Pharmacological inhibition of syntenin PDZ2 domain impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo

Article abstract of DOI:10.1002/jev2.12039

Exosomes support cell-to-cell communication in physiology and disease, including cancer. We currently lack tools, such as small chemicals, capable of modifying exosome composition and activity in a specific manner. Building on our previous understanding o

Full text of DOI:10.1002/jev2.12039

Received: 23 April 2020
DOI: 10.1002/jev2.12039
Revised: 2 November 2020
Accepted: 14 November 2020
R E SE A RC H A RT IC L E
Pharmacological inhibition of syntenin PDZ domain impairs
breast cancer cell activities and exosome loading
with syndecan and EpCAM cargo
R. Leblanc
M. Feracci
P. Ro ch e 
R. Kashyap
M. Garcia
X. Morelli
K. Barral
C. Derviaux
L. Hoffer
A.L. Egea-Jimenez
D. Kovalskyy,,*
S. Betzi
R. Ghossoub M. Platonov,
Pascale Zimmermann,
1 Equipe labellisée Ligue 2018, Centre de Recherche
en Cancérologie de Marseille (CRCM),
Aix-Marseille Université, Inserm, CNRS, Institut
Paoli-Calmettes, Marseille, France
Abstract
Exosomes support cell-to-cell communication in physiology and disease, including
cancer. We currently lack tools, such as small chemicals, capable of modifying exo-
some composition and activity in a specific manner. Building on our previous under-
standing of how syntenin, and its PDZ partner syndecan (SDC), impact on exosome
composition we optimized a small chemical compound targeting the PDZ2 domain
of syntenin. In vitro , in tests on MCF-7 breast carcinoma cells, this compound is non-
toxic and impairs cell proliferation, migration and primary sphere formation. It does
not affect the size or the number of secreted particles, yet it decreases the amounts
of exosomal syntenin, ALIX and SDC4 while leaving other exosomal markers unaf-
fected. Interestingly, it also blocks the sorting of EpCAM, a bona fide target used for
carcinoma exosome immunocapture. Our study highlights the first characterization
of a small pharmacological inhibitor of the syntenin-exosomal pathway, of potential
interest for exosome research and oncology.
2 Centre de Recherche en Cancérologie de
Marseille, Integrative Structural & Chemical
Biology, Aix-Marseille Université, Inserm, CNRS,
Institut Paoli Calmettes, Marseille, France
3 Enamine Ltd., Kyiv, Ukraine
4 Taras Shevchenko National University of Kyiv,
Kyiv, Ukraine
5 Institute of Molecular Biology and
GeneticsNational Academy of Sciences of Ukraine,
Kyiv, Ukraine
6 Department of Human Genetics, K. U. Leuven,
Leuven, Belgium
Correspondence
K E YWOR D S
drug design, exosomes, inhibitors, PDZ, syndecan, Syntenin
Pascale Zimmermann, Department ofHuman
Genetics, K. U. Leuven, B-3000, Leuven, Belgium.
Email:pascale.zimmermann@inserm.fror
pascale.zimmermann@kuleuven.be
R.L. andR.K. contributedequallytothiswork.
*Currentlyat GreeheyChildren’sCancerResearch
Institute, 8403 Floyd Curl Drive, San Antonio, TX
78229, USA

INTRODUCTION
It is now clear that cells communicate via the release of membrane-limited vesicles, collectively called extracellular vesicles (EVs)
(Kalluri & Lebleu, 2020; Mathieu, Martin-Jaular; Lavieu, & Théry, 2019; Stahl & Raposo, 2019). EVs represent complex biological
messengers, containing lipids, proteins, and nucleic acids, including miRNAs. EVs appear to be important in various physio-
pathological situations, in particular during cancer progression, with a role in metastatic niche formation and tumour immune
escape (Greening, Gopal, Xu, Simpson, & Chen, 2015; Wortzel, Dror, Kenific, & Lyden, 2019). Their use for biomarker discovery,
and as vehicles or targets of therapies is currently the focus of intense research. Pending on their origin and mechanism of
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original
work is properly cited.
© 2020 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles
J Extracell Vesicles. 2020;10:e12039.
wileyonlinelibrary.com/journal/jev2
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https://doi.org/10.1002/jev2.12039

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LEBLANC et al.
formation, specific subsets of these vesicles are generally referred to as microvesicles, exosomes, or apoptotic bodies among
others. Exosomes are ‘exocytic’ vesicles, approximately 40–100 nm in size, released from most cell types. Their biogenesis starts
by the inward budding of the limiting membrane of endosomes, generating multivesicular endosomes. Exosomes, including
exosomes derived from both the tumour and tumour-associated host cells, help composing the niches that support tumour
development, occupying a central place in tumour biology. ‘Cancer exosomes’ are thus gaining considerable attention, in basic
and applied clinical research, for their role in the modulation of the tumour microenvironment and their contribution to tumour-
host crosstalk.
Our laboratory has identified a molecular path, supported by the syndecan (SDC) heparan sulfate proteoglycans and their
adaptor protein syntenin, which regulates the biogenesis of a specific class of exosomes (Friand, David, & Zimmermann, 2015).
Syntenin is a small intracellular scaffold protein that contains two PSD-95, Discs-large, ZO-1 (PDZ) domains that bind SDC, an
N-terminal domain that binds ALIX (via three LYPxL motifs), and a short C-terminal domain regulating the conformation of the
protein and accessibility of its domains (Baietti et al., 2012; Grootjans et al., 1997; Saito, Nishioka, Iwatsuki, Kumagai, & Sasaki,
1975). The PDZ domains of syntenin bind the C-termini of the SDCs (Grootjans et al., 1997), but bind also to PIP2 (Zimmermann
et al., 2002) and several other membrane proteins, including CD63 (Latysheva et al., 2006). The binding of syntenin to PIP2
regulates the recycling of endocytosed SDCs from late endosomes back to the cell surface (Zimmermann et al., 2005). In contrast,
SDC-syntenin-ALIX binding regulates the formation of endosomal intraluminal vesicles and the secretion of CD63+ exosomes
that contain syntenin, C-terminal fragments of the SDCs (SDC-CTFs) and potential SDC-cargo such as ligand-engaged receptors
(Baietti et al., 2012). SDCs have been linked to a plethora of cellular processes, including cell–cell and cell–matrix adhesion, signal
transduction, as well as vesicular trafficking (Christianson & Belting, 2014; Couchman, 2010; Lambaerts, Wilcox-Adelman, &
Zimmermann, 2009). The expression level of syntenin are extremely high during fetal development compared to adult tissues
(Zimmermann et al., 2001). Noteworthy, gain of syntenin expression in tumour cells has been associated with the invasion and
the metastatic potential of various solid cancers (Kegelman et al., 2015).
The precise mechanisms involved remain to be further clarified, but the correlation implies that syntenin-exosomes might be
implicated in the processes of malignant cell invasion and the establishment of niches that promote tumour metastasis. Along that
line, we recently revealed that SRC oncogene functions in cell-to-cell communication by controlling the biogenesis of syntenin-
exosomes, identifying these as a specific class of pro-migratory exosomes stimulating recipient HUVEC cells. SRC kinase, phos-
phorylating both SDCs and syntenin, supports the endosomal budding and exosomal secretion of syntenin and associated cargo,
including syndecans and CD63, both binding directly to syntenin, and, in turn, components potentially bound to SDC and
tetraspanins, such as integrins and EGFR (Imjeti et al., 2017). Similarly, the syndecan-syntenin-ALIX pathway is also known to
impact on the intracellular trafficking and exosomal confinement of FGF signals (Baietti et al., 2012). Exosomal CD81, CD9 and
flotillin, in contrast, do no depend on SDC-syntenin (Ghossoub et al., 2014; Imjeti et al., 2017; Roucourt, Meeussen, Bao, Zim-
mermann, & David, 2015), underscoring the heterogeneity of exosome compositions and of mechanisms of exosome formation.
Together, all the data therefore suggest that exosome biogenesis regulated by the syntenin-syndecan pathway might significantly
contribute to tumour-host interactions.
Targeting protein–protein interactions (PPI) has emerged as a potential approach for the treatment of solid tumours or hema-
tologic malignancies (Cierpicki & Grembecka, 2015; Makley & Gestwicki, 2013; Morelli, Bourgeas, & Roche, 2011). Promising
results have been obtained with PPI inhibitors of p53 and BclxL or more recently bromodomain inhibitors (Faivre et al., 2020;
Filippakopoulos et al., 2010; Jung, Gelato, Fernández-Montalván, Siegel, & Haendler, 2015; Milhas et al., 2016; Ulucan, Eyrisch,
& Helms, 2012). PDZ interactions are intracellular PPIs that occur between so-called PDZ domains and short peptide sequences
(PDZ binding motifs). Since syntenin activity strictly depends on the functionality of its PDZ domains (Baietti et al., 2012;
Grootjans et al., 1997; Grootjans, Reekmans, Ceulemans, & David, 2000; Koo et al., 2002; Lambaerts et al., 2012; Luyten et al.,
2008; Meerschaert et al., 2007; Zimmermann et al., 2002; Zimmermann et al., 2005; Zimmermann et al., 2001), we aimed to
develop syntenin PDZ inhibitors and investigate their potential utility to control cellular phenotypes as well as exosomal-releases.
By screening a PDZ-focused fragment library and pursuing a hit-to-lead optimization strategy, we identified a small molecule
that selectively affects syntenin-exosomal releases. We also provide evidence that this inhibitor markedly affects the loading of
exosomes with EpCAM, a transmembrane glycoprotein involved in cell migration, adhesion, proliferation and differentiation,
which has been associated with the processes of tumorigenesis and metastasis of several carcinomas (Keller, Werner, & Pantel,
2019).

