Targeting Integrin-Dependent Adhesion and Signaling with 3-Arylquinoline and 3-Aryl-2-Quinolone Derivatives: A new Class of Integrin Antagonists
Targeting Integrin-Dependent Adhesion and Signaling with 3-Arylquinoline and 3-Aryl-2- Quinolone Derivatives: A new Class of Integrin Antagonists
Sandrine Fiorucci 0 1 2
Xiaochen Lin 0 1 2
Karin Sadoul 0 1 2
Guy Fournet 0 1 2
Daniel Bouvard 0 1 2
Olga Vinogradova 0 1 2
Benoît Joseph 0 1 2
Marc R. Block 0 1 2
0 1 Université Grenoble Alpes , Grenoble, France, 2 INSERM , Centre de recherche U823, Grenoble, France, 3 University of Connecticut, Storrs, United States of America , 4 Université Claude Bernard-Lyon 1, Lyon , France
1 Funding: This work was supported by Fondation ARC, INSERM, CNRS, and Université Grenoble Alpes. CNRS and Fondation ARC gave a fellowship to SF. INSERM gave bench fees
2 Editor: Guido Serini, Candiolo Cancer Institute , ITALY
We previously reported the anti-migratory function of 3-aryl-2-quinolone derivatives, chemically close to flavonoids (Joseph et al., 2002). Herein we show that 3-arylquinoline or 3-aryl2-quinolone derivatives disrupt cell adhesion in a dose dependent and reversible manner yet antagonized by artificial integrin activation such as manganese. Relying on this antiadhesive activity, a Structure-Activity Relationship (SAR) study was established on 20 different compounds to throw the bases of future optimization strategies. Active drugs efficiently inhibit platelet spreading, aggregation, and clot retraction, processes that rely on αllbβ3 integrin activation and clustering. In vitro these derivatives interfere with β3 cytoplasmic tail interaction with kindlin-2 in pulldown assays albeit little effect was observed with pure proteins suggesting that the drugs may block an alternative integrin activation process that may not be directly related to kindlin recruitment. Ex vivo, these drugs blunt integrin signaling assayed using focal adhesion kinase auto-phosphorylation as a read-out. Hence, 3-arylquinoline and 3-aryl-2-quinolone series are a novel class of integrin activation and signaling antagonists.
Competing Interests: The authors have declared
that no competing interests exist.
Many physiological and pathological processes are largely dependent on cell adhesion to the
surrounding extracellular matrix (ECM) . Integrins, the main cell adhesion receptors, are
transmembrane, heterodimeric proteins composed of a large extracellular domain that
interacts with specific ECM proteins and a short cytoplasmic tail which recruits a complex and
dynamic platform of proteins involved in both signaling and mechanical functions.
Integrins are central regulators of cell fate and have raised a great interest as therapeutic
targets [2–5]. So far many of the twenty four known integrin hetero-dimers have been targeted
due to their involvement in immunity , cancer , and processes such as platelet aggregation
or angiogenesis . On the other hand, αvβ3 integrins  and α2/α5β1  have recently been
shown to bypass tyrosine kinase inhibitors treatments leading to resistances in cancer
therapies. Some integrin inhibitors are undergoing late-stage clinical trials in cancer, inflammation,
autoimmune disorders and thrombosis treatment. Most of integrin targeting compounds,
including antagonist monoclonal antibodies, peptide derivatives and small molecule inhibitors,
were designed on a ligand-based strategy. They mimic all or part of the binding domain of
integrin substrates, often recognized through their RGD motif. Such molecules act as
competitors and efficiently prevent integrin mediated cell attachment or platelet aggregation.
Unfortunately though, they also act as partial agonists of integrin signaling , leading to significant
side effects that have, so far, reduced the use of the related therapeutic strategies [12–14]. For
platelets, the use of intravenous αIIbβ3 antagonists has been superseded by the combination of
aspirin and a P2Y12 inhibitor due to decreased bleeding risk and lower cost. Finally, treatments
with integrin antagonists often result in modification of the integrin expression pattern and
drug resistance, hence putting forward the need of inhibitors with multiple targets .
Extracellular stimuli lead to a direct interaction between the β-integrin cytoplasmic tail and
the FERM-like domain of talin, which is regarded as a major integrin activator [16, 17]. The
interaction of integrin cytosolic domain with talin unclasps the complex between α and β
subunit tails [18, 19], triggering conformational changes along transmembrane and extracellular
integrin domains. This rearrangement allows the shift between the integrin “low affinity state”,
where the extracellular domain is folded toward the membrane resulting in access hindrance
for extracellular ligands, to an extended, “high affinity state" where the interaction with the
extracellular matrix is favored. However, talin alone is not sufficient to trigger full integrin
activation. Kindlins can also interact directly with the integrin cytoplasmic tail through a
FERMlike domain  on a NPxY motif distinct from the talin binding site and act as a talin
cofactors required for effective integrin activation. Finally, it has recently been proposed that in
platelets, talin triggers the switch to the high affinity state of αIIbβ3 while kindlin-3 favors
integrin clustering , a state leading to an irreversible aggregation.
