Identification of tetrapeptides from a mixture based positional scanning library that can restore nM full agonist function of the L106P, I69T, I102S, A219V, C271Y, and C271R Human Melanocortin-4 Polymorphic Receptors (hMC4Rs)

Article abstract of DOI:10.1021/jm500064t

Human obesity has been linked to genetic factors and single nucleotide polymorphisms (SNPs). Melanocortin-4 receptor (MC4R) SNPs have been associated with up to 6% frequency in morbidly obese children and adults. A potential therapy for individuals possessing such genetic modifications is the identification of molecules that can restore proper receptor signaling and function. These compounds could serve as personalized medications improving quality of life issues as well as alleviating diseases symptoms associated with obesity including type 2 diabetes. Several hMC4 SNP receptors have been pharmacologically characterized in vitro to have a decreased, or a lack of response, to endogenous agonists such as α-, β-, and γ₂-melanocyte stimulating hormones (MSH) and adrenocorticotropin hormone (ACTH). Herein we report the use of a mixture based positional scanning combinatorial tetrapeptide library to discover molecules with nM full agonist potency and efficacy to the L106P, I69T, I102S, A219V, C271Y, and C271R hMC4Rs. The most potent compounds at all these hMC4R SNPs include Ac-His-(pI)DPhe-Tic-(pNO₂)DPhe-NH₂, Ac-His-(pCl)DPhe-Tic-(pNO₂)DPhe-NH₂, Ac-His-(pCl)DPhe-Arg- (pI)Phe-NH₂, and Ac-Arg-(pCl)DPhe-Tic-(pNO₂)DPhe-NH ₂, revealing new ligand pharmacophore models for melanocortin receptor drug design strategies.

Full text of DOI:10.1021/jm500064t

Article
pubs.acs.org/jmc
Identification of Tetrapeptides from a Mixture Based Positional
Scanning Library That Can Restore nM Full Agonist Function of the
L106P, I69T, I102S, A219V, C271Y, and C271R Human Melanocortin‑4
Polymorphic Receptors (hMC4Rs)
Erica M. Haslach,† Huisuo Huang,†,‡ Marvin Dirain,† Ginamarie Debevec,§ Phaedra Geer,§
Radleigh G. Santos,§ Marc A. Giulianotti,§ Clemencia Pinilla,∥ Jon R. Appel,∥ Skye R. Doering,‡
Michael A. Walters,‡,⊥ Richard A. Houghten,§,∥ and Carrie Haskell-Luevano*,†,‡
†Department of Pharmacodynamics, University of Florida, Gainesville, Florida 32610, United States
‡Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street SE, Minneapolis, Minnesota 55455, United States
§Torrey Pines Institute for Molecular Studies Port St. Lucie, Florida 34987, United States
∥Torrey Pines Institute for Molecular Studies, San Diego, California 92121, United Stateas
⊥The Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota 55414, United States
S
* Supporting Information
ABSTRACT: Human obesity has been linked to genetic
factors and single nucleotide polymorphisms (SNPs).
Melanocortin-4 receptor (MC4R) SNPs have been associated
with up to 6% frequency in morbidly obese children and
adults. A potential therapy for individuals possessing such
genetic modifications is the identification of molecules that can
restore proper receptor signaling and function. These
compounds could serve as personalized medications improving
quality of life issues as well as alleviating diseases symptoms
associated with obesity including type 2 diabetes. Several
hMC4 SNP receptors have been pharmacologically characterized in vitro to have a decreased, or a lack of response, to
endogenous agonists such as α-, β-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin hormone
(ACTH). Herein we report the use of a mixture based positional scanning combinatorial tetrapeptide library to discover
molecules with nM full agonist potency and efficacy to the L106P, I69T, I102S, A219V, C271Y, and C271R hMC4Rs. The most
potent compounds at all these hMC4R SNPs include Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Tic-
(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2, and Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, revealing new ligand
pharmacophore models for melanocortin receptor drug design strategies.
associated with each hMC4R SNP. The most common
molecular defects that have been identified in vitro include
the lack of the hMC4R SNP to (a) traffic to the cell membrane
surface for expression, (b) decreased endogenous agonist
binding and/or molecular recognition, and (c) reduced/absent
endogenous agonist potency and/or efficacy. Several hMC4R
SNPs have been pharmacologically characterized to possess one
or more of these molecular defects. Once these in vitro
pharmacological characterizations have been performed for
individual hMC4R SNPs, the next goal is to determine
strategies to restore normal function to the SNP as a pathway
toward the development of therapeutic approaches to return
afflicted individuals to an increased quality of life and decrease
their genetic predisposition toward an insatiable hunger and
INTRODUCTION
■
The melanocortin system is comprised of five G-protein
coupled receptors (GPCRs) that stimulate the adenylate
cyclase signal transduction pathway.1−7 The endogenous
ligands are derived by post-translational processing of the
pro-opiomelanocortin (POMC) protein by prohormone
convertases PC1 and PC2 to generate the endogenous
melanocortin agonist peptides α-, β-, and γ2-melanocyte
stimulating hormones (MSH) and adrenocorticotropin
(ACTH).8−10 The human melanocortin-4 receptor (MC4R)
has been identified by genomic wide association studies as well
as in individual morbidly obese humans to be a locus connected
to obesity.11,12 Greater than 100 single nucleotide poly-
morphisms (SNPs) have been identified to date in obese and
nonobese human control adults and children. Substantial efforts
have been undertaken to characterize these hMC4R SNPs both
physiologically and in vitro to determine molecular deficits
Received: January 13, 2014
Published: February 11, 2014
© 2014 American Chemical Society
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Figure 1. Putative locations of the I69T, I102S, L106P, A219V, C271Y, and C271R SNPs within the serpentine structure of the hMC4R.
