Synthesis and initial pharmacology of dual-targeting ligands for putative complexes of integrin αvβ3 and PAR2

Article abstract of DOI:10.1039/d0md00098a

Unpublished data from our labs led us to hypothesize that activated protein C (aPC) may initiate an anti-inflammatory signal in endothelial cells by modulating both the integrin αVβ3 and protease-activated receptor 2 (PAR2), which may exist in close proxi

Full text of DOI:10.1039/d0md00098a

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RESEARCH ARTICLE
Synthesis and initial pharmacology of dual-
targeting ligands for putative complexes of
integrin αVβ3 and PAR2†
Cite this: DOI: 10.1039/
d0md00098a
Mark W. Majewski,a Disha M. Gandhi,a Trudy Holyst,b Zhengli Wang,b
Irene Hernandez,b Ricardo Rosas Jr.,a Jieqing Zhu,bc
a
Hartmut Weilerbd and Chris Dockendorff
*
Unpublished data from our labs led us to hypothesize that activated protein C (aPC) may initiate an anti-
inflammatory signal in endothelial cells by modulating both the integrin αVβ3 and protease-activated
receptor 2 (PAR2), which may exist in close proximity on the cellular surface. To test this hypothesis and to
probe the possible inflammation-related pathway, we designed and synthesized dual-targeting ligands
composed of modified versions of two αVβ3 ligands and two agonists of PAR2. These novel ligands were
connected via copper-catalyzed alkyne–azide cycloadditions with polyethylene glycol (PEG) spacers of
variable length. Initial in vitro pharmacology with EA.hy926 and HUVEC endothelial cells indicated that
these ligands are effective binders of αVβ3 and potent agonists of PAR2. These were also used in
preliminary studies investigating their effects on PAR2 signaling in the presence of inflammatory agents,
and represent the first examples of ligands targeting both PARs and integrins, though concurrent binding
to αVβ3 and PAR2 has not yet been demonstrated.
Received 29th March 2020,
Accepted 24th June 2020
DOI: 10.1039/d0md00098a
rsc.li/medchem
concept of biased GPCR signaling9–12 suggests that the
physiologic outcomes of PAR2 activation likewise depend on
Introduction
Protease-activated receptors (PARs) are an important family of
G-protein coupled receptors (GPCRs) that possess their own
agonists: a tethered ligand at the N-terminus that is activated
upon cleavage by certain extracellular proteases.1 The subtype
PAR2 has been implicated in a range of inflammatory
processes,2 and PAR2 antagonists3 have been investigated in
cellular and animal models of inflammation, including
examples related to colitis, arthritis, obesity, and pain,4–8 as
well as for cancer metastasis.4 However, PAR2 ligands have
yet to reach clinical stages, and it is unclear if they can be
safely utilized without adversely affecting normal PAR2
signaling in a range of tissues. One approach could involve
the use of biased PAR2 ligands that may more selectively
inhibit pathological signaling.5–8 The now well-established
the cellular context (cell type), agonist/ligand-specific mode of
activation, heterodimerization with other members of the
PAR family,13,14 and on the interaction of PARs with protease-
specific co-receptors such as tissue factor or the endothelial
cell protein C receptor.15 A notable example of such cell- and
context-specific PAR signaling outcomes has been described
in animals subjected to experimental models of septic
inflammation, where disease stage-specific protective effects
of PAR1 signaling required transactivation of PAR2 on
endothelial cells.16 We reasoned that tissue and/or context-
selectivity for PAR2 signal modulation could be facilitated by
targeting putative heteromeric complexes of PAR2, especially
with increasing evidence for the existence of complexes of
PARs with other PARs or alternative receptors.13,14 We
hypothesize that these complexes can be targeted with
heterobivalent ligands composed of scaffolds established to
bind to the individual targets, connected by a suitable
spacer.17–19 We recently disclosed our first efforts in this area
targeting putative PAR1/PAR2 heteromers.20
a Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, WI,
53201-1881, USA. E-mail: christopher.dockendorff@mu.edu; Tel: +1 414 288 1617
b Blood Research Institute, Versiti, Milwaukee, WI, 53226, USA
c Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI,
53226, USA
d Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, 53226,
USA
Recently, preliminary studies from our labs measuring the
effects of several cytotoxins on both immune and endothelial
cells suggested that the integrin receptor αVβ3 and PAR2 may
play synergistic roles in mediating the anti-inflammatory
action of activated protein C (aPC), a serine protease that is a
central player in the coagulation cascade.21 aPC acts as an
† Electronic supplementary information (ESI) available: Additional assay data
(Fig. S1 and S2); assay protocols; synthetic protocols; compound characterization
data (1H NMR, 13C NMR, and LC-MS traces) (PDF). See DOI: 10.1039/
d0md00098a
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anticoagulant by downregulating the activation of thrombin,
such as fibronectin, fibrinogen, and vitronectin. We chose to
focus on two such αVβ3 ligands developed by Kessler and
coworkers,27 the macrocyclic RGD-containing peptide
cilengitide (1, Fig. 2 and 3),28,29 and the β-amido acid 2
(Fig. 3).30 Cilengitide is notable for being the first anti-
angiogenic drug candidate (which progressed to phase 2
clinical trials for glioblastoma), and 2 is noteworthy for its
high potency and excellent selectivity versus α5β1. We also
chose cilengitide for the insight provided by its X-ray
structure in complex with the extracellular segment of
αVβ3.23
Inspection of the αVβ3–cilengitide structure (Fig. 2)
showed that the valine residue of cilengitide is particularly
solvent exposed, and we reasoned that this would be an ideal
site for inclusion of the adapter required for spacer
attachment (vide infra). Docking31 of the peptidomimetic
ligand 2 to αVβ3 (Fig. 3) showed the expected binding of the
carboxylic acid of 2 to the manganese of the metal ion-
dependent adhesion site (MIDAS), and the ligand overlaid
nicely with the X-ray structure of cilengitide bound to the
same site. The isopropoxy and methoxy groups of 2 are
reasonably solvent exposed and could represent suitable
adapter positions for bivalent ligands. Kiessling reported a
similar strategy for the design of bifunctional molecules
targeting αVβ3 and anti-α-galactosyl antibodies;32 other
bifunctional cyclic RGD ligands connected to various
anticancer drugs33 and labels34–42 have also been reported.
The reported PAR2 agonists are variations of PAR2
tethered ligand peptides which are connected to the receptor
at the C-termini. Therefore, it stands to reason that it should
be possible to attach a spacer at the C-termini of these
ligands. Fatty acid (palmitoyl)-based membrane anchors have
been attached to PAR2 agonist peptides at sites proximal to
the C-termini, and these have been reported by Boitano to be
highly potent PAR2 agonists, in fact up to 200-fold higher
but it has also demonstrated
a range of potent anti-
inflammatory and cytoprotective effects, such that it was
marketed for the treatment of sepsis prior to its withdrawal.22
Its effects can be mediated by both PAR1 and PAR2, among
numerous targets. Under the assumption that αVβ3 and
PAR2 may both be activated by aPC and are both in close
proximity on the cell surface, we reasoned that it could be
possible to selectively recapitulate the anti-inflammatory
effects of aPC by targeting both αVβ3 and PAR2 with a
heterobivalent ligand (Fig. 1). Such ligands may also permit
their delivery to specific cellular targets, such as αVβ3-
expressing vascular endothelial cells. This manuscript
describes the design and synthesis of the first examples of
such ligands utilizing PAR2 agonists, along with our early
efforts to study their effects in endothelial cells.
Materials and methods
The ESI† contains all assay protocols, synthetic protocols,
and select characterization data (1H NMR, 13C NMR, and LC-
MS traces).
Results and discussion
Design of bivalent ligands
In order to engage each of the target receptors with a bivalent
ligand, it is critical to identify scaffolds that are suitably
modifiable. Ideal scaffolds should possess structure–activity
relationship (SAR) data and/or structural information that
indicate where an “adapter” can be added for connection to a
spacer to bridge the gap between the receptors. Several
scaffolds have been identified in ligands targeting either
αVβ3 or PAR2.