Need to develop new strategies for integrin-based therapies with limited side effects will
mainly rely on the design of antagonists that block both integrin/extracellular matrix
interaction and integrin signaling. Consistent with this view, it has been recently reported that
blocking the interaction between the integrin α4 subunit and paxillin with a small membrane
permeable molecule may be used in anti-inflammatory treatment . This inhibition
abrogates α4-integrin mediated responses in T-cells while maintaining integrin-independent
Herein, we describe potent integrin inhibitors with a 3-arylquinoline or a
3-aryl-2-quinolone scaffold that inhibit integrin mediated cell adhesion and integrin signaling (See
supplemental information section for structural description of the molecules). These compounds,
that share similarities with flavonoids, were initially described as cell migration inhibitors .
Indeed, 3-aryl-2-quinoline BJINT006, the lead compound of this study, strongly impacted
focal adhesions (FA) assembly and dynamics in a reversible and dose-dependent manner.
Since platelet activation is a physiological process that is strictly dependent on the integrin
αIIbβ3 , platelets were treated with BJINT small molecules to assess their putative
antithrombotic potential. As expected, the treated platelets showed impaired spreading and
aggregation and were unable to trigger efficient clot retraction. Finally, ex vivo, these drugs blunt
outside-in integrin signaling. Altogether, our results showed that 3-Arylquinoline and
3-aryl2-quinolone derivatives are integrin antagonists that might be used for integrin based therapy
such as anti-thrombotic agents.
Materials and Methods
Reagents and antibodies
Fibronectin was purified from bovine plasma as described previously , rat tail collagen I
was purchased from Becton Dickinson (Pont-de-Claix, France), vitronectin from Life Science
Invitrogen (Saint-Aubin, France) and fibrinogen from EMD Millipore (Molsheim, France).
Poly-lysine was obtained from Sigma-Aldrich (L'isle-d'Abeau, France). Monoclonal antibodies
raised against Kindlin-2 (clone 3A3) and talin (clone TA205) were purchased from
EMD-Millipore. β3 integrin mAb was obtained from Emfret (Eibelstadt, Germany). Various Alexa-488
conjugated antibodies were obtained from Invitrogen and HRP-coupled antibodies from
Biorad (Marnes-la-Coquette, France). Blebbistatin was purchased from EMD Millipore,
TRITC-phalloidin and Thrombin from Sigma-Aldrich and collagen for platelet aggregation
assays from Helena Biosciences.
Plasmids and cell lines
Talin head was inserted in pDsRed C1 plasmid (Clonetech, Saint-Germain-en-Laye, France)
and transfected in pre-osteoblasts using Lipofectamine 2000 (Invitrogen) according to provider
procedure. GFP-Kindlin-2 expressing pre-osteoblasts were obtained from newborn mice
sacrificed by decapitation as previously described . Kindlinfl/fl preosteoblasts were isolated from
corresponding mouse strain generated by EMMA and transfected par ERT2Cre plasmid .
FAK -/- pre-osteoblasts were prepared from FAKfl/fl mice, immortalized with SV40 large T
antigen followed by gene deletion after infection with adenoviruses expressing the Cre
In France since February 2013, according to the European Directive 2010–63, research
using animal models is subject to authorization from the Ministry of Research. Each project
authorization includes ethics opinion issued by the ethics committee for animal experiments
joined by the animal facility. The ethics committee of Grenoble, ComEth-Grenoble is
registered with the National Committee of Reflection on Ethical Animal Experiments of the
Ministry of Research under number 12 (CEEA No. 12). The main task of the committee is to issue
reasoned opinion on the ethics of experimental projects proposed by the experimenters. The
committee has validated this project
Cells were fixed with 4% PFA, platelets with 4% neutral formalin (Sigma-Aldrich). They were
permeabilized with 0.2% Triton X100 in PBS and incubated with appropriate primary
antibodies in PBS- supplemented with 10% goat serum- 0.1% Tween. After 3 washes, the coverslips
were incubated with appropriate secondary antibodies. Cells were mounted in Mowiol solution
and imaged on an inverted microscope (Axioimager, Carl Zeiss S.A.S., LePecq, France).
Cell adhesion was assayed as previously described (Yan, 2008). Briefly, 96-wells plates were
coated with 5 μg/mL of fibronectin overnight. Washed twice with PBS, the wells were then
blocked in 1% BSA for 1 h at 37°C. Cells (1.104/well) in suspension were incubated with or
without BJINT molecule for half an hour at 37°C before being allowed to adhere for 30 min.
Cells were washed with PBS and fixed in 10% methanol– 10% acetic acid before staining in
0.5% Crystal Violet (Sigma Aldrich)- 10% methanol for 10 min at room temperature. Wells
were rinsed three times before dissolving the dye in 10% acetic acid at 37°C for 15 min.
The ability of 3-arylquinoline or 3-aryl-2-quinolone derivatives to detach spread cells was
assayed by modifying the spreading assay protocol. Plates were coated as described above. Cells
were allowed to spread for 1 h at 37°C then the medium was removed and replaced by medium
containing the tested molecule at the desired concentration. Cells were treated for 1 h then
washed before fixation and coloration as described above.