Figure 2. Summary of the previously reported in vitro pharmacological characterization of the L106P hMC4R SNP as compared with the wild-type
(WT) hMC4R control stably expressed in HEK293 cells.13,19 (A) Fluorescence activated cell sorting (FACS) demonstrating that both the WT and
L106P hMC4R proteins are expressed within the cell at approximately the same levels (white box) yet reduced cell surface expression is observed for
the L106P hMC4R (black box). (B) Represents the total specific binding counts per minute (cpm) of radiolabeled I125‑NDP-MSH agonist binding
to the cells stably expressing the WT and L106P hMC4Rs. (C) Illustrates the ligand binding affinity curves of the WT and L106P hMC4Rs
competing I125-NDP-MSH and unlabeled NDP-MSH in a dose−response fashion that result in the same IC50 values, within experimental error. (D)
Illustrates the pharmacological agonist dose−response curves for the endogenous melanocortin agonists α-, β-, and γ2-MSH and ACTH at the L106P
hMC4R.
obesity. Toward this objective, we hypothesized that we could
identify molecules that could target hMC4R SNPs that are
expressed at the cell surface but do not respond normally to the
endogenous agonists α-, β-, and γ2-MSH and ACTH. Different
experimental approaches are available including a rational
ligand design/discovery strategy,13 however, this is limited by
the known structure−activity relationships (SAR) and the
assumptions and limitations of our current knowledge. An
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Table 1. Amino Acid Sequences of the Endogenous and Synthetic Melanocortin Ligands Characterized at the Wild-Type (WT)
and L106P hMC4R
name
α-MSH
amino acid sequence
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
Ala-Glu-Lys-Lys-Asp-Glu-Gly-Pro-Tyr-Arg-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Pro-Lys-Asp
Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly
Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Tyr-Pro-Asn
Ac-His-DPhe-Arg-Trp-NH2
β-MSH
γ2-MSH
ACTH(1-24)
(1) JRH887-9
(2) JRH420-12
(3) JRH322-18
peptide
Ac-Anc-DPhe-Arg-Trp-NH2
Ac-His-(pI)DPhe-Arg-Trp-NH2
a
a
WT hMC4R agonist EC50 (nM)
L106P hMC4R agonist EC50 (nM)
α-MSH
0.65 0.19
0.42 0.13
73 24
50% @ 1 μM
356 53
β-MSH
γ2-MSH
2660 370
40% @ 1 μM
191 15
ACTH(1-24)
(1) JRH887-9
(2) JRH420-12
(3) JRH322-18
0.65 0.15
0.93 0.3
2.17 0.82
1.25 0.25
60 12
52 10
a
Previously published values from refs 13 and 19. The mean of at least three independent experiments the standard error of the mean (SEM) is
provided. A % indicates that at the highest concentration tested, some stimulatory response was observed, but not the full efficacy observed for the
nonreceptor dependent forskolin control.
alternative unbiased approach involves the screening of
synthetic combinatorial libraries that are designed with no
preconceived ligand SAR assumptions. This approach has
previously been described for the discovery of novel ligands,14
and in particular for μ, δ, and κ opioid receptors15 as well as
formyl peptide receptors (FPR1 and FPR2)16 among others.
To test this hypothesis and validate this experimental
approach for polymorphic hMC4 receptors, the L106P
hMC4R (Figure 1) was selected as a rigorous target for the
study herein. The L106P hMC4R SNP was identified in an
individual included in a 350 patient study of severe early onset
obesity (including 108 control patients) and pharmacologically
characterized in vitro to modify cell surface expression levels as
well as affect ligand function.17,18 Studies by our laboratory
pharmacologically characterized this L106P hMC4R SNP to
possess reduced cell surface expression, normal NDP-MSH
ligand binding affinity, and significantly impaired endogenous
agonist potency (Figure 2 and Table 1).19 Upon the basis of the
location at the juncture of the receptor TM domain and the
extracellular portion, as well as the substitution from the flexible
aliphatic Leu amino acid to the constrained Pro residue that is
known to be a “helix breaker,” it could be envisioned that this
particular SNP might be modifying the putative receptor
binding pocket important for molecular recognition and ligand
accessibility crucial for the formation of the ligand−receptor
complex required for initiating the intracellular signal trans-
duction process. Further characterization of the L106P hMC4R
in attempts to identify ligands based upon rational design
approaches13 resulted in the discovery that the tetrapeptides
JRH887-9 (1), JRH420-12 (2), and JRH322-18 (3) possessed
nM full agonist potencies whereas the endogenous agonists did
not (Table 1).20,21 The tetrapeptides were designed based upon
the common melanocortin core His-Phe-Arg-Trp conserved
sequence (Table 1) present in all the endogenous melanocortin
agonists reported to date. These core tetrapeptide residues have
been widely accepted to contain the critical pharmacophore
domain for melanocortin receptor selectivity (versus other
GPCRs) and agonist induced cAMP signal transduction. Thus,
this polymorphic L106P hMC4R presented itself as an
opportunity to test our hypothesis. Herein, we present the
successful application of this approach to identify molecules
that can not only restore full functional signaling and efficacy of
the L106P hMC4R SNP but results in the discovery of
previously unidentified chemotypes for further melanocortin
based drug discovery and development efforts. It is worthy of
note that some of these chemotypes are more potent than the
lead tetrapeptides previously reported in the literature. As it has
been established that hMC4R SNPs constitute 1−6% of the
genetic modifications identified in morbidly obese (BMI >
30%) human patients and to further explore the applicability of
the newly discovered tetrapeptides to extend to other hMC4
polymorphic receptors that do not respond normally to the
endogenous agonists, the additional five hMC4R SNP
receptors, I69T, I102S, A219V, C271Y, and C271R, distributed
at different positions within the protein (Figure 1), were
examined with the eight selected L106P “hit” ligands.
Melanocortin receptor subtype selectivity profiles at the
mouse MC1R, MC3R, MC4R, and MC5R was also
characterized for the eight “hit” tetrapeptides.
RESULTS AND DISCUSSION
■
The wild type and L106P, I69T, I102S, A219V, C271Y, and
C271R hMC4R stably expressing HEK293 cells previously
characterized by our laboratory were used for the studies
presented herein.13,19,22 A mixture based positional scanning
combinatorial library (TPI 924), consisting of the Ac-
tetrapeptide-NH2 template composed of 60 different amino
acids (comprised of natural L-amino acids, their D-isomers, and
unnatural amino acids) at each position and containing a total
of 12960000 tetrapeptides was screened at the L106P hMC4R
at 100 μg/mL concentrations in a 96-well plate format. Figure 3
illustrates the results from the primary agonist functional screen
with the average of duplicate wells that have been normalized to
the protein content of each well as well as the average of
quadruple wells for the nonreceptor dependent forskolin
control. Forskolin was selected as a positive control as it is
well established to stimulate a cAMP signal transduction
response directly and is not dependent upon the melanocortin
receptor. Results with an agonist stimulation response greater
than 0.7 were considered “hits” at the indicated position. These
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Figure 3. Illustration of the primary screening results of the TPI 924 tetrapeptide library at the L106P hMC4R. The primary screen was assayed
using a rough approximation at 100 μg/mL concentrations. The X-axis represents the amino acid residue that was held constant at that position (O)
of the tetrapeptide library with the three remaining positions composed of mixtures of the 60 amino acids (X), and the Y-axis represents the
functional agonist activity observed. The agonist activity (average of duplicate wells) was determined using a β-galactosidase colorimetric reporter
gene bioassay that has been normalized to both relative protein content as well as the maximal value observed for the nonreceptor dependent
forskolin control (average of four wells). A value of 1 indicates a result that is able to generate the same maximal stimulation level observed for the
forskolin control. The dotted line for each position indicates the criteria of >0.7 that was used as the cut-off point for classification as deconvolution
“hits”.
are summarized in Table 2. The threshold of 0.7 was selected to
increase the chances for the identification of potent full agonist
compounds while balancing the resource considerations of
time, labor, and expense regarding the number of individual
ligands that would need to be synthesized and analytically and
pharmacologically characterized.