MATERIALS AND METHODS
.
Compound library and in silico selection
As a source of screening compounds, we used the PDZ targeted library (1185 compounds at the time) developed at Enamine to
cover all PDZ domains with published structures (https://enamine.net/hit-finding/focused-libraries/ppi-library). For this study,
compounds from the PDZ targeted library were selected with two approaches. First, a random subset of 118 compounds was

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selected which represented 10% of the library. The second subset (an extra 21 compounds) resulted from docking of the PDZ
library to crystal structures of syntenin PDZ2, yielding compounds selected specifically against the syntenin PDZ2 domain. For
that, two crystal structures of the PDZ2 domain (PDB 1W9O, 1W9E) with bound peptides were selected for the docking stage.
The 1W9E structure includes a small rearrangement of the site (essentially Phe213) allowing the binding of a larger C-terminal
hydrophobic residue (Val in 1W9O and Phe in 1W9E). The Schrödinger suite [http://www.schrodinger.com] was used for the
modelling stage. Binding sites were defined as 20 Å cube centred on the carboxylic carbon from C-terminal residue of the bound
peptide. Compounds from the PDZ-targeted library were converted into 3D format using LigPrep routine. The Glide docking
tool was employed for the virtual screening of the library to both binding sites. Standard precision level of accuracy was used
and a maximum of five poses per ligand were allowed during the virtual screening stage. The final selection of compounds was
based on three criteria: score from Glide scoring function, visual inspection of poses and ability of a compound to partially
mimic binding mode of the C-terminal residue from corresponding peptide ligand. At least one hydrogen bond with backbone
atoms of Val209 or Phe211 was required. A total of 25 compounds were finally selected, after the visual analysis stage, to define
the second set of compounds to be experimentally evaluated. In total, 139 non-redundant compounds were selected in silico for
further analysis.
.
Molecular biology
Human syntenin1 cDNA was cloned into the pGEX-5X vector (residues 1–298 or syntenin full-length (FL) for HTRF assays)
(Amersham Pharmacia Biotech AB) or into the pETM11 vector (residues 113–273 of syntenin used for crystallization) (EMBL
Heidelberg) to produce N-Terminal GST or 6xHis tagged fusion proteins respectively. Human GRASP55 tandem PDZ and human
Erbin PDZ domain were cloned into the Gateway pDEST15 prokaryotic expression vectors (Invitrogen) intended to produce the
corresponding N-terminal GST-tagged fusion protein, as previously described (Milhas et al., 2016). For BIAcore experiments, the
constructs pAviTag_N-His_syntenin tandem + Cterm (residues 107–298), pAviTag_N-His_syntenin tandem (residues 107–275),
pAviTag_N-His_GRASP tandem (residues 1–209) and pAviTag_N-His_GST tagged with 6xHis at the N-terminus, followed by
the biotinylation site (AviTag peptide, GLNDIFEAQKIEWHE), were generated by recombination between the pAviTag_N-His
vector (Lucigen) and the PCR amplification product from appropriate cDNA libraries, using primers including 18 nucleotides
of overlap with the ends of the vector (sense 5′-CCG AGC ACT CCT CCT ACC and antisense 5′-GTG GCG GCC GCT CTA
TTA). All open reading frames were verified by DNA sequencing.

PROTEIN PRODUCTION AND PURIFICATION
xHis-syntenin (aa –) for crystallization studies
.
Competent E.coli (ER2566 strain) cells were transformed with the pETM-11 vector (EMBL Heidelberg) allowing expression of
N-terminally His-tagged proteins. Bacteria were plated on LB agar plates with 50 μg/ml Kanamycin. The culture and induction
were performed in TYB medium (5-6 h induction at 30◦C after addition of 0.5 mM IPTG). The pellet was re-suspended
in 30 ml of lysis buffer (25 mM Hepes pH 7.4, 150 mM NaCl, 1 μg/ml DNAse I Recombinant RNase free (Qiagen), 0.013 g
Lysozyme (Sigma) and 1 tablet of protease inhibitor EDTA free (Roche)) and kept on ice for 30 min. After sonication, the
suspension was transferred to JA 20 tubes and centrifuged at 12,000 × g at 4◦C for 45 min. The supernatant was applied
to a HistrapTMHP columns 17-5247-01 (GE Healthcare) equilibrated with 20 ml Buffer A (25 mM Hepes pH 7.4, 150 mM
NaCl and 20 mM Imidazole). The purification was performed on the Akta Explorer (Amersham Pharm Biotech). His-tagged
proteins were eluted with 30 ml of an imidazole gradient (20–500 mM, Buffer B, 25 mM Hepes pH 7.4, 150 mM NaCl and
500 mM Imidazole). 6xHis-tagged proteins were fully recovered within the first 10 ml of elution. The His-tag was removed
using TEV protease at 4◦C for 16 h for crystallization trials. Purification was finalised using size exclusion gel chromatography
with a HiLoad 16/600 Superdex 75pg (GE Healthcare) column in buffer containing 10 mM Hepes pH 7.4, 150 mM NaCl and
1 mM DTT.
.
GST-GRASP and GST-Erbin PDZ domains for HTRF experiments
The production of N-terminal GST-tagged GRASP55 fusion proteins and N-terminal GST-tagged Erbin were accomplished by an
induction 18 h at 25◦C with 0.4 mM IPTG in E. coli BL21 (DE3) bacteria cells transformed with the purified pDEST15-GRASP55
or pDEST15-Erbin expression plasmids (Invitrogen). Fusion proteins were recovered from the cell lysates by conventional affinity
chromatography on Glutathione resin using an Akta Explorer system (Amersham Pharm Biotech).

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LEBLANC et al.
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GST-syntenin full length for HTRF experiments
Competent E. coli (ER2566 strain) cells were transformed with the human syntenin1 pGEX-5X expression vector (GE Health-
care). Expression of N-terminally GST-tagged syntenin-1 full length was induced overnight at 30◦C and by the addition of 0.4 mM
IPTG (Fisher Scientific). Protein was purified using GSTrap4B columns 28-4017-45 (GE Healthcare).
.
Biotinylated xHis tag fusion proteins for BIAcore experiments
Competent Biotin XCell F’ cells were transformed with pAviTag_N-His_syntenin tandem + Cterm, pAviTag_N-His_syntenin
tandem, pAviTag_N-His_GRASP tandem or pAviTag_N-His_GST expression vectors. Expression of the protein was induced
overnight at 25◦C by addition of rhamnose, arabinose and biotin at final concentrations of 0.2%, 0.01% and 50 μM, respectively.
Proteins were purified using HistrapTMHP columns 17-5247-01 (GE Healthcare).
.
HTRF screen
HTRF assays were performed in white 96Well Small VolumeHiBase Polystyrene Microplates (Greiner) with a total working vol-
ume of 100 μl, as described previously (Milhas et al., 2016). Briefly, compounds were firstly dispensed into the wells at 400 μM for
the primary screen or with serial DMSO dilutions for IC50 measurement assays. Primary screening assays have been performed in
monoplicate while IC50 measurements were performed in triplicates. All HTRF reagents were purchased from CisBio Bioassays
and reconstituted according to the supplier protocols. The plates were spun for 3 min at 500 × g and then incubated at 4◦C for 16
h. The HTRF signals were recorded on a POLAR star Omega plate reader (BMG Labtech) with an excitation filter at 337 nm and
fluorescence wavelength measurement at 620 and 665 nm, an integration delay of 60 μs and an integration time of 400 μs. Results
were analysed with a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)] × 104. Percentage of inhibition was
calculated using the following equation: % inhibition = [(compound signal)–(min signal)]/[(max signal)–(min signal)] × 100,
where ‘max signal’ is the signal ratio with the compound vehicle alone (DMSO) and ‘min signal’ the signal ratio without protein
B. For IC50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation:
Y = 100/(1+((X/ IC50)ˆHill slope)).
.
Surface plasmon resonance experiments
All SPR experiments were conducted with a BIAcore T200 instrument (GE Healthcare), at 25◦C. A total of 8000 resonance
units (RU) of biotinylated ligands (N-terminal 6xHis tagged biotinylated syntenin tandem, syntenin tandem with C termi-
nus, GRASP55 tandem or GST as control) were immobilized on a streptavidin-sensor chip (GE Healthcare). Analytes (C58 and
SyntOFF compounds) were perfused at 30 μl per min in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween20)
at different concentrations. The reference channel was N-terminal 6xHis tagged biotinylated GST protein. The injection time
was 240 s, long enough for the sensorgrams to reach equilibrium, and the dissociation time was 240 s. To carry out these SPR
experiments, we employed the method known as ‘single-cycle kinetics’ described in 2006 by Karlsson and co-workers (Karlsson,
Katsamba, Nordin, Pol, & Myszka, 2006). It consists in injecting the analyte at increasing concentrations, without regeneration
steps between each sample injection to avoid that folded-proteins can be damaged after regeneration steps. Equilibrium disso-
ciation constants (KD) were calculated by fitting the data to a simple Langmuir binding isotherm using GraphPad Prism. For
determination of the apparent KD, signals obtained at equilibrium were plotted as a function of compound concentration. Cog-
nate peptides for syntenin (19mer SDC4 peptide, sequence YDLGKKPIYKKAPTNEFYA) and GRASP55 (19mer JAM-C peptide,
sequence NYIRTSEEGDFRHKSSFVI) were used to confirm the functionality of the immobilized proteins.
.
Chemistry
All commercial reagents were purchased from Sigma-Aldrich company. Furthermore, all dry solvents were obtained via Sigma-
Aldrich with Sure/Seal system and regular solvents were obtained via Sigma-Aldrich at technical grade. Analytical thin layer
chromatography (TLC) of the reactions was performed on silica gel 60F 254 aluminium plates (Merck) of 0.2 mm thickness with
appropriate solvents. The spots were examined with UV light (λ = 254 nm). Preparative flash column chromatographie’s were
performed using silica gel (Merck) G60 230–240 under compressed air. The 1H NMR and 13C NMR spectra were determined with
a BRUCKER AMX 250 MHz or BRUKER Avance III nanobay 400 MHz. The chemical shifts are reported in ppm and coupling
constants (J) are reported in hertz. Reaction monitoring and purity of compounds were recorded by analytical Agilent Infinity