Cell spreading on various substrates
Coverslips were coated either with 10 μg/mL fibronectin or vitronectin in PBS at 4°C overnight
or with 20 μg/mL collagen for 1 h at 37°C in 0.2M acetic acid. The coated coverslips are then
blocked with 1% BSA for 1 h at 4°C. Cells expressing GFP-kindlin-2 were incubated in PBS 5%
BSA for 45 min at 37°C before being allowed to spread on the coated coverslips overnight in
DMEM with 10% fibronectin free FCS. Medium is then replaced with medium containing the
tested molecule or the corresponding volume of DMSO. The cells were incubated 1 h at 37°C
before prior fixation.
Spreading on poly-lysine versus fibronectin
Cells spread on fibronectin (integrin dependent) or poly-lysine (integrin independent)
35-mm-diameter cell culture dishes were coated overnight at 4°C with 10 μg/mL fibronectin or
100 μg/mL poly-lysine. Fibronectin coated dishes were then blocked with 1% BSA while
polylysine coating was continued for 1 h at 37°C. Then the PLL coated dishes were dried for at least
3 hours before use. Cells were incubated in 5% BSA for 1 h at 37°C before being centrifuged
and resuspended in DMEM with 10% fibronectin free FCS and allowed to spread on dishes for
2 h. Medium was then removed and replaced with DMEM with fibronectin free serum
containing BJINT006 at 12.5 or 25 μM. Cells were incubated for 1 h at 37°C before fixation. Remaining
cells were then manually counted on 15 microscope fields.
Live/dead cell toxicity assays
Cells in D-MEM supplemented with 10% fetal calf serum were incubated with 50 μM of BJINT
molecules or DMSO for 1 h at 37°C. 106 cells were centrifuged and washed two times with PBS
then resuspended with 200 μL of PBS. Then 1 μL of freshly prepared 1 μM Calcein AM solution
in PBS was added. Incubation was performed for 45 min at 37°C. Then 5 μL of propidium
iodide solution (10 mg/mL) were added. After homogenization 0.5 mL of cold PBS was added
and cells were immediately analyzed by FACS (FL1 vs FL3 channels).
Human platelet-rich plasma concentrates from anonymous donors were obtained from the
French national blood bank, Grenoble Branch, and used accordingly to the European rules and
approved by Grenoble University Ethical committee. Human PRP and PPP were prepared as
previously described . Briefly, non-therapeutic buffy coats were diluted with an equal
volume of PBS and centrifuged 10 min at 400g at RT. The upper phase corresponding to the PRP
was collected, and a part of the lower erythrocyte rich fraction is kept for the clot retraction
Platelets resuspended in PBS were preincubated for 30 min with BJINT006 at the tested
concentrations. A number of 1.106 platelets/well/400 μL were then seeded into 24-wells plates
containing fibrinogen- or collagen-coated coverslips, centrifuged (3 min, 600g, RT) and placed in
an incubator for 30 min before fixation.
For aggregation assays PRP was adjusted to a platelet concentration of 3x108/mL with PPP and
different concentrations of BJINT drugs or vehicle were added and preincubated for 30 min.
Platelet aggregation was induced by adding 50 uL of collagen (20 μg/mL in 0.9% NaCl) or ADP
(10 μg/mL) to 150 μL PRP and evaluated using an APACT 4004/LABiTec aggregometer.
PRP was adjusted to 3x108 platelets/mL with PPP, 400 μL were pipetted into an aggregation
tube and 2 μL of an erythrocyte rich fraction (see PRP preparation) was added for color
contrast. After an initial 30 min incubation at 37°C coagulation was induced by adding 4 μL of
thrombin (20 U/mL, 0.1% BSA) and mixing with a plastic inoculation loop. After 10 min the
clots were removed, and placed into new tubes containing 400 μL PBS. Pictures were taken for
visual inspection and the remaining extruded serum volume was measured for quantitative
evaluation of clot retraction.
Expression and purification
Cloning, expression, and purification the β3 cytoplasmic tail in non- and
mono-phosphorylated forms have been described previously [18, 29]. To produce 15N-labeled proteins, cells
were grown in M9 minimal medium containing 15NH4Cl (1.1 g/L) as the sole source of
nitrogen. Expression of GST tagged β1, β3, F2/F3 talin domain, or kindlin-2 FERM domains were
purified by affinity on Glutathion-Sepharose 4B (GE heathcare). Biotinylation of purified
proteins was carried out using EZ link NHS kit from ThermoScientific according to the
GST β1 and β3 cytoplasmic tails were expressed in the BL21-CodonPlus–RIL E. coli strain.
Bacteria were disrupted in a buffer made of 50 mM TRIS Cl pH 7.5, 1% (w:v) Triton X100; 150
mM NaCl, 5 mM MgCl2, 2 mM DTT and anti-protease mix (Roche) by sonication. The lysate
was clarified by centrifugation at 14000 rpm in a JA20 rotor (Beckman) and used immediately
or frozen in liquid nitrogen and stored at -80°C. Glutathione coupled Sepharose beads (160 μL
of bead suspension) were washed with the bacteria lysis buffer, mixed with 1 mL of bacterial
lysate, and incubated overnight at 4°C. Beads were washed 3 times with cell lysis buffer and
resuspended in 1 mL of this buffer before use.