Of first importance is to validate the overall screening
experiment. This validation is supported by the fact that this
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parallel approaches. First, we synthesized tetrapeptides with the
amino acids identified at the respective positions from the
tetrapeptide library screening (Tables 2 and 3) into the
tetrapeptide template Ac-His-DPhe-Arg-Trp-NH2 (1). Using
this approach, we did not identify any new sequences with
agonist EC50 values less than 40 nM at the L106P hMC4R.
The second approach was to utilize the deconvolution of the
positional scanning library, where the individual tetrapeptides
were selected for synthesis by combining the defined
functionalities of the most active mixtures at each position
(Figure 3 and Table 2). This was done by testing the active
mixtures from the library in a dose−response manner in order
to identify the most active mixtures at each of the four positions
of the tetrapeptide library. By ranking these mixtures at each
position by the activities, one can then select the amino acid
that is defined in each active mixture at each position. In this
case, three amino acids were selected from position 1 (Tic, His,
Arg), two amino acids from position 2 [(pI)DPhe, (pCl)-
DPhe], two amino acids from position 3 (Tic, Arg), and three
amino acids from position 4 [(pNO2)DPhe, (pI)Phe, Tic],
Figure 4. The combinations of these amino acids were used to
design a set of tetrapeptides and resulted in 36 (3 × 2 × 2 × 3)
unique sequences. These tetrapeptides were synthesized, and
the results of the agonist pharmacology are summarized in
Table 4 for the L106P and wild type control hMC4Rs. This
series of tetrapeptides resulted in identification of the
tetrapeptides Ac-His-(pI)DPhe-Arg-(pI)Phe-NH2 (1981-11,
5), Ac-Arg-(pI)DPhe-Tic-(pNO2)DPhe-NH2 (1981-13, 6),
and Ac-Arg-(pI)DPhe-Arg-(pI)Phe-NH2 (1981-17, 7) possess-
ing 2−4 nM full agonist EC50 values at the L106P hMC4R and
Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2 (1981-7, 4) that
possessed a 10 nM full agonist EC50 value at the L106P
hMC4R, as it could be anticipated based upon the results of the
primary screen (Figure 3). Also, peptides containing (pCl)-
Table 2. Summary of the Primary Screening Deconvolution
Hits at Each Position That Resulted in the Criterion of a
Stimulatory Response >0.7
a
Ac-AA1
His
AA2
Arg
AA3
AA4-NH2
Trp
Lys
Arg
Tic
Arg
DPhe
Tic
Trp
(pCl)DPhe
(pI)DPhe
(pCl)Phe
(pCl)DPhe
(pI)Phe
(pNO2)DPhe
Nal
DArg
Tyr
Tic
DTic
(pCl)DPhe
(pI)DPhe
(3I)Tyr
a
AA represents amino acid and the position in the tetrapeptide
template.
mixture based positional scanning library approach identified
active mixtures that contained amino acids in defined positions
that corresponded to Ac-His-DPhe-Arg-Trp-NH2 (1) and Ac-
His-(pI)DPhe-Arg-Trp-NH2 (3) tetrapeptides that were
previously reported to stimulate the L106P hMC4R at nM
concentrations.13 The Anc residue of tetrapeptide 2 was not
included in the library screened. Furthermore, additional
mixtures defined with other residues were consistent with
previously reported SAR studies of similar tetrapeptides at the
mouse melanocortin receptors.20,21,23−25 The library screening
also identified the mixtures fixed with (pI)DPhe and (pCl)-
DPhe at the second amino acid position of the tetrapeptide as
active, confirming previous in vitro SAR studies20,21 and in vivo
feeding studies26 demonstrating the Ac-His-(pI)DPhe-Arg-Trp-
NH2 (3) tetrapeptide as a pharmacological tool and implicating
a role for the centrally expressed MC3R to be involved in the
regulation of food intake.26 At this juncture, we took two
Table 3. Summary of the Individually Synthesized Tetrapeptides Incorporating Amino Acids Identified from the Primary Screen
a
into the Ac-His-DPhe-Arg-Trp-NH2 Melanocortin Agonist Template
compd
EMH reference peptide
sequence
WT hMC4R agonist EC50 (nM)
L106P hMC4R agonist EC50 (nM)
1
control (JRH887-9)
EMH4-91
Ac-His-DPhe-Arg-Trp-NH2
2.6 1.6
215 83
124 28
Ac-Arg-DPhe-Arg-Trp-NH2
Ac-Trp-DPhe-Arg-Trp-NH2
Ac-Tyr-DPhe-Arg-Trp-NH2
Ac-DArg-DPhe-Arg-Trp-NH2
Ac-His-Arg-Arg-Trp-NH2
1.06 0.24
EMH4-92
32
6.2
40
8
2
5
170 114
EMH4-93
540 195
EMH4-94
1242 360
4940 770
6240 506
2096 695
8230 4810
4940 1980
17920 1150
4440 830
4910 2630
EMH4-95
75 19
40 16
EMH4-96
Ac-His-DPhe-Lys-Trp-NH2
EMH4-97
Ac-His-DPhe-Arg-(pCl)Phe-NH2
Ac-His-DPhe-Arg-(pCl)DPhe-NH2
Ac-Tic-DPhe-Arg-Trp-NH2
17
46 26
15
297 59
48
5
EMH4-98
EMH4-99
7
EMH4-100
EMH4-101
EMH4-102
EMH4-103
EMH4-104
EMH4-105
EMH4-106
EMH4-107
EMH4-108
EMH4-109
EMH4-110
Ac-DTic-DPhe-Arg-Trp-NH2
Ac-(pCl)DPhe-DPhe-Arg-Trp-NH2
Ac-(pI)DPhe-DPhe-Arg-Trp-NH2
Ac-(3I)Tyr-DPhe-Arg-Trp-NH2
Ac-His-(pCl)DPhe-Arg-Trp-NH2
Ac-His-(pI)DPhe-Arg-Trp-NH2
Ac-His-DPhe-Tic-Trp-NH2
4
76 35
1.9 0.40
0.28 0.04
1.3 0.47
204 73
43
7
12
3
48 16
97 48
18800 5170
>100,000
Ac-His-DPhe-Arg-Tic-NH2
>10,000
Ac-His-DPhe-Arg-(pI)Phe-NH2
Ac-His-DPhe-Arg-(pNO2)Phe-NH2
Ac-His-DPhe-Arg-Nal(1′)-NH2
5.3 1.3
195 20
680 150
23000 9400
4280 1120
28
1
a
The indicated errors represent the standard error of the mean determined from at least three independent experiments; >10000 and >100000
indicates that agonist or antagonist activity was not observed for these compounds at up to 10 and 100 μM concentrations respectively.
