Design and Evaluation of Heterobivalent PAR1-PAR2 Ligands as Antagonists of Calcium Mobilization

Article abstract of DOI:10.1021/acsmedchemlett.8b00538

A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1-PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders.

Full text of DOI:10.1021/acsmedchemlett.8b00538

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Design and Evaluation of Heterobivalent PAR1−PAR2 Ligands as
Antagonists of Calcium Mobilization
Mark W. Majewski,†,§ Disha M. Gandhi,†,§ Ricardo Rosas, Jr.,† Revathi Kodali,‡ Leggy A. Arnold,‡
and Chris Dockendorff*,†
†Department of Chemistry, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53201-1881, United States
‡Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin, Milwaukee,
Wisconsin 53211, United States
S
* Supporting Information
ABSTRACT: A novel class of bivalent ligands targeting putative protease-
activated receptor (PAR) heteromers has been prepared based upon
reported antagonists for the subtypes PAR1 and PAR2. Modified versions
of the PAR1 antagonist RWJ-58259 containing alkyne adapters were
connected via cycloaddition reactions to azide-capped polyethylene glycol
(PEG) spacers attached to imidazopyridazine-based PAR2 antagonists.
Initial studies of the PAR1−PAR2 antagonists indicated that they inhibited
G alpha q-mediated calcium mobilization in endothelial and cancer cells
driven by both PAR1 and PAR2 agonists. Compounds of this novel class
hold promise for the prevention of restenosis, cancer cell metastasis, and
other proliferative disorders.
KEYWORDS: Bivalent ligand, protease-activated receptor, PAR1 antagonist, PAR2 antagonist, calcium mobilization, metastasis,
restenosis
rotease-activated receptors (PARs) are a unique family of
class A G-protein-coupled receptors (GPCRs) that are
PAR1antagonistsmaypreventthrombosisviainhibitionofPAR1
on platelets, while concurrently interfering with endothelial
barrier integrity by inhibiting normal PAR1 signaling in
endothelium. This has inspired our recent efforts to identify
biased ligands that may inhibit or activate only a subset of PAR1-
mediated signaling pathways.13−15
An alternative therapeutic approach could utilize hetero-
bivalent ligands to selectively target the differential signaling
mediated by PAR heteromeric complexes (Figure 1). To our
knowledge, no ligands selective for PAR heteromers have been
reported. Evidence has been accumulating in recent years that
GPCRs frequently form oligomers in vitro, but the in vivo
P
activatedbyextracellularproteases,whichrevealatetheredligand
at the N-terminus.1,2 Self-activation of PARs by their tethered
ligand agonists leads to a wide range of cellular responses, in part
due to the fact that PARs are highly expressed in different tissues
with varying signaling components; they can also be activated by
different proteases that cleave at different locations on the N-
terminus.3 The unusual complexity of PAR-mediated signaling is
further increased by the potential for PARs and other GPCRs to
form multireceptor complexes in cell membranes.4 More
specifically, evidence has emerged in recent years that PARs
form homomeric and heteromeric complexes among the four
different PAR subtypes, and the signals mediated by these
complexes are distinct from those of the monomers.5−7
Additionally, the N-terminus of one PAR subtype can directly
activate a neighboring PAR.8
The range of productive and pathological signaling for which
PARs have been implicated has prompted their study as potential
targets for numerous indications, including thrombosis, inflam-
mation, stroke, kidney disease, reperfusion injury, and cancer cell
metastasis (vide infra). Despite their therapeutic promise, to our
knowledge only a few PAR modulators have thus far reached
clinical stages, all as antithrombotic agents inhibiting platelet
activation.9−12
Figure 1. Proposed heterobivalent PAR ligands.
The present lack of clinical PAR modulators for other
indications may reflect in part the pleiotropic signaling of PARs,
which may complicate the selective inhibition of pathological
signalswithoutadverselyaffectingnormalsignaling.Forexample,
Received: November 7, 2018
Accepted: December 3, 2018
Published: December 3, 2018
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
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relevance of these oligomers has been harder to quantify, in part
duetoalackofadequate chemical tools.4 The concept ofbivalent
ligands for multimeric GPCRs was pioneered by Portoghese
nearly 40 years ago,16 and this strategy has been used to generate
compoundsprimarilyforthestudyofCNSGPCRs.17,18 Recently,
promising in vivo data have been obtained that suggest that
bivalent ligands can modulate GPCRs in a manner distinct from
that of their monovalent counterparts.19−22 This approach holds
particularpromiseforPARs,asthedifferentialtissueexpressionof
PAR subtypes could permit tissue-selective targeting of PAR
heteromers using heterobivalent ligands. This is best exemplified
by the fact that PAR2 is not expressed on human platelets;
therefore,wereasonedthatPAR2-directedheterobivalentligands
could permit the selective modulation of signaling by PAR2-
containing heteromers in vascular tissues without adversely
affecting platelet activation and hemostasis.
