline substitution had improved
potency, but it lost all selectivity as it activates hMC1R (EC50= 4.6 nM) and hMC3R (EC50= 5.0
nM) with almost the same potency. The leucine substitution on compound 4 did affect its binding
to hMC3R and hMC4R, as they only showed weak binding and no activity on these receptors.
However, the potency to hMC1R (EC50= 604 nM) is also decreased by 76ꢀfold. A double
replacement of His5 to Pro5 and Arg7 to Leu7 (compound 6) didn’t show synergistic effects, as
compound 6 lost all activities for any melanocortin receptor. The loss of potency is also seen in
the triple replacements of His5 to Pro5; Arg7 to Leu7; Trp8 to Phe8 (compound 7). The
replacements of H5P and W6F (compound 3) also showed loss of activities at all melanocortin
receptors. The purpose of our design was achieved with a double replacement of Arg7 to Leu7
and Trp8 to Phe8 (compound 5, [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2). Compound 5 ([Leu3, Leu7,
Phe8]ꢀγꢀMSHꢀNH2) has 1.8ꢀfold
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Table 2. Competitive binding assay and cAMP assay results of γꢀMSH analogues
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4
5
6
7
8
9
hMC1R
hMC3R
aIC50, nM
hMC4R
%ꢂmax
hMC5R
aIC50, nM
%ꢂmax
effect
%ꢂmax
effect
%ꢂmax
effect
no. aIC50, nM
%BE
bEC50, nM
%BE
bEC50, nM
aIC50, nM
%BE
bEC50, nM
%BE
bEC50, nM
effect
1
2
3
4
5
6
7
8
9
0.30±0.02
82±7
100
100
0
3.0±0.2
4.6±0.9
>1000
604±74
4.5±0.4
NA
70
5.0±0.6
4.1±0.4
18±2
100
100
33
300±6
5.0±0.4
NA
6
6.0±1
5.0±0.4
NB
100
100
0
630±70
23±3
18
3.5±0.5
18±3
100
100
0
NA
6
100
100
100
100
0
100
0
90
16±1
NA
82
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
NB
NA
0
NB
137±11
24±3
100
100
29
17±1
26
NA
0
>1000
210±5
NB
47
NA
0
NB
0
NA
0
>1000
117±24
>1000
118±14
56±3
45
>1000
NA
50
58
71±8
54
NB
0
NA
0
16±2
32
0
0
NA
0
0.42±0.07
NB
32
NA
0
1.1±0.1
>1000
23±2
32
>1000
40±6
100
100
100
100
100
100
100
100
100
47
>1000
521±72
>1000
>1000
700±89
520±50
2.0±0.2
6.7±1
0.8±0.1
100
100
100
100
100
38
6.7±0.9
360±42
20±1
36
200±25
128±13
444±69
178±23
710±70
65±12
1.4±0.1
2.1±0.6
0.2±0.04
100
78
0
NA
0
52
73
62
36±1
47
>1000
NA
100
0
100
100
100
100
100
100
100
31±6
69
86
100
100
100
76
254±41
>1000
2200±200
330±57
2.4±0.3
18±2
38
10 42±7
8±1.8
>1000
71±10
40±10
2.0±0.02
30±3.9
3.3±0.3
53
68±11
760±80
45±5
100
100
100
100
100
100
60
NA
0
11 1000±20
12 1.2±0.2
13 0.5±0.01
14 0.4±0.01
15 0.01
300±30
70±10
1.5±0.1
0.7±0.01
0.01
100
100
100
100
100
100
100
100
100
100
550±60
390±25
1.9±0.2
8.1±1.5
1±0.3
100
73
100
100
100
100
100
100
1.2±0.2
5±1
100
100
100
0.4±0.02
2.2±0.5
a IC50 is the concentration of peptide at 50% specific binding (N = 4). %BE is the percentage of [125I] NDPꢀαꢀMSH displacement at 10 ꢁM. NB means that 0% of [125I] NDPꢀαꢀMSH displacement was
observed at 10 ꢁM. EC50 is the effective concentration of peptide that could generate 50% maximal intracellular cAMP accumulation (N = 4). Act% is the percentage of cAMP produced at a ligand
concentration of 10 ꢁM, in relation to MTꢀII. NA indicates 0% cAMP accumulation observed at 10 ꢁM. The peptides were tested over a range of concentrations from 10−10 to 10−5 M. Compounds 1,
11ꢀ15
were
previously
published
data
and
are
included
for
comparison.