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multiple polymorphic receptors for ligands identified for the
L106P hMC4R, the identification of ligands that restore agonist
activity of more than one polymorphic receptor could be
explored directly using the screening results of a combinatorial
library with the polymorphic hMC4Rs of interest.
Melanocortin Receptor Subtype Profiles at the Mouse
MC1R and MC3-5Rs and Unanticipated Structure
Activity Relationships (SAR). The mouse melanocortin
receptor isoforms, versus the human, were selected to
characterize differences in ligand−receptor subtype profiles as
any interesting compounds in our laboratory are examined
subsequently in vivo in the wild-type and/or melanocortin
knockout receptor mouse models.26,34−39 Table 6 summarizes
the selected eight tetrapeptides at the mouse MC1, MC3, MC4,
and MC5 receptors. All these tetrapeptides are full agonists
with potencies ranging from ca. 0.5 to 94 nM at these mouse
melanocortin receptors examined. As both the MC3 and MC4
receptors are expressed in the hypothalamus of the brain,
selectivity between these two isoforms could be important for
the interpretation of physiological in vivo data. The
tetrapeptides in Table 6 exhibited modest MC4R versus
MC3R selectivity profiles with the 4, 5, and 6 possessing 15−
17-fold selectivity. At the mMC1R, which is expressed primarily
in the skin, the full agonist potency EC50 values of these
compounds range from ∼3 to 40 nM. At the mMC3R, which is
expressed both centrally and peripherally, the full agonist
potency EC50 values range from 12 to 94 nM. At the mMC4R,
expressed primarily in the brain and CNS, full agonist potency
EC50 values range from 0.7 to less than 8 nM. At the mMC5R,
which is the most widely expressed subtype both in the brain,
CNS, and periphery, full agonist potency EC50 values range
from 0.45 to ∼5 nM and possessed the greatest ligand potency
versus the other receptor isoforms.
The tetrapeptide 3 containing the single (pI)DPhe at the
second position has been previously reported to result in partial
agonist/antagonist mMC3R and full agonist mMC4R pharma-
cological profiles.21,24 Additionally, in the NDP−MSH 13
amino acid peptide template, the (pI)DPhe7 (α-MSH
numbering) containing peptide was reported to result in partial
agonist/antagonist pharmacological profiles at both the hMC3
and hMC4 receptors.40 Incorporation of the (pI)DPhe into a
chimeric AGRP−melanocortin template resulted in a mMC3R
antagonist with partial agonist/antagonist pharmacology at the
mMC4R.41 Despite the previously reported melanocortin
ligand SAR, the four multiple substitution containing
tetrapeptides possessing the (pI)DPhe at the second position
examined at the mouse MCRs resulted in full agonists at the
mMC3R (Table 6). Furthermore, on the basis of melanocortin
receptor mutagenesis studies and the majority of ligand SAR,
presence of the Arg residue has been previously hypothesized as
important for ligand potency, molecular recognition, and
putative ligand−receptor interactions important for agonist
induced signal transduction.42−56 However, it was noted in a
few reports that the Arg residue could be replaced by Ala or
other residue/side chain modifications and still retain agonist
functionality at the melanocortin receptors.25,55,57 Herein, the 4
and 8 are further unanticipated examples of melanocortin
ligands; specifically, they are tetrapeptides that do not contain
the Arg side chain moiety and are still full agonists at the
melanocortin receptors. Surprisingly, these two tetrapeptides
are not only sub-nM to nM full agonists at the mMC1,
mMC3−5Rs (Table 6), but they also possess nM full agonist
potency at all the six the polymorphic hMC4Rs examined in
Figure 4. Summary of the key amino acid structures used in this study.
DPhe at the second position resulted in the tetrapeptide Ac-
Arg-(pCl)Phe-Tic-(pNO2)DPhe-NH2 (2073-18, 10) that pos-
sessed ca. 3 nM full agonist efficacy at the L106P hMC4R while
the Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2 (2073-16, 9), Ac-
Arg-(pCl)DPhe-Arg-(pI)Phe-NH2 (2073-22, 11), and Ac-His-
(pCl)DPhe-Tic-(pNO2)DPhe-NH2 (2073-12, 8) possessed 10,
13, and 15 nM full agonist EC50 values at the L106P hMC4R,
respectively. Thus, we have provided conclusive experimental
data to support the hypothesis that novel and potent molecules
could be identified to target hMC4R SNPs that are expressed at
the cell surface but do not respond normally to the endogenous
agonists α-, β-, and γ2-MSH and ACTH using an unbiased
mixture based positional scanning combinatorial library screen-
ing approach.
Effects at Other Polymorphic hMC4Rs. Our laboratory
has previously reported the side-by-side pharmacological
comparison of 70 polymorphic hMC4Rs.13,19,22 Because the
above tetrapeptides 4−11 were identified as possessing full
agonist nM potency at the L106P polymorphic hMC4R, we
wanted to evaluate these selected ligands at other polymorphic
hMC4Rs that did not respond normally to the endogenous
agonists (Table 5). The I69T,17,22,27 I102S,13,19,28−30
A219V,22,31 C271Y,13,17−19,32,33 and C271R17,22 hMC4Rs
were selected (Figure 1) to be characterized with the eight
tetrapeptides summarized in Table 5. These polymorphic
hMC4R amino acid substitutions are located in distinct regions
of the receptor and putatively have different molecular features
important for the hMC4R molecular recognition and functional
agonist stimulation. Four (4, 8, 9, and 10) of the eight
tetrapeptides could stimulate nM to μM full agonist functional
response at these additional five polymorphic hMC4Rs. The
other four tetrapeptides were able to stimulate a full agonist
response at the I69T, I102S, and A219V hMC4Rs with 6, a
weak μM full agonist at the C271Y hMC4R. Clearly, the
C271Y/R polymorphic receptors are the most challenging of
the polymorphic hMC4Rs examined in this study to restore full
agonist nM efficacy with the tetrapeptides examined. Although
we present here the determination of agonist activity at
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Table 4. Summary of the Individually Synthesized Tetrapeptides Based upon the Screening Data Obtained from the Positional
a
Scanning Library TPI924
a
The indicated errors represent the standard error of the mean determined from at least three independent experiments. A % indicates that at the
highest concentration tested, some stimulatory response was observed, but not the full efficacy observed for the nonreceptor dependent forskolin
control; >100000 indicates that agonist or antagonist activity was not observed for these compounds at up to 100 μM concentrations.