synthesis of an alkyne-tethered version of RWJ-58259, which
retained significant potency at PAR1.15 Based on the SARs
published for the imidazopyridazine reported by Vertex, we
elected to prepare carboxyl-substituted versions of these PAR2
antagonists using the reported synthetic route (Scheme 1).33 3-
Scheme1. SynthesisofPAR2AntagonistwithCarboxylicAcid
Adapter Based on Reported Imidazolopyridazines
Mueller and colleagues presented evidence that thrombin-
enhanced migration of melanoma cells is driven by PAR1 and
PAR2, in a manner consistent with the “direct transactivation” of
PAR2 by thrombin-activated PAR1.23 Bar-Shavit has also
reported synergistic effects of PAR1 and PAR2 activation in
tumor development24,25 Kuliopulos reported that signaling by
PAR1−PAR2 heteromers may be implicated in restenosis, the
dangerousnarrowingofbloodvesselswhichoccursinasignificant
fraction of patients after percutaneous interventions (PCIs).26
We hypothesize that heterobivalent antagonists may act
selectively at putative PAR1−PAR2 heteromers and thus may
act to inhibit cancer cell metastasis and proliferative processes
such as restenosis, while minimizing impacts on normal PAR
signaling. PAR1 antagonists have beeninvestigated for restenosis
in animal models,27,28 and individual PAR129,30 and PAR231
antagonists have been studied in cancer cell metastasis and
invasion. To our knowledge, however, none of these studies has
progressed to clinical stages for these indications. One
complication may arise from the fact that the tethered ligand of
PAR1 can directly transactivate a neighboring PAR2 receptor,
which could possibly happen even in the presence of a PAR1
antagonist.8 Together, these observations make the preparation
ofmultivalentPARligandsanattractivestrategywithbenefitsthat
may extend beyond the administration of multiple separate
ligands.Hereinwedescribeourinitialeffortstowardthesynthesis
and study of heterobivalent ligands targeting PAR1 and PAR2.
The primary considerations for the design of heterobivalent
ligands are as follows: 1. What monovalent ligands should be
used? 2. What spacers should be used to connect them? 3. Where
should the spacer be attached to each ligand? Based on its
published SAR data and potential for diverse modifications, we
elected to utilize the PAR1 antagonist RWJ-58259 as the first
PAR1 scaffold for our bivalent ligands27,32 and an imidazopyr-
idazine scaffold reported by Vertex as the PAR2 antagonist.33 To
connect the ligands, we elected to first utilize commercially
available polyethylene glycol (PEG)-based spacers with a variety
of lengths, particularly for their beneficial physicochemical
properties, especially increased water solubility. Also beneficial
is the fact that bivalent ligands with PEG spacers with excess
“slack”maypaylessofanentropicpenaltyuponbindingrelativeto
those with more hydrophobic spacers,34 presumably because
hydrophilic PEGs can maintain flexibility in water and do not
require highly ordered solvation shells. The use of commercially
available amino-PEG-azide spacers permits convenient copper-
catalyzed alkyne/azide cycloadditions (CuAAC), suitable for the
connection of two appropriately functionalized ligands via the
PEGspacerunderdiverseconditions.35 Werecentlyreported the
Amino-6-chloropyridazine (1) was subjected to Suzuki coupling
with 4-fluorophenylboronic acid to yield the biarylamine 2.
Bromination followed by alkylation with bromomethylpyruvate
yielded the imidazopyridazine 4. Suzuki coupling with the
propenylboronic ester A gave 5, and hydrogenation provided the
ester6.HydrolysisfollowedbycouplingwithpiperazineByielded
amide 8. Removal of the Boc group, coupling with succinic acid
monomethyl ester, and hydrolysis of the resulting ester gave
carboxylic acid 10a. PAR2 antagonists containing an arene
substituentinplaceoftheisopropylgroupof10aandacyclohexyl
carboxylicacidinplaceoftheeasternethylenegroupwereamong
numerous analogs previously reported;33 therefore, carboxylic
acid 10b was additionally prepared (Scheme S1). Both of the
carboxylic acids 10a and 10b were confirmed to be highly potent
antagonistsofPAR2(videinfra),soweproceededtocouplethem
to a variety of PEG-based spacers (Schemes 2 and S2). Amide
couplingwithamino(PEG)azides,followedbyCuAAC36 withthe
previously synthesized RWJ-58259-based alkyne DG-207,15
yielded heterobivalent ligands 13a−d. Control compounds
15−18 composed of the monovalent ligands with attached PEG
spacers are depicted in Figure S1.
