Potent agonists of the protease activated receptor2 (PAR2)

Article abstract of DOI:10.1021/jm1013049

Novel peptidomimetic pharmacophores to PAR₂ were designed based on the known activating peptide SLIGRL-NH₂. A set of 15 analogues was evaluated with a model cell line (16HBE14o-) that highly expresses PAR ₂. Cells exposed to the PAR₂ activating peptide with N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH₂) initiated increases in intracellular calcium concentration ([Ca²⁺]_i EC₅₀ = 0.84 μM) and in vitro physiological responses as measured by the xCELLigence real time cell analyzer (RTCA EC₅₀ = 138 nM). We discovered two selective PAR₂ agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca²⁺ EC₅₀ = 1.77 μM, RTCA EC₅₀ = 142 nM) and compound 2 (6-aminonicotinyl; Ca²⁺ EC₅₀ = 2.60 μM, RTCA EC₅₀ = 311 nM). Unlike the previously described agonist, these novel agonists are devoid of the metabolically unstable 2-furoyl modification and thus provide potential advantages for PAR₂ peptide design for in vitro and in vivo studies. The novel compounds described herein also serve as a starting point for structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists and antagonists of PAR ₂.

Full text of DOI:10.1021/jm1013049

ARTICLE
pubs.acs.org/jmc
Potent Agonists of the Protease Activated Receptor 2 (PAR2)
Scott Boitano,†,‡ Andrea N. Flynn,†,‡ Stephanie M. Schulz,†,‡ Justin Hoffman,†,‡ Theodore J. Price,§ and
Josef Vagner*,‡
†Arizona Respiratory Center and Department of Physiology, University of Arizona, 1501 N. Campbell Avenue, Tucson, Arizona 85724,
United States
‡The BIO5 Research Institute, University of Arizona, 1657 E. Helen Street, Tucson, Arizona 85721, United States
§Department of Pharmacology, University of Arizona, 1501 N. Campbell Avenue, Tucson, Arizona 85724, United States
S Supporting Information
b
ABSTRACT: Novel peptidomimetic pharmacophores to PAR2 were designed based on the known
activating peptide SLIGRL-NH2. A set of 15 analogues was evaluated with a model cell line
(16HBE14o-) that highly expresses PAR2. Cells exposed to the PAR2 activating peptide with
N-terminal 2-furoyl modification (2-furoyl-LIGRLO-NH2) initiated increases in intracellular calcium
concentration ([Ca2þ]i EC50 = 0.84 μM) and in vitro physiological responses as measured by the
xCELLigence real time cell analyzer (RTCA EC50 = 138 nM). We discovered two selective PAR2
agonists with comparable potency: compound 1 (2-aminothiazol-4-yl; Ca2þ EC50 = 1.77 μM, RTCA
EC50 = 142 nM) and compound 2 (6-aminonicotinyl; Ca2þ EC50 = 2.60 μM, RTCA EC50 = 311 nM).
Unlike the previously described agonist, these novel agonists are devoid of the metabolically
unstable 2-furoyl modification and thus provide potential advantages for PAR2 peptide design for in
vitro and in vivo studies. The novel compounds described herein also serve as a starting point for
structure-activity relationship (SAR) design and are, for the first time, evaluated via a unique high
throughput in vitro physiological assay. Together these will lead to discovery of more potent agonists
and antagonists of PAR2.
’ INTRODUCTION
Significant tools used to study PARs are small peptides or
peptidomimetics that mimic the ligand binding properties of the
tethered ligand exposed by proteolysis of the N-terminus from
the natural receptor (reviewed in ref 1b): SFLLRN-NH2 acti-
vates PAR1; human-derived SLIGKV-NH2 and murine-derived
SLIGRL-NH2 activate PAR2; GYPQVN-NH2 activates PAR4.
