Inhibiting extracellular vesicles formation and release: a review of EV inhibitors

Figures & data

Figure 1. Exosomes are released from intracellular compartments known as multi-vesicular bodies (MVBs). MVBs biogenesis is associated with two different mechanisms: ESCRT-dependent and ESCRT-independent pathways.

ESCRT is a multi-molecular machinery composed of different proteins able to interact with ubiquitylated cargos and promote the formation of Intra-Luminal Vesicles (ILVs). The ESCRT pathways seems to be associated to RAS, a small GTPases able to orchestrate different cellular processes including exosomes production and cell proliferation, differentiation, adhesion and migration. In order to function properly, RAS must be linked to a lipid chain, achieved by the activity of specific enzymes called farnesyltransferases. Manumycin A is proposed to be able to inhibit these latter enzymes; thus, in turn, it is able to prevent RAS activation and function, preventing the formation of exosomes through the ESCRT pathway. The ESCRT-independent pathway is orchestrated by neutral sphingomyelinases, a family of enzymes able to convert sphingomyelin, present inside lipid raft, in ceramide. The resulting ceramides, interacting with each other, form large microdomains inducing the budding and the formation of ILVs into MVBs. GW4869 is a potent neutral sphingomyelinases inhibitor that preventing the formation of ILVs is able to block exosomes production.

Table 1. Most commonly used compounds reported to prevent EVs (MVs or exosomes) release and their biological properties.

Name	Chemical structure	Chemical formula and IUPAC name	MOLECULAR WEIGHT	SOLUBILITY (25°C)	MeSh PHARMACOLOGICAL CLASSIFICATION	BIOLOGICAL TARGETS & CONCENTRATION USED
Calpeptin		H30N2O4 Benzyl N-[(2S)-4-methyl-1-oxo-1-[[(2S)-1-oxohexan-2-yl]amino]pentan-2-yl]carbamate	362.47 g/mol	DMSO: 72 mg/mL (198.64 mM) Ethanol: 72 mg/mL (198.64 mM) Water: insoluble	Cystein proteinase inhibitors	Calpains, involved in MVs production. Concentrations used: 0–300 µM in vitro 10 mg/Kg of body weight in vivo^[Citation52–Citation56,Citation58]
Manumycin A		C31H38N2O7 (2E,4E,6R)-N-[(1S,5S,6R)-5-hydroxy-5-[(1E,3E,5E)-7-[(2-hydroxy-5-oxocyclopenten-1-yl)amino]-7-oxohepta-1,3,5-trienyl]-2-oxo-7-oxabicyclo[4.1.0]hept-3-en-3-yl]-2,4,6-trimethyldeca-2,4-dienamide	550.652 g/mol	DMSO: ~10 mg/mL Ethanol: ~5 mg/mL	Anti-Bacterial Agents Enzyme Inhibitors	Small GTPases Ras, involved in Exosomes generation and release Concentrations used: 0–5 µM but also the nanomolar scale, in vitro.^[Citation60–Citation62]
Y27632		C14H21N3O 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide	247.342 g/mol	DMSO: 64 mg/mL warmed (199.83 mM) Ethanol: Insoluble Water: 14 mg/mL (43.71 mM)	Anti-hypertensive agents; muscle relaxants; central enzyme inhibitors	ROCK1 and ROCK2 involved in MVs production. Concentrations used: 0–30 µM in vitro^[Citation63–Citation67]
D-Pantethine		C22H42N4O8S2 (2R)-N-[3-[2-[2-[3-[[(2R)-2,4-dihydroxy-3,3-dimethylbutanoyl]amino]propanoylamino]ethyldisulphanyl]ethylamino]-3-oxopropyl]-2,4-dihydroxy-3,3-dimethylbutanamide	554.718 g/mol	DMSO: insoluble Water: 100 mg/mL (180.27 mM)	A11-Vitamins	Cholesterol synthesis, fundamental for MVs generation. Concentrations used: both in µM and in mM scale in vitro 150 mg/Kg body weight or 30 mg per mouse in vivo^[Citation69–Citation72]
Imipramine		C19H24N2 3-(5,6-dihydrobenzo[b][1]benzazepin-11-yl)-N,N-dimethylpropan-1-amine	280.415 g/mol	DMSO: 60 mg/mL (198.12 mM) Water: 50 mg/mL	Adrenergic uptake inhibitors; anti-depressive agent, tricyclic	Acid sphingomyelinase (aSMase) involved in MVs generation. Concentrations used: 0–25 µM in vitro^[Citation35,Citation74,Citation75] 20 mg/Kg of body weight in vivo^[Citation102]
GW4869		C30H30Cl2N6O2 (E)-3-[4-[(E)-3-[4-(4,5-dihydro-1H-imidazol-2-yl)anilino]-3-oxoprop-1-enyl]phenyl]-N-[4-(4,5-dihydro-1H-imidazol-2yl)phenyl]prop-2-enamide	577.51 g/mol	DMSO: 0.2 mg/mL Ethanol: insoluble Water: insoluble	NA	Membrane neutral sphingomyelinase (nSMase) involved in Exosomes generation and release. Concentrations used: 0–40 µM in vitro 1–100 mg/Kg of body weight in vivo^[Citation34,Citation38,Citation62,Citation76–Citation86]