During the course of our studies, Marshall and colleagues
reported several X-ray crystal structures of modified PAR2,
including a cocrystal of PAR2 with the inhibitor AZ3451 at an
allosteric site at the side of the receptor.37 The structural
similarities between 10a and AZ3451 led us to hypothesize that
they may share a binding site on PAR2, which was supported by
rigid protein docking studies (Figure 2a)38 and is consistent with
the allosteric mode of inhibition recently reported by Fairlie for
the Vertex33 imidazopyridazine I-191.39 Compound 10a docks
into the lipophilic pocket at the side of PAR2 in a similar pose to
AZ3451, with both molecules possessing a heterocyclic nitrogen
capable of accepting a hydrogen bond from Tyr210.
B
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Scheme 2. Assembly of Heterobivalent PAR1−PAR2 Antagonists
from N = 3 on a single plate, fitted using four-variable nonlinear
regression with GraphPad Prism v. 5. Error bars represent
standard error of the mean (SEM) for the three measurements at
each concentration.
The attachment of PEG spacers to the PAR1 antagonist RWJ-
58259andthePAR2antagonist10awastolerated,albeitwith5-to
70-fold decreases in potency. Half-maximal inhibitory concen-
tration (IC50)for theinhibitionofTFLLRN-NH2 (5 μM)PAR1-
mediatediCa2+ mobilizationincreasedfrom0.02to0.1μMforthe
PEG spacer-linked control 17 (Figure 3a). Compound 10a
Figure 2. (A) X-ray structure of modified PAR2 with bound allosteric
ligandAZ3451(green)anddockedligand10a(pink).(B)Novelbivalent
ligand20 and controlcompound19 possessinga PEG-aminoalkylamide
spacer.
The position of this binding site within the cell membrane
suggests that the hydrophilic PEG spacer may be counter-
productive and could explain the greatly decreased potency at
PAR2 of our PEG-linked PAR2 antagonists. We addressed this
potential issue by synthesizing modified spacers containing a
terminal aminoalkylamide that could possess a higher affinity for
the cell membrane above the putative PAR2 binding site (Figure
2b and Scheme S3).
The ligands were tested in our intracellular calcium
mobilization assay (iCa2+) using the transformed human
endothelial cell line EA.hy926, and this assay was also suitable
for studying the signaling of the breast cancer cell line MDA-MB-
231, which expresses both PAR1 and PAR2. Adherent cells were
cultured in 96-well plates to confluence over ∼48 h, then loaded
with the fluorescent calcium binding dye Fluo-4/AM, according
to our reported protocol.15 Addition of the antagonists, followed
by the relevant PAR peptide agonists TFLLRN-NH2 (PAR1),
SLIGKV-NH2 (PAR2), or SFLLRN-NH2 (PAR1/2), permitted
the measurement of inhibition of Gq-driven signaling from both
PAR1 and PAR2. All concentration−response curves show data
Figure 3. Inhibition of TFLLRN-NH2 (5 μM) PAR1-mediated iCa2+
mobilizationby monovalentPAR1ligandRWJ-58259andspacer-linked
control 17 (A) in EA.hy926 endothelial cells and (C) in MDA-MB-231
cancer cells; inhibition of SLIGKV-NH2 (5 μM) PAR2-mediated iCa2+
mobilization by monovalent PAR2 ligand 10a and spacer-linked control
15 (B) in EA.hy926 endothelial cells and (D) in MDA-MB-231 cancer
cells.
inhibited SLIGKV-NH2 (5 μM) PAR2-mediated iCa2+ mobi-
lizationwithanIC50 of0.1μM,andthePEGspacer-linkedcontrol
15 was significantly less potent (IC50 = 7 μM, Figure 3b). This is
consistent with a report from Bunnett (published during revision
of this manuscript) that describes an imidazopyridazine PAR2
antagonist that suffers an approximately 1000-fold drop in
potencyuponattachmentofaPEGtetheratasimilarsite.40 These
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compoundsalsoshowed goodpotenciesinanalogousassayswith
MDA-MB-231 cancer cells (Figures 3c,d).