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high performance liquid chromatography (Column Zorbax SB-C18 1.8 μM (2.1 × 50 mm); Mobile phase (A: 0.1% FA H2O, B: 0.1%
FA CH3CN, Time/%B: 0/10, 4/90, 7/90, 9/10, 10/10); Flow rate 0.3 ml/min with DAD at 254 nM. All tested compounds yielded
data consistent with a purity of ≥95%. Low-resolution mass spectra were obtained with Agilent SQ G6120B mass spectrometer
in positive and/or negative electrospray modes. Resolution Mass Spectra (HRMS) were obtained on a SYNAPT G2-S WATERS
mass spectrometer.
.
Synthesis of -(-chlorophenylthio)propanoic acid 
To a stirred solution of 4-chlorothiophenol 1 (1 eq, 5.0 mmol) and sodium hydroxide (2.4 eq, 12.0 mmol) in water, aqueous solution
of 3-chloropropionic acid was added dropwise (1.7 eq, 8.5 mmol). The mixture was refluxed during 3 h. After completion, 1N HCl
solution was added dropwise at 4◦C until pH 1, to afford a crude precipitate which was filtered and dried. The crude precipitate
was purified by column chromatography eluting with dichloromethane-methanol (100 to 98:2) to afford pure compound . 1H
NMR (CDCl3, 250 MHz) δ = 7.35–7.08 (m, 4H), 3.07 (t, J = 7.2 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H). 13C NMR (CDCl3, 63 MHz)
δ = 175.58, 132.74, 132.16, 130.80, 128.18, 33.13, 28.39. LC/MS (ESI): 215.7 [M-H]–; white powder. Yield = 75%.
.
Synthesis of (S)-methyl -(-(-chlorophenylthio)propanamido)--methylbutanoate
cSyntOFF
To a stirred solution of propionic acid derivative  (1 eq, 1.0 mmol) and L-valine methyl ester hydrochloride (1 eq, 1.0 mmol) in dry
dichloromethane, N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (2 eq, 2.0 mmol) and triethylamine (2 eq,
2 mmol) were added. The mixture was stirred under argon atmosphere during 16 h at room temperature. After completion, the
resulting mixture was washed twice with 1N HCl solution, once with water, once with brine, dried over Na2SO4, and evaporated
under vacuum. The crude residue was purified by column chromatography eluting with dichloromethane-methanol (100 to 98:2)
to afford pure compound cSyntOFF. 1H NMR (DMSO-d, 300 MHz) δ = 8.19 (bd, J = 8.1 Hz, 1H), 7.38 (d, J = 8.9 Hz, 2H), 7.35
(d, J = 9.0 Hz, 2H), 4.26–4.12 (m, 1H), 3.63 (s, 3H), 3.14 (t, J = 7.1 Hz, 2H), 2.53 (t, J = 7.1 Hz, 2H), 2.00 (m, 1H), 0.87 (d, J = 6.6 Hz,
3H), 0.86 (d, J = 6.7 Hz, 3H). 13C NMR (DMSO-d, 75 MHz) δ = 172.47, 170.86, 135.62, 130.89, 130.45, 129.41, 57.87, 52.06, 34.91,
30.39, 28.83, 19.40, 18.72; LC/MS (ESI): 328.63 [M-H]–, 330.1 [M+H]+; white solid. Yield = 62%.
.
Synthesis of (S)--(-(-chlorophenylthio)propanamido)--methylbutanoic acid SyntOFF
To a stirred solution of ester intermediate derivative  (1eq, 0.5 mmol) in THF at 0◦C, aqueous solution of LiOH (10 eq, 5.0 mmol)
was added dropwise. The resulting mixture was stirred 1 h at room temperature. After completion, THF was removed under
vacuum. Then, 1N HCl solution was added dropwise until pH 2–3 and the aqueous layer was extracted with ethyl acetate. The
organic layer was washed once with water, once with brine, dried over Na2SO4, and evaporated in vacuo to afford crude residue
SyntOFF which does not require further purification. 1H NMR (DMSO-d, 300 MHz) δ = 12.51 (bs, 2H), 8.04 (d, J = 8.5 Hz, 1H),
7.38 (d, J = 9.2 Hz, 2H), 7.34 (d, J = 9.3 Hz, 2H), 4.16 (dd, J = 8.4 Hz and 6.0 Hz, 1H), 3.14 (t, J = 7.1 Hz, 2H), 2.54 (t, J = 7.0 Hz,
2H), 2.03 (m, 1H), 0.87 (bd, J = 6.6 Hz, 6H); 13C NMR (DMSO-d, 75 MHz) δ = 173.41, 170.71, 135.69, 130.86, 130.42, 129.40, 57.62,
34.99, 30.34, 28.91, 19.58, 18.48. LC/MS (ESI): 314.6 [M-H]–, 316.2 [M+H]+; HRMS (TOF, ESI+) calculated for C14H19NO3SCl
[M+H]+ 316.0774, found 316.0776; white solid. Yield = 89%.
.
X-ray crystallography
Crystallization trials were performed using the hanging drop vapour diffusion method at 4 and 12◦C. The apo–Syntenin PDZ
protein crystallized in 0.1 M Sodium Acetate pH 4.6/0.2 M Ammonium Acetate and 22% PEG 3350 at a protein concentration
of 6 mg/ml. Syntenin PDZ crystals were soaked in mother liquors containing 5 mM of SyntOFF or 5 mM C compounds (5%
DMSO) for 4 h at 12◦C. Crystals were flash-frozen in mother liquor containing 10% glycerol. Data set of syntenin PDZ - SyntOFF
and Syntenin PDZ - C were collected at the ID30B ESRF and processed using X-ray Detector Software (XDS).
.
Compound C
Data set were collected at a resolution of 1.91 Å and cut at 2.0 Å. The space group was assigned to P1 with four proteins and two
C compounds per asymmetric unit. The phase was solved by molecular replacement using the structure of the PDB entry
1W9E as a template and the program PHASER. Models were rebuilt using COOT and refined with REFMAC5.