IUPAC = International Union of Pure and Applied Chemistry.

Figure 2. Calpains, once activated through calcium binding, can activate different cellular processes including cell migration (through their interaction with Talin and FAK), cell invasion (thorough the promotion of MMP release and activation), and MVs formation and release (through their activity on cytoskeletal proteins including cortactin). Calpain inhibitors, of which calpeptin is one of the most studied, are under investigation for clinical application.

Table 2. EV separation and characterisation methods used in the studies discussed in this review.

Cell type	EV type (as reported by authors)	Separation methods	Characterisation methods
Platelets^[Citation52]	Microparticles	No isolation	FACS analysis with Annexin V and CD41a
Platelets^[Citation53]	Microparticle	Differential centrifugation	FACS analysis with Annexin V and CD41a
Platelets^[Citation54]	Microvesicles	(a) 0.2 µm filtration (b) Ultracentrifugation	Radio-labelled membrane glycoprotein quantification
Platelets^[Citation55]	Microparticles	No isolation	FACS analysis for GP IIb-IIIa and GP Ib
HEK293, SH-SY5Y^[Citation56]	Microvesicles	Ultracentrifugation	Live-cell confocal microscopy and FACS with Annexin A1
PC3^[Citation58]	Microvesicles	Differential centrifugation	NTA, TEM and FACS analysis with Annexin V
F11, HeLa, rat dorsal root ganglion, mouse neuroblastoma hybrid cells^[Citation60]	Exosomes	Differential ultracentrifugation	NTA, Immunoblots for: Tsg101, CD63
RWPE1, CRPC, C4-2B^[Citation61]	Exosomes	(a) Differential ultracentrifugation (b) ExoEasy Maxi kit	qNano, NTA, NanoFACS with CD63, Immunoblots for: Tsg101, CD63, CD9, CD81, Alix and GADPH
BUMPT^[Citation62]	Exosomes	Differential ultracentrifugation	NTA, TEM, Immunoblots for: CD63, Tsg101
Hela, MDAMB231, U87, NIH3T3[^Citation39]	Microvesicles	(a) Differential ultracentrifugation (b) Filtration with Millipore Steriflip PVDF-filter	Confocal microscopy and SEM on MVs budding cells. Immunoblots for: Flottilin-2, tTG, actin, IκBα
HMEC-1^[Citation63]	Microparticles	No isolation	FACS with Annexin V Coulter Epics XL for size evaluation
hCMEC/D3^[Citation64]	Microparticles	Differential centrifugation	FACS with Annexin V and CD-105
HCAECs^[Citation65]	Microparticles	Differential centrifugation	FACS with CD62E, CD105, CD51, CD106, CD31 and CD54
Immortalised human umbilical vein endothelial cells^[Citation66]	Microparticles	Differential centrifugation	FACS with Annexin V, CD73 and CD144
HUVEC^[Citation67]	Microparticles	Differential centrifugation	FACS with Annexin V
MCF-7^[Citation70]	Microparticles	Differential centrifugation	FACS with Annexin V
HPMECs, HUVECs^[Citation71]	Microparticles	Differential centrifugation	FACS with Annexin V and CD144
MBECs and HUVECs^[Citation72]	Microparticles	Differential centrifugation	FACS with Annexin V and ICAM-1
UAMS-32P^[Citation74]	Microvesicles	Differential centrifugation	NTA, BCA
PC3, MCF-7^[Citation75]	Exosomes and Microvesicles	Differential ultracentrifugation	NTA, FACS with Annexin V and CD63
Human Glioblastoma cells^[Citation35]	Microparticles	(a) Annexin V immunoaffinity (b) Differential ultracentrifugation	FACS with Annexin V Immunoblots for HSP70, CD63
Oli-neu cells^[Citation13]	Exosomes	Differential ultracentrifugation	Immunoblots for Alix, CD63
MDA-MB-231, SKBR3^[Citation34]	Microvesicles and exosomes	Differential ultracentrifugation	NTA, TEM, Immunoblots for: Alix, GADPH, HSP70, Tsg101, Syntenin, CD81, CD9
GT1-7^[Citation38]	Exosomes	Differential ultracentrifugation	TEM, qNano, Immunoblots for: Tsg101, Flottilin-1, Acetylcholinesterase activity
Neonatal rat cardiac fibroblast^[Citation76]	Exosomes	Differential centrifugation	TEM, Immunoblots for: CD9, CD63, Tsg101, HSP70, Calnexin
SW620, 18Co, CAFs^[Citation77]	Exosomes	(a) Total Exosomes Isolation kit (b) Differential ultracentrifugation	TEM, Immunoblots for: CD81
L3.6lp cells, fibroblast, Isolated CAFs^[Citation78]	Exosomes	ExoQuick	TEM, DLS, Beckman coulter Delsa Nano S Particle Analyser
HSC, LX-2^[Citation79]	Exosomes	Not reported	Not reported
PC9, A549^[Citation80]	Exosomes	ExoQuick	TEM, Immunoblots for: CD63
SKOV3, ESC (endothelial stromal cells)^[Citation81]	Exosomes	ExoQuick	Immunoblots for with: DNMT1, CD9, CD63
B16BL6^[Citation82]	Exosomes	Differential ultracentrifugation	TEM, qNano, Immunoblots for: Alix, HSP70, CD81, Calnexin
LNCap, 22Rv1, PC3, PWR-1E, MDA PCa 2b, RC77T/E and RC77N/E^[Citation83]	Exosomes	(a) ExoQuick (b) 0.22 µm filter followed by ultracentrifugation	NTA, TEM, Immunofluorescence for CD63, Rab5
RAW 264.7^[Citation85]	Exosomes	Differential ultracentrifugation	DLS, Immunoblots for: CD81, CD63, GADPH
Mov, Rov, N2a^86	Exosomes	Differential ultracentrifugation	TEM, Immunoblots for: Tsg101, Alix, Calnexin
PC3^[Citation87]	Microvesicles	Differential ultracentrifugation	NTA, TEM, Immunoblots for: CD63, Alix
HUVECs^[Citation89]	Microparticles	No Isolation	FACS with CD31 and CD42
K562, HUVECs, CCD-27Sk^[Citation90]	Exosomes	Differential ultracentrifugation	TEM, Immunoblots for CD63, CD81, Tsg101
K562[^92]	Exosomes	Differential ultracentrifugation	Acetylcholinesterase activity, Immunoblots for HSP70
CT26, EL4, NIH/3T3, H23^[Citation93]	Exosomes	Differential ultracentrifugation	TEM, acetylcholinesterase activity
Isolated monocytes^[Citation95]	Microvesicles	Differential centrifugation	FACS with Annexin V
DLBCL cell lines SU-DHL-4, Balm3, OCI-Ly1, OCI-Ly3^[Citation97]	Exosomes	Differential ultracentrifugation	Western Blot with Flottilin-2 and CD63
PC3^[Citation99]	Microvesicles	Differential centrifugation	NTA, Guava EasyCyte microcapillary FACS with Annexin V
HeLa, Panc 1, PC3, A293^[Citation100]	Exosomes	Differential ultracentrifugation	Acetylcholinesterase activity, TEM, FACS with CD9, CD54, LAMP1, HSP70, Immunoblots for HSP70, HSP90, LAMP1