The inability of the PAR2 ligand 15 to inhibit SFLLRN-NH2 to
any measurable degree suggests that its signaling is largely driven
byPAR1withEA.hy926cells. Inhibitionwiththebivalentligands
derived from the PAR2 ligand 10b was inferior to those derived
from 10a. These data are included in the Supporting Information
(Figures S2−S4).
As described above, we proceeded to prepare bivalent ligands
possessing spacers of different lengths, from shortest (21 atoms,
13a)tolongest(72atoms,13d).Tosupporttheirpotentialutility
forfutureinvivoexperiments,thestabilityof13dwasmeasuredin
mouse plasma, and a half-life of 5.6 h was determined (see
Supporting Info for details). All four ligands showed good
inhibition of PAR1-driven (TFLLRN-NH2) calcium mobiliza-
tioninEA.hy926cells,withcomparableIC50srangingfrom0.03to
0.18μM(Figure4a). Theefficacyofthelongestligand(13d)was
The bivalent ligand 13d was next studied in the breast cancer
cell line with all three agonists (Figure 6). It was most effective at
Figure 6. Inhibition of TFLLRN-NH2 (5 μM) PAR1-mediated,
SLIGKV-NH2 (5 μM) PAR2-mediated, and SFLLRN-NH2 (3.16 μM)
PAR1/2-mediated iCa2+ mobilization of 13d in MDA-MB-231 breast
cancer cells.
Figure 4. Inhibition of TFLLRN-NH2 (5 μM) PAR1-mediated iCa2+
mobilization (A) and SLIGKV-NH2 (5 μM) PAR2-mediated iCa2+
mobilization (B) by bivalent ligands 13a−d in EA.hy926 endothelial
cells.
the inhibition of TFLLRN-NH2-driven calcium mobilization,
with an IC50 of 0.14 μM and excellent efficacy. The IC50 for
inhibition of SFLLRN-NH2 was 3.3 μM, with lower efficacy. The
inhibition of SLIGKV-NH2 by 13d was lessefficient, with an IC50
estimated at 3.5 μM, but with no inhibition observed at
submicromolar concentrations. The bivalent ligand possessing a
more hydrophobic spacer (20, Figure 2b) was also tested for its
possibleimprovedactivityatPAR2;unfortunately, attachmentof
this spacer did not improve the inhibition of PAR2-mediated
signaling (Figures S6−S8).
Finally,13dwastestedforitsabilitytoinhibitthenaturalPAR1-
and PAR2-activating proteasesthrombin (Figure S9) andtrypsin
(Figure S10). Compound 13d inhibited 1 nM thrombin with an
IC50 of 0.36 μM in Ea.hy926 cells, but interestingly, the spacer-
linked control 17 was ∼10× more potent. The PAR2 antagonist
10awasapotentinhibitorof5nMtrypsin(IC50 =0.002μM), but
attachment of a spacer (15) abrograted its activity, and the
bivalent ligand 13d also showed no inhibition of trypsin.
We have reported the first examples of bivalent PAR1−PAR2
antagonists, and we demonstrated that the optimal compound
13d can perform similarly to the combination of monovalent
control ligands in the inhibition of intracellular calcium
mobilization in the transformed endothelial cell line EA.hy926,
as well as the epithelial breast cancer cell line MDA-MB-231.
However, the decrease in potency observed upon attachment of
PEG-based spacers to the monovalent PAR1 and PAR2 ligands
means that the ligands are less potent than the combination of
monovalent ligands without spacers attached, though there is
always a significant disadvantage of dosing multiple drugs to
obtain a desired therapeutic effect. These considerations may be
less important than the ability of a heterobivalent ligand to
selectively inhibit a putative heteromeric receptor complex that
drivesapathologicaleffect,suchasmetastasis,withoutinterfering
withhealthycellsignaling.Wehavenotyetobtainedevidencethat
the bivalent ligands disclosed here selectively inhibit putative
PAR1−PAR2 complexes; an example of such evidence would be
biphasic concentration−response curves, where the ligands
inhibit a population of heteromers at lower concentrations.