Pre-osteoblast cells (90% confluent) expressing either GFP kindlin-2, or DsRed talin head in
10 cm Petri dishes were washed with 10mL/dish of cold PBS, then lysed by 1 mL/dish with cell
lysis buffer (10 mM PIPES pH 6.8, NaCl 100 mM, Na2VO4 1 mM, Na2PO4,7H2O 50 mM, 0.1%
(w:v) Deoxycholate, Sucrose 150 mM, 0.5% (w:v) Triton X100, anti-protease mix). The lysate
was clarified by centrifugation at 13600 rpm for 15 min.
Beads and cell lysates were pre-incubated separately with 50 mM of BJINT derivatives or
vehicle for 1 h with agitation at 4°C, then 250 μL of bead suspension were mixed with 0.6 mL of
cell lysate and incubation was continued for 4 h at 4°C. After 3 washes in cell lysis buffer the
beads were drained and mixed with 15 μL of Laemli sample buffer and elutes proteins were
analysed by SDS-PAGE and Western blotting with anti kindlin or anti talin head primary
Solid phase binding assays
GST tagged recombinant proteins were expressed in the BL21-CodonPlus–RIL E. coli stain,
purified on glutathione-Sepharose 4B beads according to classical procedures, frozen in liquid
nitrogen, and stored at -80°C. GST β1 and β3 cytoplasmic tails were biotinylated using the
EZlink SulfoNHS biotinylation kit (Themo Scientific) according to the manufacturer
All stages were carried out at room temperature. 96 wells plates (Maxisorp Nunc) were
coated with 100 μL/well of 10 μg/mL of GST, GST-talin-F2/F3 domain, or GST
kindlin2-FERM domain for 1 h. The wells were post coated with 200 μL/well of PBS supplemented
with 3% (w:v) BSA for another hour. Then GST-β integrin tails in the cell lysis buffer (100 μL/
well) were introduced at increasing concentrations and let to incubate 1 h. After 3 washes with
PBS (200 μL/well), 100 μL/well of streptavidin HRP ELISA grade (BioRad) in PBS 3% BSA
were added and incubated for 1 h. Finally after 3 more washes with PBS, integrin tail detection
was achieved using an ABTS solution (Vectastain) according to the manufacturer instructions
and absorbance was measured at 405 nm. Coating with plain GST allowed us to determine the
non-specific binding and this signal was subtracted from the signals obtained with GST-talin
or GST-kindlin-2 domains. It is noteworthy that specific binding was only significant in the
presence of non-ionic detergent.
Integrins are important receptors involved in cell migration by orchestrating the clustering of
proteins at or near the plasma membrane. We generated mesenchymal cell line
(pre-osteoblasts) expressing an important integrin regulator, kindlin-2, fused to eGFP to dynamically
monitor adhesive structures such as FAs or focal complexes. Immunofluorescence labeling
using a monoclonal anti-kindlin-2 antibody recognizing both endogenous and exogenous
kindlins shows a perfect co-localization, indicating that the distribution of both proteins was
identical (S1 Fig). To improve FA assembly, the cells were seeded on fibronectin coated coverslips
and allowed to spread for 90 minutes in presence of DMSO, the drug vehicle. Under these
conditions the cells were nicely spread and displayed typical peripheral focal adhesions. However,
addition of BJINT006 to the medium triggered cell rounding up at the lowest concentrations
tested, along with a clear kindlin-2 delocalization from FAs (Fig 1A). Increasing the drug
concentration finally resulted in a dose dependent cell detachment which was estimated using
Crystal Violet staining as described under Materials and Methods. Similar results were
observed using 3-aryl-2-quinolone BJINT 011 (Fig 1B), a closely related drug with the lateral
side chain positioned differently on the molecule backbone, while in the absence of this lateral
side chain on 3-aryl-2-quinolone BJINT020, the molecule exhibited no effect on cells even
when used at the high concentration of 50 μM (not shown). To rule out any possible artifacts
due to drug toxicity, BJINT treated cells were subjected to Calcein AM/propidium iodide
Fig 1. BJINT derivatives promote focal adhesion disassembly and cell detachment in a reversible and dose dependent manner. (A) Suppression of
cell spreading under BJINT006 treatment. Pre-osteoblasts expressing GFPkindlin-2 were spread on fibronectin coated coverslips (5 μg/mL) for 1 h 30 at
37°C before adding the BJINT drugs and incubation was continued for 1 h. (left and central panels) Reversibility of BJINT006 after 1 hour drug wash-out
(right panel). (B) Cells were treated with increasing BJINT006 and 011 concentrations and adhesion was compared to untreated cells (control) or cells
treated with vehicle (DMSO). Cells were spread for 1h30, and then treated for 1h. After fixation, they were stained with Crystal Violet (0.5% w:v) and optical
density was read at 620 nm. (C) Cells in suspension were treated for 1h at 37°C then let to attach and spread on fibronectin coated dishes for 1 h 30. They
were compared to cells treated with vehicle (DMSO) or untreated (control). Adherent cell numbers were estimated as described above.