a
Table 5. Summary of the Selected Tetrapeptides at Other hMC4R Polymorphic Receptors
L106P
hMC4R
agonist
I69T
hMC4R
agonist
I102S
hMC4R
agonist
A219V
hMC4R
agonist
C271Y
hMC4R
agonist
C271R
hMC4R
agonist
WT hMC4R
agonist
EC50(nM)
compd
structure
EC50(nM)
EC50(nM)
EC50(nM)
EC50(nM)
EC50(nM)
EC50(nM)
b
1
Ac-His-DPhe-Arg-Trp-NH2
0.93 0.3
0.24 0.04
0.14 0.02
0.26 0.07
0.29 0.03
0.40 0.20
0.45 0.13
0.21 0.08
0.19 0.08
191 15
10 1.4
2.9 0.3
2.6 0.3
4.4 0.9
40.7 5.1
3.8 0.22
1.5 0.4
4.1 1.5
3.4 0.5
4.3 0.5
2.9 0.3
5.2 0.7
8.2 1.2
720 28
14 2.3
4.0 0.5
2.9 0.2
3.5 0.6
3.0 1.7
9.78 1.12
4.5 0.34
3.2 0.5
6.1 0.93
14 1.4
1200 110
190 42
1600 1060
295 90
>100000
4
Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2
Ac-His-(pI)DPhe-Arg-(pI)Phe-NH2
Ac-Arg-(pI)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Arg-(pI)DPhe-Arg-(pI)Phe-NH2
Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2
Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Arg-(pCl)DPhe-Arg-(pI)Phe-NH2
5
>100000
6
19650 6410
>100000
>100000
7
>100000
8
15
10
3
3
2.8 0.5
6.1 1.7
4.2 0.4
82
7
130
8
9
20
6
260 120
470 260
>100000
210 72
3390 2460
>100000
10
11
3.4 0.3
13 0.4
4.4 0.7
13 1.9
17
6
a
The indicated errors represent the standard error of the mean determined from at least three independent experiments. >100000 indicates that
b
agonist or antagonist activity was not observed for these compounds at up to 100 μM concentrations. Previously reported values in references 13
and 19.
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a
Table 6. Summary of the Selected Tetrapeptides at the Mouse Melanocortin Receptors for Subtype Selectivity Profiles
mMC1R EC50
(nM)
mMC3R EC50
(nM)
mMC4R EC50
(nM)
mMC5R EC50
(nM)
peptide
structure
NDP-MSH
Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe- Arg-Trp-Gly-Lys-Pro-Val-NH2
Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2
Ac-His-(pI)DPhe-Arg-(pI)Phe-NH2
0.088 0.031
2.9 0.8
12 3.8
2.7 1.3
40 11
0.33 0.08
0.12 0.01
0.26 0.05
0.84 0.15
0.45 0.13
1.1 0.24
3.0 1.3
4
35
5
2.4
1
5
11.7 1.0
0.70 0.13
2.7 1.2
4.3 1.1
5.2 1.1
3.1 0.2
6
Ac-Arg-(pI)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Arg-(pI)DPhe-Arg-(pI)Phe-NH2
47
22
8
9
7
8
Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2
4.0 1.2
19 0.9
22 16
55 13
19
94 16
21
0.63 0.2
2.4 0.6
9
8
10
11
Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Arg-(pCl)DPhe-Arg-(pI)Phe-NH2
7.6
2
1.0 0.1
24
7
4
7.4 1.1
5.1 0.9
a
The indicated errors represent the standard error of the mean from at least three independent experiments.
Figure 5. Summary of the Pipeline Pilot experimental design modular approach that was utilized in this study.
this study (Tables 4 and 5). To further probe their ligand
biophysical properties in attempts to identify a common ligand
feature between these Arg deficient ligands and the other
tetrapeptides examined in this study, the following computa-
tional biophysical analysis was performed.
hydrogen bonds between the ligand and receptor important for
the L−R complex activation of the agonist signal transduction
pathway. Remarkably however, 4 and 8, the most potent overall
compounds at all the polymorphic hMC4 receptors examined
herein, do not possess an Arg side chain. Thus, on the basis of
the studies performed herein, it is difficult to conclude if these
ligands are interacting at the receptors within the orthosteric
binding pocket or at alternative allosteric receptor binding sites
that may overlap, or be distinct from, the orthosteric site.
Furthermore, putative ligand−receptor interactions may be
either overlapping or distinct for each ligand at the different
polymorphic hMC4R and mouse receptor subtypes. Additional
studies, outside the scope of the current work, would need to
be performed to determine the molecular mechanism for these
putative ligand−melanocortin receptor molecular recognition,
binding, and agonist stimulation interactions.