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enhanced potency for the
hMC1R (EC50= 4.5 nM) than
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the
(compound
selectivity to hMC1R is over
template
compound
<1min
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10).
The
>14 days
16ꢀfold.
Furthermore,
compound 5 ([Leu3, Leu7,
Phe8]ꢀγꢀMSHꢀNH2) is only
enhanced potency for the
hMC1R (EC50= 4.5 nM) than
Figure 1a NDPꢀαꢀMSH induced pigmentation on the lizard. Left, before
injection; Right, after injection in 1 min.
the
(compound
selectivity to hMC1R is over
16ꢀfold. Furthermor
template
compound
10). T<h1emin
<1day
e,
compound 5 ([Leu3, Leu7,
Phe8]ꢀγꢀMSHꢀNH2) is only
Figure 1b [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 induced pigmentation on the
lizard. Left, before injection; Right, after injection in 1 min.
a partial agonist with 54%
maximal activation on the
hMC4R. The template compound (compound 10), on the other hand, is a full agonist on the
hMC4R. These results suggest that [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 is an ideal peptide with
strong potency and selectivity for the hMC1R.
Human serum stability. To examine the stability of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 in
comparison to γꢀMSH and NDPꢀMSH, serum stability assays were performed. The endogenous
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ligand γꢀMSH has the shortest halfꢀlife for around 5 min. Comparing to γꢀMSH, compound 5 has
an improved serum stability of 17.5 min. The elongated halfꢀlife is possibly due to the
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replacement of methionine on the γꢀMSH template. NDPꢀMSH, which contain
longest halfꢀlife in human serum of around 30 min.
DꢀPhe, has the
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In vivo study results Pigmentation studies of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 in the Anolis
carolinensis show it is a super potent agonist in vivo. The black color appeared in one minute
after injection (i.p.:2ug/g). (Figure 1b) This is exactly the same response of injection of the same
dose of NDPꢀαꢀMSH (Figure 1a). In comparison, intraperitoneal injection of the vehicle didn’t
produce any pigmentation effect (data not shown). The green color was able to resume less than
24 hours after [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 injection but not for the NDPꢀαꢀMSH. It took two
weeks to resume the green color for the NDPꢀαꢀMSH (Figure 1a). The much longer halfꢀlife of
the NDPꢀαꢀMSH in vivo is probably due to two unꢀnatural amino acids, Nle, and DꢀPhe, which
are not recognized by proteases in vivo, thus resulting in biological stability. However, [Leu3,
Leu7, Phe8]ꢀγꢀMSHꢀNH2 are composed of all of natural amino acids. This indicate that [Leu3,
Leu7, Phe8]ꢀγꢀMSHꢀNH2 will be ideal to be used in a skin care product to induce skin
Figure 2. Conformational Studies of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 . Figure 2a. Left, Stereo view of lowest energy conformation
search derived structure of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 . Figure 2b. Right, Ramachandran plot of lowest energy conformation
search derived structure of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH . Showing most of the amino acids are in the 310 helix region except for
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pigmentation and protect against melanoma during sun exposure. The natural color can be
resumed in one day with a lower dosage. Furthermore [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 is highly
selective for hMC1R and therefore avoids possible side effects due to activation of the other
hMCRs subtypes.
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Conformational Study of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2
The lowest energy conformation search via Macro Model (Schrodinger) revealed a very
constrained sphereꢀlike structure for the [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2. (Figure 2a). The major
force for the constraint is hydrophobicity due to the presence of dominant hydrophobic amino
acids in this peptide. The Ramachandran plot shows that all of the amino acids are in the region
of a 310 helix. (Figure 2b) except for the Gly 4, Phe6 and Asp9. These three amino acids are in the
turn region.
Molecular docking studies
The initial hMC1R structure applied for this study is from previous published work from the
Mosberg lab32. First we searched for a potential binding site with Maestro SiteMap and identified
the one with the highest druggability score of 1.06 (Figure 3). We then performed a flexible
docking simulation with Maestro Glide targeting that pocket.