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LEBLANC et al.
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Compound SyntOFF
Data set were collected at a resolution of 2.01 Å and cut at 2.2 Å. The space group was assigned to P1 with four proteins and
four SyntOFF compounds per asymmetric unit. The phase was solved by molecular replacement using the structure of the PDB
entry 1W9E as a template and the program PHASER. Models were rebuilt using COOT and refined with REFMAC5. Both datasets
showed translational Non-Crystallographic Symmetry (TNCS) that was handled by PHASER during the molecular replacement
step.
.
Cell culture and reagents
MCF-7 cell lines were purchased from the American Type culture collection (Manassas, VA, USA). Cells were grown in DMEM-
F12 (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Eurobio) at 37◦C under 5% CO2. Syntenin CRISPR/Cas9 knock-
out (SyntKO) MCF-7 cells were generated following the procedures as previously described (Imjeti et al., 2017). Briefly, SDCBP-
specific gRNA oligos sgRNASDCBP1 targeting exon1 (F: CACCgCTATCCCTCACGATGGAAGT; R: AAACACTTCCATCGT-
GAGGGATAGc) were cloned into the pX458 two-in-one CRISPR targeting vector (Addgene, Cambridge, MA, USA), individu-
ally transfected into MCF7 cells and sorted for GFP expression after 48 h. Single cell clones were grown and screened by western
blotting for syntenin expression. One individual clone has been used in the in vitro experiments. The cDNA encoding mCherry-
syntenin was derived from the eGFP-syntenin cDNA construct (Grootjans et al., 1997), by restriction-ligation. The expression
vector for Ce-RAB5(Q79L) was received from W. Annaert (K.U. Leuven, Belgium). The CAY10594 inhibitor (used at 10 μM) was
purchased from Santa Cruz (sc-223874).
.
Cancer cell viability
The effects of the compounds C and SyntOFF were tested both on MCF-7 and MCF-7 SyntKO cells. Briefly, the cells were
treated with increasing concentrations of the compounds for 48 h and early/late apoptosis was measured by using the FITC
Annexin V apoptosis detection kit with 7-AAD (Biolegend, San Diego, CA, USA) according to the manufacturer. Annexin V
positive but 7-AAD negative (early apoptotic cells) and Annexin V positive and 7-AAD positive (late stage apoptosis) was deter-
mined by using FACS LSRII flow cytometer (BD Biosciences) and data were analysed with Flowjo software (Tree Star).
.
Exosomes and total cell lysates
For comparative analyses, exosome-enriched fractions were collected from equivalent amounts of culture medium, conditioned
by equivalent amounts of cells. MCF-7 cells were treated with indicated compounds or with DMSO 0.2% as control, in medium
containing exosome-depleted FCS (10%). After 16 h, cell media were collected and exosomes were isolated by three sequential
centrifugation steps at 4 ◦C: 10 min at 500 × g, to remove cells; 30 min at 10,000 × g, to remove cell debris; and 1h30min at
100,000 × g, to pellet exosomes (exosome-enriched fraction), followed by one wash with 1400 μl of PBS1X (100,000 × g, 1 h),
to remove soluble serum and secreted proteins. Exosomal pellets were then re-suspended in 100 μl of PBS1X. The lysates from
corresponding cultures were cleared by centrifugation at 1500 rpm for 5 min and then resuspended in lysis buffer (TrisHCL
pH 7.4 30 mM, NaCl 150 mM, 1% NP40 (IGEPAL), 1 μg/ml aprotinin, 1 μg/ml leupeptin). Although only little variations were
observed from sample to sample, exosomal amounts loaded for the western blot were normalized according to the number of
parent cells from where exosomes were secreted.
.
Western blots
The proteins were heat-denaturated in Laemmli sample buffer, fractionated in 12.5% or 15% gels by SDS–PAGE and electro-
transferred to nitrocellulose membrane. Membranes were stained with Ponceau red and immunoblotted with the indicated
primary antibodies for: CD9 (1/5000), CD63 (1/5000), CD81 (1/5000) and ADAM10 (1/1000) antibodies provided by E. Rubin-
stein (Charrin et al., 2001) (Université Paris-Sud UMRS_935, Villejuif); syntenin (Homemade (Kashyap et al., 2015); 1/3000),
Tubulin (Sigma-Aldrich; 1/10,000), ALIX (Homemade (Baietti et al., 2012); 1/500), HSP70 (Santa Cruz Biotechnology; 1/500),
Flotillin-1 (BD Biosciences; 1/1000), EGFR (Cell Signaling; 1/1000), EpCAM (Santa Cruz Biotechnology; 1/200), Fibronectin
(BD Biosciences; 1/5000), Src (Cell signaling; 1/1000), TSP1 (Lab Vision; 1/100), SDC1 intracellular domain (Homemade (Baietti
et al., 2012); 1/1000), SDC4 intracellular domain (Abnova; 1/2000), and HRP-conjugated secondary antibodies (Mouse or Rabbit,
Thermofisher scientific; 1/10,000). Signals were visualized using Amersham ECL Prime Western Blotting Detection Reagent
(GE Healthcare).

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.
Nanoparticle tracking analysis (NTA)
Concentrations and size distributions of exosomal fractions (100,000 x g pellet) isolated from the same number of cells were
measured at a dilution of 1:50 in 500 μl of PBS1 with the Nanosight NS300 instrument (Malvern), which was equipped with a
488 nm laser and a sCMOS camera. Three videos of 60 s each were recorded for each sample at 25◦C and -used to calculate mean
values of particle concentration.
.
Wound-healing assay
For migration assays, cells were treated with 1 ng/ml of mitomycin (Sigma M4287) overnight to inhibit cell division. Treated cells
were plated in Ibidi culture-inserts (Ibidi Cat-80206) for 12 h to reach 90%–95% of confluence with or without compounds (100
μM). A wound was created by removing inserts from the dishes. Medium with or without compounds (100 μM) was refreshed to
remove dead cells and the cells were observed under an inverted light microscope (Leica SP2) equipped with a camera. Images
were taken by MetaMorph software every 10 min for 24 h. Measurements of cell velocity were calculated using the MetaMorph
software.
.
Soft agar colony formation assay
Agarose was melted in water and mixed with DMEM-F12 medium pre-incubated at 37◦C to reach a 0.6% or a 0.36 % final
concentration. A lower layer of 0.6% agarose was deposited in each well of a 96-well plate except that the first and last rows and
columns of the plate were filled with PBS to avoid drying. Agarose was allowed to cool for 15 min before the upper layer containing
different concentrations of cells, a final concentration of 0.36% agarose and compounds (100 μM) was added. Compounds were
added only once at the beginning of experiment. Each condition was tested in triplicate. Colonies were kept at 37◦C in a 5%
CO2 incubator and daily observed under microscope. At the end of the experiment, cells were tested for their ATP content, an
indicator of their metabolic activity, using the Promega kit G7570. In this assay, ATP is used as a co-factor for a luciferase reaction.
Signals were analysed by measuring luminescence with a LUMIstar luminometer (BMG Labtech).
.
Mammosphere formation assay
MCF-7 and MCF-7 SyntKO cells were treated with increasing concentrations of compounds C, SyntOFF (50-100μM) or
DMSO 0.2% as control. After 24 h, cells were trypsinized, re-suspended in normal growth media and washed three times in
PBS to remove serum. Cell concentration was determined using the LUNA-FL Automated Fluorescence Cell Counter (Logos
Biosystem) and cells were then seeded at 1000 cells/ml in 6-well Ultra low Adherence plates (Corning Inc., Corning, NY) in
MEBM medium (Lonza) supplemented with 5 μg/ml insulin (Sigma-Aldrich), 20 ng/ml recombinant epidermal growth factor
(Sigma-Aldrich), 1X of B27 supplement (Invitrogen, Carlsbad, CA, USA), 0.5 μg/ml hydrocortisone (Sigma-Aldrich), 1X of β-
mercaptoethanol and 1% penicillin/streptomycin (Gibco). Fresh medium was added into the plates every 3–4 days. The number
of spheres (diameter > 50 μm) was evaluated under microscope on days 7–10. Sphere formation was calculated as the number of
spheres divided by the original number of cells seeded and normalized to control.

RESULTS
.
In vitro evaluation of a PDZ-focused compound library
The search strategy for PDZ2 syntenin inhibitors was based on the PDZ targeted library developed from Enamine compound
collection. The PDZ targeted library was developed to cover virtually all PDZ domains. Consequently, to create the syntenin
PDZ2 focused library we used two independent paths: docking of the PDZ targeted library against structures of syntenin PDZ2
available in Protein Data Bank and a random selection from the PDZ targeted library. The docking has produced 21 virtual
hits, which were added to 118 randomly selected compounds (10% of the pan PDZ library). The resultant set of 139 compounds,
syntenin PDZ library was subjected to experimental validation. The primary screen was performed at 400μM using a previously
validated Homogeneous Time Resolved Fluorescence (HTRF) assay (Milhas et al., 2016). HTRF is based on Fluorescence
Resonance Energy Transfer (FRET) between two fluorescent reagents. In the present case, Anti-GST-Terbium antibody and
streptavidin-bound Dynamic2 were used as donor and acceptor respectively. Interactions between GST PDZ-fusion proteins
and biotinylated peptides containing PDZ binding motifs were challenged with the potential inhibitors. In parallel, control

 of 
LEBLANC et al.
F IG U R E  Biochemical assays identify five structurally related active compounds amongst 139 in silico selected potential syntenin PDZ2 inhibitors. (a)
Dot plot illustrating the percentage of inhibition of the syntenin-SDC2 (Y-axis) and syntenin-Frizzled 7 (X-axis) interactions, as determined experimentally by
HTRF for the 139 potential inhibitors selected in silico. Compounds that interfere with the FRET signal in a non-specific manner were previously excluded from
the analysis. See table S1, column ’GST-biot’ for more details (b) Chemical structure of the five structurally related compounds (C, C, C, C and C)
inhibiting syntenin PDZ interactions. See table S1 for more details. (c) Selectivity of syntenin PDZ inhibitors. Values correspond to the ratio of the signal in
the absence of compound (FRET for the interaction) divided by ratio of the signal in the presence of 400μM compound. Compound number (column 1), effect
on biotinylated-GST (column 2), on syntenin-SDC2 or syntenin-Frizzled 7 (column 3–4), on GRASP55-JAM-B or GRASP55-JAM-C (column 5 or 6), and on
Erbin-ERBB2 or Erbin-P0071 interactions (column 7 or 8). Biotinylated GST (GST-biot) was used to test for the effects of the compounds on the FRET signal
(irrespective of effects on any interaction). Values indicate minor effects (below 10 % difference) validating the approach for each individual compound. HTRF
assays on GRASP55 and Erbin PDZ interactions were performed to address the selectivity of the compounds. Note that C selectively affects syntenin PDZ
interactions, while compound C also inhibits GRASP-JAM-B interaction
experiments were performed with a biotinylated-GST to verify that compounds were not interfering with the FRET signal in
a non-specific manner. To identify inhibitors of syntenin PDZ interactions, we tested for the effect of the 139 compounds on
syntenin-SDC2 and syntenin-Frizzled7 complexes (Figure 1a; Table S1). When the HTRF signal dropped below 0.9, inhibition
was considered as effective. Five structurally related compounds (valine–like analogues mimicking the last hydrophobic residue
of a PDZ binding motif), namely C, C, C, C and C (Figure 1b) were identified to inhibit syntenin PDZ interactions
while having no significant effect on control (biotinylated-GST) FRET signals. Noteworthy, these compounds had no effect on
other PDZ complexes like GRASP55-JAM-B or GRASP55-JAM-C and Erbin-Erbb2 or Erbin-P0071 (Figure 1c). Interestingly,
compounds C and C specifically inhibit Syntenin-PDZ complexes, respectively, to an extent of 40% and 60% (Figure 1c).
To validate and characterize the binding mode of the most potent compound C with its target protein, syntenin crystals were
soaked with the compound. X-ray crystal structure of C bound to syntenin was resolved (PDB code 6R9H) and revealed that
C is located within the syntenin-PDZ2 domain (Figure 2a). The phenyl ring of fragment C shows a strong face-to-face
π-stacking with Phe213, its nitrogen atom making a hydrogen-bonding interaction with the backbone carbonyl of Phe211 and
its carboxylic acid making the expected canonical hydrogen-bonding interactions with the backbone nitrogen of Val209, Gly210
and Phe211. C was further characterized in dose-response HTRF assays testing syntenin-SDC2 interaction and the IC50 was
determined to be 350 μM (Figure 2b).
The binding of C to syntenin was also validated in surface plasmon resonance experiments (BIAcore) and in this exper-
imental setting apparent KD values were determined to be 886 μM and 1026 μM for syntenin-PDZ tandem plus C-terminal
domain and syntenin-PDZ tandem (without C-terminal domain) respectively (Figures 2c & 2d). Consistent with the selec-
tivity of C for syntenin, no binding could be observed in BIAcore between this compound and GRASP55-PDZ tandem
(Figure 2e).