However, these short peptides activate cognate receptors with
lower potency compared with native tethered ligands, which
require lower binding energy as a consequence of the covalent
link to the receptor. Also, untethered activating peptides have the
potential to react across PARs. For example, the PAR1 activating
peptide SFLLRN-NH2 can also activate PAR2.5 In lieu of this
caveat, activating peptides/peptidomimetics provide useful tools
for establishment of SAR and rational drug design because they
limit off-target effects that are often a complication of natural
protease activation and allow for a more potent and specific
activation of individual PARs. Indeed, the most potent PAR2
agonist reported to date was developed by modifying a known
activating peptide sequence.6 The N-terminal serine of SLIGRL-
NH2 was substituted with 2-furoyl, and the resulting analogue
Protease-activated receptors (PARs) 1-4 are a family of four
G-protein-coupled receptors (GPCRs) that are self-activated by
a tethered sequence exposed by proteolysis of the extracellular
domain.1 The four members of the GPCR family of PARs are
expressed on a wide variety of cell types. PARs are activated by
proteolysis in response to endogenous and exogenous proteases
and can contribute to both cellular homeostasis and pathology.1,2
In the case of PAR2, the exposed peptides such as SLIGVK-NH2
(human) or SLIGRL-NH2 (murine) remain tethered on the
receptor and activate an orthosteric binding site located on
second loop of the receptor.1 The different N-termini of the
PARs present substrates for a variety of proteases that create
selective activation mechanisms for signal transduction. This
enzyme-linked self-activation is limited to PARs among GPCRs.3
There are a variety of enzymes that can expose the tethered
ligand on PAR, but a key difference between PAR2 and the other
PARs is the lack of activation by thrombin.4 As an obvious
consequence of its activation mechanism, PAR2 is associated with
pathologies with a strong protease release. The involvement of
PAR2 in inflammatory diseases such as arthritis, lung inflamma-
tion (asthma), inflammatory bowel disease, sepsis, and pain
disorders makes PAR2 an attractive target for drug intervention.
Received: October 11, 2010
Published: February 04, 2011
r
2011 American Chemical Society
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Figure 1. Ca2þ signaling induced by novel agonists in the presence or absence of PAR2: typical experiments showing the average [Ca2þ]i ((SEM)
plotted over time for all cells (∼90) in a single experiment following agonist exposure. In each experiment 5 μM agonist was added (denoted by line) to
PAR2 expressing kNRK cells (top) or plasmid expressing control kNRK cells (bottom). In the PAR2-transfected cells, agonists induce rapid and robust
changes in [Ca2þ]i. However, in the absence of PAR2 overexpression, [Ca2þ]i response is minimal. Similar traces are observed with PAR2-transfected/
nontransfected HeLa cells (not shown).
2-furoyl-LIGRLO-NH2 is 10-25 times more efficient at increas-
ing [Ca2þ]i in human and rat PAR2 expressing cells.6,7 The
development of 2-furoyl-LIGRLO-NH2 significantly moved re-
search toward a better understanding of PAR2 agonist activity
and established a platform for rational peptidomimetic design.
Such peptides, peptidomimetics, and other small molecules8 have
been demonstrated to have increased specificity and affinity for
PAR2 and have been used to both activate PAR2 and, in high
concentrations, desensitize cells to subsequent PAR2 responses
in model cells.6,7,9 Besides therapeutic intervention, agonists of
PAR2 can serve as tools for SAR and for defining the cellular and
molecular signaling mechanisms that may contribute to pathology.
Although 2-furoyl-LIGRLO-NH2 has been useful in many
PAR2 cellular and molecular studies, the furane ring is not consid-
ered as a safe structural element for drug design.10 Furane-
containing drugs (including the structurally similar 2-acetyl furane
and at least eight other drugs like the diuretic drug furosemide)
can be metabolically activated by cytochrome P450 to form
electrophilic intermediates (γ-ketocarboxylic acids) by oxidative
opening of the furane heterocycle. These reactive metabolites
have been shown to bind covalently to liver proteins and cause
hepatotoxicity in vivo. Because furane-based structures present a
potential risk for hepatotoxicity, substitutes for the furane in
2-furoyl-LIGRLO-NH2 may provide a more optimal drug design
paradigm.