Positional Scanning Deconvolution. The results pre-
sented herein offer an excellent comparison of the usage of the
data that result from screening a positional scanning
combinatorial library. In particular, while the use of these
data to make single-substitutions to a known ligand did result in
increases in activity, no resulting ligand was >10-fold more
active than the Ac-His-DPhe-Arg-Trp-NH2 (1) ligand control
(Table 3). In contrast, proper deconvolution of the positional
scanning library resulted in compounds representing an order
of magnitude, or greater (up to 80-fold) improvement in
activity. Indeed, while a 1-tail Fisher’s Exact Test categorizing
ligands based on improvement in activity over Ac-His-DPhe-
Arg-Trp-NH2 (1, EC50 < 215 nM) could not distinguish
between the single substitution ligands and the positional
scanning deconvolution ligands (p = 0.718), an analogous 1-tail
Fisher’s Exact Test categorizing ligands based on an order of
magnitude improvement in activity over Ac-His-DPhe-Arg-Trp-
NH2 (EC50 < 21.5 nM) showed a significant positive difference
in the positional scanning deconvolution of ligands over the
single substitution ligands (p = 0.021), Figure 7. These results
are not surprising because the individual peptides resulting
from a positional scanning deconvolution are precisely those
which the most active mixtures have in common and are
therefore the most likely to be driving the activity in those
mixtures. In contrast, a single substitution essentially implies
total independence of activity between positions, which need
Tetrapeptide Computational Analysis of Biophysical
Properties. Computational modeling studies of a previous
tetrapeptide SAR study had allowed us to hypothesize
differences in the ligand electrostatic surface biophysical
properties as a correlation with melanocortin receptor
pharmacology as opposed to differences in putative ligand−
receptor interactions.21 Thus, we wanted to apply a similar
approach for the tetrapeptides examined herein in attempts to
identify any potential biophysical properties that might facilitate
the development of a new ligand design hypothesis to explain
the observed receptor pharmacology data that could be
subsequently tested. Using Pipeline Pilot, the tetrapeptides
were input as SMILES strings and processed as indicated in
Figure 5. For the NDP−MSH, 1 and 3 control peptides, and
the 36 TPI combination tetrapeptides, the following biophysical
properties were calculated and summarized in the Supporting
Information: ALogP, number of H acceptors, number of atoms,
number of rotatable bonds, number of rings, number of
aromatic rings, Log D, molecular surface area, minimized
energy, and molecular 3D SASA. These properties were then
individually evaluated to determine if there was any correlation
with either polymorphic hMC4Rs and/or mouse melanocortin
receptor subtype functional pharmacology profiles using the
GraphPad Prism software. There were no observed correlations
in activity with respect to molecular weight, number of H bond
donors or acceptors, number of aromatic rings, molecular
surface area, and 3D solvent accessible surface area. Figure 6
illustrates the electrostatic surface area for the control Ac-His-
DPhe-Arg-Trp-NH2 (1), Ac-His-(pI)DPhe-Arg-Trp-NH2 (3),
and Ac-His-(pCl)DPhe-Arg-Trp-NH2 (12) control tetrapep-
tides as well as the eight tetrapeptides (4−11) that were tested
at all the polymorphic hMC4Rs and mouse MCRs examined in
this study (Tables 3−6). It is relatively easy to envision how the
tetrapeptide Arg amino acid side chain (shown in blue) could
be putatively interacting with the key acidic melanocortin
receptor resides Glu (TM2) and two Asp residues (TM3)
previously postulated to form a network of ionic and potential
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Figure 6. Electrostatic surface area for the control Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9 1), Ac-His-(pI)DPhe-Arg-Trp-NH2 (EMH4-105 3), and
Ac-His-(pCl)DPhe-Arg-Trp-NH2 (EMH4-104 12) control tetrapeptides as well as the 8 tetrapeptides (4−11) that were tested at all the
polymorphic hMC4Rs and mouse MCRs examined in this study (Tables 5 and 6). The His/Arg residue at the first position is oriented at the top of
the molecule, and the Arg/amino acid side chain at the third position is oriented down. The electrostatic surfaces were calculated using Maestro 9.5
(Schrodinger) with red (−5.0) to blue (+5.0) using the solute dielectric constant of 10 and the solvent dielectric constant of 80.
̈
not be the case. In fact, the resulting data clearly shows two
different structural families associated with different combina-
tions of amino acids in the third and fourth positions (Table 7);
while four of the six peptides having Arg at the third and
(pI)Phe in the fourth position were more active than Ac-His-
DPhe-Arg-Trp-NH2, and likewise, four of six of the peptides
having Tic at the third position and (pNO2)DPhe in the fourth
position, were more active than Ac-His-DPhe-Arg-Trp-NH2,
not a single positional scanning peptide tested was more active
than Ac-His-DPhe-Arg-Trp-NH2 when the third and fourth
position combinations were reversed (Arg in the third with
(pNO2)DPhe in the fourth or Tic in the third with (pI)Phe in
the fourth). This activity driven by a multipositional
combination of amino acids perfectly illustrates the need for
a full positional scanning deconvolution approach. It should be
noted that the two potent arginine lacking peptides 4 and 8 are
members of the Tic in the third position and (pNO2)DPhe in
the fourth position family, perhaps further supporting the
hypothesis of a different ligand−protein binding mode for these
peptides as discussed in the previous section.
CONCLUSIONS
■
The study performed herein, using a nonbiased tetrapeptide
ligand discovery positional scanning library approach, has
identified four tetrapeptide ligands that were able to restore full
nM agonist potency to the L106P, I69T, I102S, A219V, C271Y,
and C271R polymorphic hMC4Rs identified in obese human
patients, expressed at the cell surface, and did not respond
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Table 7. Tetrapeptide Structural Families Based on Position
a
3 and 4 Substitutions
L106P hMC4R
agonist EC50
peptide
sequence
(nM)
P3 Arg and P4 (pI)Phe
1981-5
Ac-Tic-(pI)DPhe-Arg-(pI)Phe-NH2
Ac-His-(pI)DPhe-Arg-(pI)Phe-NH2
Ac-Arg-(pI)DPhe-Arg-(pI)Phe-NH2
Ac-Tic-(pCl)DPhe-Arg-(pI)Phe-NH2
Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2
Ac-Arg-(pCl)DPhe-Arg-(pI)Phe-NH2
440 43
2.9 0.3
4.4 0.9
380 78
1981-11
1981-17
2073-10
2073-16
2073-22
10
3
13 0.4
P3 Tic and P4 (p NO2)DPhe
2073-1
1981-7
1981-13
2073-6
2073-12
2073-18
Ac-Tic-(pI)DPhe-Tic-(pNO2)DPhe-NH2
280 53
10 1.4
2.6 0.3
310 50
Figure 7. Comparison of the single substitution (Table 3) and
positional scanning deconvolution approaches (Table 4) using the
L106P hMC4R pharmacological data. (A) When considering only
improvements on the activity of the known ligand Ac-His- DPhe-Arg-
Trp-NH2 (1, EC50 < 215 nM), there is no statistical difference
between single substitution and positional scanning deconvolution; 5
of the 20 single substitution compounds had an EC50 < 215,
comparable to the 8 of the 36 positional scanning deconvolution
compounds (Fisher’s Exact Test, 1-tail, p = 0.718). (B) When
considering improvements an order of magnitude or greater over the
known ligand (EC50 < 21.5 nM), the difference is readily apparent and
statistically significant; none of the 20 single substitution compounds
had an EC50 <21.5 nM, but 8 of the 36 positional scanning
deconvolution compounds did (Fisher’s Exact Test, 1-tail, p = 0.021).
Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Arg-(pI)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Tic-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2
15
3
3.4 0.3
P3 Arg and P4 (p NO2)DPhe
1981-4
Ac-Tic-(pI)DPhe-Arg-(pNO2)DPhe-NH2
657 70
293 122
655 50
1981-10
1981-16
2073-9
Ac-His-(pI)DPhe-Arg-(pNO2)DPhe-NH2
Ac-Arg-(pI)DPhe-Arg-(pNO2)DPhe-NH2
Ac-Tic-(pCl)DPhe-Arg-(pNO2)DPhe-NH2
Ac-His-(pCl)DPhe-Arg-(pNO2)DPhe-NH2
Ac-Arg-(pCl)DPhe-Arg-(pNO2)DPhe-NH2
710 240
3210 2030
1100 320
2073-15
2073-21
P3 Tic and P4 (pI)Phe
normally to the endogenous melanocortin receptor agonists.
Two tetrapeptides that were the most potent at the
polymorphic hMC4Rs examined in this study, 4 and 8 did
not contain an Arg amino acid previously postulated as
important for melanocortin ligand−receptor molecular recog-
nition and agonist potency/efficacy. The results of this study
have generated new SAR and pharmacophore templates for the
further development of melanocortin receptor molecular
probes and potential therapeutic molecules. One can also
envision using the positional scanning library approach to
identify ligands specific for each SNP based upon deconvolu-
tion specifically for each SNP that was screened.
1981-2
1981-8
1981-14
2073-7
2073-13
2073-19
Ac-Tic-(pI)DPhe-Tic-(pI)Phe-NH2
70% @ 10 μM
570 50
Ac-His-(pI)DPhe-Tic-(pI)Phe-NH2
Ac-Arg-(pI)DPhe-Tic-(pI)Phe-NH2
Ac-Tic-(pCl)DPhe-Tic-(pI)Phe-NH2
Ac-His-(pCl)DPhe-Tic-(pI)Phe-NH2
Ac-Arg-(pCl)DPhe-Tic-(pI)Phe-NH2
2230 490
2670 382
790 290
215 55
a
Selected peptides from positional scanning deconvolution were
sorted by functionality in the 3rd and 4th positions. The indicated
errors represent the standard error of the mean determined from at
least three independent experiments. A % indicates that at the highest
concentration tested, some stimulatory response was observed, but not
the full efficacy observed for the nonreceptor dependent forskolin
control.
EXPERIMENTAL SECTION
■
The TPI 924 N-acetylated C-amidated tetrapeptide positional
scanning library is composed of four sublibraries in which each of
the four positions are defined with a single amino acid (O) with the
three remaining positions made up of a mixture of 60 different L-, D-,
and unnatural amino acids (X). The 60 different amino acids are Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser,
Thr, Val, Trp, Tyr, DAla, DAsp, DGlu, DPhe, DHis, DIle, DLys,
DLeu, DMet, DAsn, DPro, DGln, DArg, DSer, DThr, DVal, DTrp,
DTyr, Nle, DNle, Cha, DCha, PyrAla, DPyrAla, ThiAla, DThiAla, Tic,
DTic, (pCl)Phe, (pCl)DPhe, (pI)Phe, (pI)DPhe, (pNO2)Phe,
(pNO2)DPhe, 2-Nal, 2-DNal, β-Ala, ε-Aminocaproic acid, Met[O2],
dehydPro, and (3I)Tyr.58,59 Thus, there are 60 acetylated tetrapeptide
mixtures per positional sublibrary totaling 240 acetylated tetrapeptide
mixtures, each made up of 216000 acetylated tetrapeptides, with the
library containing a diversity of 12960000 acetylated tetrapeptides.
The 240 aceylated tetrapeptide mixtures were synthesized using the
solid-phase simultaneous multiple peptide synthesis (SMPS) approach
on p-methylbenzhydrylamine (MBHA) polystyrene resin.60 Mixture
positions (X) were prepared using chemical mixtures of t-Boc
protected amino acids, yielding close to equimolar coupling of each
amino acid as previously described.61 Acetylation was achieved by
treating the resin bound tetrapeptide mixtures with excess amounts of
acetyl imidazole (40× excess) in dimethylformamide (0.3M) over-
night. Completion of all coupling reactions, amino acid and
acetylation, were monitored by ninhydrin. Side chain deprotection
and cleavage from the resin support were achieved using low-HF and
high-HF procedures.62,63 The 240 mixtures were individually extracted
with 95% acetic acid in water, lyophilized, and resuspended three
additional times using 50:50 (acetonitrile:water). The 240 mixture
samples were then individually brought up in 50% DMF/water at a
final concentration of 20 mg/mL. Individual acetylated tetrapeptides
identified from the results of the library screening were synthesized
using the SMPS approach, and the purity (>95%) and identity of each
compound were confirmed by LCMS (Supporting Information).
The EMH single tetrapeptide synthesis was performed using
standard 9-fluorenylmethoxycarbonyl (Fmoc) methodology in a CEM
Discover SPS microwave peptide synthesizer.64,65 The amino acids
Fmoc-His(trt), Fmoc-DPhe, Fmoc-Arg(Pbf), Fmoc-Trp(Boc), Fmoc-
Tyr(tBu), Fmoc-DArg(Pbf), Fmoc-Tic (Synthetech), Fmoc-DTic
(Synthetech), Fmoc-(pCl)DPhe, Fmoc-(pI)DPhe (Synthetech),
Fmoc-(3I)-Tyr(tBu) (Anaspec), Fmoc-Lys(Boc), Fmoc-(pCl)Phe,
Fmoc-(pNO2)DPhe, and Fmoc-Nal(1′) were purchased from Peptides
International (Louisville, KY, USA) unless specified. The tetrapeptides
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were assembled on Rink-amide-p-methylbenzylhydrylamine (Rink-
amide-MBHA, 0.37meq/g substitution, 0.3mmol scale). The resin was
placed in a reaction vessel (25 mL CEM reaction vessel) and allowed
to swell for 2 h to overnight in dichloromethane (DCM). All reagents
were ACS grade or better. The Fmoc protecting groups were removed
using 20% piperidine (Sigma-Aldrich) in N,N-dimethylformamide
(DMF) outside the instrument for 2 min and then deprotection
solution was washed away. Additional 20% piperidine/DMF solution
was added to the resin and further deprotected using the following
conditions: temperature = 75 °C, power = 30 W (W), time = 4 min
with nitrogen bubbling the solution. After 3−5 min of a cooling down
period, the reaction vessel was removed from the instrument to
continue with synthesis. Amino acid coupling (3-fold excess) was
accomplished using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HBTU, 3-fold excess) with a 6-fold
addition of N,N-diisopropylethylamine (DIEA) and the desired amino
acid (3-fold excess) dissolved in minimum DMF (DCM was not used
in microwave synthesis). The microwave synthesizer conditions for
amino acid coupling conditions varied for cysteine and histidine (50
°C, 30W, 5 min), arginine (75 °C, 30 W, 10 min), and for all the other
amino acids used (75 °C, 30W, 5 min). After a 3−5 min cool down
period, the resin was washed and the method of deprotection and
coupling was repeated until the desired chain was synthesized.