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 of 
F IG U R E  Characterization of C58-syntenin interaction. (a) Details of the interaction of compound C with the PDZ tandem of syntenin as determined
by crystallographic approach (PDBID: 6R9H). Structures are represented as ribbons while compounds are represented as balls and sticks. Hydrogen bonds are
represented as black dashes. Water molecules seen in the cavity are shown as blue spheres. One water molecule establishes a direct hydrogen bond with C58 and
is highlighted as a cyan sphere. (b) Determination, by HTRF assays, of the half maximal inhibitory concentration (IC50) of the SDC2 cognate peptide (black)
and compound C (red) for syntenin-SDC2 complexes. (c) BIAcore sensorgrams (subtracted for GST reference channel) illustrating the binding of increasing
concentrations (as indicated) of compound C (50μM -1000 μM) to N-terminally biotinylated-syntenin PDZ tandem immobilized on a streptavidin sensorchip
(d) Langmuir graph showing the binding (in resonance units, RU) of compound C, at equilibrium as observed in BIAcore (compound C was in PBS),
to biotinylated-syntenin PDZ tandem (open squares) or biotynlated-syntenin PDZ tandem plus c-terminal domain (plain dots), as a function of compound
C concentration. (e) BIAcore sensorgrams (subtracted for GST reference channel) illustrating the absence of binding of compound C (50μM -1000 μM) to
biotinylated GST-GRASP PDZ tandem
.
In vitro validation of syntenin C inhibitor
In wild-type MCF-7 breast carcinoma cells and in MCF-7 cells with (CRISPR/Cas9-engineered) syntenin-KO (SyntKO), com-
pound C is not toxic at concentrations up to 100μM (Figure 3a). While such concentration of C decreases MCF-7 cell
migration, no such effect was observed in MCF-7 cells that are KO for syntenin (MCF-7 SyntKO) (Figure 3b), documenting the

 of 
LEBLANC et al.
F IG U R E  Compound C58 impairs MCF7 cell proliferation, migration and primary sphere formation, pending on syntenin expression. (a) Wild-type
MCF-7 cells (MCF-7 cells) and MCF-7 cells with syntenin knock-out (CRISPR-Cas9 approach / MCF-7 SyntKO cells) were treated with increasing concentration
(0.1-100μM) of compounds C or C (taken as negative control) for 48h. Cell viability was assessed by flow cytometry. (b) (Upper part) Wide-field micrographs
illustrating wound healing assays performed on MCF-7 and MCF-7 SyntKO cells treated with vehicle (PBS) or compound C (100μM). We monitored the ability

LEBLANC et al.
 of 
biological specificity of C effects. C also decreases colony formation (Figure 3c) and mammosphere formation (Figure 3d)
in wild-type MCF-7 cells. Interestingly, syntenin-KO in MCF-7 cells mimics the effects of C treatment (Figure 3b micrographs,
d). Taken together, our data indicate that syntenin function can be selectively and specifically inhibited by a small compound tar-
geting its PDZ2 domain. Yet, SDC2 cognate peptide outperforms our best chemical compound by a factor 10 in HTRF (Figure 2b)
indicating room for improvement.
.
Hit optimization increases the affinity for syntenin-PDZ domain
In an attempt to optimize C affinity for syntenin, we synthesized 16 analogues covering three different structural modifications
(thioether vs. ether, chlorine vs. hydrogen and valine vs. alanine moieties) and tested their ability to impair the formation of
syntenin-SDC2 complex, using HTRF assays. Yet, this approach failed to identify any better compound. Surprisingly, a ’thioether
to ether’ analogue proved to be almost inactive, highlighting the difficulty to favourably interact with the protein backbone using
our chemical core. An alternative strategy was therefore considered, aimed at both optimizing strain energy of the fragment and
decreasing desolvation penalty upon binding from the protein side. Molecular modelling studies, relying on the X-ray structure of
C (Figure 2a) and MOE/Pymol tools, suggested that addition of one carbon spacer in the compound structure could maintain
both the canonical binding mode and, the face-to-face π-stacking with Phe213, while slightly increasing the distance between the
hydrophobic thioether-chlorophenyl moiety and the polar protein backbone. Moreover, better torsion angles, closer to optimal
geometry (60◦), with favourable staggered conformation on the flexible carbohydrate moiety, were also anticipated for this C
analogue.
A new compound with one carbon spacer, further called SyntOFF, was synthesized in three steps from the commercial four-
chlorothiophenol (Figure 4a, Figure S1). A first conversion to 3-(4-chlorophenyl thio)propanoic acid by S-alkylation gave deriva-
tive , which was condensed with the L-valine methyl ester hydrochloride leading the methyl ester intermediate cSyntOFF.
Then, cSyntOFF was hydrolysed to the desired compound SyntOFF with an excellent yield. SyntOFF accordingly inhibited the
syntenin-SDC2 interaction by 96% at 400 μM in HTRF, while the methyl ester intermediate and very close analogue cSyntOFF
proved to be inactive (Figure 4a & 4b, Figure S1). The X-ray crystal structure of compound SyntOFF in complex with syn-
tenin was also resolved (PDB code 6RLC) indicating, as anticipated, a canonical binding mode with the carboxylate bind-
ing loop (Val109/Gly210/Phe211) into syntenin-PDZ2 domain (Figure 5a). The small structural modification, consisting in the
of cells to migrate into the scratch by photographing the same area at the beginning of the experiment (0h left) or 24 h later (right) with an inverted microscope
equipped with a digital camera. (Lower part). Wound closure was expressed relative to control (vehicle treated cells), bars represent mean value SD of three
independent experiments. Statistical analysis was performed using Student’s t-test (***P < 0.001). C. (Upper part) Representative micrographs showing MCF-7
cells colony formation in soft agar comparing samples pretreated with vehicle (PBS) or compound C (100μM). The number of colonies was evaluated after 7
days. (Lower part) The bar graph indicates the percentage of luciferase activity relative to control (vehicle). Values correspond to mean sd of three independent
experiments. Statistical analysis was performed using Student’s t-test (**P < 0.01). (d) MCF7 and MCF7 SyntKO cells were treated with vehicle (DMSO 0.2%) or
with compound C (50 & 100μM) and seeded at the same density to form mammospheres. Micrographs are representative of the mammospheres formed after
after seven days in culture (upper part). The number of spheres (diameter > 50 μm) was evaluated under a microscope on days 7–10. Three independent experi-
ments were performed. Sphere formation was calculated as the number of spheres divided by the original number of cells seeded and normalized to control sd.
Statistical analysis was performed using the one-way analysis of variance (ANOVA) with a Bonferroni posttest (*P < 0.05; **P < 0.01; ***P < 0.001) (lower part)
F IG U R E  SyntOFF, a potent C58 derivative. (a) Chemical synthesis scheme of the compound SyntOFF and its intermediary cSyntOFF. Yields are indicated
in percentage (b) Activities of 400 μM SyntOFF and cSyntOFF (also further used as negative control) as determined in HTRF assay testing for inhibition of
SDC2-syntenin interaction. Values correspond to mean sd of three independent experiments. Statistical analysis was performed using the one-way analysis of
variance (ANOVA) with a Bonferroni posttest (***P < 0.001)