Having shown that the molecules were able to interfere with focal adhesion stability we
asked whether it could prevent cell adhesion. Cells were first incubated in suspension with
increasing drug concentrations and then let to attach and spread for 90 minutes. Quantification
of the number of cells attached to the plates indicated that the compounds 006 and 011 not
only triggered FA disassembly but were also able to prevent their formation within the same
concentration range (Fig 1C).
To assess whether the molecules action was irreversible or not, medium containing
BJINT006 was removed and the cells were extensively washed before being allowed to spread
for 1 hour. After the drug wash-out, the cells were able to assemble FAs again, showing that
BJINT006 inhibitory activity was reversible and that the drug is likely nontoxic during the time
course of incubation (Fig 1A). In addition we observed that the BJINT006 dependent inhibition
of adhesion was independent of the ECM protein used to coat coverslips, since cell rounding
up and detachment was also observed on either type I collagen or vitronectin (S3 Fig). This
lack of specificity suggested that the drugs target a component or regulatory mechanism
common to all adhesive structures, thus are likely to act intra-cellularly.
The activity of BJINT family members is closely related to their structure
To address the structure-activity relationship of 3-arylquinoline and 3-aryl-2-quinolone
derivatives, compounds with modifications on their lateral positions were synthesized according to
the strategy described in (S4 Fig). Twenty different compounds were tested using the adhesion
assay described above. It appeared that the presence of a tertiary amine on R3 is required for
BJINT family members to impede cell adhesion (Fig 2). Higher steric occupation around the
amine may alter molecule activity depending on the lateral chain attachment to the backbone.
Modification of the lateral chain length has little effect on the compound activity whereas
increasing chain rigidity (BJINT018) seems to impair its activity, as does the suppression of R1
and R2 methoxy- substitutions. Whether longer chains can be introduced at these positions has
yet to be investigated. This opportunity may raise the perspective of coupling the molecule to a
fluorochrome or another relevant tool to further study the activity of the compounds.
Therefore, further key experiments were carried out with BJINT006 and BJINT011 as active
compounds and BJINT020 as negative control. The structural differences in these two active
molecules allowed assessing the importance of the lateral chain positioning on the main
backbone of the molecule.
3-Arylquinoline and 3-aryl-2-quinolone derivatives inhibit FA assembly
independently on the integrin Src/FAK signaling axis and cell contractility
FA disassembly caused by 3-arylquinoline and 3-aryl-2-quinolone derivatives may be of direct
or indirect nature. The effect due to impairment of function or recruitment of the structural
components, such as integrin receptors themselves, or linkers to cytoskeleton, including talins,
kindlins, vinculin, is considered direct. The indirect action might occur through the
perturbation of integrin signaling pathways, which include the activation state of Src-family kinases. On
SYF cells (lacking Src, Fyn and Yes expression) as well as on FAK-/- immortalized
pre-osteoblasts, addition of BJINT 006 resulted in cell detachment (Fig 3A). This rules out any indirect
effect in the drug action relying on FAK and Src-family kinases, two major actors in integrin
Along with adhesive structures, the actin cytoskeleton and cell contractility machinery are
key players of cell migration . Reciprocally, cell contractility dramatically impacts on
adhesive structure assembly and patterning. To test whether BJINT006 adhesion inhibition depends
on contractility, we treated GFP-kindlin-2 expressing pre-osteoblasts with a combination of
Fig 2. The activity of BJINT family members is closely related to their structure. Structure-activity relationship of 3-arylquinolines and
3-aryl2-quinolones on cell spreading and attachment. BJINT003, BJINT006 and derivatives impair cell spreading and attachment mediated by focal adhesions.
The activities have been estimated using at least three independent experiments where cells have been spread on fibronectin, fixed and stained with Crystal
BJINT006 and Blebbistatin, a specific myosin-II inhibitor , (Fig 3B). Blebbistatin at 10 μM
was added to cells during their spreading and BJINT006 was added 2 h later. When cell
contractility was inhibited with Blebbistatin, the cells displayed a greater projected area compared
to untreated cells. However, BJINT006 treatment for two hours still resulted in cell rounding
up, suggesting that myosin II is not the primary target of the drug and that the molecule is
active even when integrin clustering cannot be triggered by inner tension.
Integrin engagement in cell adhesion is necessary for BJINT full activity
Next, we wondered whether integrins or associated proteins might be directly targeted by the
molecule. Indeed, if the protein platform, recruited around integrin cytoplasmic tails, or the
integrins themselves were the molecular target of BJINT drugs, these compounds should be
ineffective on cell adhesion when this latter process does not require integrin engagement with
the ECM substrate. To address this question, we studied BJINT006 and BJINT020 adhesion
inhibition on cells spread on fibronectin and compared it to cells attached to poly-lysine, a
substrate known to support cell adhesion in a non-specific, integrin-independent manner .
When seeded on poly-lysine, the cells, although being adherent, did not spread on the
substrate. Neither BJINT006 nor BJINT020 addition at the concentration of 25 μM significantly
affected cell attachment to poly-lysine, while cell attachment on fibronectin was reduced by
more than 90% by BJINT006 (Fig 3C). Consistent with the results described above, addition of
BJINT020 did not significantly impair cell adhesion on both substrates. These results strongly
suggested that the drugs specifically impaired integrin related adhesive structures. Specificity
was further confirmed by the Mn2+ artificial switch of the integrins to the high affinity state
that antagonized both BJINT006 and BJINT011 action (Fig 3D), while BJINT020 was once
again ineffective on cell adhesion, independent on the experimental conditions used. These
latter results suggested that the adhesion defect observed upon drug treatment was due to a lack
of integrin activation.