Despite the potential for drug development from known
peptide/peptidomimetic PAR2 agonists as starting points for
drug discovery (e.g., SLIGVK-NH2, SLIGRL-NH2 and 2-furoyl-
LIGRLO-NH2), there are limited reports on SAR5,8b,11 and a
limited selection of small molecule compounds that can be used
as tools to understand the contribution of PAR2 to cellular and
tissue function. We hypothesized that the 2-furoyl residue could
be substituted by other metabolically stable heterocycles at the
N-terminus of the peptide. Consistent with this hypothesis, Barry
et al.8b has presented a small compound library based on a X-
LIGRLI-NH2 hexapeptide motif. Results using a Ca2þmobiliza-
tion cellular assay to evaluate the X-LIGRLI-NH2 library showed
that (i) no heterocyclic substitution was better than 2-furoyl-
LIGRLI-NH2, (ii) aliphatic residues lead to less Ca2þmobilization,
(iii) aromatic residues were required, but electron donating
effects of the heteroatom can potentiate the activity (2-furoyl-
LIGRLI-NH2, EC50 = 0.16 μM, compared to 4-phenyl-LIGRLI-
NH2, EC50 = 0.8 μM, and (iv) steric effects can diminish activity
(2-furoyl-LIGRLI-NH2, EC50 = 0.16 μM, compared to 2-benzo-
furanyl-LIGRLI-NH2, EC50 = 0.25 μM; phenyl-LIGRLI-NH2,
EC50 = 0.8 μM, compared to 4-biphenyl-LIGRLI-NH2, EC50
=
15.8 μM). These results supported our hypothesis that a meta-
bolically stable modification of 2-furoyl-LIGRLO-NH2 can be
found. We focused our initial design on truncation and confor-
mational constraint of the peptide chain in an effort to move
toward small molecule drug design for PAR2. Here, we present
potential PAR2 ligands based on the shortened pentapeptide
sequence X-LIGRL-NH2.
’ RESULTS
Synthesis. Compound peptidomimetics were synthesized
using standard Fmoc chemistry on Rink amide resin.12 Com-
pounds were cleaved off the resin with TFA-scavenger cocktails
and purified by PR-HPLC and/or size-exclusion chromatogra-
phy. All compounds were 95%þ pure from analytical HPLC and
expected MS results.
Agonist Design. Our goal was to design potent and selective
agonists of PAR2 that contain a metabolically stable substitution
of the furane ring at the N-terminus. Furthermore, we strived to
modify the peptide portion of the agonist into a peptidomimetic
structure to increase stability for systemic applications. In con-
trast to a previous reported library used to help guide our
design,8b we used the LIGRL-NH2 pentapeptide instead of the
more active but longer hexapeptides X-LIGRL-NH2.
Ca2þ Mobilization Assay. A set of 15 compounds based on
the X-LIGRL-NH2 sequence were first screened in model
epithelial cells (16HBE14o-) by monitoring changes in [Ca2þ
]
i
in response to an initial compound dose of 100 μM (based on
activity of native SLIGRL-NH2 peptide activation). [Ca2þ]i was
determined for 80-100 cells using the Ca2þ-sensitive fluores-
cent dye fura-2 and digital imaging microscropy.13 To quantify
the response, cells were determined to be “activated” if their
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Table 1. Ca2þ Mobilization and in Vitro Physiological (RTCA) Assays
[Ca2þ]i measurements
% response
% response % response
xRTCAb
a
R-CO-Leu-Ile-Gly-Arg-Leu-NH2
100 μM
10 μM
2.5 μM
EC50 (μM)
EC50 (nM)
2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn(Aloc)-NH2
2-aminothiazol-4-yl
100 ( 0
97.8 ( 1.0
96.2 ( 1.4
93 ( 2.0
93.6 ( 3.3
67.5 ( 7.5
55 ( 11.6
27.1 ( 6.9
16.5 ( 5.3
0.84 ( 0.08
1.77 ( 0.24
2.60 ( 0.32
3.50 ( 0.36
6.59 ( 1.7
138 ( 13
142 ( 18
311 ( 26
ND
1
2
3
4
100 ( 0
6-aminonicotinyl
100 ( 0
6-aminopyridin-2-yl
99.3 ( 0.7
93.0 ( 0.3
94.4 ( 2.6
86.9 ( 4.1
4-aminophenyl
ND
a The structures do not contain C-terminal Orn as 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn(Aloc)-NH2. b EC50 = concentration of compound that was able to
generate 50% maximal intracellular activity. Values are expressed ( SEM (N g 2 for 100 μM; N g 4 for all others in Ca2þ assay; data are expressed %
response ( SEM; N g 8 in the RTCA assay). ND = not determined.
[Ca2þ]i reached 200 nM or more (from a resting value of 50-
75 nM) in a 3 min time period. The percentage of activated cells
for each experiment was determined. Compounds 11-16 lacked
activity in the Ca2þ mobilization assay (0% activated cells) at
100 μM dose and were eliminated from further screening.
Compounds 5-10 activated cells at doses of 100 μM but failed
to activate cells at doses of 2.5 μM (for details see Supporting
Information Table S1). Compounds 1-4 displayed activated
Ca2þ responses in a significant percentage of cells (>5%) at doses
of e2.5 μM. EC50 values were calculated for these four agonists.
As a point of reference, SLIGRL-NH2 has an approximate EC50
of >40 μM in this assay (data not shown).
Because there are a variety of mechanisms that can lead to
[Ca2þ]i mobilization in epithelial cells, we examined the speci-
ficity of the novel compounds for PAR2. We redeployed the Ca2þ
mobilization assay in either low expressing PAR2 HeLa cells and
HeLa cells transfected with human PAR2 (HeLa-PAR2) or low
expressing kNRK and cells transfected with human PAR2
Figure 2. Physiological signaling induced by novel agonists. Dose
response curves were calculated based on area under the curve for 2 h
of agonist exposure and calculated from 1/2 log dose steps from 10 μM
(kNRK PAR2). We found that 2-furoyl-LIGRLO-NH2 and the
down to 10 nM for each agonist.
novel compounds 1 and 2 were able to quickly stimulate
increases in [Ca2þ]i in HeLa-PAR2 cells (not shown) or kNRK-
PAR2 cells (Figure 1). In the absence of PAR2 overexpression,
agonist-induced [Ca2þ]i changes were minimized and represen-
tative of background PAR2 expression in these cell types (Figure 1).
From these data we conclude that compounds 1 and 2 represent
novel, potent, and selective full agonists at PAR2.
designed and synthesized a set of 15 analogues to investigate
these N-terminal modifications and their ability to activate PAR2
(Scheme 1). The analogues were first evaluated using a Ca2þ
mobilization screen. This initial screen indicated a subset of N-
terminal modifications that approached the potency of the furan
analogue 2-furoyl-LIGRLO-NH2 with modifications that have
structural similarity to be used in further SAR.
In Vitro Physiological Response Assay. Novel agonists 1
and 2 with Ca2þ mobilization assay EC50 values approaching that
determined for 2-furoyl-LIGRLO-NH2 were evaluated along
with 2-furoyl-LIGRLO-NH2 in an in vitro physiological response
assay using the xCELLigence real time cell analyzer (RTCA,
Table 1).14 The RTCA records changes in impedance (reported
as a cell index) over a prolonged time course in a noninvasive
system. These readings represent a physiological output that ref-
lects the combined cellular effects of multiple signaling pathways
such as those activated by PAR2.1,4 Each agonist was applied to
the cells over an appropriate dose range and cell index was
monitored every 15 s for 2 h. We found that agonist activity was
similar among the three compounds, with 2-furoyl-LIGRLO-
NH2 and compound 1 displaying nearly identical RTCA EC50
values and compound 2 displaying less potent activity (Figure 2).
We found that aliphatic modifications such as diglycolyl (15),
1-piperidinethyl (14), and 2-cyanomethyl (10) were ineffective
(structures are depicted in Scheme 1). Heteroaromatic substit-
uents also did not guarantee PAR2 activity as documented for
indole derivatives (11 and 13) or 2-hydroxypyridinyl (12). How-
ever, a subset of heteroaromatic substituents (5-9) elicited
[Ca2þ]i activation at the highest doses tested (100 μM) but
failed at doses shown to be effective for highest activity com-
pounds (e.g., 10 or 2.5 μM). In this manner, they were as effective
as the native peptide sequence (SLIGRL-NH2). The most potent
agonists created (1-4) share a common structural feature, nitro-
gen based heterocycle (thiazole or pyridine) with an amino
group orientated in the ortho position to the nitrogen (Table 1).
2-aminothiazol-4-yl (1) induced Ca2þ mobilization EC50 (1.77
μM) that was higher but not significantly different from that
induced by 2-furoyl-LIGRLO-NH2 (Ca2þ EC50 = 0.84 μM). In
contrast, 6-aminonicotinyl (Ca2þ EC50 = 2.6 μM) (2) and
6-aminopyridin-2-yl (3) (Ca2þ EC50 = 3.5 μM) derivatives
’ DISCUSSION
To date, there has not been a comprehensive SAR study on
N-terminal modifications of short pentapeptide sequences (e.g.,
X-LIGRL-NH2) built as agonists to PAR2. Therefore, we have
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Scheme 1. Solid-Phase Synthesis of Compound 1 and Structures of PAR-2 Agonistsa
a (a) (i) Fmoc/tBu synthetic strategy; (ii) 10% piperidine in DMF, 2 þ 20 minutes; (iii) HBTU coupling of 2-aminothiazole-4-carboxylic acid;
(b) (i) 91% TFA, 3% thioanisole, 3% 1,2-ethanedithiol, 3% water, 4 h; (ii) ether extraction; (iii) HPLC and SEC purifications.
induced Ca2þ mobilization EC50 values that were significantly
higher than that observed for 2-furoyl-LIGRLO-NH2. In 4-ami-
nophenyl (4), the nitrogen atom was removed from the aromatic
ring of the nicotinyl residue and the Ca2þ mobilization EC50
dropped to 6.5 μM. Other isomers of aminonicotinic acid
analogues such as 6-aminopyridin-4-yl (6) and 6-aminopyridin-
5-yl (7) or 3-aminotriazine (8) were ineffective at 2.5 μM. From
this short peptapeptide SAR we conclude that both the amino
group and the aromatic nitrogen contributed to the PAR2 Ca2þ
signaling agonistic activity.
of PAR2 agonists than traditional single pathway analyses (e.g.,
Ca2þ mobilization).
’ EXPERIMENTAL SECTION
Synthesis. Peptidomimetics were prepared as previously published
by solid-phase synthesis as summarized in Scheme 1 on Rink amide
TentaGel resin (0.23 mmol/g) using Fmoc/tBu synthetic strategy and
standard DIC and HBTU or HCTU activations.12c,15 The synthesis was
performed in fritted syringes using a Domino manual synthesizer
obtained from Torviq (Niles, MI). N-Terminal heterocyclic acids were
obtained from Sigma-Aldrich or TCI and coupled using HBTU activa-
tion. For compound 4, the Fmoc-protected version (Fmoc-4-amino-
benzoic acid) was used and otherwise was acid coupled as purchased. All
compounds were fully deprotected and cleaved from the resin by
treatment with 91% TFA (3% water, 3% EDT, and 3% TA). After ether
extraction of scavengers, compounds were purified by HLPC and/or
size-exclusion chromatography (Sephadex G-25, 0.1 M acetic acid) to
>95% purity. All compounds were analyzed for purity by analytical
HPLC and MS by ESI or MALDI-TOF.
Because PAR2 activation can lead to downstream signaling
pathways in addition to Ca2þ signaling, we evaluated the most
potent PAR2 agonists on 16HBE14o- cells using RTCA, an in
vitro physiological assay. This high-throughput assay detects
changes in cell membrane interaction with a tissue culture surface
by continually measuring impedance across the tissue culture
surface.14 We selected the two high potency novel agonists (1
and 2) and compared them to the most potent PAR2 agonist
described to date, 2-furoyl-LIGRLO-NH2. Both novel agonists
(1 and 2) stimulated an increase in cell index in a dose-dependent
manner and reached their peak responses within 30 min (not
shown). Each compound induced the greatest increase in cell
index at a dose of 3 μM, which is approximately 1/10 the concen-
tration required for a maximum response to the native ligand
SLIGRL-NH2 (not shown). Physiological signaling outputs
induced by novel agonists (1 and 2) and 2-furoyl-LIGRLO-
NH2 are plotted as dose response curves in Figure 2. The
calculated physiological EC50 values are summarized in Table 1.
The physiological EC50 of the novel agonist 1 was 142 nM, nearly
identical to that of 2-furoyl-LIGRLO-NH2 (138 nM). A signifi-
cantly higher physiological EC50 (311 nM) was determined for
compound 2. Both agonists (1 and 2) achieved equal Emax values
equivalent to that of the 2-furoyl-LIGRLO-NH2,6 indicating that
these novel ligands are full agonists at PAR2. This RTCA analysis
demonstrates potent activation of model epithelial cells by known
and novel PAR2 agonists and presents a high-throughput in vitro
physiological assay that allows better comparison of full activity
Ca2þ Mobilization Assay. Model epithelial cells (16HBE14o-,
HeLa, kNRK) were grown on matrix coated coverslips for 5-7 days with
appropriate growth and selection medium. Cells were loaded with the
Ca2þ sensitive dye fura-2 and evaluated for intracellular calcium con-
centration ([Ca2þ]i) over 3 min using digital imaging microscopy.13,16
For calculation of Ca2þ mobilization EC50, response was quantified by
the percent of cells responding in the field of view with an increase of
[Ca2þ]i of g200 nM. Compounds were initially tested at 100 μM and
down to 250 nM, as appropriate (Table 1, Supporting Information Table
S2). To demonstrate specificity, the average [Ca2þ]i among all cells
((SEM) in the field of view was calculated over time with [Ca2þ
]
i
measured every 0.6 s (Figure 1).
In Vitro Physiological Response Assay. 16HBE14o- cells
were plated onto 96-well E-plates (Roche) and grown in fully supple-
mented growth medium to 95% confluence overnight at 37 °C, 5% CO2,
and monitored for proliferation using the xCELLigence real time cell
analyzer (RTCA; Roche Diagnostics).14a Prior to the experiment, well
cultures were washed and replaced with 100 μL of modified Hank’s
balanced saline solution (HBSS) prewarmed to 37 °C and allowed to
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ARTICLE
come to room temperature (30-45 min). Each well was supplemented
with 100 μL of HBSS containing appropriate agonists to measure seven
concentrations for three agonists in quadruplicate. Additional wells
(eight) were used for HBSS controls (0 mM agonists). Concentrations
were from 10 μM to 10 nM in 1/2 log steps. Relative impedance in each
well was monitored every 15 s over 2 h. For evaluation purposes, relative
impedance at any given time is expressed as a “cell index.” Cell index is
defined as (Zi - Zo)/(15 Ω), where Zi is impedance at a given time
point during the experiment and Zo is impedance before the addition of
the agonist. Average cell index for each agonist/dose (n = 4) was graphed
over time. Physiological EC50 values were calculated from the area under
the curve values.
thioanisole; THF, tetrahydrofuran; TIS, triisopropylsilane; trifluor-
oacetic acid; TFA, trifluoroacetic acid
’ ADDITIONAL NOTE
Abbreviations used for amino acids and designation of peptides
follow the rules of the IUPAC-IUB Commission of Biochemical
Nomenclature in J. Biol. Chem. 1972, 247, 977−983.
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Supporting Information. Solid phase synthesis, purifica-
b
tion and characterization procedures, spectral and HPLC data,
Ca2þ mobilization, and in vitro physiological assays. This mate-
rial is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (520) 626-4179. Fax: (520) 626-4824. E-mail: vagner@
email.arizona.edu.
’ ACKNOWLEDGMENT
The authors thank the Department of Chemistry, University
of Arizona, and more specifically Arpad Somogy for mass spec-
trometry results. The authors also thank Renata Patek and
Zhenyu Zhang for technical assistance, Daniel X. Sherwood for
his program that allowed for quick quantification of Ca2þ data,
and Cara L. Sherwood for her help in the laboratory in getting
this work going. This work is part of a multiprincipal investigator
collaboration between J.V., T.J.P., and S.B. This work was sup-
ported in part by the following grants: NIEHS Superfund
Research Grant ES 04940 (S.B.), SRC Project 425.024 (S.B.),
NIH Grant R01NS065926 (T.J.P.), Technology and Research
Initiative Fund from Arizona State Proposition 301 (J.V.), and
NIH Training Grant T32-HL007249 (A.N.F.). S.M.S. is a UA
UBRP scholar (Grant HHMI 52005889).
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’ ABBREVIATIONS USED
Aloc, allyloxycarbonyl; Boc, tert-butyloxycarbonyl; BB, bromo-
phenol blue; CH3CN, acetonitrile; DCM, dichloromethane; DI,
deionized; DIPEA, diisopropylethylamine; DMF, N,N-dimethyl-
formamide; DIC, diisopropylcarbodiimide; DMEM, Dulbecco’s
modified Eagle medium; Fmoc, fluorenylmethoxycarbonyl; FT-
ICR, Fourier Transform ion cyclotron resonance; ESI-MS, elec-
trospray ionization mass spectrometry; EDT, 1,2-ethanedithiol;
Et2O, diethyl ether; HBSS, Hank’s balanced saline solution
buffered with 25 mM Hepes; HCTU, O-[1H-(6-chlorobenzo-
triazol-1-yl)(dimethylamino)ethylene]uranium hexafluorophos
phate N-oxide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid; HOBt, N-hydroxybenzotriazole; HOCt, 6-chloro-
1-hydroxybenzotriazole; GPCR, G-protein-coupled receptor;
MALDI-TOF, matrix assisted laser desorption ionization time
of flight; PAR2, protease-activated receptor type 2; Pbf, 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl; SPPS, solid-phase pep-
tide synthesis; RP-HPLC, reverse-phase high performance liquid
chromatography; RCTA, xCELLigence real-time cell analyzer; TA,
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