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LEBLANC et al.
F IG U R E  Characterization of SyntOFF biological activity and interaction with syntenin. (a) Details of the interaction of compound SyntOFF with the
PDZ tandem of syntenin as determined by crystallographic approach (PDBID: 6RLC). Structures are represented as ribbons while compounds are represented
as ball and stick. Hydrogen bonds are represented as black dashes. Water molecules seen in and around the cavity are shown as blue spheres. (b) Determination,
by HTRF assays, of the half maximal inhibitory concentration (IC50) of the SDC2 cognate peptide (black) and SyntOFF (blue) for syntenin-SDC2 complexes
(c) Plot comparing the binding of compound C and SyntOFF to biotinylated-syntenin PDZ tandem plus C-terminal domain, as a function of compound
concentration. Values correspond to binding at equilibrium as observed in BIAcore (compounds were in DMSO). (d) MCF7 and MCF7 SyntKO cells were
treated with increasing concentration (0.1-100 μM) of SyntOFF for 48h. Cell viability was assessed by flow cytometry. 10 μM H2O2 treatment was used as
positive control. (e) MCF7 cells (left panel) and MCF7 or MCF7 SyntKO (right panel) cells were treated with vehicle (Ctrl, DMSO 0.2%) or with SyntOFF (50
& 100 μM, 100 μM right panel) and seeded at the same density to form mammospheres. The number of spheres (diameter > 50 μm) was evaluated under a
microscope on days 7–10. Three independent experiments were performed. Sphere formation was calculated as the number of spheres divided by the original
number of cells seeded and normalized to control sd. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with a Bonferroni
posttest (*P < 0.05; **P < 0.01; ***P < 0.001)

LEBLANC et al.
 of 
introduction of a single methylene group as a spacer, resulted in a > 10-fold improvement of the affinity as compared
to C (IC50 = 37 μM). SyntOFF reached the reference ligand efficiency (Hopkins, Groom, & Alex, 2004) threshold of
0.30 kcal/mol/atom, starting from the C moderate 0.24 kcal/mol/atom value, highlighting the optimal introduction of an
additional one carbon spacer in the structure of the C (compare Figure 5b and 2b). SyntOFF appeared selective for syntenin,
not affecting Erbin-P0071 (Figure S2a) or GRASP55-JAM-B complexes in HTRF (Figure S2b). In BIAcore, using DMSO as vehi-
cle, SyntOFF displayed a KD value of 130 μM for syntenin-PDZ tandem plus C-terminal domain (Figure 5c). In cellular assays,
SyntOFF, like C, is non-toxic up to 100 μM (Figure 5d). Stability properties were evaluated (Table S2), it appears that SyntOFF
is stable in PBS pH7.4 (solubility > 7.3 mM) and also in murine plasma (81% of remaining compound after 2h). Moreover,
SyntOFF decreases MCF-7 mammosphere formation more efficiently than does C (Figure 5e, left to compare to Figure 3d).
Of note, SyntOFF had no effect when MCF-7 cells were KO for syntenin expression (Figure 5e, right).
.
Syntenin-PDZ domain inhibitor regulates exosome cargo
Next, we investigated the effect of C, SyntOFF and the inactive control compound cSyntOFF (Figure 4a) on the production
and the composition of exosomes. As it was previously shown that the PLD2 inhibitor (CAY10594; 10 μM) affects the secretion of
exosomal markers including syntenin (Ghossoub et al., 2014), we included this enzymatic inhibitor as control, in parallel to our
syntenin-PDZ inhibitors. Nanoparticle tracking analyses (NTAs) revealed that none of the present syntenin-PDZ2 inhibitors
(100 μM) affects the size or the number of secreted nanoparticles, whereas PLD2 inhibitor clearly decreases their number (Figure
S3a and S3b).
Exosomal fractions released by cells treated with syntenin-PDZ2 or PLD2 inhibitors were then analysed by western blot,
investigating the effect of the different compounds on both syntenin-dependent and -independent exosomal markers (Figure 6a).
The amounts of exosomal syntenin, ALIX and SDC4 C-terminal fragment (CTF) were significantly decreased when cells were
treated with 10 μM PLD2 inhibitor or 100 μM SyntOFF, but not with 100 μM C58 or the control compound cSyntOFF (Figure 6b).
PLD2 inhibitor also markedly decreased exosomal markers like CD9, CD81 and Flotillin-1, but that was not the case for SyntOFF
(Figure 6c). Noteworthy, cellular levels remained unaffected, regardless of the treatment (Figure S4a and S4b). Finally, we also
examined whether syntenin inhibitors affect the loading of exosomes with signalling or adhesion proteins (Figure 6a). Clearly,
both SyntOFF and PLD2 inhibitor markedly decrease the incorporation of EpCAM and c-Src into exosomes (Figure 6d), but
leave fibronectin (Figure 6d), EGFR, ADAM10 or TSP1 (Figure S5a) signals unaffected. Here again, drugs did not impact on the
cellular levels (Figure S4c and S5b). Taken together, our data indicate that inhibition of syntenin PDZ2 domain, while having
no major effect on exosome number or cellular protein levels, can impact on the secretion of specific exosome cargo. They also
highlight that EpCAM loading into exosomes depends on syntenin.

DISCUSSION
In this study, we have developed a novel syntenin inhibitor, SyntOFF, which specifically binds syntenin, and serves as an antago-
nist of syntenin-PDZ2 domain, interfering with cell migration, proliferation and mammosphere formation, as well as inhibiting
the loading of exosomes with specific cargo.
By screening 139 in silico-selected compounds, we first identified a family of five structurally related compounds as potential
inhibitors of syntenin PDZ interactions. All compounds similarly affected the interaction of syntenin with Frizzled 7 and SDC2
(Figure 1), two well-documented interactors of syntenin PDZ2 domain (Egea-Jimenez et al., 2016; Grembecka et al., 2006). As
shown in Figure 1, these five inhibitor candidates share, at one end, a common pharmacophore (a valine-like residue) mimicking
the last hydrophobic residue at the C-terminus of cognate peptides. They also present, on another end, a variously substituted
aromatic core. The C inhibitor was selected for further investigations because it yielded the highest extinction of the HTRF
signal. In complementary biophysical experiments, C proved to selectively bind to syntenin (Figure 2b-c) and not to GRASP55
PDZ domains (Figure 2e). Moreover, co-crystallization experiments indicated that C binds syntenin PDZ2 but not PDZ1
domain (Figure 2a). Noteworthy, when used at a concentration of 100 μM (three times below the IC50 calculated by HTRF), C
exerts biological effects on cells expressing syntenin, but not on cells where syntenin was depleted by CRISPR-Cas9 approach
(Figure 3). These data suggest that small compounds (or drug fragments) can achieve biological specificity. Interestingly, C
had no toxic effects at working concentrations (Figure 3a). Because C displays an IC50 one order of magnitude higher than the
cognate peptide SDC2 (Figure 2b), we surmised there must be room for improvement. Yet, classical chemical approaches, relying
on the growing concept from Fragment Based Drug Discovery completely failed. However, an optimization strategy, based on
crystallographic data, enabled the development of SyntOFF where the introduction of an additional methylene group (Figure 4)
improved the affinity 10-fold compared to C (compare Figure 2b & 5b). As expected, SyntOFF also interacts with syntenin
PDZ2 and not PDZ1 domain according to crystallography data (PDB 6RLC and Figure 5a). Consistent with the gain in affinity,
SyntOFF appeared more effective than C in cellular cancer assays (Figure 5e). SyntOFF was also non-toxic at concentrations

 of 
LEBLANC et al.
F IG U R E  SyntOFF regulates the loading of exosomes with syntenin-dependent cargo and EpCAM. (a) Schematic representation of an exosome high-
lighting which markers were previously shown to rely on syntenin (red, syntenin pathway) or to be unaffected by syntenin (blue, syntenin independent) for their
secretion in MCF7 exosomes. The scheme also represents additional exosome cargo (cargo proteins, among which Fibronectin, EGFR and EpCAM investigated
in this study). (b-d) MCF7 cells were treated with DMSO vehicle (Ctrl) or the indicated compound (CAY10594 10 μM; cSynOFF, C, or SyntOFF at 100 μM) for
16 h before analysis. Total cell lysates (See supplementary figure S4) and the corresponding exosomes were analyzed by western blot, tracing different markers, as
indicated. Histograms represent mean signal intensities ( SEM) in exosomes, relative to controls (Ctrl). Data were obtained from n independent experiments,
as indicated. Statistical analysis was performed using the one-way analysis of variance (ANOVA) with a Bonferroni posttest (*P < 0.05; **P < 0.01; ***P < 0.001)

LEBLANC et al.
 of 
where it was biologically active (Figure 5d). As observed for C, SyntOFF biological effects were solely significant on cells
expressing syntenin (Figure 5e). Of note, PDZ interactions are promiscuous (Ivarsson, 2012) and PDZ binding motifs are known
to bind multiple PDZ proteins. Therefore, the selectivity and the biological specificity observed for C and SyntOFF were
not anticipated. Interestingly, although it is unclear to date which ligands might directly and efficiently interact with syntenin
PDZ1 domain (Grembecka et al., 2006), Fisher’s group recently developed a syntenin-PDZ1 domain-targeted small molecule. This
molecule reduces the invasive potential of glioblastoma cells (Kegelman et al., 2017), as well as the invasiveness and the migration
of prostate cancer cells (Das et al., 2019). We thus have evidence that syntenin PDZ interactions can be targeted by small organic
chemicals, be it with modest affinity in the same range that the cognate peptides. Besides, such chemicals compounds are non-
toxic at concentrations where they display biological effects and specificity.
When used at 100 μM, SyntOFF, but not C or the negative control compound cSyntOFF (unable to make the canonical H-
bonds network with the carboxylate binding loop into syntenin PDZ2 domain) reduced the amounts of exosomal syntenin, ALIX
and SDC4 CTF (Figure 6b). Moreover, SyntOFF does not seem to affect other exosome populations (Figure 6c). These results
confirm the success of the early stages of our optimization and illustrate that SyntOFF significantly alters the syntenin-dependent
exosomal pathway. Yet, to meet current standards in drug discovery, we need to improve affinity by one order of magnitude. For
these reasons, we will continue to work on syntenin PDZ2 chemical inhibition and optimization of compounds to improve their
molecular and pharmacological properties by performing a modelling-assisted fragment growing process.
SyntOFF specifically targets and antagonizes the Syntenin-PDZ2 domain in vitro, which might explain why our compound
suffices for effectively and specifically inhibiting syntenin-SDC, including exosomal pathways. Interestingly, we noticed that
pharmacological inhibition of the syntenin-PDZ2-domain does not affect the number of the nanoparticles secreted, whereas
our group had previously shown that syntenin controls the number of exosomes made/released. Indeed, using syntenin RNAi-
treated MCF-7 cells, Baietti et al. had observed that loss of syntenin leads to a ∼50% reduction in the number of particles with a
diameter of 30–110 nm. This effect was further validated by confocal fluorescence spectroscopy assessing the effects of syntenin
overexpression on the detection of mGFP–CD63 in exosomes, with a two to three-fold increase in the number of the fluorescent
peaks. These results suggest that the syntenin-PDZ2 inhibitor affects syntenin-exosomal loading without altering the endosomal
membrane budding. The sequestration of cargo in specific endosomal membrane domains and the budding of these membranes
to create intraluminal vesicles and multi-vesicular endosomes are processes that mechanistically are closely intertwined. Yet the
finding with the inhibitor is consistent with the notion that the stoichiometry between membrane unit area, membrane cargo
and budding machinery is not constant, the PDZ2 inhibitor leading to the production of relatively ‘cargo-depleted’ intralumi-
nal vesicles, and from there ‘cargo-depleted’ exosomes. That notion (of vesicles of heterogeneous compositions) is commonly
accepted in the fields of virology, studying the assembly of viruses and defective, virus-like particles. Likely, more potent PDZ2
inhibitors, more effective at thinning and disrupting syndecan-syntenin lattices on endosomal membranes, will ultimately prove
to also affect exosome numbers. Alternatively, exosome uptake mechanisms and net exosome accumulations in peri-cellular
spaces might also be exosome- and cargo-specific, and differentially depend on syntenin functions in recipient cells. Further
investigations are needed to clarify these possibilities.
Consistent with our previous work, we noticed that pharmacological inhibition of the syntenin-PDZ2-domain markedly
decreases the incorporation of c-Src into the exosomes (Imjeti et al., PNAS 2017). Finally, our results strongly suggest that
EpCAM is sorted to exosomes via the syntenin machinery (Figure 6d), depending on determinants the path has in common
with syntenin-mediated SDC sorting. However, EpCAM finding must be confirmed, also using complementary approaches,
and it remains to be established how this sorting proceeds: that is, whether direct interactions with syntenin are involved (i.e.
via PDZ2, the PDZ-BM of EpCAM composing alternative bait for syntenin), and whether, like that of CD63 (Baietti et al.,
2012), EpCAM sorting to exosomes might depend on the assistance of SDC (CD63 binding primarily via PDZ1, SDC via PDZ2).
EpCAM was originally identified as a marker for carcinoma and its role includes diverse processes such as signalling, cell
migration, proliferation, and differentiation (Keller et al., 2019). Some studies suggest that the levels of exosomal EpCAM could
constitute a biomarker of ovarian and pancreatic cancers (Im et al., 2014) (Castillo et al., 2018). Proteomic analysis of EVs isolated
from colon carcinoma cell-derived organoids even revealed a distinct population of exosomes according to EpCAM expression
and showed a colocalization of EpCAM with CD44 and claudin 7, proteins that are known to promote tumour progression
(Tauro et al., 2013). In accordance with previous data from our laboratory concerning syntenin-dependent cargo (SRC, FGF,
Frizzled 7), this result reinforces the notion of the syntenin-SDC exosomal pathway as an important player in cancer progression.
Overall, we show that Syntenin-PDZ2 is a promising therapeutic target for inhibiting oncogenic processes and mostly tumour
exosomal communication. This study confirmed that syntenin is strongly involved in the loading of exosomes with cargo. Given
the versatility of the SDC co-receptor functions, this potentially pertains to a plethora of pro-oncogenic proteins that support
tumorigenesis and metastatic dissemination. Likely, also further investigations about pro-tumour miRNA possibly connected
with syntenin and its cargo will appear essential for a better understanding of the role of cancer exosomal communication.
AC K NOW L E D G E M E N T S
We thank F. Lembo for preparation of the plasmid for crystallization studies and Prof. Em. G. David for critical review of the
manuscript. This work was supported by grants from the Concerted Actions Program of the KU Leuven (GOA/12/016), the

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LEBLANC et al.
National Research Agency (ANR, Investissements d’Avenir, A∗MIDEX project ANR-11-IDEX-0001-02), the Fund for Scientific
Research–Flanders (Fonds Wetenschappelijk Onderzoek—Vlaanderen Grants G.0846.15 and G0C5718N), the Institut National
du Cancer (INCa, subvention 2013–105), the Belgian Foundation against cancer (STK, FA/2014/294), the French National
Research Agency (ANR-18-CE13-0017, Project SynTEV), the French Infrastructure for Integrated Structural Biology (FRISBI)
ANR-10-INSB-05-01 with the use of the AFMB laboratory X-ray facility. The authors acknowledge the European Synchrotron
Radiation Facility (ESRF) as well as the SOLEIL synchrotron (Proxima 1 and Proxima 2 beamlines) for provision of synchrotron
radiation facilities. This project has received funding from the European Union’s Horizon 2020 research and innovation program
under the Marie Sklodowska-Curie grant agreement No 747025. R. Kashyap and M. Garcia were recipient of La Ligue French
Foundation for Cancer Research. RL was the recipient of the ARC French Foundation for Cancer Research.
C ON F L IC T S OF I N T T E R E ST
The authors declare no competing interests.
AU T HOR C ON T R I BU T ION S
All authors provided support for the writing. R. Leblanc carried out HTRF and cell biological work, and wrote the manuscript;
R. Kashyap performed HTRF and cell biology experiments; K. Barral contributed to structure–activity relationship studies, crys-
tallization experiments, X-ray complex structures resolution, and organic syntheses; D. Kovalskyy designed the PDZ focused
library; A.L. Egea-Jimenez did the SPR experiments and protein production for crystallization studies; M.Feracci contributed
to crystallization studies and associated calculations; M. Garcia performed chemistry experiments and associated analyses; C.
Derviaux contributed to automated HTRF experiments; S. Betzi contributed to protein purification and crystallization studies;
R. Ghossoub contributed to cell biology experiments; M. Platonov designed the PDZ focused library; P. Roche helped with the
in silico analyses; X. Morelli supervised and conceived the project; L. Hoffer analysed the structural data and carried out the
computer-aided design of organic compounds during the optimization stage; Pascale Zimmermann supervised and conceived
the project, and wrote the article.
R E F E R E NC E S
Baietti, M. F., Zhang, Z., Mortier, E., Melchior, A., Degeest, G., Geeraerts, A., … David, G. (2012). Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.
Nature Cell Biology, , 677–685.
Castillo, J., Bernard, V., San Lucas, F. A., Allenson, K., Capello, M., Kim, D. U., … Alvarez, H. (2018). Surfaceome profiling enables isolation of cancer-specific
exosomal cargo in liquid biopsies from pancreatic cancer patients. Annals of Oncology : Official Journal of the European Society for Medical Oncology / ESMO,
, 223–229.
Charrin, S., Le Naour, F., Oualid, M., Billard, M., Faure, G., Hanash, S. M., … Rubinstein, E. (2001). The major CD9 and CD81 molecular partner. Identification
and characterization of the complexes. The Journal of Biological Chemistry, , 14329–14337.
Christianson, H. C., & Belting, M. (2014). Heparan sulfate proteoglycan as a cell-surface endocytosis receptor. Matrix Biology, , 51–55.
Cierpicki, T., & Grembecka, J. (2015). Targeting protein-protein interactions in hematologic malignancies: Still a challenge or a great opportunity for future
therapies? Immunological Reviews, , 279–301.
Couchman, J. R. (2010). Transmembrane signaling proteoglycans. Annual Review of cell and Developmental Biology, , 89–114.
Das, S. K., Kegelman, T. P., Pradhan, A. K., Shen, X. -. N., Bhoopathi, P., Talukdar, S., … Fisher, P. B. (2019). Suppression of Prostate Cancer Pathogenesis Using
an MDA-9/Syntenin (SDCBP) PDZ1 Small-Molecule Inhibitor. Molecular Cancer Therapeutics, , 1997–2007.
Egea-Jimenez, A. L., Gallardo, R., Garcia-Pino, A., Ivarsson, Y., Wawrzyniak, A. M., Kashyap, R., … Zimmermann, P. (2016). Frizzled 7 and PIP2 binding by
syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling. Nature Communications, , 12101.
Faivre, E. J., Mcdaniel, K. F., Albert, D. H., Mantena, S. R., Plotnik, J. P., Wilcox, D., … Shen, Y. (2020). Selective inhibition of the BD2 bromodomain of BET
proteins in prostate cancer. Nature, , 306–310.
Filippakopoulos, P., Qi, J., Picaud, S., Shen, Y., Smith, W. B., Fedorov, O., … Bradner, J. E. (2010). Selective inhibition of BET bromodomains. Nature, ,
1067–1073.
Friand, V., David, G., & Zimmermann, P. (2015). Syntenin and syndecan in the biogenesis of exosomes. Biology of the Cell, , 331–341.
Ghossoub, R., Lembo, F., Rubio, A., Gaillard, C. B., Bouchet, J., Vitale, N., … Zimmermann, P. (2014). Syntenin-ALIX exosome biogenesis and budding into
multivesicular bodies are controlled by ARF6 and PLD2. Nature Communications, , 3477.
Greening, D. W., Gopal, S. K., Xu, R., Simpson, R. J., & Chen, W. (2015). Exosomes and their roles in immune regulation and cancer. Seminars in Cell & Devel-
opmental Biology, , 72–81.
Grembecka, J., Cierpicki, T., Devedjiev, Y., Derewenda, U., Kang, B. S., Bushweller, J. H., & Derewenda, Z. S. (2006). The binding of the PDZ tandem of syntenin
to target proteins. Biochemistry, , 3674–3683.
Grootjans, J. J., Reekmans, G., Ceulemans, H., & David, G. (2000). Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ
domains of syntenin. The Journal of Biological Chemistry, , 19933–19941.
Grootjans, J. J., Zimmermann, P., Reekmans, G., Smets, A., Degeest, G., Durr, J., & David, G. (1997). Syntenin, a PDZ protein that binds syndecan cytoplasmic
domains. Proceedings of the National Academy of Sciences of the United States of America, , 13683–13688.
Hopkins, A. L., Groom, C. R., & Alex, A. (2004). Ligand efficiency: a useful metric for lead selection. Drug Discovery Today, , 430–431.
Im, H., Shao, H., Park, Y. I.l, Peterson, V. M., Castro, C. M., Weissleder, R., & Lee, H. (2014). Label-free detection and molecular profiling of exosomes with a
nano-plasmonic sensor. Nature Biotechnology, , 490–495.
Imjeti, N. S., Menck, K., Egea-Jimenez, A. L., Lecointre, C., Lembo, F., Bouguenina, H., … Zimmermann, P. (2017). Syntenin mediates SRC function in exosomal
cell-to-cell communication. Proceedings of the National Academy of Sciences of the United States of America, , 12495–12500.
Ivarsson, Y. (2012). Plasticity of PDZ domains in ligand recognition and signaling. Febs Letters, , 2638–2647.