Integrins are receptors that mediate cell adhesion, and
their role in blood clotting, angiogenesis, bone remodeling,
and immune responses has prompted the investigation of
their ligands for the prevention or treatment of thrombosis,
cancer, osteoporosis, and inflammatory diseases.24–26 Many
of the integrin ligands that have been developed mimic the
conserved RGD binding domain of natural protein ligands
Fig. 1 Design of αVβ3–PAR2 bivalent ligands targeted to putative
Fig. 2
X-ray structure of cilengitide bound to αVβ3.23 Solvent
receptor complexes.
accessible surface is colored red, rendered using MacPyMOL v.1.8.6.
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cycloadditions (CuAAC) (Scheme 1).46,47 This was prepared
starting with a solid-phase peptide synthesis (SPPS) using an
automated microwave peptide synthesizer, though Yamada
and Shimizu report an alternative protocol optimized for
cyclic RGD peptides.48 A chlorotrityl resin, preloaded with
glycine, was coupled sequentially with the Fmoc-protected
amino
acids
Pbf-arginine,49
propargyl-glycine,
D-
phenylalanine, and t-Bu-aspartate, using COMU® as the
coupling reagent.50 The peptide was cleaved from the resin
using acetic acid in 2,2,2-trifluoroethanol/DCM, leaving the
Pbf and t-Bu protecting groups intact. The resulting linear
peptide 4 was cyclized with PyBOP at 0 °C to yield the
protected macrocycle
conjugation.
5 with alkyne adapter ready for
Additionally, we elected to replace the isoproxy group of
the peptidomimetic αVβ3 ligand 2 with a suitable alkyne for
connection to spacers (Scheme 2). The commercially available
protected β-amino acid 6 was converted to methyl ester 7,
then the phenol was deprotected via hydrogenolysis to give
phenol 8. Following Kessler's reported synthesis of 2,30 this
was alkylated with 3-bromo-1-pentanol to give alcohol 9, which
was oxidized with Dess–Martin periodinane (DMP) (providing
aldehyde 10) and subjected to a reductive amination reaction
with 2-amino-4-methoxypyridine (11) to give amine 12.
Removal of the Boc group with HCl, followed by amide
coupling with the benzoic acid 14 yielded amide 15, which
was hydrolyzed to yield the desired acid 16, possessing a
pendant alkyne.
Fig. 3 Docked structure of Kessler's αVβ3 ligand (yellow) overlaid on
the X-ray structure of αVβ3–cilengitide (green). The manganese ion of
the binding site is shown as a gray sphere. Docking was performed
with FITTED31 and results visualized using MacPyMOL v.1.8.6.
than the parent peptides.43 Very recently and subsequent to
our development of the ligands reported here, researchers at
AstraZeneca reported a model for the binding of the PAR2
agonist peptide SLIGKV to PAR2,44 using as a starting point
the X-ray structure of a modified version of PAR2 complexed
with the antagonist AZ8838.45 These results all suggested that
it should be feasible to attach a spacer to the C-termini of
peptidic PAR2 agonists without losing activity to a punitive
degree.
Synthesis of PAR2 ligands
We selected several reported PAR2 peptide agonists of varying
lengths to be included in our set of ligands. It should be
noted that ligands with variable PAR2 signal biases have been
reported; for example, Hollenberg reported peptides that could
activate intracellular calcium mobilization without activating
mitogen-activated protein kinase (MAPK) in human embryonic
kidney (HEK) cells, and peptides that activated both.5 More
recently, Fairlie reported peptides with varying biases for
Synthesis of αVβ3 ligands
We proceeded to synthesize an analog of cilengitide with the
valine residue replaced by propargyl glycine. We omitted the
proximal N-methyl group for synthetic convenience, which is
not strictly required for decent binding to αVβ3, but
improves selectivity and presumably stability.29 The propargyl
group, which we expect to be solvent-exposed in the ligand-
bound complex, permits convenient, late stage connection to
azide-containing spacers using copper-catalyzed alkyne–azide
PAR2-driven
calcium
mobilization
and
ERK1/2
phosphorylation.7
Scheme 1 Synthesis of alkynyl-cilengitide analog 5.
Scheme 2 Synthesis of alkynyl-modified Kessler ligand 15.