To further confirm that bioactive BJINT components inhibit integrin activation and/or
clustering, we investigate the drug action on physiological and well characterized processes, namely
platelet spreading, clot retraction, and platelet aggregation, that are largely dependent on αIIbβ3
activation. Indeed, the switch from low to high affinity states is required for platelets to spread
and aggregate. Furthermore, αIIbβ3 provides the physical link between the platelet cytoskeleton
and the fibrin network, which is essential for clot retraction. Therefore, we first checked
whether BJINT006, 011 and 020 were able to prevent platelet spreading on fibrinogen.
Similarly to what was found with pre-osteoblasts, platelet spreading was impaired by BJINT006 and
011 in a dose dependent manner but not by 020 (Fig 4A left panel). However, platelets
adhesion was not affected by the drug, consistent with the known ability of αIIbβ3 to interact with
immobilized fibrinogen independently of integrin activation (Fig 4A right panel).
Platelet aggregation requires αIIbβ3 linkage with fibrinogen. Indeed, in the presence of
BJINT006 and BJINT011, collagen-induced platelet aggregation was completely abolished
(Fig 4B right panel). This result clearly fits with experimental data showing that β1 knock out
Fig 3. BJINT derivative inhibition of cell adhesion is integrin dependent. (A) Pre-osteoblasts, FAK-/- MEFs, or MEFs devoid of Src, Yes, Fyn kinase
expression (SYF cells) were allowed to spread for 1 h 30 on fibronectin coated multi-well plates in presence of increasing concentrations of BJINT006.
Adhesion was inhibited similarly in all cell lines. (B) Pre-osteoblasts expressing GFP-kindlin2 spread for 1 h on fibronectin coated coverslips were treated with
10 μM Blebbistatin. BJINT006 at 12.5 or 25 μM in DMSO was then added directly into the cell medium and cell culture was continued for 2 h. Cells were fixed
in 4% PFA. Cell contractility inhibition by Blebbistatin does not prevent cell rounding up after BJINT006 treatment. (C) Cells were seeded on fibronectin or
poly-lysine for 2 h before 1 h incubation with BJINT006, 011, or 020. The cell adhesion on poly-lysine was not significantly modified by BJINT treatment in
contrast to cells spread on fibronectin. (D) MnCl2, an artificial activator of integrins was introduced into the cell culture medium at the concentration of 1 μM
prior addition of BJINT derivatives. It efficiently prevents cell adhesion inhibition by BJINT006 and 011.
completely abolished aggregation in response to soluble collagen . When aggregation was
induced with ADP (Fig 4B left panel), the initial aggregation was normal but limited and
platelets could not sustain aggregation over time suggesting that the secretory phase of aggregation
Fig 4. BJINT006 and 011 inhibit αIIbβ3 mediated platelet activation related processes. (A) Platelet adhesion and spreading on collagen were carried out
as described under Materials and Methods. Quantification of spreading and adhesion upon BJINT006, 011, and 020 treatments is shown on left and right
panels, respectively. (B) Collagen- and ADP -induced aggregation kinetics are shown. Aggregation assays are carried out in the presence or absence of
BJINT derivatives (50μM). (C) Clot retraction is induced by thrombin addition to PRP as described under Materials and Methods. BJINT006, 011 but not
BJINT020, prevents proper clot retraction at the concentration of 100 μM. (D) The extruded serum volume was measured 10 minutes after thrombin addition
to quantify clot retraction.
did not occur. BJINT molecules are hydrophobic and must undergo a phase partitioning
between membranes and cytosol as a prerequisite of their action. Therefore BJINT
pharmacodynamics are likely slow. Consistent with this view, a 15 min preincubation of platelets with
the drugs enable us to block aggregation to a similar level but with a twofold lower drug
concentration (200 to 100 μM). Compared to ADP induction, collagen induction of platelet
aggregation is slower and may provide sufficient time for the drugs to act. Finally, we studied the
ability of treated platelets to achieve clot retraction which arises from the transmission of
actomyosin forces to the fibrin network through activated αIIbβ3. At high concentrations of
BJINT006 and 011 but not BJINT020 (100 μM), the clots fail to retract (Figs 4C and 2D).