LEBLANC et al.
 of 
Jung, M., Gelato, K. A., Fernández-Montalván, A., Siegel, S., & Haendler, B. (2015). Targeting BET bromodomains for cancer treatment. Epigenomics, , 487–501.
Kalluri, R., & Lebleu, V. S. (2020). The biology, function, and biomedical applications of exosomes. Science, , eaau6977.
Karlsson, R., Katsamba, P. S., Nordin, H., Pol, E., & Myszka, D. G. (2006). Analyzing a kinetic titration series using affinity biosensors. Analytical Biochemistry,
, 136–147.
Kashyap, R., Roucourt, B., Lembo, F., Fares, J., Carcavilla, A. M., Restouin, A., … Ghossoub, R. (2015). Syntenin controls migration, growth, proliferation, and
cell cycle progression in cancer cells. Frontiers in Pharmacology, , 241.
Kegelman, T. P., Wu, B., Das, S. K., Talukdar, S., Beckta, J. M., Hu, B., … Fisher, P. B. (2017). Inhibition of radiation-induced glioblastoma invasion by genetic and
pharmacological targeting of MDA-9/Syntenin. Proceedings of the National Academy of Sciences of the United States of America, , 370–375.
Kegelman, T. P., Das, S. K., Emdad, L., Hu, B., Menezes, M. E., Bhoopathi, P., … Fisher, P. B. (2015). Targeting tumor invasion: the roles of MDA-9/Syntenin.
Expert Opinion on Therapeutic Targets, , 97–112.
Keller, L., Werner, S., & Pantel, K. (2019). Biology and clinical relevance of EpCAM. Cell Stress, , 165–180.
Koo, T. H., Lee, J. -. J., Kim, E-Mi, Kim, K. -. W., Kim, H. D.o, & Lee, J. -. H. (2002). Syntenin is overexpressed and promotes cell migration in metastatic human
breast and gastric cancer cell lines. Oncogene, , 4080–4088.
Lambaerts, K., Van Dyck, S., Mortier, E., Ivarsson, Y., Degeest, G., Luyten, A., … Zimmermann, P. (2012). Syntenin, a syndecan adaptor and an Arf6 phos-
phatidylinositol 4,5-bisphosphate effector, is essential for epiboly and gastrulation cell movements in zebrafish. Journal of Cell Science, , 1129–1140.
Lambaerts, K., Wilcox-Adelman, S. A., & Zimmermann, P. (2009). The signaling mechanisms of syndecan heparan sulfate proteoglycans. Current Opinion in
Cell Biology, , 662–669.
Latysheva, N., Muratov, G., Rajesh, S., Padgett, M., Hotchin, N. A., Overduin, M., & Berditchevski, F. (2006). Syntenin-1 is a new component of tetraspanin-
enriched microdomains: Mechanisms and consequences of the interaction of syntenin-1 with CD63. Molecular and Cellular Biology, , 7707–7718.
Luyten, A., Mortier, E., Van Campenhout, C., Taelman, V., Degeest, G., Wuytens, G., … Zimmermann, P. (2008). The postsynaptic density 95/disc-large/zona
occludens protein syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling. Molecular Biology of the Cell, , 1594–1604.
Makley, L. N., & Gestwicki, J. E. (2013). Expanding the number of ‘druggable’ targets: Non-enzymes and protein-protein interactions. Chemical Biology & Drug
Design, , 22–32.
Mathieu, M., Martin-Jaular, L., Lavieu, G., & Théry, C. (2019). Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell
communication. Nature Cell Biology, , 9–17.
Meerschaert, K., Bruyneel, E., De Wever, O., Vanloo, B., Boucherie, C., Bracke, M., … Gettemans, J. (2007). The tandem PDZ domains of syntenin promote cell
invasion. Experimental Cell Research, , 1790–1804.
Milhas, S., Raux, B., Betzi, S., Derviaux, C., Roche, P., Restouin, A., … Morelli, X. (2016). Protein-Protein Interaction Inhibition (2P2I)-Oriented chemical library
accelerates hit discovery. ACS Chemical Biology, , 2140–2148.
Morelli, X., Bourgeas, R., & Roche, P. (2011). Chemical and structural lessons from recent successes in protein-protein interaction inhibition (2P2I). Current
Opinion in Chemical Biology, , 475–481.
Roucourt, B., Meeussen, S., Bao, J., Zimmermann, P., & David, G. (2015). Heparanase activates the syndecan-syntenin-ALIX exosome pathway. Cell Research,
, 412–428.
Saito, K., Nishioka, K., Iwatsuki, K., Kumagai, K., & Sasaki, M. (1975). The effect of pentazocine on PHA-stimulated human lymphocyte transformation. The
Tohoku Journal of Experimental Medicine, , 229–231.
Stahl, P. D., & Raposo, G. (2019). Extracellular vesicles: exosomes and microvesicles, integrators of homeostasis. Physiology, , 169–177.
Tauro, B. J., Greening, D. W., Mathias, R. A., Mathivanan, S., Ji, H., & Simpson, R. J. (2013). Two distinct populations of exosomes are released from LIM1863
colon carcinoma cell-derived organoids. Molecular & Cellular Proteomics, , 587–598.
Ulucan, O., Eyrisch, S., & Helms, V. (2012). Druggability of dynamic protein-protein interfaces. Current Pharmaceutical Design, , 4599–4606.
Wortzel, I., Dror, S., Kenific, C. M., & Lyden, D. (2019). Exosome-mediated metastasis: communication from a distance. Developmental Cell, , 347–360.
Zimmermann, P., Meerschaert, K., Reekmans, G., Leenaerts, I., Small, J. V., Vandekerckhove, J., … Gettemans, J. (2002). PIP(2)-PDZ domain binding controls
the association of syntenin with the plasma membrane. Molecular Cell, , 1215–1225.
Zimmermann, P., Tomatis, D., Rosas, M., Grootjans, J., Leenaerts, I., Degeest, G., … David, G. (2001). Characterization of syntenin, a syndecan-binding PDZ
protein, as a component of cell adhesion sites and microfilaments. Molecular Biology of the Cell, , 339–350.
Zimmermann, P., Zhang, Z., Degeest, G., Mortier, E., Leenaerts, I., Coomans, C., … David, G. (2005). Syndecan recycling [corrected] is controlled by syntenin-
PIP2 interaction and Arf6. Developmental Cell, , 377–388.
SU PP ORT I NG I N F OR M AT ION
Additional supporting information may be found online in the Supporting Information section at the end of the article.
How to cite this article: Leblanc R, Kashyap R, Barral K, et al. Pharmacological inhibition of syntenin PDZ2 domain
impairs breast cancer cell activities and exosome loading with syndecan and EpCAM cargo. J Extracell Vesicles.
2020;10:e12039. https://doi.org/10.1002/jev2.12039