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Fig. 4 PAR2 peptide agonists synthesized via SPPS. All side chains
were kept protected prior to conjugation steps.
widely used PAR2 agonist hexapeptide reported by McGuire
(termed 2-furoyl-LIGRLO-NH2) was synthesized.52 Finally, the
protected 13-mer 26 (termed P3R-13) and 24-mer 27 (termed
P3R-24) were synthesized, which correspond to the
N-terminal 13 and 24 residue tethered peptides of aPC-
cleaved PAR3. These peptides were reported by Mosnier to
promote barrier protection in endothelium,53 and prior work
from Hansen and coworkers suggested that related PAR3
tethered ligands could activate either PAR1 or PAR2 in Jurkat
T-cells.54
Scheme 3 Synthesis of the acid derivative of the PAR2 agonist AY77.
AY77 is a dipeptide with C-terminal amide reported by
Fairlie to be a potent PAR2 agonist for calcium mobilization
(EC50 = 33 nM in human PAR2-expressing CHO cells).51 We
selected it as our smallest PAR2 agonist that may be suitable
for conjugation, and with its relatively simple structure, larger
quantities would be easily accessible. We synthesized it as the
C-terminal acid via solution phase (Scheme 3), rather than
with the solid phase protocol reported for the preparation of
the C-terminal amide. The acid moiety of N-Boc
cyclohexylglycine 17 was protected via allylation, then
N-deprotected and coupled with cyclohexylalanine 20 to
generate the protected dipeptide 21. The Boc group was
removed and the amine was coupled with isoxazole-acid 22,
then the allyl group was removed with catalytic palladium to
yield the desired acid 24a.
Assembly of dual-targeting ligands
Synthesis of the dual-targeting ligands and their respective
monovalent spacer-linked controls proceeded according to
the protocols outlined in Schemes 4 to 6. The carboxylic acid
(24a) corresponding to AY77 (24b) was coupled using HATU
to commercially available amino-PEG–azide spacers (28a–d)
of four widely different lengths (termed informally as short,
medium, long, and extra-long). These spacers are 24, 30, 36,
and 75 atoms long; as a comparison, optimal bivalent ligands
for GPCR heteromers have been reported by Portoghese to
have 16 to 22 atom spacers for mu opioid/cholecystokinin 2
receptor heteromers,55 and by Gmeiner to have 88 atom
spacers for bivalent ligands bridging dopamine 2/neurotensin
S1 receptor heteromers.56 The resulting amides 29a–d were
subjected to CuAAC reaction with the protected alkynyl-
Longer PAR2 agonist peptides were prepared via SPPS
analogously to 5, using chlorotrityl resin, automated
microwave reactor, and cleaving the peptides from the resin
with protected side chains intact (Fig. 4). In this manner, the
protected and C-terminal acid version (25b) of the potent and
Scheme 4 Assembly of dual-targeting ligands derived from the cilengitide analog and AY77.
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Scheme 5 Assembly of dual-targeting ligands derived from the cilengitide analog and 2-furoyl-LIGRLO-NH2.
cilengitide analog 5, generating the triazoles 31b–d,
respectively. Improved results were obtained when using the
ligand TBTA reported by Fokin.57 Global deprotection with a
cocktail of TFA and carbocation scavengers generated the dual-
targeting ligands 32b–d. Alternatively, the azide 29a was
subjected to CuAAC with methyl propargyl ether to generate the
control compound 30, composed of the PAR2 agonist with a
triazole-capped spacer. All final compounds and intermediates
in Schemes 4 to 6 were purified via preparative HPLC.
In a similar fashion, the protected McGuire peptide 25b
was used to generate cilengitide analog-conjugated dual-
targeting ligands 36b–d, as well as the spacer-linked control
compound 34b (Scheme 5). We were unsuccessful in
generating in pure form several ligands derived from the
longer peptides P3R13 (26) and P3R24 (27), as the protected
versions of these suffered from extremely low solubility after
coupling reactions, and the products were not successfully
isolated after normal- or reverse-phase chromatography.
Finally, the alkynylated analog of Kessler's peptidomimetic
(16) was subjected to CuAAC with several PAR2 agonists,
including AY77 (Scheme 6). Compound 37a is the first dual-
targeting ligand we prepared using 16, with our early
pharmacology data reported here.