Altogether, these experiments showed that, as expected for an antagonist of integrin activation,
BJINT006 and 011 exhibited a strong inhibition of platelet aggregation and are potential
Talin and kindlin recruitment onto the β cytoplasmic tail is regarded as the end point of
integrin activation . Therefore we studied the possible competition between the small molecules
and these proteins for the interactions with the integrin intracellular domain. Pull-down assays
were carried out to investigate the interaction between overexpressed DsRed-talin head domain
or endogenous kindlin-2, overexpressed GFP-kindlin-2, and GST-β3 or GST-β1 cytoplasmic
domains in presence or absence of BJINT006 or 020. Under our experimental conditions, no
differences could be observed in talin head/β3 or β1 integrin interactions whatever the drug
used, while kindlin-2 interaction with the β3 cytoplasmic domain but not β1 was reduced up to
70% by BJINT 006, but not BJINT020, consistent with ex vivo results (Fig 5A). However, in
pull down assays, molar ratios between integrins and cytoplasmic partners are not controlled
and we could not exclude that under our experimental conditions, drug inhibition on β1
interactions with its partners might have been blunted by an excess of ligand. In addition, since the
drugs were added into the cytosol, one cannot exclude an additional effect of these drugs onto
an upstream or alternative regulatory mechanisms of talin and kindlin recruitment. Therefore,
we designed a solid phase binding assay of purified biotinylated integrin tails fused to GST
onto immobilized purified GST tagged kindlin-2 FERM domain or talin F2/F3 domain.
Unspecific binding was estimated using plain GST. This assay allowed the measurement of typical
saturation curves (S4 Fig) and to determine the integrin tail concentrations under which the
interaction with the partner should be sensitive to a competitive inhibitors. Under these
experimental conditions, the overall drug inhibition of the binding of kindlin-2 FERM domain or of
talin F2/F3 domain on β1 or β3 tails were either absent or quite small even at 50 μM, (Fig 5B).
NMR studies to detect a direct interaction of BJINT 006 on the β3 cytoplasmic domain
exhibited very small shifts that were identical for all amino acids, suggesting a non-specific
interaction (S6 Fig). On the other hand, ITC experiments did not reveal any interaction (not shown).
Altogether, these data suggested that BJINT compounds may not specifically interact with
integrin tails. Therefore one could conclude that BJINT molecules interfere with integrin
activation events upstream or alternative to talin and kindlin recruitment.
BJINT derivatives inhibit outside-in integrin signaling
Many biases with currently available integrin antagonists originate from their ability to trigger
outside-in signaling while they efficiently inhibit inside-out signaling and subsequent
cellmatrix or cell-cell interactions. Since BJINT derivatives target integrin tails, we wondered
whether they were able to hamper integrin outside-in signaling. As read-out we looked at the
Fig 5. BJINT derivatives interfere with the binding of talin and kindlin to integrin cytoplasmic tails. (A) Pull-down assays with GST-β3 and GST-β1 tails
bound to glutathione beads incubated for 4 h with cells lysates. Associated talin head and kindlin was revealed Western blotting using anti kindlin and anti
talin head antibodies. Chemiluminescence was monitored by Biorad imager. (B) Summary of 6 solid phase binding assays of biotinylated integrin tails to talin
(F2/F3 domain) and kindlin-2 (FERM domain) under non saturable conditions and in the presence of 50 μM BJINT derivatives.
auto-phosphorylation of FAK, one of the earliest events of integrin signaling using the
established procedure described in . Briefly, HeLa cells were re-suspended in the medium to
switch off integrin signaling, then specific integrin signaling was switched on again by adding
the activating β1 integrin monoclonal antibody TS2/16 in presence or absence of the drug. In
that way, the action of BJINT molecules could not be attributed to an indirect effect due to cell
Fig 6. BJINT derivatives blunt integrin outside-in signaling. Auto-phosphorylation of FAK, one of the earliest integrin dependent signaling events was
used as readout of integrin signaling activity. 107 HeLa cells were harvested and incubated for 1 h at 37°C in D-MEM, then suspended at the concentration of
106 cells/mL as described in  with TS2/16 monoclonal antibody and BJINT molecules or DMSO. Cells were centrifuged and lysed in Laemmli's sample
buffer and analyzed by Western blotting.
detachment. After one hour in suspension, phosphorylation of tyrosine 397 still could be
detected in cell lysates, although this level was slightly increased upon addition of the β1
activating monoclonal antibody TS2/16. BJINT006 and 011, but not 020, completely abolished
FAK auto-phosphorylation and likely all the downstream stages of integrin signaling (Fig 6).
The data presented indicate that the previously described inhibition of cell migration by
3-arylquinoline and 3-aryl-2-quinolone derivatives was likely due to the ability of these compounds
to alter the integrity of structures relying on integrins, as visualized by GFP-kindlin-2
delocalization. Conversely to Kindlin-1 and -3, kindlin-2 is universally expressed and constitutes a
choice marker of focal adhesions whatever the cell line used. Since integrin activation was
largely described to be dependent on the recruitment of kindlin-2 [36, 37], delocalization of
GFP-kindlin-2 appeared as a pertinent read-out. Kindlin-3 is preferentially expressed in blood
cell lineage. A decrease in its expression in humans causes type III leukocyte adhesion
deficiency (LAD-III), which is associated with an inability to activate integrins on platelets and
leukocytes and manifests as susceptibility to bleeding and infections. However, kindlin-2 was
shown to be able to activate β3 integrins at least ex vivo , indicating that both proteins have
a similar function regarding β3 integrin activation. Conversely, kindlin-3 was shown to be
unable to compensate kindlin-2 loss for α5β1 activation in fibroblasts .