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Figure 3. Binding site of hMC1R for the [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2
Molecule docking revealed a very hydrophobic binding pocket for the hMC1R. A docking score
of ꢀ11.32 kcal/mol was achieved and key interactions between [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2
and the hMC1R were identified (Figure 4). [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 showed an
antiparallel betaꢀsheet conformation with the modified pharmacophore, His5ꢀPhe6ꢀLeu7ꢀPhe8, at
the turn region (Supplementary Figure 4). His5 and Phe6 interact with the transmembrane
segment 6 (TM6) of the hMC1R. His5 is stabilized by πꢀπ stacking interactions with Thr262 and
His260 inside the hMC1R. Phe6 has πꢀπ stacking interactions with both Trp254 and Phe257.
Both His260 and Phe257 have been shown by previous mutagenesis studies to be important for
melanocortin receptor activation28, 30. Phe8 interacts with Cys125 on the TM3 domain. Other
residues on [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 interact with the TM1 and TM3. The relative
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movement on the TM6 was shown to be critical for G protein coupled receptor activation33. Our
results indicated that the pharmacophore of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 interacts with TM6
to generate its movement while other residues on [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 anchor to other
transmembrane segments on hMC1R. It has to be noted that this peptide maintains all the
important interactions as reported from previous studies. The key for its high affinity and
selectivity lies in the fact that it introduces new types of interactions. It also has many hydrogen
bond interactions, indicative of stronger binding.
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We also emphasize that the evaluation of the binding site where our ligand was bound, led to an
increased druggability score of 1.21 suggesting that the peptide targeted a highly druggable part
of the binding site.
Molecular interactions between [Leu3, Leu7,
Phe8]ꢀγꢀMSHꢀNH2 and hMC1R.
Interacting
residue on
compound 5
Interacting
residue on
hMC1R
Distance
Type of
interaction
His5 NE2
His5
Thr262
His260
Trp254
Phe257
Phe277
Phe280
Cys125
Ser126
Asp117
Phe45
1.14 Å
5.12 Å
3.68 Å
3.16 Å
5.32 Å
3.99 Å
2.79 Å
2.62 Å
1.79 Å
3.55 Å
Hꢀbond
PiꢀPi stacking
PiꢀPi stacking
PiꢀPi stacking
PiꢀPi stacking
PiꢀPi stacking
Hꢀbond
Phe6
Phe6
Tyr1
Tyr1
Leu7 N
Asp9 OD1
Arg10 NH2
Phe11
Hꢀbond
Salt bridge
PiꢀPi stacking
Figure 4. Molecular interactions between the modified pharmacophore ꢀHisꢀPheꢀLeuꢀPheꢀof [Leu3, Leu7,
Phe8]ꢀγꢀMSHꢀNH2 and the hMC1R.
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Molecular interactions between compound 1 and
hMC1R.
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Interacting
residue on
compound 1
Interacting
residue on
hMC1R
Distance
Type of
interaction
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His N
Arg NE
Arg NH2
Arg NH2
Arg NH1
Trp
Ile276
Glu94
2.30Å
1.93 Å
2.27 Å
2.31 Å
2.72 Å
5.02 Å
1.90 Å
Hꢀbond
Hꢀbond
Glu94
Hꢀbond
Thr124
Asp121
Phe179
Asp121
Hꢀbond
Hꢀbond
PiꢀPi stacking
Hꢀbond
Trp N
Figure 5. Molecular interactions between the pharmacophore ꢀHisꢀDꢀNaI(2’)ꢀArgꢀTrpꢀ of compound 1 and the hMC1R.
Furthermore, we tested compound 1 for comparison purposes and after having it docked inside
the receptor cavity and analyzing its interactions we note that compound 1 has a slightly higher
and therefore worse docking score (ꢀ11.08 kcal/mol) compared to [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀ
NH2. It also presents less interactions with the receptor, although most of them are hydrogen
bonds as shownin Table 4. Surprisingly, only the pharmacophore of compound 1 (HisꢀDꢀNaI(2’)ꢀ
ArgꢀTrp) interacts with hMC1R (Figure 5) whereas in [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 the
majority of its amino acids are participating in interactions. In [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2
we see more different types of interactions which can partially explain its enhanced selectivity
towards the hMC1R.