First, the binding of Alexa Fluor 647-labeled fibronectin to
HEK293FT cells transfected with αVβ3 was measured in the
presence of several dual-targeting ligands (Fig. 5A). Cells
expressing αVβ3 were also treated with an antibody to β3
integrin (AlexaFluor 488-labeled AP3), which was used to sort
cells via flow cytometer, prior to fluorescence measurement.
Both of the cilengitide-based ligands 32d (MWM-321) and 36d
(MWM-319) substantially inhibited fibronectin (Fn) binding at a
concentration of 5 μM, at a similar level to the established
integrin binder cilengitide (cRGD). Though concentration–
response curves have not yet been generated, these early results
confirm that the attachment of the spacer to the cilengitide
analog does not appear to adversely impact its binding to αVβ3.
Since our focus is on endothelial cells, we repeated this
experiment with vitronectin coated plates and HUVEC, since
HUVEC also express fibronectin-binding α5β1. Cell binding was
qualitatively inhibited by 32d, 36d, and 37a (DG-258) (Fig. 5B).
Integrin binding assays
To confirm that our modifications to the integrin ligands were
well tolerated, we performed two preliminary binding assays.
Scheme 6 Assembly of dual-targeting ligands derived from Kessler's
ligand and AY77.
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Fig.
7
Stimulation of calcium mobilization in EA.hy926 cells by
cilengitide analog–McGuire peptide PAR2 agonists. Error bars indicate
SEM (n ≥ 3). % stimulation values were normalized to the response
with 100 μM SLIGKV-NH2 (100%).
The release of calcium from the endoplasmic reticulum in
response to Gq signaling leads to diverse effects in different
cell types, and this calcium mobilization is easily quantified
using calcium binding dyes that can be absorbed into cells.
Such assays are widely used to characterized GPCR activation,
in this case via Gq. All curves were fitted using data from n ≥
3 measurements on a single 96 well plate, with the error bars
indicating the standard error of the mean (SEM). The
cilengitide analog–AY77-based compounds from Scheme 4
were studied first in EA.hy926 cells (Fig. 6). The control
compound composed of AY77 with attached PEG spacer (30)
had only moderately decreased potency (EC50 = 0.89 μM)
relative to AY77 (24b, EC50 = 0.27 μM), with similar efficacy.
The ligand with the medium length spacer (32b) provided the
best results with comparable potency to AY77 (EC50 = 0.56
μM), with significantly increased efficacy. The ligands with
the long (EC50 = 0.88 μM) and extra-long (EC50 = 2.5 μM)
spacers possessed lower potencies in this assay.
Concentration–response curves generated with the dual-
targeting ligands containing the McGuire peptide (2-furoyl-
LIGRLO-NH2) are shown in Fig. 7. Interestingly, all of the
spacer-linked agonists were more potent than the parent
compound 25c (EC50 = 0.35 μM). The potencies were similar
for the spacer-linked control 34b (EC50 = 0.073 μM) and the
medium, long, and extra-long ligands 36b, 36c, and 36d
(EC50 = 0.062, 0.084, and 0.13 μM, respectively). Again, the
dual-targeting ligand with the medium length PEG spacer (28
atoms, including one nitrogen at each end) was optimal. We
speculate that the longer 2-furoyl-LIGRLO-NH2 likely has its
Fig. 5 A) Inhibition of AlexaFluor 647-labeled fibronectin (Fn) binding
to αVβ3-containing HEK293 cells was measured in the presence of the
ligands cilengitide (cRGD), 33d (MWM-321) and 36d (MWM-319). The
binding was measured in buffer containing 1 mM CaCl2 plus 1 mM
MgCl2 (Ca2+/Mg2+) or 0.2 mM CaCl2 plus 2 mM MnCl2 (Ca2+/Mn2+).
Data are mean SEM (n = 4). Two-tailed t-test was used to compare
the Ca2+/Mn2+ condition with inhibitors and without inhibitors. MFI =
mean fluorescence intensity. B) Binding of HUVEC (stained with DAPI,
5 μg mL−1) to vitronectin-coated plates, and inhibition of binding by 50
μM cRGD, MWM-319, MWM-321, and DG-258. See ESI† for details.
Scale bar = 400 μm.
Calcium mobilization assays
Next, we studied the ability of our dual-targeting ligands to
activate PAR2 by measuring Gq-driven calcium mobilization
in adherent endothelial cells, according to our reported plate
reader protocol using the calcium sensing dye Fluo-4/AM.58
Fig.
6 Stimulation of calcium mobilization in EA.hy926 cells by
cilengitide analog–AY77 PAR2 agonists. Error bars indicate SEM (n ≥ 3).
% stimulation values were normalized to the response with 100 μM
SLIGKV-NH2 (100%).
Fig.
8
Stimulation of calcium mobilization in HUVEC by dual-
stimulation values were
targeting PAR2 agonists 32c and 36c.
normalized to the response with 100 μM SLIGKV-NH2 (100%).
%
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C-terminus closer to the mouth of the receptor than AY77,
and is thus better able to tolerate the addition of the PEG-
based spacer.
Several dual-targeting ligands were also tested in cultured
human umbilical vein endothelial cells (HUVEC) (Fig. 8). The
results were very similar compared to the EA.hy926 cells, with
ligand derived from Kessler's peptidomimetic αVβ3 inhibitor
and the PAR2 agonist AY77 (37a, DG-258). In these cases,
there was no statistical difference in potency (EC50 = 1.2 μM
for 37a alone) with or without the addition of 10 μM
cilengitide, and with or without pretreatment for 2 h with 10
μg mL−1 LPS (Fig. S2†).
the ligand derived from the McGuire peptide (36c, EC50
=
0.09 μM) approximately 10-fold more potent than the
analogous ligand with AY77 (32c, EC50 = 1.0 μM).
Conclusions
We have reported
a toolbox of bifunctional molecules
We did not observe any significant increase in potency for
the dual-targeting ligands over the monovalent controls,
which would support the existence of αVβ3–PAR2 complexes,
or at least the two receptors residing within the span of the
longest ligands (possessing 72 atom spacers). Nonetheless,
we performed competition studies to test for bivalency by
adding a competing monovalent ligand. If a dual-targeting
ligand shows increased binding affinity and activity due to
concurrent interactions with two receptors (e.g. within a
heteromeric complex), we would expect binding and activity
to decrease upon addition of a competing monovalent ligand.
In our studies, the activities of our dual-targeting ligands at
PAR2 were measured (via calcium mobilization) in the
presence of the αVβ3 ligand cilengitide (Fig. 9). No decrease
in potency of the dual-targeting ligand 32d (composed of the
cilengitide analog and AY77, joined by the extra-long PEG
spacer) was observed in EA.hy926 cells upon addition of 10
μM cilengitide, although a decrease in efficacy was observed
at the highest concentrations.
We reasoned that putative integrin–PAR complexes may
only emerge under conditions of cellular stress, and evidence
suggests that PAR2 plays an important role in the
inflammatory responses to lipopolysaccharide (LPS),59 the
bacterial cell membrane component.60 Concentration–
response curves with EA.hy926 cells were measured with 32d
after a 2 h pretreatment with 10 μg mL−1 LPS. No significant
differences in potency were noted, with or without the
addition of cilengitide (Fig. 9). Similar results were obtained
with the dual-targeting ligand derived from the McGuire
peptide (36d, Fig. S1†).
designed to target both integrins and PARs. Consistent with
the results of Kiessling32 and others, known αVβ3 ligands
could be modified with the attachment of spacers without
precluding integrin binding. Similarly, inspired by the prior
results of Boitano,43 PAR2 agonists could be successfully
modified by attachment of spacers without decreases in
potency for the activation of calcium mobilization in
endothelial cells. In preliminary experiments, we did not
observe obvious changes in concentration–responses in
cultured endothelial cells that would be diagnostic of the
presence of a population of putative αVβ3–PAR2 complexes
that could be selectively activated by our dual-targeting
ligands. However, investigations in cellular models of
inflammation are pending with these and related dual-
targeting ligands in various cell types. These compounds
have the potential to characterize and modify signaling
unique to specific cell surface receptor complexes, and these
or related ligands may have diagnostic and therapeutic
applications in the future, including integrin-mediated
delivery of PAR2 ligands to specific tissues or cell types.
Funding sources
C. D. thanks the National Heart, Lung, and Blood Institute
(NHLBI) (R15HL127636); C. D. and R. R. thank Marquette
University for a Summer Faculty Fellowship and Regular
Research Grant; C. D. and H. W. thank the Clinical and
Translational Science Institute of Southeast Wisconsin
(8UL1TR000055); H. W. thanks NHLBI (R01HL133348); and
J. Z. thanks NHLBI (R01HL131836) for support of the work
described here.