Our experiments ruled out the possibility that BJINT inhibition of cell adhesion was directly
linked to cell contractility, FAK, and Src signaling. In addition, the antagonistic action of Mn2+
(a specific activator of integrin receptors) together with the drug insensibility of cells attached
on Poly-lysine point to the targeting of integrin activation by BJINT through an indirect but
common process on inside-out mechanism resulting in the lack of specificity toward
extracellular matrix components. In turn, the switch to the integrin low affinity conformation would
result in the disruption of the adhesive structures.
This indirect effect is suggested by the lack of any detected interaction of BJINT molecules
with integrin tails and the absence of an inhibitory effect of the drugs on the binding of the
purified tails to purified integrin partners such as kindlins and talin, although we cannot rule
out the possibility that full length talin or kindlin-2 may behave differently than the F2/F3 talin
domain and kindlin-2 FERM domain, respectively. In addition, since both pull downs and
solid phase assays were carried out in detergent to minimize non-specific binding, the
hydrophobic nature of these small molecules may account for their trapping into micelles, thus
resulting in a strong decrease in their actual efficient concentration. In vivo, membranes play a
major role in integrin activation and their interaction with cytoplasmic partners, and may also
strongly modulate BJINT inhibitory activity. For instance, under physiological conditions, not
only talin head domain, but also talin tail may participate to the integrin activation process,
which also requires the disruption of the saline bridge and separation of α and β cytoplasmic
domains. In addition, it was recently reported that talin interaction with β3 tail occurs in two
waves, one which triggers external ligand binding but is not involved in outside-in signaling
the other one that is dependent on Gα13 which selectively mediates outside–in signaling .
Therefore it is conceivable that BJINT molecules only impair this second interacting process
while pull down assays only monitor the first one. Phosphoinositide phosphate have also been
reported to play a role in kindlin-2 activity . Finally, while evidence has recently been
provided that talin head and kindlin can interact at the same time on integrin tail , the
spatiotemporal roles of these two major players are yet to be unraveled.
Upstream or alternative direct targets of BJINT derivatives can be envisioned. For instance,
kindlin-2 biding to Integrin linked kinase (ILK) pseudokinase complex was reported to play a
major role in focal adhesion localization of the protein . Other possible candidates are
adaptors proteins allowing the membrane recruitment of the Rap1-interacting adapter
molecule (RIAM) as already demonstrated for αIIbβ3 integrins . Finally, the drug may strengthen
the interaction with endogenous inhibitors in the α cytoplasmic tail such as sharpin .
Whatever the mechanism, it is clear that BJINT molecules blunt both inside-out and
outside-in integrin signaling with a broad receptor spectra. As expected for such a mechanism,
BJINT derivatives block the integrin dependent processes in platelets, and are therefore
potential anti-thrombotic agents. For ADP triggered aggregation however, a limited initial phase of
aggregation was still present. Recently it has been proposed that αIIbβ3 activation is triggered
by talin while kindlin favors clustering . According to this mechanism, and since BJINT
derivatives seem to favor rather than inhibit talin/β3 and blocks kindling β3 tail interaction, it is
conceivable that they only block the integrin clustering required for sustaining platelet
aggregation over time.
SAR studies with twenty members of the 3-arylquinoline and 3-aryl-2-quinolone series
shed some light on important structural features required for the improvement of the molecule
efficiency. Higher affinity compounds will allow structural studies that should help optimizing
the BJINT family members to reach the submicromolar affinity, thus opening the doors to
therapeutic uses. By blocking the integrin cytoplasmic face, this new class of molecules is
expected to minimize integrin outside-in signaling that is a major drawback of all
RGDmimetic agents presently used in therapy. Inhibiting integrin signaling has long been thought
to be a potent way to suppress unwanted downstream signaling pathways . The proof of
concept that such a strategy can be achieved has been provided a couple of years ago when
α4integrin/paxillin interaction has been inhibited by a small molecule (Kummer et al., 2010).
Herein, we showed using FAK auto-phosphorylation as readout that bioactive BJINT
derivatives efficiently blunt integrin outside-in signaling.
Our work provides the first example of small molecules able to cross the plasma membrane
and impair integrin both inside-out and outside-in signaling. It demonstrates that
3-arylquinoline and 3-aryl-2-quinolone derivatives can be efficiently used to block a physiological process,
i.e. platelet activation.
S1 Fig. Colocalization of GFP-kindlin-2 and kindlin-2.
S2 Fig. Toxicity of BJINT derivatives: Analyses of 3 representative molecules.
S3 Fig. BJINT006 cell adhesion inhibition is not specific to an ECM substrate.
S4 Fig. Synthesis strategy for BJINT010-BJINT019.
S1 Methods. Chemical general procedure, synthesis and characterizations.
We thank Pr. Benoît Polack (Grenoble Hospital) for granting us access to the aggregometer
facility, Pr. David Crichley and Dr. Yizheng Tu for providing Talin and kindlin-2 constructs,
respectively, Dr. C. Albiges-Rizo and Dr. L. Lafanechere (IAB Grenoble) for fostering us
workConceived and designed the experiments: SF KS DB OV BJ MRB. Performed the experiments:
SF XL KS GF BJ MRB. Analyzed the data: SF KS OV MRB. Contributed reagents/materials/
analysis tools: DB OV GF BJ. Wrote the paper: SF KS DB BJ MRB.
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