FACS = fluorescent activated cell sorting; TEM = transmission electron microscopy; NTA = nanoparticle tracking analysis. Some studies have used two methods of EV separation, we have denoted these (a) and (b).

Figure 3. Rho-associated protein kinases (ROCK) are serine-threonine kinases involved in cytoskeleton re-organisation. Once activated by multiple stimuli, ROCK regulate the shape and the movement of the cells, through the activation of Adducin or ERM (ezrin, radixin and moesin), but they can also interact with MLC (myosin light chain) and LIMK (LIM kinases, able to inactivate cofilin, fundamental for actin filament stabilisation) both involved in MVs release. Y27632 is a competitive inhibitor of both ROCK1 and ROCK2 and by blocking these proteins it can inhibit MVs release.

Figure 4. MVs present unique lipid characteristics, including an enrichment of sphingomyelin and ceramide, thus every enzyme able to interfere with membrane composition, i.e. calpains, scramblases and acid sphingomyelinases, plays a key role in MVs biogenesis. Acid sphingomyelinases convert sphingomyelin into ceramide, a cone-shaped rigid lipid that forms micro-domains inside the cell membrane, inducing the budding of MVs. Imipramine, a well-known anti-depressant, can promote membrane fluidity by acting on aSMases, thus preventing MVs generation.

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