We also did not observe evidence of bivalency or increased
potency after pretreatment with LPS using the dual-targeting
Notes
Earlier versions of this manuscript were submitted to the
preprint server ChemRxiv.61
Abbreviations
Boc
tert-Butoxycarbonyl
cRGD
Cilengitide
COMU (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)
dimethylamino-morpholino-carbenium
hexafluorophosphate
Fig.
pretreated with LPS by dual-targeting PAR2 agonist 32d (MWM-321),
with and without cilengitide as competing ligand. Cells were
pretreated with 10 μg mL−1 LPS for 2 h prior to addition of ligand(s). %
stimulation values were normalized to the response with 100 μM
SLIGKV-NH2 (100%).
9 Stimulation of calcium mobilization in EA.hy926 cells
a
CuAAc Copper-catalyzed alkyne/azide cycloadditions
DAPI
DCE
4′,6-Diamidino-2-phenylindole
1,2-Dichloroethane
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DCM
DIEA
Dichloromethane
N,N-Diisopropylethylamine
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DPPF
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FDA
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U.S. Food and Drug Administration
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Fluorenylmethyloxycarbonyl
HATU Hexafluorophosphate azabenzotriazole tetramethyl
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HOBt
1-Hydroxybenzotriazole
HUVEC Human umbilical vein endothelial cells
IC50
Half-maximal inhibitory concentration
Intracellular calcium mobilization
iCa2+
MIDAS Metal ion-dependent adhesion site
PAR
Pbf
Protease-activated receptor
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate
SEM
SPPS
TBTA
Standard error of the mean
Solid phase peptide synthesis
Tris(benzyltriazolylmethyl)amine
Author contributions
Conceived the project: C. D., H. W. Designed compounds: C.
D. Designed synthetic routes: C. D., M. W. M., D. M. G.
Synthesized and characterized compounds: M. W. M., D. M.
G., T. H., R. R. Cultured cells: I. H., M. W. M., D. M. G., R. R.
Performed assays: M. W. M., R. R., Z. W. Analyzed data: M. W.
M., C. D., Z. W., J. Z., H. W. Wrote the manuscript: M. W. M.,
C. D. Prepared ESI:† M. W. M., D. M. G., R. R., C. D., Z. W., J. Z.
11 L. Tan, W. Yan, J. D. McCorvy and J. Cheng, Biased Ligands
of
G
Protein-Coupled Receptors (GPCRs): Structure-
Functional Selectivity Relationships (SFSRs) and Therapeutic
Potential, J. Med. Chem., 2018, 61, 9841–9878.
Conflicts of interest
12 T. Kenakin, Biased Receptor Signaling in Drug Discovery,
Pharmacol. Rev., 2019, 71, 267–315.
13 H. Lin, A. P. Liu, T. H. Smith and J. Trejo, Cofactoring and
Dimerization of Proteinase-Activated Receptors, Pharmacol.
Rev., 2013, 65, 1198–1213.
There are no conflicts of interest to declare.
Acknowledgements
14 F. Gieseler, H. Ungefroren, U. Settmacher, M. D. Hollenberg
and R. Kaufmann, Proteinase-activated receptors (PARs) -
We thank Prof. Nicolas Moitessier (McGill University) for
providing the FITTED® software used for docking studies and
Dr. Sheng Cai (Marquette University) for assistance with LC-
MS and NMR instruments. We thank ACD/Labs (NMR
processing) and ChemAxon (NMR prediction and compound
property prediction) for software. We thank the Nowick lab
(UC-Irvine) for sharing detailed protocols for solid-phase
peptide synthesis via their webpage.
focus
on
receptor-receptor-interactions
and
their
physiological and pathophysiological impact, Cell Commun.
Signaling, 2013, 11, 86.
15 J. Disse, H. H. Petersen, K. S. Larsen, E. Persson, N. Esmon,
C. T. Esmon, L. Teyton, L. C. Petersen and W. Ruf, The
Endothelial Protein
C Receptor Supports Tissue Factor
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