DISCUSSION AND CONCLUSIONS
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Seeking effect treatments of melanoma is very hot in current era of science and technology.
However, prevention of melanoma has been neglected. We are providing a very useful natural
AA made peptide here and it will be more applicable and benefit for most of people. In this
study, we first successfully developed a natural aa made peptide, [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀ
NH2, which is a potent selective hMC1R agonist. The composition of canonical amino acids
ensure that the peptide can be easily degraded into natural building block, amino acids. The use
of only natural amino acids versus unnatural can alleviate most safety concerns. The high
selectivity of the [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 for the hMC1R and shorter halfꢀlife provides a
safer and reduced sideꢀeffect agent. As peptides have been widely used in cosmeceuticals as
active ingredients and can be applied transdermally34, [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 is suitable
as a safe skin care product for the prevention of melanoma skin cancer.
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Compared to our previously developed peptide (compound 1), [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀ
NH2 seems to have less selectivity to hMC1R in terms of EC50. However, compound 1 is also an
antagonist at the hMC3R, hMC4R and hMC5R as suggested by its strong binding affinity to
those MCRs. The binding affinity of compound 1 to the hMC1R (IC50= 0.3 nM) is only 12ꢀfold
stronger than hMC5R (IC50= 3.5 nM). As hMC5R regulates sebum production in the skin35,
compound 1 may cause hMC5R antagonism and decreased production of sebum. On the other
hand, [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 doesn’t have detectable binding to hMC5R in our
biological assays. Binding of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 to the hMC3R and hMC4R is also
weak as evidenced by high IC50 and low % binding. More importantly, MC4R is exclusively
expressed in the central nervous system36. Long linear peptides such as NDPꢀαꢀMSH can’t pass
through the bloodꢀbrain barrier to reach the brain37. With similar length and sequence as NDPꢀαꢀ
MSH, compound 5 is unlikely to reach the brain and interfere with MC4R’s function. As a result,
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compound 5 is selective to hMC1R and less likely to interfere with physiological processes
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regulated by other MCRs.
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The serum stability assay showed a halfꢀlife of 17.5 min for [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 in
human serum. It’s worth noting that the skin pigmentation effect lasts longer than the peptide’s
halfꢀlife in serum. Even though the halfꢀlife of NDPꢀαꢀMSH in human serum is 30 min, the
European standard is to have NDPꢀαꢀMSH in a controlledꢀrelease implant and subcutaneously
administrated to EPP patients once every two months38. It was further confirmed with our in vivo
assay that the pigmentation effect would last for less than 24 hours. It’s worth to mention that
even though pigmentation effect in lizards is mostly regulated by melanosome dispersion rather
than melanogenesis, the melanosome dispersion process in coldꢀblooded animals is also
regulated by the MC1R/cAMP pathway39. [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 is expected to
effectively induce short period skin pigmentation in human, and thus can be used in skin care
products for short term melanoma prevention.
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In our in silico study, we discovered that hMC1R has a very hydrophobic binding pocket.
The modified pharmacophore of [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2, HisꢀPheꢀLeuꢀPhe, has
enhanced hydrophobicity compared to the natural MSHs pharmacophore (HisꢀPheꢀArgꢀPhe)
which contributes to hMC1R selectivity. Computational chemistry combined with chimeric
receptors studies verify our hypothesis that enhanced hydrophobicity of the MSHs
pharmacophore can increase the hMC1R selectivity. The newly designed hMC1R selective
agonist, [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 fits perfectly within the hydrophobic binding pocket of
hMC1R provides new insights for future drug design for the hMC1R.
Molecular docking is a very useful tool in structural biology and computer aided drug
design, especially for GPCR based drug design owing to the limited crystal structures available
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(none of the melanocortin receptors) and the dynamic environment of the membrane protein. The
major goal of ligand ꢀ receptor docking is to evaluate the feasible binding geometries of a
putative ligand with a target protein of known threeꢀdimensional structure. Normally, docking
calculations can help obtain an idea about the binding affinity of the ligand. It also provides
useful insights regarding the interactions between ligand and receptors and their roles. Here we
study the [Leu3, Leu7, Phe8]ꢀγꢀMSHꢀNH2 and hMC1R interaction by conjugating the study of
biological information from multiple mutagenesis, chimeric receptor studies of the hMC1R and
molecular docking.
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Finally, structureꢀbased drug design has become a useful approach for current drug discovery.
In our long ꢀterm peptideꢀbased drug development, peptide truncation and amino acid scans have
been used to discover the major pharmacophore. Conformational constraints were applied to
produce numerous stable and selective melanotropins, and the threeꢀdimensional structure of
ligands using NMR spectroscopy combined with computational based drug design have led to
several selective compounds. Peptide mimetic studies also led to selectivity and potency.
However, few of these strategies led to a drug. In this study, we first used a strategy by applying
useful information from multiple mutagenesis and chimeric receptor studies along with
computational chemistry to rationally design a hMC1R peptide ligand using canonical amino
acids. The economic favorability of canonical amino acids and straightforward synthesis strategy
can provide lower cost for mass production. Our strategy represents the rational design to fulfill
specific requirements for developing skin care products for short term skin pigmentation and
melanoma prevention.
EXPERIMENTAL SECTION
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Synthesis. NαꢀFmocꢀamino acids were obtained from Bachem, NovaBiochem, and Advanced
ChemTech. The side chain protecting groups were Boc and tBu [FmocꢀAsp(tBu)ꢀOH, Fmocꢀ
Trp(Boc)ꢀOH, FmocꢀArg(pbf)ꢀOH, FmocꢀHis(trt)ꢀOH, FmocꢀSer(tBu)ꢀOH and FmocꢀTyr(tBu)ꢀ
OH]. FmocꢀRink amide resin was purchased from Polymer Laboratories. Organic solvents and
reagents were purchased from Aldrich and used without further purification. All peptides were
synthesized by the NꢀFmoc solidꢀphase peptide strategy using DIEA and HCTU as the coupling
reagents.Rink amide resin (0.65 mmol/g) or 2ꢀchlorotrityl resin (1mmol/g) was placed into the 5
mL polypropylene syringe with the frit on the bottom and swollen in DCM (2 mL) and DMF (2
mL) for one hour. The Fmoc protecting group on the Rink linker was removed by 20%
piperidine in DMF. After 20 min the solution of piperidine was removed and the resin was
washed with DMF (2 mL, 4 times) and DCM (2 mL, 4 times). NꢀFmoc amino acid (3 equiv) and
HCTU (3 equiv) were dissolved in DMF, and then DIEA (3 equiv) was added. The coupling
mixture was transferred into the syringe with the resin and shaken for 30 mins. Coupling
completion was monitored with a Kaiser test. The coupling mixture was removed and the resin
was washed with DMF (2 mL, 4 times) and DCM (2 mL, 4 times). NꢀFmoc groups were
removed with 20% piperidine in DMF in 20 min. Each coupling and deprotection step was
repeated until a linear peptide was assembled. The final wash of the resin was done with DMF (2
mL, four times) and DCM (2 mL, four times). The product was cleaved from the resin with a
mixture of 95% TFA, 2.5% TIPS, and 2.5% water during 3h. Side chain protecting groups were
removed during the cleavage step as well. The cleaved mixture was evaporated on a rotary
evaporator, and the crude peptide was dissolved in H2O/methanol and purified by HPLC.
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The peptide was lyophilized and purified by preparative RPꢀHPLC on a C18 bonded silica
column (Vydac 218TP152022, 250 _ 22 mm, 15ꢀ20 ím, 300 Å). A C18 analytical column (YMCꢀ
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Pack ODSꢀAM 150 _ 4.6 mm, Sꢀ3 ím, 120A) was used to analyze the purity, eluted with a linear
gradient of acetonitrile (gradient, 2ꢀ80% B in A over 30 min, flow rate 0.8 mL/min. System 1:
solvent A, 0.1% TFA in water; solvent B, 0.08% TFA in acetonitrile. System 2: solvent A, 1%
formic acid in water; solvent B, 1% formic acid in methanol) and aqueous 0.1% TFA (v/ v). The
major peak of all compounds accounted for ≥95% of the combined total peak area monitored by
a UV detector at 254 nM.
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Biological Assays. Competitive binding assays with [125I]ꢀNDPꢀMSH and adenylate cyclase
assays were performed using previously described protocols26, 41ꢀ43
.
Pigmentation Study Lizards Anolis carolinensis were purchased from Carolina online. Peptide
samples were dissolved in saline at the concentration of 1mM. Lizards were anesthetized by
diethyl ether before injection. The total amount of peptide was through i.p. injection with 3ug/g
of each lizard. The strategy follows previous publications44ꢀ48
.
Serum Stability Assay. Compound 5 and 15 were dissolved at 10 mg/mL as stock solution. 1 mL
of RPMI supplemented with 25% of human serum were allocated into a 1.5 ml tube and warmed
up to 37°C before adding 5 uL of peptide stock solution. 100 uL of the reaction solution is
removed from time 0, 10 min, 20 min, 30 min and 40 min. 200 uL of 96% ethanol were added for
precipitation of serum proteins. The reaction sample was cooled (4°C) for 15 min and then spun
at 18,000 g (Eppendorf centrifuge) for 2 min. The reaction supernatant was then analyzed using
RPꢀHPLC on a 5um 25 × 0.4ꢀcm Vydac Cꢀ18 column. A linear gradient from 100% buffer A, to
50–50% of buffer A and buffer B, is used over 30 min. The absorbance was detected at 280 nm.
Area under the expected peak is expected to be proportional to the amount of remaining peptide.
The halfꢀlife was estimated by fitting the data into an exponential decay curve.
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Molecular modeling experiments employed MacroModel version 10.5 equipped with Maestro
10.5 graphical interface (Schrödinger, LLC, New York, NY, 2016) installed on a Linux Red Hat
11 system, and were performed as previously described. Peptide structures were built into
extended structures with standard bond lengths and angles, and they were minimized using the
OPLS3 force field and the PolakꢀRibier conjugate gradient (PRCG). Optimizations were
converged to a gradient RMSD less than 0.05 kJ/Å mol or continued until a limit of 50,000
iterations was reached. Aqueous solution conditions were simulated using the continuum
dielectric water solvent model (GB/SA). Extended cutꢀoff distances were defined at 8Å for Van
der Waals, 20Å for electrostatics and 4 Å for Hꢀbonds.
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Conformational profiles of the cyclic peptides were investigated by the hybrid Monte Carlo/Low
Frequency Mode (MCMM/LMCS) procedure as implemented in Macromodel using the energy
minimization parameters as described above. MCMM torsional variations and Low Mode
parameters were set up automatically within Maestro graphical user interface. A total of 20,000
search steps were performed and the conformations with energy difference of 50 kJ/mol from the
global minimum were saved. Interatomic dihedral angles were measured for each peptide
analogue using the Maestro graphical user interface, and they are described in Table 3.
(Supplement Material).
Molecular Docking Molecular Docking Studies using the Glide programs (version 7.0,
Schrodinger, LLC, New York, 2016). To analyze the docking results and execute the protocol,
the Maestro user interface (version10.5, Schrodinger, LLC, New York, 2016) was employed.
Docking was performed using the SP (Standard Precision Mode) protocol. This includes 1.
Preparation of Protein. The protein was subjected to energy minimization using Schrodinger
implementation of OPLS3 force field. 2. Preparation of Ligand. The ligand was prepared using
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the LigPrep 3.7 module of the Schrodinger suite using the standard protocol with OPLS3 force
field. 3. Active Site Prediction. We employed Sitemap (version 3.8) to search for potential
binding sites. Sitemap applies theoretical methods and predicts the most accurate binding site.
Again, we used Sitemap after we had docked our ligand to evaluate the binding site. 4. Grid
generationꢀ docking calculation. Glide used a series of hierarchical filters to search for possible
locations for the ligand in the active site region of the receptor. For the gridꢀbased ligand
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docking, the receptor grid generation process was used. A grid box of 30 × 30 × 30ꢂ with a
default inner box (10 × 10 × 10ꢂ 3) was centered on the corresponding ligand. The receptor grid
was defined as an enclosed box at the centroid of the ligand. Lastly, we performed a flexible
docking calculation using the “Standard Precision” Glide algorithm and after the postꢀdocking
minimization we kept the pose with the best docking score.
Preparation of Protein and Ligand. The hMC1R model was obtained from Chai et al.32,
imported and prepared by a multistep process through the protein preparation wizard of Maestro.
The protocol was especially used to obtain the optimized and minimized energy conformation of
the protein. First, we assigned bond orders and added hydrogen atoms. Water molecules which
did not participate in interactions were removed. Following the above steps of preparation, the
protein was subjected to energy minimization using Schrodinger implementation of OPLS3 force
field. The structure for compound 5 and compound 1 was built in 3D coordinates, minimized in
Macromodel to obtain the lowest energy conformation, and retaining the geometry was then
prepared using the LigPrep 3.7 module of the Schrodinger suite using the standard protocol with
OPLS3 force field.
Active Site Prediction
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We employed Sitemap (version 3.8) to search for potential binding sites. Sitemap applies
theoretical methods and predicts the most accurate binding site. The OPLS3 force field generates
site points, possible for ligand interaction within the protein. Sitemap searches for positions
favorable for a donor, acceptor, and hydrophobic group to be present in the receptor.
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We used Sitemap to identify the top ranked potential receptor binding sites keeping all
parameters as default. Five sites with different site scores were obtained as output and the site
with the highest score (SiteScore and Dscore) was selected.
We again used Sitemap after we had docked our ligand to evaluate the binding site based on the
druggability score (Dscore). Druggability is a term used in drug discovery to describe
a biological target that is known to or is predicted to bind with high affinity to a drug. If that
score is higher than 0.75, the target is considered to be druggable.
Molecular docking studies
We performed molecular docking studies using the Glide program (version 7.0, Schrödinger,
LLC, New York, 2016). To analyze the docking results and execute the protocol, the Maestro
user interface (version 10.5, Schrödinger, LLC, New York, 2016) was employed.
We used the set of site points from the proposed binding site to generate a grid box for the
docking calculation. The receptor grid was defined as an enclosing box at the centroid of the
ligand. Then we performed a flexible docking calculation using the “Standard Precision” Glide
algorithm and after the postꢀdocking minimization we kept the pose with the best docking score.
SUPPORTING INFORMATION
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Physiochemical and spectral data (1H NMR, mass spectrometry), HPLC chromatograms,
hMC1R binding site prediction, overall conformation of compound 5 bound to hMC1R
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Molecular formula strings and some data
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AUTHOR INFORMATIONCorresponding Author
** To whom correspondence should be addressed. Phone: (520) 621ꢀ8617. Fax: (520) 621ꢀ
8407. Eꢀmail: mcai@email..arizona.edu. Present Addresses:1306 E. University Blvd, Tucson,
AZ 85721. USA
ORCID
Yang Zhou: 0000-0001-5199-156X
Minying Cai: 0000-0001-9504-2091
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. *These authors contributed equally.
M. C. designed the experiments. M. C. performed the conformational study and in vivo study. Y.
Z. performed the bioassays. S. M., J. R. S. and V. H. synthesized the compounds. I. Z. performed
the docking studies. Y. Z., M. C., S. M. and I. Z. wrote the manuscript.
Notes
The authors declare no conflict of interests.
ACKNOWLEDGMENT
*These authors contributed equally.
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These studies were supported in parts by grants from the U.S. Public Health Service, National
Institutes of Health, DK017420, GM 108040 and Proof of Concept grant from the University of
Arizona Tech Launch Transfer.
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ABBREVIATIONS USED
Abbreviations used for amino acids and designation of peptides follow the rules of the IUPACꢀ
IUB Commission of Biochemical Nomenclature in J. Biol. Chem.40. The following additional
abbreviations are used: AAA, amino acid analysis; Boc, tertꢀbutyloxycarbonyl; Fmoc,
fluorenylmethoxycarbonyl; Fmo, fluorenylmethyl; Bzl, benzyl; tBu, tertꢀbutyl; CH3CN,
acetonitrile; DCM, dichloromethane; DIEA, diisopropylethylamine; DMF, N,Nꢀ
dimethylformatmide; DIC, diisopropyl carbodiimide; HOBt, Nꢀhydroxybenzotriazole; Nal(2),
naphthylalanine; TFA, trifluoroacetic acid; TIPS, triisopropylsilyl; SPPS, solidꢀphase peptide
synthesis; RPꢀHPLC, reversedphase highꢀperformance liquid chromatography; hMC1R, human
melanocortinꢀ1 receptor; MSH, melanocyteꢀstimulating hormone;
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186x134mm (96 x 96 DPI)
ACS Paragon Plus Environment