After each amino acid coupling and Fmoc deprotection step, the
peptide sequence was monitored using the Kaiser/ninhydrin test.66 All
peptides were N-terminally acetylated with a 3:1 mixture of acetic
anhydride and pyridine that bubbled with the resin bound peptide for
30−45 min. Final peptide cleavage from the resin and amino acid side
chain protecting group removal was performed using 95% trifluoro-
acetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% water for
2−3 h at room temperature. After cleavage and side chain
deprotection, the solution was concentrated and the peptide was
precipitated and washed using cold (4 °C), anhydrous diethyl ether.
Ligands were purified by reverse phase-high performance liquid
chromatography (RP-HPLC) using a Shimadzu chromatography
system with a photodiode array detector and a semipreparative RP-
HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 cm × 25
cm) and lyophilized. The purified peptides were analyzed using RP-
HPLC with an analytical Vydac C18 column (Vydac 218TP104). The
purified peptides were at least >95% pure as determined by RP-HPLC
in two diverse solvent systems (10% acetonitrile in 0.1% trifluoroacetic
acid/water and a gradient to 90% acetonitrile over 35 min or 10%
methanol in 0.1% trifluoroacetic acid/water and a gradient to 90%
methanol over 35 min). Molecular mass was determined by mass
spectrometry (Voyager-DE Pro, University of Florida Protein Core
Facility), see Supporting Information.
Bioassay. cAMP Based Functional Bioassay. Peptide ligands were
dissolved in DMSO at a stock concentration of 10−2 M and stored at
−20 °C until assayed. HEK-293 cells stably expressing the mutant and
wild-type melanocortin receptors were transiently transfected with 4
μg of CRE/β-galactosidase reporter gene as previously de-
scribed.13,19,42,67 Briefly, 5000−15000 post transfection cells were
plated into collagen treated 96-well plates (Nunc) and incubated
overnight. Forty-eight hours post-transfection, the cells were
stimulated with 50 μL of peptide (10−5−10−12 M for single
tetrapeptides in dose−response or ca. 100 μg/mL for screening) or
forskolin (10−4 M) control in assay medium (DMEM containing 0.1
mg/mL BSA and 0.1 mM isobutylmethylxanthine) for 6 h. [For
screening, each plate was visually inspected under a microscope to
determine if cells were healthy or had been killed during the
compound stimulation process. All the cells were observed to be
normal and healthy after the 6 h stimulation in the screening assays
involving the TPI 924 library.] The assay media was aspirated, and 50
μL of lysis buffer (250 mM Tris-HCl pH = 8.0 and 0.1% Triton X-
100) was added. The plates were stored at −80 °C overnight. The
plates containing the cell lysates were thawed the following day.
Aliquots of 10 μL were taken from each well and transferred to
another 96-well plate for relative protein determination. To the cell
lysate plates, 40 μL of phosphate-buffered saline with 0.5% BSA was
added to each well. Subsequently, 150 μL of substrate buffer (60 mM
sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM β-
mercaptoethanol, 2 mg/mL ONPG) was added to each well and the
plates were incubated at 37 °C. The sample absorbance, OD405, was
measured using a 96-well plate reader (Molecular Devices). The
relative protein was determined by adding 200 μL of 1:5 dilution Bio
Rad G250 protein dye:water to the 10 μL of cell lysate sample taken
previously, and the OD595 was measured on a 96-well plate reader
(Molecular Devices). Data points were normalized to forskolin and the
relative protein content. The EC50 values represent the mean of three
or more independent experiments. The EC50 estimates, and their
associated standard errors of the mean, were determined by fitting the
data to a nonlinear least-squares analysis using the PRISM program
(v4.0, GraphPad Inc.).
Ligand Biophysical Computational Analysis. Pipeline Pilot 8.5
was used to calculate a series of biophysical properties on the
tetrapeptides examined in this study. These calculated values were
used to probe the structure−activity relationship. A protocol
developed in Pipeline Pilot allowed for the importation of molecular
data, calculation of the biophysical properties, and output into an Excel
format (Figure 5). The protocol started with the importation of the
tetrapeptide SMILES from an Excel file. From this, Pipeline Pilot
converted the SMILES into a 2D molecular representation. The ligand
preparation component ionized each of the peptides to a pH of 7.4,
fixed bad valencies, and generated a 3D structure. The next three
components calculated ALogP, molecular weight, number of H bond
donors and acceptors, number of rotatable bonds, number of rings and
aromatic rings, Log D at pH 7.4, and molecular surface area. Following
these calculations, 3D coordinates were assigned to each peptide. The
energy was then minimized using a maximum number of 1000 steps
and a convergence energy difference of 0.0001, and the 3D coordinates
were then updated. The surface area and volume component then
calculated the molecular 3D solvent accessible surface area. All of the
calculated properties were then assembled into an Excel file and
analyzed using GraphPad Prism 4. The measured functional activity
for each of the peptides was plotted as a −Log10(EC50) value with
respect to the above-described biophysical properties.
ASSOCIATED CONTENT
■
S
* Supporting Information
Pipeline Pilot biophysical properties and analytical information
for the single tetrapeptides synthesized and characterized in this
study. Purity for these compounds is >95%. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 612-626-9262. Fax: 612-626-3114. E-mail: chaskell@
umn.edu. Address: Department of Medicinal Chemistry,
University of Minnesota, 308 Harvard Street SE, Minneapolis,
Minnesota, 55455, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work has been supported in part by the Minnesota
Supercomputing Insititute and NIH grants RO1DK064250
(C.H.L.), R01DK091906 (C.H.L.), and RO1DA031370
(R.A.H.), and the State of Florida, Executive Office of the
Governor’s Office of Tourism, Trade, and Economic Develop-
ment (R.A.H.).
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