However, the ability to detect low concentrations of such
complexes is limited by the precision of the calcium mobilization
assay. Receptor binding assays with labeled ligands are planned
higher than the others at the highest concentrations. All four
ligandshadmuchlowerpotencyandefficacyasPAR2antagonists,
usingSLIGKV-NH2 asagonist(Figure4b).AnIC50 couldonlybe
estimated from the concentration−response curve fitted to the
data from 13d, with an IC50 of 3.3 μM, and with only partial
inhibitionatthehighestconcentration(31.6μM).Thedecreased
potencies and efficacies of the bivalent ligands at PAR2,
particularly comparing these ligands to the PEG-linked PAR2
antagonist controls, may reflect a higher affinity of the bivalent
ligandsforPAR1,and/orahigherreceptordensityforPAR1,such
that ligands may be binding significantly to monomeric PAR1.
Next, the bivalent ligands 13a−d were tested for their abilityto
inhibit the PAR1/2 agonist SFLLRN-NH2, dosed at 3.16 μM
(Figure 5a). Again, the ligand with the longest spacer (13d)
proved to be most potent, with an IC50 of 0.15 μM. It was also
compared to the control compounds composed of monovalent
PAR1 (17) and PAR2 (15) ligands with spacers and their
combination (Figure 5b). The combination of equal concen-
trations of 17 and 15 gave an IC50 of 0.32 μM, resulting in an
inhibition profile very similar to that of the bivalent ligand 13d.
Figure 5. Inhibition of SFLLRN-NH2 (3.16 μM) PAR1/2-mediated
iCa2+ mobilization by bivalent ligands 13a−d (A) and individual and
codosed monovalent control compounds (17 + 15) in EA.hy926
endothelial cells (B).
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thatwouldbemoresuitableforthedetectionoflowpopulationsof
heteromers, and studies on PAR-driven cancer cell migration are
also underway. The main weakness of the present ligands is their
decrease in potency at PAR2 upon attachment of spacers to the
PAR2 antagonist scaffolds, and therefore, we are presently
investigating alternative PAR2 antagonists and spacers. Bivalent
PAR1−PAR2antagonistsareanovelclassofligandsthatmayhold
promisefortheinhibitionofcancercellmetastasis,restenosis,and
other proliferative diseases.
assistance with the stability study, and Dr. Sheng Cai (Marquette
University) for assistance with LC−MS and NMR instruments.
WethankACD/Labs(NMRprocessing)andChemAxon(NMR
prediction and property prediction) for providing access to
software.
ABBREVIATIONS
■
Boc,tert-butoxycarbonyl;CuAAc,copper-catalyzedalkyne/azide
cycloadditions; DCE, 1,2-dichloroethane; DCM, dichloro-
methane; DIEA, N,N-diisopropylethylamine; DMAP, 4-dime-
thylaminopyridine; DPPF, 1,1′-bis(diphenylphosphino)-
ferrocene; EDC, 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide; GPCR, G-protein coupled receptor; HATU,
hexafluorophosphate azabenzotriazole tetramethyl uronium;
HOBt, 1-hydroxybenzotriazole; IC50, half-maximal inhibitory
concentration; iCa2+, intracellular calcium mobilization; MI,
myocardial infarction; PAR, protease-activated receptor; PCI,
percutaneous interventions; PyBOP, (benzotriazol-1-yloxy)-
tripyrrolidinophosphonium hexafluorophosphate; SEM, stand-
ard error of the mean; TBTA, tris(benzyltriazolylmethyl)amine
ASSOCIATED CONTENT
* Supporting Information
TheSupportingInformationisavailablefreeofchargeontheACS
Publications website at DOI: 10.1021/acsmedchem-
lett.8b00538.
■
S
Additional abbreviations; Schemes S1−S3; Figures S1−
S10; assay protocols; plasma stability assay protocol and
datafor13d;syntheticprotocols;andcharacterizationdata
(1H NMR, 13C NMR, and LC−MS chromatograms)
(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: christopher.dockendorff@mu.edu. Tel: 1-414-288-
1617.
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6394430.v1.
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ACKNOWLEDGMENTS
■
We thank Dr. Hartmut Weiler, Irene Hernandez, and Trudy
Holyst (Blood Research Institute) for assistance with cell culture
and assay troubleshooting, Dr. Michael Dwinell and Donna
McAllister(MedicalCollegeofWisconsin)forhelpfuladviceand
a sample of MDA-MB-231 cells, Prof. Nicolas Moitessier for
providing access to FITTED docking software, Dr. Rachel Jones
(Medical College of Wisconsin) for advice regarding the CuAAC
reaction, Elliot DeMilo (Univ. Wisconsin-Milwaukee) for
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DOI: 10.1021/acsmedchemlett.8b00538
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX