EP3074044A2 - Inhibiteurs de synaptojanine-2 et leurs utilisations - Google Patents

Inhibiteurs de synaptojanine-2 et leurs utilisations

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Publication number
EP3074044A2
EP3074044A2 EP14827537.3A EP14827537A EP3074044A2 EP 3074044 A2 EP3074044 A2 EP 3074044A2 EP 14827537 A EP14827537 A EP 14827537A EP 3074044 A2 EP3074044 A2 EP 3074044A2
Authority
EP
European Patent Office
Prior art keywords
group
compound
moiety
cancer
synj2
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14827537.3A
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German (de)
English (en)
Inventor
Yosef Yarden
Nir Ben-Chetrit
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yeda Research and Development Co Ltd
Original Assignee
Yeda Research and Development Co Ltd
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Filing date
Publication date
Priority claimed from PCT/IL2013/050986 external-priority patent/WO2014083567A2/fr
Application filed by Yeda Research and Development Co Ltd filed Critical Yeda Research and Development Co Ltd
Publication of EP3074044A2 publication Critical patent/EP3074044A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024—Esters of saccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7032—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a polyol, i.e. compounds having two or more free or esterified hydroxy groups, including the hydroxy group involved in the glycosidic linkage, e.g. monoglucosyldiacylglycerides, lactobionic acid, gangliosides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention in some embodiments thereof, relates to cancer therapy and more particularly, but not exclusively, to compounds, compositions and methods for preventing tumor metastasis, and for treating cancer.
  • PI(4,5)P 2 phosphatidyl-inositol 4,5-bisphosphate
  • PI3K phosphatidylinositol 3- kinase
  • PI(4,5)P 2 regulates multiple proteins controlling endocytosis and actin dynamics [Saarikangas et al., Physiol Rev 90, 259-289 (2010)], but its levels are stringently controlled by two additional types of enzymes: phospholipase C (PLCy) promotes PI(4,5)P 2 hydrolysis, which activates cofilin (an actin-severing protein) and drives mammary cell migration [van Rheenen et al., Cell Biology 179, 1247-1259 (2007)], and synaptojanin-2.
  • PLCy phospholipase C
  • Synaptojanin-2 is an inositol polyphosphate 5 -phosphatase, which dephosphorylates the D5 position of the inositol ring. Dephosphorylation by SYNJ2 controls glioma cell migration [Chuang et al., Cancer Research 64, 8271-8275 (2004); Malecz et al., Curr Biol 10, 1383-1386 (2000)]. In addition homozygous mutations in SYNJ2 were identified in certain prostate cancer samples [Rossi et al., Cancer Genet Cytogenet 161, 97-103 (2005)].
  • the flavonoid ampelopsin has been reported to inhibit growth and metastasis of prostate cancer [Ni et al., PLoS ONE 7, e38802 (2012)].
  • the flavonoid chrysin has been reported to suppress survival and metastasis of mouse breast cancer cells [Lirdprapamongkol et al., Oncol Rep 30, 2357-2364 (2013)].
  • flavonoids for which an anti-invasive or anti-metastatic activity towards tumors has been reported include (-)-epigallocatechin-3-gallate, (-)-epigallocatechin, (-)-epicatechin-3-gallate, (-)- epicatechin, genistein/genistin, silibinin, nobiletin, quercetin, anthocyanin, luteolin, apigenin, myricetin, tangeritin, kaempferol, glycitein, licoricidin, daidzein and naringenin [Weng & Yen, Cancer Metastasis Rev 31, 323-351 (2012); Kawabata et al., Biosci Biotechnol Biochem 69, 307-314 (2005)].
  • a method of preventing tumor metastasis comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula I: X-L-[(Y)i-(Z)j]-(L-X)k
  • i, j and k are each independently 0 or 1, wherein at least one of i, j and k is 1;
  • L is absent or is a linking moiety
  • X is an aryl group substituted by one or more group selected from the group consisting of hydroxy, thiohydroxy, alkoxy, aryloxy, thioalkoxy and thioaryloxy;
  • Z is selected from the group consisting of a monosaccharide moiety, a disaccharide moiety, a shikimate moiety and a quinate moiety;
  • Y is a bicyclic moiety having the general formula II:
  • B is absent or is O, S, R 7 , CH, CH 2 , C-0-R 2 , C-S-R 2 , C-N(R 8 )-R 2 , CH-0-R 2 , CH-S-R 2 or CH-N(R 9 )-R 2 ;
  • R 1 -R5 are each independently selected from the group consisting of hydrogen, methyl, aryl and a covalent bond with an L, Z or X moiety described herein;
  • R5-R9 are each independently selected from the group consisting of hydrogen and alkyl
  • the dashed line denotes a saturated or unsaturated bond, wherein when the dashed line denotes a saturated bond, B is O, CH 2 or CH-0-R 2 , and when the dashed line denotes an unsaturated bond, B is CH or C-0-R 2 ,
  • a method of preventing tumor metastasis comprising administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula III:
  • E is selected from the group consisting of hydrogen and substituted or non- substituted benzyl
  • a method of preventing tumor metastasis comprising administering to a subject in need thereof a therapeutically effective amount of a compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof, thereby preventing tumor metastasis.
  • a method of preventing tumor metastasis comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of Compound 12 in Table 1 herein, chlorhexidine and pyrvinium, and pharmaceutically acceptable salts thereof, thereby preventing tumor metastasis.
  • a compound having the general formula I hereinabove for preventing tumor metastasis.
  • a compound having the general formula III hereinabove for preventing tumor metastasis.
  • a compound listed in Table 1 herein, or a pharmaceutically acceptable salt thereof, for preventing tumor metastasis for preventing tumor metastasis.
  • a compound selected from the group consisting of Compound 12 in Table 1 herein, chlorhexidine and pyrvinium, and pharmaceutically acceptable salts thereof, for preventing tumor metastasis.
  • a method of treating cancer comprising, administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula I hereinabove, and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, thereby treating cancer.
  • a method of treating cancer comprising, administering to a subject in need thereof a therapeutically effective amount of a compound having the general formula III hereinabove, and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, thereby treating cancer.
  • a method of treating cancer comprising, administering to a subject in need thereof a therapeutically effective amount of a compound listed in Table 1 herein or a pharmaceutically acceptable salt thereof, and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, thereby treating cancer.
  • a method of treating cancer comprising, administering to a subject in need thereof a therapeutically effective amount of:
  • a compound having the general formula III hereinabove and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, for treating cancer.
  • a compound listed in Table 1 herein or a pharmaceutically acceptable salt thereof and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, for treating cancer for treating cancer.
  • a compound selected from the group consisting of Compound 12 in Table 1 herein, chlorhexidine and pyrvinium, and pharmaceutically acceptable salts thereof,
  • kits for the treatment of cancer or prevention of cancer metastasis comprising a packaging material packaging a compound as described herein and an inhibitor of a cell surface receptor associated with an onset or progression of cancer.
  • a method of inhibiting synaptojanin-2 comprising contacting the synaptojanin-2 with an effective amount of a compound having the general formula I hereinabove, thereby inhibiting synaptojanin-2.
  • a method of inhibiting synaptojanin-2 comprising contacting the synaptojanin-2 with an effective amount of a compound having the general formula III hereinabove, thereby inhibiting synaptojanin-2.
  • a method of inhibiting synaptojanin-2 comprising contacting the synaptojanin-2 with an effective amount of a compound listed in Table 1 herein or a pharmaceutically acceptable salt thereof, thereby inhibiting synaptojanin-2.
  • a method of inhibiting synaptojanin-2 comprising contacting the synaptojanin-2 with an effective amount of a compound selected from the group consisting of Compound 12 in Table 1 herein, chlorhexidine and pyrvinium, and pharmaceutically acceptable salts thereof, thereby inhibiting synaptojanin-2.
  • the abovementioned method is effected ex vivo.
  • the abovementioned method is for treating a disease or disorder in which inhibition of synaptojanin-2 is beneficial.
  • the metastasis is EGF dependent.
  • the tumor is a breast cancer tumor.
  • the cell surface receptor associated with the onset or progression of cancer is a receptor tyrosine kinase.
  • the receptor tyrosine kinase is an ErbB receptor.
  • the ErbB receptor is Epidermal Growth Factor Receptor (EGFR).
  • EGFR Epidermal Growth Factor Receptor
  • the cancer is breast cancer.
  • the inhibitor of the cell surface receptor associated with the onset or progression of cancer is an antibody.
  • the inhibitor of the cell surface receptor associated with the onset or progression of cancer is a small molecule inhibitor.
  • B is CH, CH 2 , C-0-R 2 , or CH-0-R 2 .
  • the aryl is a phenyl
  • X is a hydroxylated phenyl group selected from the group consisting of trihydroxyphenyl, dihydroxyphenyl, hydroxyphenyl, methoxydihydroxyphenyl, and methoxy hydroxyphenyl;
  • X is selected from the group consisting of 3,4,5-trihydroxyphenyl, 3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl, 3,5- dihydroxyphenyl, 2,3 -dihydroxyphenyl, 6-methoxy-2,4-dihydroxyphenyl, 3-methoxy-4- hydroxyphenyl, 3 -hydroxy -4-methoxyphenyl and 4-hydroxyphenyl.
  • i 1
  • Ri is selected from the group consisting of methyl, phenyl and a covalent bond.
  • Ri is a bond with X, or Ri is phenyl.
  • R 2 is selected from the group consisting of hydrogen and a covalent bond.
  • R 3 is selected from the group consisting of hydrogen, methyl and a covalent bond.
  • R 4 and R 5 are each independently selected from the group consisting of hydrogen and a covalent bond.
  • the dashed line denotes a saturated bond
  • the carbon atom attached to Ri is chiral and is in an (S) configuration
  • j is 0, and X is selected from the group consisting of 3,4,5-trihydroxyphenyl, 3,4-dihydroxyphenyl, 2,3 -dihydroxyphenyl and 4- hydroxyphenyl.
  • Ri and R4 are each independently a covalent bond to Z, L or X.
  • Z is a monosaccharide or disaccharide, being attached to Y, L or X via a glycosidic bond.
  • i is 0.
  • j is 0 and k is 1.
  • each X is independently selected from the group consisting of 2,4-dihydroxyphenyl, 3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl and 6-methoxy -2,4-dihydroxyphenyl.
  • j is 1.
  • Z is selected from the group consisting of a monosaccharide, shikimate and quinate.
  • the monosaccharide is attached to L or X at the 1 -position and/or 6-position of the monosaccharide
  • the shikimate and the quinate are attached to L and/or X at a 3-position and/or 5-position of the shikimate or the quinate.
  • a sum of i, j and k is 2.
  • the compound has the general formula:
  • D is a terpenoid derivative selected from the group consisting of:
  • Figures 1A-I show that EGF promotes invasive growth of mammary cells and induces a specific set of genes.
  • Figure 1A - MCF10A cells were plated in the absence of growth factors and allowed to form clusters. Seventy-two hours later, cells were treated with the indicated growth factors (each at 10 ng/mL) and phase contrast images were taken 24 hours later (scale bar, 50 ⁇ ).
  • Figure IB - MCF10A cells were plated in migration or invasion chambers, as indicated, in the presence of the indicated ligands (10 ng/mL), and 18 hours later cells that migrated to the lower compartment were stained with crystal violet (left panel). Shown are quantifications of migration and invasion signals, normalized to the effect of EGF treatment. Data represent mean ⁇ S.D.
  • Figure 1C - MCF10A cells were plated in transwell inserts in EGF-containing medium, without or with the inhibitors AG-1478 (1 ⁇ ), U0126 (5 ⁇ ), or Wortmannin (200 nM), and allowed to migrate for 18 hours. Data represent mean ⁇ S.D. of triplicates. The experiment was repeated twice.
  • Figure ID - A list of 425 genes specifically induced in human mammary MCF10A cells by EGF (and not by serum) [Amit et al., Nat Genet 39, 503-512 (2007)], was intersected with genes that were up- regulated in the context of metastasis of MDA-MB-231 cells (1,597 genes) [Minn et al., Nature 436, 518-524 (2005)].
  • One of the 23 overlapping genes encodes the 5'- phosphatidylinositol lipid phosphatase Synaptojanin-2 (SYNJ2).
  • Figure IE - MCF10A cells were infected with lentiviral particles encoding LacZ (Ctrl) or SYNJ2-GFP (SYNJ2-OX). Expression levels of the endogenous SYNJ2 and the SYNJ2-GFP fusion protein were determined by immunoblotting, and equal protein loading was confirmed by probing for tubulin.
  • Figure IF - The Ctrl and SYNJ2-OX clones of MCFIOA cells were plated in migration chambers (5 x 10 4 cells/well) in the absence (NT) or presence of EGF (10 ng/mL) and allowed to migrate for 22 hours. Migrating cells that reached the other side of the filter were stained with crystal violet and images were taken.
  • Figure 1G - MCFIOA cells were transfected with siRNA control (siCtrl) or siRNA directed to SYNJ2 (siSYNJ2), and protein levels of SYNJ2 were determined 36 hours later by immunoblotting. Equal protein loading was confirmed by immunoblotting for Ras-GAP.
  • Figure 1H - The cells presented in G were plated in migration chambers (5 x 10 4 cells/well) in the absence (NT) or presence of EGF (10 ng/mL) and allowed to migrate for 22 hours. Migrating cells that reached the lower face of the filter were stained with crystal violet and images were captured.
  • Figure II - Confluent cultures of MCFIOA cells were treated with the indicated siRNAs. Once monolayers formed, they were subjected to an automated scratching system that monitors the rate of scratch closure.
  • Figures 2A-E show that transcriptional induction of SYNJ2 by EGF promotes invasive growth.
  • Figure 2A Serum-starved MCFIOA cells were stimulated with EGF (20 ng/mL) or serum (5 %), and SYNJ2 mRNA expression was assayed by using microarrays or RT-qPCR.
  • Figure 2B - MCFIOA cells were stimulated with EGF, extracted and immunoblotted as indicated.
  • FIG. 2D-E - MCFIOA cells were cultured for 22 hours in migration or invasion chambers (5-6 x 10 4 cells/well) in the absence (NT) or presence of EGF (10 ng/mL). Cells that reached the filter's bottom were stained and filter's coverage quantified (mean ⁇ S.D ).
  • Figures 3A-G show inducible translocation of SYNJ2 to the leading edge accompanies mammary cell migration and invasion.
  • Figure 3 A - MDA-MB-231 cells were infected with lentiviral particles encoding LacZ (Ctrl) or a V5-tagged SYNJ2 (SYNJ2-V5), along with control shRNA (shCtrl) or an shRNA directed against SYNJ2 (shSYNJ2). Protein levels of V5-SYNJ2 and endogenous SYNJ2 were determined by immunoblotting. Equal protein loading was confirmed by immunoblotting for AKT.
  • Figure 3B Phase images (left panels) and invasion images (right panels) of MDA-MB- 231 cells stably over-expressing SYNJ2, or LacZ as control.
  • the invasive capacities were determined in triplicates using an invasion assay, and invading cells were quantified and normalized to control (Ctrl). Scale bar, 50 ⁇ .
  • Figure 3C - MDA-MB- 231 cells were transfected with siRNA oligonucleotides directed to SYNJ2 (or siCtrl). Following 36 hours, protein levels of SYNJ2 were determined by immunoblotting. Equal protein loading was confirmed by immunoblotting for Ras-GAP.
  • Figure 3D Cells from C were plated in migration or invasion chambers and incubated for 18 hours.
  • FIG. 3E MDA-MB-231 cells transiently expressing GFP-SYNJ2 were plated on glass coverslips and stimulated with TGFa (10 ng/mL). Time-lapse microscopy photos were taken (every 10 seconds). The images shown are inverted, with black spots representing SYNJ2 and its assembly at the base of lamellipodia. Scale bar, ⁇ .
  • Figure 3F - MDA-MB-231 cells were immunostained for endogenous SYNJ2 and F-actin using TRITC-phalloidin. The squared area is magnified. Scale bar, ⁇ .
  • Figure 3G - MCF10A cells were stimulated with EGF for 18 hours, and then immunostained for endogenous SYNJ2 and counter- stained for F-actin using TRITC-phalloidin. Scale bar, 10 ⁇ .
  • Figures 4A-F show that the catalytic activity of SYNJ2 is essential for invasive growth.
  • FIGS 4C-D - shSYNJ2-expressing MDA-MB-231 cells were infected with WT SYNJ2 (shSYNJ2+SYNJ2 WT ) or with a catalytically disabled mutant (shSYNJ2+SYNJ2 ⁇ ). Cells were either extracted and immunoblotted as indicated, or they were allowed to invade for 18 hours in invasion chambers. Images of the invaded cells and their normalized quantification are shown (mean ⁇ S.D).
  • Figure 4E - show scanning electron micrographs of shCtrl and shSYNJ2 cells grown on fibronectin. Scale bar, 2 ⁇ .
  • Figure 4F Images of F-actin in the indicated MDA-MB-231 cells stained with phalloidin and DAPI. Z-axis sections (lines) and magnified areas are shown. Arrowheads mark swollen structures. Scale bar, 10 ⁇ .
  • Figures 5A-H show the subcellular localization of SYNJ2.
  • Figure 5A MDA- MB-231 cells expressing GFP-SYNJ2 were transfected with an RFP-Clathrin and plated on fibronectin-coated plates. Using spinning-disc microscopy, cells were imaged every five seconds. Arrowheads mark a newly formed leading edge. Scale bar, 5 ⁇ .
  • Figure 5B Representative time frames depicting assembly and disassembly of SYNJ2 at the leading edge (upper two rows) and underneath the cell body. For the lower rows, cells were transfected with a mCherry-lifeACT plasmid and plated on collagen. Thereafter, cells were imaged at 1 minute intervals.
  • FIG. 5C Cells were simultaneously imaged by TIRF and epifluorescence microscopy and signals converted into kymographs (x-axis). Arrowheads mark signal initiation. Scale bar, 5 ⁇ .
  • Figure 5D Cells were imaged using spinning disc confocal microscopy 5 minutes before and 5 minutes after treatment with Dyngo-4a (30 ⁇ ; a Dynamin-2 inhibitor). Scale bar, 5 ⁇ .
  • Figure 5E MDA-MB-231 cells stably expressing GFP-SYNJ2 were pre-incubated with Dyngo-4a (30 ⁇ ; 30 min), or with solvent (DMSO).
  • FIG. 5F Cell lysates were subjected to immunoprecipitation with anti-GFP antibodies (or with no antibody; -Ab), and then immunoblotted, along with a sample (5%) of the cell lysate, with the indicated antibodies.
  • Figure 5F - Cells were plated on fibronectin, fixed and immunostained for endogenous Racl . Scale bar, 10 ⁇ .
  • Figure 5G - Cells were imaged using confocal microscopy 5 minutes prior to and 5 minutes after a 30min-long treatment with NSC- 23766 (5 ⁇ ). Scale bar, 5 ⁇ .
  • Figure 5H - MDA-MB-231 cells were treated with the indicated siRNA oligonucleotides. Cell extracts were blotted for SYNJ2 and Ras-GAP. GTP-Racl levels were determined using an ELISA-based assay (Cytoskeleton).
  • Figures 6A-D show SYNJ2 localization to the leading edge is distinct from caveolins distribution and depends on F-actin, cholesterol and PI3K.
  • Figure 6A - MDA- MB-231 cells expressing GFP-SYNJ2 and co-expressing RFP-Cavl were simultaneously imaged over time, and signals converted into kymographs (x- and y- axis). Note the transient nature of SYNJ2 assemblies and stable appearance of Caveolin 1. Scale bar, 5 ⁇ .
  • Figure 6B - The left panel depicts the distribution (% of pits versus lifetime) of 150 randomly selected SYNJ2 assemblies, imaged as in Figure 5 A (5 second intervals, single plane, spinning disk confocal).
  • FIG. 6C The right panel depicts the average ( ⁇ SEM) relative intensity of assemblies that showed a 55 seconds lifetime.
  • Figure 6C MDA-MB-231 cells stably-expressing GFP-SYNJ2 were treated with M CD (10 mM, 15 minutes) or with Wortmannin (500 nM, 15 minutes). Images of the same selected cells were captured every 6 seconds, either prior to or following treatment, and signals were converted into kymographs (representing the squared insets in the left panels). Scale bar, 20 ⁇ .
  • Figure 6D - MDA-MB-231 cells stably co- expressing GFP-SYNJ2 and lifeACT-mCherry were treated with LatrunculinB (1 ⁇ , 15 minutes). Images were acquired either prior to or following treatment. Scale bar, 5 ⁇ .
  • Figures 7A-E show SYNJ2 depletion arrests EGFR in intracellular vesicles
  • Figure 7A - MCF10A cells stably expressing shRNA control (shCtrl) or shRNA specific to SYNJ2 (shSYNJ2) were extracted three days after plating in EGF-containing medium. Immunoblots were probed for SYNJ2, EGFR, phosphorylated tyrosine 1068 of EGFR (pEGFR), phosphorylated ERK (pERK), and Ras-GAP, as a loading control.
  • Figure 7B - MCF10A cells were transfected with siRNA control, or siRNA directed against SYNJ2, in the presence of EGF.
  • FIG. 7C Three derivatives of MDA-MB-231 cells were immunostained for EGFR and counterstained for DAPI and F-actin: (i) cells in which SYNJ2 was knocked-down (shSYNJ2; left column), (ii) the same cells infected by lentiviral gene transfer corresponding to the catalytically-dead form (shSYNJ2+SYNJ2 ; middle column), and (iii) cells in which SYNJ2 was knocked-down and the wild type form was introduced by infection (shSYNJ2 +SYNJ2 WT ; right column). Scale bar, 20 ⁇ .
  • Figure 7D Ubiquitinated EGFR levels (densitometry).
  • Figure 7E Ubiquitinated EGFR levels (densitometry).
  • Figures 8A-I show that SYNJ2 regulates EGFR trafficking and chemotaxis.
  • Figure 8A Whole extracts of MDA-MB-231 cells transfected with the indicated siRNAs were immunoblotted as indicated.
  • Figure 8B FACS (left) and 125 I-EGF binding (right; in triplicates) analyses of surface EGFR in the indicated MDA-MB-231 subclones.
  • Figure 8C shCtrl and shSYNJ2 cells were grown on fibronectin and immunostained for EGFR and F-actin. Bar, 20 ⁇ .
  • Figure 8D Rose plots of tracks of shCtrl and shSYNJ2 MDA-MB-231 cells, which migrated in chemotaxis chambers upon exposure to an EGF gradient. The red tracks indicate cells migrating toward EGF.
  • Figure 8E Starved MDA-MB-231 derivatives were treated with EGF (10 ng/mL) and cell ly sates were subjected to immunoprecipitation and immunoblotting as indicated.
  • Figure 8F Cells were cultured as in C and immunostained for active EGFR (pY1045) and F-actin. Bar, 10 ⁇ .
  • Figure 8G The indicated MDA-MB-231 derivatives were treated with EGF (10 ng/ml) for 5 hours and extracts immunoblotted as indicated.
  • Figure 8H The indicated MDA-MB-231 derivatives were exposed to Alexa Fluor 488- Tfn (25 ⁇ g/ml; 5 min), acid-washed to remove surface-bound ligands, and images taken at the indicated intervals. Normalized fluorescence signals are shown. Bar, 10 ⁇ .
  • Figures 9A-D show that SYNJ2 is necessary for both vesicular trafficking and focal adhesion formation.
  • Figure 9A - MDA-MB-231 derivatives (shCtrl and shSYNJ2) were fixed and stained for EEAl, F-actin and nuclei (DAPI). Scale bar, 10 ⁇ .
  • FIG 9C MDA-MB-231 cells were treated with siCtrl and siSYNJ2 for 48 hours and then immunostained for integrin beta-1 and phosphorylated EGFR.
  • Figure 9D Immunofluorescence analysis of MDA- MB-231 derivatives for paxillin, nuclei (DAPI), and F-actin (using TRITC-phalloidin). The paxillin signal was quantified in cytoplasmic regions relative to focal adhesions, and the numbers of focal adhesions per cell were also quantified. In addition, the shapes of focal adhesions were quantified by determining deviations from a perfect circle (eccentricity). Scale bar, 10 ⁇ .
  • Figures 10A-F show that SYNJ2 depletion perturbs phosphoinositide homeostasis, inflates early endosomes and disassembles focal adhesions.
  • Figure 10B - MDA-MB-231 derivatives were immunostained for Rab5, F- actin and nuclei (DAPI). Images were quantified for the size and number of Rab5- positive vesicle, as well as for the average cell area. Scale bars, 10 ⁇ .
  • Figure IOC Phosphoinositides extracted from 3 H-phosphoinositol labeled derivatives of MDA-MB- 231 cells, were separated by chromatography and their levels determined in three different experiments (signals normalized to shCtrl cells).
  • Figure 10D shCtrl and shSYNJ2 MDA-MB-231 cells were probed for pY1068-EGFR, Paxillin and F-actin (co- localization signal are white). Scale bar, 10 ⁇ .
  • Figure 10E shCtrl and shSYNJ2 MDA-MB-231 cells were seeded. Unattached cells were removed 20 min later and attached cells were imaged and quantified for surface area.
  • FIG. 10F MDA-MB-231 cells, stably expressing shCtrl or shSYNJ2, were plated on RTCA E-plates and realtime impedance measurements were recorded in 5 sec intervals for 80 min, and then in 10 min intervals for additional 80 min. Means of 2 replicates ( ⁇ S.D.) are shown.
  • Figures 11 A-G show that SYNJ2 regulates protease secretion and invadopodium assembly.
  • Figure 11A shCtrl and shSYNJ2 MDA-MB-231 cells were cultured in Matrigel for 5 days, fixed and immunostained for MMP-9. Signal intensities were converted into heat-maps and plotted against distance from colony cores. Arrowheads mark spheroid boundaries. Bar, 50 ⁇ .
  • Figure 1 IB Supernatants from control MDA- MB-231 cells and cells stably overexpressing SYNJ2 were analyzed in triplicates for MMP-2 and MMP-9 activity using gelatin zymography.
  • Figure 11C MDA-MB-231 cells stably expressing GFP-SYNJ2 were plated onto coverslips pre-coated with cross- linked fluorescent gelatin. Three hours later, cells were probed for GFP and F-actin, and invadopodial structures detected (arrowheads). Bar, 10 ⁇ .
  • Figure 11D MDA-MB- 231 cells overexpressing SYNJ2 (SYNJ2-OX), as well as cells pre-treated with siCtrl or siSYNJ2 oligonucleotides, were plated on coverslips pre-coated with cross-linked fluorescent gelatin and invadopodial structures were quantified in three independent experiments.
  • Figure HE Invadopodial structures of MDA-MB-231 cells treated with the indicated siRNAs were detected by gelatin degradation, as well as by staining for F- actin or TKS5. Arrowheads (z-axis images) mark invadopodia. Bar, 10 ⁇ .
  • Figure 1 IF - MDA-MB-231 cells expressing siCtrl or siSYNJ2 were plated on gelatin-coated coverslips and processed as in C using phalloidin and antibodies to the phosphorylated form of EGFR (tyrosine 1068). Scale bar, 10 ⁇ .
  • Figure 11G Media conditioned over 3 days by the indicated MDA-MB-231 derivatives were examined using an ELISA- based assay for EGF-like ligands.
  • Figures 12A-G show that SYJN2 regulates matrix degradation and invadopodia assembly.
  • Figure 12A The indicated siRNA-treated MDA-MB-231 cells were plated in triplicates, cultured for 3 days and their conditioned media were separated electrophoretically using a gelatin (0.1%) embedded gel, followed by protein staining to quantify MMP-2 and MMP-9 proteolytic activity.
  • Figure 12B Co- immunoprecipitation analysis using GFP-conjugated beads and cleared extracts of MDA-MB-231 cells stably expressing GFP-SYNJ2.
  • FIG 12C MDA-MB-231 cells stably expressing GFP-SYNJ2 were transfected with a RFP-Cortactin plasmid and plated on collagen plates. Live-cell image analysis was performed forty-eight hours later, and representative snapshot images of both peripheral and central cell areas were captured. Scale bar 5 ⁇ .
  • Figure 12D The indicated derivatives of MDA-MB-231 cells were transfected with a plasmid encoding a Myc-tagged PH domain of Tappl (a PI(3,4)P 2 binder) and 48 hours later they were plated on gelatin-coated surfaces.
  • Figure 12F An antibody to CD44 was used for FACS analysis of surface expression by shCtrl and shSYNJ2 cells. Indicated are the fractions of cells corresponding to the framed regions.
  • Figure 12G - MDA-MB-231 cells pre-treated with si Ctrl or siSYNJ2 were plated onto FITC-gelatin coated glass coverslips and incubated for 3 hours. Cells were then fixed and immunostained for MTl-MMP, and counter stained for F-actin with TRITC-phalloidin. Scale bar, 10 ⁇ .
  • Figures 13A-H show that the enzymatic activity of SYNJ2 propels metastatic spread of mammary tumor cells.
  • Figure 13 A The indicated derivatives of RFP- expressing MDA-MB-231 cells (2xl0 6 /mouse) were implanted in the fat pad of female SCID mice (10-11 per group). Tumor size (mean ⁇ S.D.) was measured 2 and 6 weeks post implantation.
  • Figures 13B-C Metastases that appeared six weeks post- implantation in axillary and distant lymph nodes ( Figure 13B), or lungs ( Figure 13C), are shown.
  • Asterisks mark p values: * ⁇ 0.05, ** ⁇ 0.01 and *** ⁇ 0.001.
  • FIGS 13D- F - Control (LacZ) and SYNJ2-overexpresseing (SYNJ2-OX) RFP-labelled MDA-MB- 231 cells were implanted in animals as in A and tumor size (Figure 13D), as well as metastases to lymph nodes (Figure 13E) and lungs ( Figure 13F) were quantified 6 and 8 weeks post implantation.
  • Figures 13G-H - The indicated MDA-MB-231-RFP derivatives were injected either intravenously (1.5xl0 5 per mouse; tail vein), or in the mammary fat pad (2.5xl0 6 per mouse) of 5-week old female SCID mice. Four weeks later, lungs from mice injected into the vein were examined for RFP signals (left and middle panels).
  • Peripheral blood was collected from the fat pad-treated group four weeks later. Samples were purified on a gradient of ficoll and the numbers of RFP- positive circulating tumor cells (CTC) were scored per lxlO 6 FACS readings and normalized to tumor weight.
  • CTC RFP- positive circulating tumor cells
  • Figure 14 is an in vivo imaging of local and distant lymph node metastases.
  • Figure 15 is a working model depicting the integrated action of SYNJ2 in cell migration and invasion.
  • EGFR-loaded recycling endosomes position active receptors at the ventral membrane, and this is followed by local activation of PI3K.
  • Phosphorylation of membranal PI(4,5)P 2 by PI3K generates PI(3,4,5)P 3 , which is dephosphorylated by SYNJ2 to PI(3,4)P 2 .
  • the latter recruits TKS5, which anchors Cortactin and nucleates actin polymerization.
  • SYNJ2 controls delivery of adhesion molecules like CD44, and proteases like MT1-MMP, to degrade the extracellular matrix (ECM) and establish new invasive structures, the invadopodia.
  • ECM extracellular matrix
  • EGFR delivery to the cell periphery leads to breakdown of PI(4,5)P 2 by SYNJ2 (and phospholipase C), which locally activates Dynamin and actin severing enzymes like Cofilin to dissolve cortical actin fibres and initiate actin-filled, integrin-rich protrusions called lamellipodia.
  • the horizontal arrow marks the direction of cell migration. Color-coded segments of the plasma membrane denote specific PI phospholipids.
  • Figures 16A-C show that SYNJ2 is highly expressed in aggressive breast tumors.
  • Figure 16A Immunohistochemistry and tissue microarrays were used to stratify 331 invasive breast carcinomas according to SYNJ2 abundance (high, medium and low). The relative fraction of tumors is presented according to clinical subtypes.
  • Figure 16B Representative images of SYNJ2 staining demonstrating intensities and patterns (magnified in the right column) observed in a luminal case (an asterisk marks expression by endothelial cells as control), and both basal-like and HER2- overexpressing breast tumors.
  • Figure 16C Kaplan-Meier curves stratified according to SYNJ2 mRNA expression in cohorts of 286 (left; GSE2034) or 99 (right; GSE19783) breast cancer patients.
  • FIG. 17 A depicting an exemplary assay for detecting binding of a molecule (black circle) labeled by a fluorescent probe (white circle) to a large molecule (ellipse), wherein binding results in an increase in fluorescence polarization following excitation by polarized light, and a bar graph (FIG.
  • Figure 18 depicts the amino acid and nucleic acid sequences of the Flag-TAPPl PH domain-His that was cloned into pET28 plasmid and expressed in E.coli.
  • the first TAPPl-PH domain is marked in yellow.
  • Figure 19 presents images of MDA-MB-231 cell spheroids seeded in 5 % Matrigel, 0 or 96 hours after being treated with 2 ⁇ pyrvinium pamoate (Compound 18) or carrier (DMSO).
  • FIG. 20A-B present a plot (FIG. 20A) and images (FIG. 20B) showing migrating MCF10A cells in the presence of various concentrations (in the range of 0.125-10 ⁇ ) pyrvinium pamoate (Compound 18) or carrier (DMSO)
  • FIG. 20A shows mean ⁇ standard deviation, and distribution of experimental values, with p values for differences between 1 an 2 ⁇ pyrvinium pamoate and DMSO control
  • FIG. 20B shows representative results from a corresponding experiment).
  • Figure 22 is a plot showing the tumor mass of MDA-MB-231 breast cancer cells in mice 6 weeks after beginning to inject the mice twice per week with the exemplary compound P-3195 (Compound 2) or carrier (DMSO) (mean ⁇ standard deviation, and distribution of experimental values; injection in each mouse began when tumor volume was approximately 3x3x3 mm 3 ).
  • P-3195 Compound 2
  • DMSO carrier
  • FIG. 23A-B present images (FIG. 23A) and a plot (FIG. 23B) showing metastases that appeared in the lungs six weeks post-implantation of MDA-MB-231 breast cancer cells in mice treated by injection of the exemplary compound NP-3195 (Compound 2) or carrier (DMSO)
  • FIG. 23A shows representative samples
  • FIG. 23B shows mean ⁇ standard deviation, and distribution of experimental values
  • Figure 24 presents images of fluorescent-labeled EGFR (endothelial growth factor receptor) in MDA-MB-231 cells subjected to knockdown of SYNJ2 (shSYNJ2) or control knockdown (shCtrl) (upper row) and in naive cells (bottom row) treated with 0.78 ⁇ pyrvinium pamoate (Compound 18) or with carrier (DMSO) (blue staining of nuclei with DAPI is used for contrast).
  • the present invention in some embodiments thereof, relates to cancer therapy and more particularly, but not exclusively, to compounds, compositions and methods for preventing tumor metastasis, and for treating cancer.
  • synaptojanin-2 SYNJ2
  • SYNJ2 synaptojanin-2
  • the present inventors substantiated their finding in vitro, in animals and in patient specimens. Specifically, employing EGF-stimulated mammary cells the present inventors link the lipid phosphatase synaptojanin 2 (SYNJ2) to an invasive phenotype, and relate high SYNJ2 to short survival rates of cancer patients. Knockdown of SYNJ2 robustly impaired metastasis of mammary tumor cells in an animal model. In vitro, SYNJ2-depleted cells exhibited derailed trafficking of EGFR and integrins, resulting in deformed focal adhesions, arrested lamellipodia and disappearance of invadopodia.
  • SYNJ2 lipid phosphatase synaptojanin 2
  • the present inventors have further screened a variety of small molecules and uncovered a group of small molecule which act as inhibitors of SYNJ2.
  • the uncovered group of small molecules was found to possess common structural features which provide SYNJ2 inhibitory activity. Accordingly, small molecules which may be used to prevent tumor metastasis as described herein are disclosed.
  • Table 1 depicts chemical structures and activity of molecules which selectively inhibit SYNJ2 enzyme activity.
  • compounds which selectively inhibit SYNJ2 enzyme activity can be characterized as comprising one or two hydroxylated phenyl groups attached (optionally via a short linking moiety) to one another or to a core moiety which comprises a benzopyran derivative and/or a saccharide or saccharide-like moiety such as shikimate or quinate.
  • FIG. 19-24 show that exemplary SYNJ2 inhibitors depicted in Table 1 (Compound 18 in Figures 19-20B, Compound 12 in Figure 21, and Compound 2 in Figures 22-23B) inhibit cancer cell invasion (Figure 19) and migration (Figures 20A-20B) in vitro, and tumor growth (Figures 21-22) and metastasis ( Figures 23A-23B) in mice.
  • Figure 24 further shows that Compound 18 causes redistribution of endothelial growth factor to within the cell.
  • hydroxylated phenyl groups and saccharides and saccharide-like moieties effect selective SYNJ2 inhibition by being similar in structure to inositol derivatives which are the natural substrates of SYNJ2, in that they comprise a six-membered ring substituted by hydroxy groups.
  • a method of preventing tumor metastasis comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of synaptojanin 2 (SYNJ2) as described herein, thereby preventing tumor metastasis.
  • SYNJ2 synaptojanin 2
  • a compound described herein as a SYNJ2 inhibitor for use in preventing tumor metastasis.
  • a compound described herein as a SYNJ2 inhibitor in the manufacture of a medicament for preventing tumor metastasis.
  • the compound is Compound 2 ( P-3195).
  • tumor metastasis refers to a malignant tumor spreading out of its primary location to other parts of the body, e.g., breast cancer which metastasizes to the lungs. Tumor metastasis often involves migration of tumor cells.
  • cancer and “tumor” are interchangeably used.
  • the term refers to a malignant growth or tumor caused by abnormal and uncontrolled cell proliferation (cell division).
  • preventing refers to arresting, halting, inhibiting the metastatic process or progression and subsequent metastasis.
  • a method of treating cancer comprising, administering to a subject in need thereof a therapeutically effective amount of an inhibitor of synaptojanin 2 (SYNJ2) described herein and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, thereby treating cancer.
  • SYNJ2 synaptojanin 2
  • a compound described herein as a SYNJ2 inhibitor, and an inhibitor of a cell surface receptor associated with an onset or progression of cancer for use in treating cancer.
  • a compound described herein as a SYNJ2 inhibitor and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, in the manufacture of a medicament for treating cancer.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • subject refers to a mammal (e.g., human), for example, one who has been diagnosed with cancer.
  • synaptojanin-2 or SYNJ2 refers to synaptic inositol-1,4,5- trisphosphate 5-phosphatase 2, EC 3.1.3.36, a ubiquitously expressed inositol polyphosphate 5-phosphatase.
  • inhibitor of synaptojanin-2 As used herein the phrases "inhibitor of synaptojanin-2", “synaptohanin-2 inhibitor” and variations thereof refer to a molecule which decreases activity of SYNJ2.
  • the compound which is a SYNJ2 inhibitor has the general formula I:
  • i, j and k are each independently 0 or 1, wherein at least one of i, j and k is 1;
  • L is absent or is a linking moiety (e.g., from 1 to 3 atoms in length);
  • X is an aryl group substituted by one or more hydroxy group and/or one or more analog of a hydroxy group selected from the group consisting of thiohydroxy, alkoxy, aryloxy, thioalkoxy and thioaryloxy;
  • Z is selected from the group consisting of a monosaccharide moiety, a disaccharide moiety, a shikimate moiety and a quinate moiety;
  • Y is a bicyclic moiety having the general formula II:
  • B is absent or is O, S, R 7 , CH, CH 2 , C-0-R 2 , C-S-R 2 , C-N(R 8 )-R 2 , CH-0-R 2 , CH-S-R 2 or CH-N(R 9 )-R 2 ;
  • Ri-R 5 are each independently selected from the group consisting of hydrogen, methyl, aryl (e.g., phenyl) and a covalent bond with an L, Z or X moiety as described herein, if present;
  • R5-R9 are each independently selected from the group consisting of hydrogen and alkyl (e.g., Ci -4 alkyl); and
  • the dashed line denotes a saturated or unsaturated bond, wherein when the dashed line denotes a saturated bond, B is O, S, R 7 , CH 2 , CH-S-R 2 , CH-N(R 9 )-R 2 or CH-0-R 2 , and when the dashed line denotes an unsaturated bond, B is CH, C-S-R 2 , C- N(R 8 )-R 2 or C-0-R 2 .
  • the compound comprises two X moieties which may be the same or different.
  • any optional embodiment described herein for any one or more of the variables X, Y, X, L, i, j, k, A, B, D, E and R 1 -R5 is intended to be combined in any possible combination with any of the optional embodiments for the remaining variables described herein, unless explicitly indicated otherwise.
  • B is CH, CH 2 , C-0-R 2 , or CH-0-R 2 .
  • the linking moiety is a bond.
  • the aryl is a phenyl
  • the aryl is a hydroxylated aryl. In some embodiments, the aryl is a hydroxylated phenyl.
  • hydroxylated aryl and “hydroxylated phenyl” refer to an aryl group or a phenyl group, respectively, substituted by any number of hydroxy groups (and optionally also one or more alkoxy or aryloxy groups, preferably methoxy groups), whereas the term “hydroxyphenyl” refers to a phenyl group substituted by one hydroxy group, and is also referred to as phenol.
  • X is a hydroxylated phenyl group selected from the group consisting of trihydroxyphenyl (dihydroxyphenol), dihydroxyphenyl (hydroxyphenol), hydroxyphenyl (phenol), methoxydihydroxyphenyl, and methoxy hydroxyphenyl;
  • X is selected from the group consisting of 3,4,5- trihydroxyphenyl (an exemplary trihydroxyphenyl); 3,4-dihydroxyphenyl, 3,5- dihydroxyphenyl, 3, 5 -dihydroxyphenyl and 2,3 -dihydroxyphenyl (exemplary dihydroxyphenyls); 6-methoxy-2,4-dihydroxyphenyl (an exemplary methoxydihydroxyphenyl); 3-methoxy-4-hydroxyphenyl and 3-hydroxy-4- methoxyphenyl (exemplary methoxyhydroxyphenyls); and 4-hydroxyphenyl (an exemplary hydroxyphenyl).
  • X comprises at least two hydroxy or methoxy groups. In some embodiments, X comprises at least two hydroxy groups. In some embodiments, X comprises at least three hydroxy groups, for example, wherein X is trihydroxyphenyl. Without being bound by any particular theory, it is believed that the number of hydroxy groups (at least up to 3 hydroxy groups) is associated with SYNJ2 inhibition. Thus, as exemplified herein in Table 1 in the Examples section, addition of a hydroxy group to a phenyl moiety of catechin-7-gallate, resulting in gallocatechin-7-gallate, decreased the IC50 of the compound more than 3-fold.
  • Ri is selected from the group consisting of methyl, phenyl and a covalent bond. In some embodiments, Ri is a covalent bond.
  • Ri is a bond with an X, that is, an L at this position is absent, or alternatively, Ri is phenyl. In such embodiments, a substituted or non- substituted phenyl is attached to the Y moiety at the Ri position.
  • Moieties and compounds in which a substituted or non-substituted phenyl is attached to the Y moiety at the Ri position are referred to herein as "flavonoid" moieties and compounds.
  • R 2 is selected from the group consisting of hydrogen and a covalent bond. In exemplary embodiments, R 2 is a bond to a 3,4-dihydroxyphenyl moiety.
  • R 2 when B is C-0-R 2 , R 2 is not hydrogen. In some embodiments, when B is C-0-R 2 , R 2 is a bond with a Z. In some embodiments, the Z is a disaccharide moiety (e.g., mannosyl galactose) or a monosaccharide attached to -L-X (e.g., a galloyl-substituted rhamnosyl).
  • a disaccharide moiety e.g., mannosyl galactose
  • a monosaccharide attached to -L-X e.g., a galloyl-substituted rhamnosyl
  • R 3 is selected from the group consisting of hydrogen, methyl and a covalent bond. In exemplary embodiments, when R 3 is a covalent bond, the bond is with an L linking moiety which is CH 2 .
  • R 4 is selected from the group consisting of hydrogen and a covalent bond.
  • R4 is a covalent bond with X or L, and X is 3,4,5- trihy droxyphenyl .
  • R4 is a covalent bond with Z (e.g., when Y is part of a flavonoid moiety).
  • Z is a monosaccharide (e.g., glucosyl). In some embodiments, the monosaccharide is attached at the 1 -position thereof. In exemplary embodiments, Z is 1-glucosyl (e.g., ⁇ - ⁇ -glucosyl).
  • R 5 is selected from the group consisting of hydrogen and a covalent bond. In some embodiments, R 5 is hydrogen.
  • R 5 when R 5 is a covalent bond, the bond is with L, and L is
  • the dashed line denotes a saturated bond, such that at least one of the two carbon atoms connected by the dashed line is chiral.
  • the carbon atom attached to Ri is chiral and is in an (S) configuration.
  • the carbon atom attached to Ri is in an (S) configuration
  • the carbon atom attached to Ri is chiral and is in an (R) configuration.
  • the Z moiety described herein has a structure of a molecule (e.g., a monosaccharide, disaccharide, shikimate or quinate molecule) in which a hydroxy group is replaced with a Y moiety as described herein, and/or a hydrogen atom (e.g., a hydrogen atom of a hydroxy group) is replaced by an L or X moiety as described herein.
  • a molecule e.g., a monosaccharide, disaccharide, shikimate or quinate molecule
  • a hydrogen atom e.g., a hydrogen atom of a hydroxy group
  • the monosaccharide is a hexose.
  • the disaccharide comprises two hexose moieties.
  • the hexose is a D-hexose.
  • Exemplary D-hexoses include D-glucose, D-mannose and D-galactose.
  • at least a portion of the monosaccharide or disaccharide comprises a pyranose, that is, at least a portion in solution form a six-membered ring comprising 5 carbon atoms and one oxygen atom.
  • the pyranose form may be in equilibrium with a non-pyranose form (e.g., an aldehyde form) of the saccharide.
  • the disaccharide comprises two pyranose moieties.
  • a Z moiety which is a saccharide moiety is attached (e.g., to Y, L or X) via a glycosidic bond.
  • glycosidic bond refers to a bond at a position of the saccharide which comprises a hemiacetal (derived from an aldehyde) or hemiketal (derived from a ketone) group in the unbound saccharide molecule, for example, the 1 - position in glucose, mannose, and galactose.
  • a glycosidic bond stabilizes a cyclic (e.g., pyranose) form of a monosaccharide, which comprises a six- membered ring substituted by multiple hydroxy groups, which is associated with SYNJ2 inhibition.
  • Suitable monosaccharides include glucose (e.g., D-glucose), mannose (e.g., L-mannose, D-mannose), galactose (e.g., D-galactose) and rhamnose (e.g., L-rhamnose).
  • the monosaccharide is a glucose or rhamnose moiety, e.g., attached via a glycosidic bond.
  • the disaccharide is mannose (e.g., L-mannose) moiety attached to a galactose (e.g., D- galactose) moiety, e.g., wherein the mannose is attached via a mannose glycosidic bond and the galactose moiety is attached to a Y, L or X moiety described herein via a galactose glycosidic bond.
  • mannose e.g., L-mannose
  • galactose e.g., D- galactose
  • one of the positions is the 6-position of the saccharide (e.g., wherein the other position is a glycosidic bond as described herein).
  • D-glucose is a particularly suitable monosaccharide, as it typically assumes a configuration wherein the free hydroxy groups are attached to a pyranose ring at equatorial positions (rather than axial positions), which renders the hydroxy groups more accessible to the environment, and accords with the structure of phosphatidyl inositol, in which almost all of the hydroxy groups are in an equatorial position.
  • shikimate and “shikimic acid” each refer to 3,4,5- trihydroxycyclohex-l-ene-l-carboxylic acid and to pharmaceutically acceptable salts thereof, including any stereoisomer thereof.
  • the terms refer to the (3R,4S,5R) stereoisomer.
  • the terms “quinate” and “quinic acid” each refer to 1,3,4,5- tetrahydroxycyclohexanecarboxylic acid and to pharmaceutically acceptable salts thereof, including any stereoisomer thereof.
  • the terms refer to a (3R,4S,5R) stereoisomer.
  • the terms refer to the (1 S,3R,4S,5R) stereoisomer.
  • shikimate and quinate are similar in structure to pyranose monosaccharides, in that they comprise a six-membered ring substituted by multiple hydroxy groups.
  • Z comprises a single ring, that is, Z is a monosaccharide, shikimate or quinate.
  • i and j are 0, and k is 1.
  • L is not absent, such that the two X moieties are linked by a linking moiety.
  • the X moieties are different from one another.
  • each X is independently selected from the group consisting of 2,4-dihydroxyphenyl, 3,4-dihydroxyphenyl, 3,5-dihydroxyphenyl and 6- methoxy-2,4-dihy droxyphenyl .
  • each X is 3, 4-dihydroxy phenyl and 6- methoxy-2,4-dihy droxyphenyl .
  • one X is 3,5-dihydroxyphenyl
  • the other X is selected from the group consisting of 2,4-dihydroxyphenyl, 3,4- dihy droxyphenyl, and 6-methoxy-2,4-dihy droxyphenyl.
  • one X is 3,5-dihydroxyphenyl and the other X is 2,4-dihydroxyphenyl.
  • the dashed line in Y denotes a saturated bond (e.g., wherein the carbon atoms connected by the dashed line exhibit stereochemistry as described herein).
  • each X is independently selected from the group consisting of 3,4,5-trihydroxyphenyl, 3, 4-dihy droxyphenyl, 2,3 -dihy droxyphenyl and 4- hy droxyphenyl.
  • each X is independently selected from the group consisting of 3,4,5-trihydroxyphenyl, 3, 4-dihy droxyphenyl and 2,3 -dihy droxyphenyl.
  • an L which is CH 2 is attached to an X which is 2,3- dihydroxyphenyl, such that -L-X is 2,3-dihydroxybenzyl.
  • R 2 and R 4 are covalent bonds.
  • R 2 is a bond to a 3,4-dihydroxyphenyl moiety
  • R 4 is a bond to a galloyl moiety.
  • Ri and R 2 are covalent bonds.
  • Ri is a bond to a 3,4-dihydroxyphenyl or 3-methoxy-4-hydroxyphenyl moiety
  • R 2 is a bond to a galloyl, disaccharide or galloyl-substituted monosaccharide moiety.
  • Ri and R 4 are covalent bonds.
  • Ri is a bond to a 3,4-dihydroxy or 3,4,5-trihydroxyphenyl moiety
  • R 4 is a bond to a galloyl or monosaccharide (e.g., glucosyl) moiety.
  • R 3 and R 5 are covalent bonds.
  • each of R 3 and R 5 is a bond with an L that is CH 2 (e.g., a bond with 2,3- dihydroxybenzyl).
  • Ri is phenyl.
  • each X is independently selected from the group consisting of 3,4,5-trihydroxyphenyl, 3,4-dihydroxyphenyl and 4-hydroxyphenyl.
  • L is absent.
  • X is attached at the Ri position of Y, such that the compound is a flavonoid.
  • any chiral carbon atom attached to the dashed line in general formula II is in an (S) configuration.
  • B is CHOH
  • R 3 is hydrogen or methyl
  • R 4 is hydrogen
  • X is selected from the group consisting of 3,4,5- trihydroxyphenyl, 3,4-dihydroxyphenyl and 3-hydroxy-4-methoxyphenyl.
  • Z is a monosaccharide (e.g., a hexose), shikimate or quinate.
  • the monosaccharide is in a pyranose configuration.
  • Glucose e.g., D-glucopyranose
  • Z is an exemplary monosaccharide.
  • Z comprises a monosaccharide
  • the monosaccharide is attached to an L and/or X via an oxygen atom at a 1 -position and/or 6-position of the monosaccharide.
  • the shikimate or quinate is attached to an L and/or X via an oxygen atom at a 3-position and/or 5-position of the shikimate or quinate.
  • the L moieties in the above formula are different from one another.
  • the L moieties are the same.
  • the X moieties in the above formula are different from one another.
  • the X moieties are different, and the L moieties are different.
  • the X moieties are different, and the L moieties are the same.
  • the X moieties are the same. In some embodiments, the X moieties are the same, and the L moieties are different. In exemplary embodiments, the X moieties are the same, and the L moieties are the same.
  • Z comprises a monosaccharide
  • the monosaccharide is attached to an L and/or X via oxygen atoms at the 1 -position and 6-position of the monosaccharide.
  • the shikimate or quinate is attached to an L and/or X via oxygen atoms at a 3-position and 5-position of the shikimate or quinate.
  • each -L-X can independently be bound to the Y moiety or Z moiety in the Y-Z core.
  • At least one -L-X is bound to the Y moiety.
  • one -L-X is bound to the Y moiety, such that the compound has the general formula:
  • an X moiety is attached to the Y moiety at the Ri position, such that the compound comprises a flavonoid moiety attached to the Z moiety.
  • Z is 1-glucosyl (e.g., attached to the R 2 or R 4 position of Y).
  • the compound comprises a flavonoid moiety and k is 0, such that the compound is a flavonoid glucoside.
  • Z is substituted or non-substituted 1-rhamnosyl (e.g., attached at the R 2 position of Y).
  • the 1-rhamnosyl is substituted at the 2-position thereof.
  • k is 1, and the 1-rhamnosyl is substituted by -L-X (as described herein).
  • the 1-rhamnosyl is substituted at the 2-position by galloyl.
  • i, j and k are each 1, such that the compound comprises at least 4 cyclic moieties, namely, 2 X moieties described herein, a Y moiety and a Z moiety described herein.
  • a carbon atom in the Z moiety which is attached to the Y moiety is adjacent to a carbon atom in the Z moiety which is attached to -L-X, for example, wherein a 1 -position of Z is attached to Y and a 2-position is attached to -L-X.
  • a configuration is more compact, allowing for a shorter distance between the -L-X and Y, and therefore relatively similar in structure (and activity) to compounds wherein Y is attached directly to at least one -L-X (e.g., as described herein).
  • At least one of i, j and k is 0, such that the compound comprises a total of no more than 3 X, Y and Z moieties.
  • the sum of i, j and k is 2, such that the compound comprises a total of 3 X, Y and Z moieties.
  • a compound wherein the sum of i, j and k is 2 is a flavonoid attached to Z (e.g., a monosaccharide) or -L-X at the R4 position (with an X moiety also being present at the Ri position, as described herein).
  • the compound described herein as an SNJ2 inhibitor has the general formula III:
  • E is selected from the group consisting of hydrogen and substituted or non- substituted benzyl.
  • the compound comprises a glucose moiety, that is, the stereochemistry of Formula III is such that the pyranose moiety depicted therein is a glucopyranose moiety).
  • the compound has the general formula:
  • the compound described herein as an SNJ2 inhibitor comprises a monosaccharide moiety (e.g., a glucose moiety), for example, a monosaccharide moiety as depicted in general formula III, attached to a terpenoid moiety.
  • the terpenoid is attached to the monosaccharide via a glycosidic bond (e.g., at the 1 -position of the monosaccharide).
  • the terpenoid moiety is a monoterpene derivative, that is, it comprises 10 carbon atoms derived from two isoprene units.
  • Exemplary terpenoid moieties include moieties a), b), c) and d) in general formula III herein.
  • Moieties a), c) and d) are exemplary monoterpene derivatives, as described herein.
  • the benzyl is non- substituted.
  • E is hydrogen
  • the compound described herein as an SYNJ2 inhibitor is a compound depicted in Table 1 in the Examples section herein, or an analog or derivative thereof, or a pharmaceutically acceptable salt thereof.
  • the inhibitor is a compound depicted in Table 1 (or a pharmaceutically acceptable salt thereof) and characterized therein as having an IC50 toward sSYNJ2 of no more than 10 ⁇ .
  • the IC50 is no more than 7 ⁇ .
  • the IC50 is no more than 5 ⁇ .
  • the IC50 is no more than 4 ⁇ .
  • the IC50 is no more than 3 ⁇ .
  • the IC50 is no more than 2 ⁇ .
  • the compound is selected from the group consisting of chlorhexidine, pyrvinium and Compound 12 as depicted below, analogs and derivatives thereof, and pharmaceutically acceptable salts thereof.
  • Pyrvinium pamoate is an exemplary pyrvinium salt.
  • the compound is Compound 12.
  • the compound is a pharmaceutically acceptable salt of pyrvinium (e.g., pyrvinium pamoate).
  • the compound may be in a form of a salt, for example, a pharmaceutically acceptable salt, and/or in a form of a prodrug.
  • the phrase "pharmaceutically acceptable salt” refers to a charged species of the parent compound and its counter-ion, which is typically used to modify the solubility characteristics of the parent compound and/or to reduce any significant irritation to an organism by the parent compound, while not abrogating the biological activity and properties of the administered compound.
  • a pharmaceutically acceptable salt of the compounds described herein may optionally be an acid addition salt comprising at least one basic (e.g., amine) group of the compound which is in a positively charged form (e.g., an ammonium ion), in combination with at least one counter-ion, derived from the selected acid, that forms a pharmaceutically acceptable salt.
  • a basic (e.g., amine) group of the compound which is in a positively charged form e.g., an ammonium ion
  • the acid addition salts of the compounds described herein may therefore be complexes formed between one or more amino groups of the drug and one or more equivalents of an acid.
  • the acid addition salts may include a variety of organic and inorganic acids, such as, but not limited to, hydrochloric acid which affords a hydrochloric acid addition salt, hydrobromic acid which affords a hydrobromic acid addition salt, acetic acid which affords an acetic acid addition salt, ascorbic acid which affords an ascorbic acid addition salt, benzenesulfonic acid which affords a besylate addition salt, camphorsulfonic acid which affords a camphorsulfonic acid addition salt, citric acid which affords a citric acid addition salt, maleic acid which affords a maleic acid addition salt, malic acid which affords a malic acid addition salt, methanesulfonic acid which affords a methanesulfonic acid (mesylate) addition salt, naphthalenesulfonic acid which affords a naphthalenesulfonic acid addition salt, oxalic acid which affords an oxalic acid addition salt,
  • the acid or base additions salts can be either mono-addition salts or poly-addition salts.
  • addition salt refers to a salt in which the stoichiometric ratio between the counter-ion and charged form of the compound is 1 : 1, such that the addition salt includes one molar equivalent of the counter-ion per one molar equivalent of the compound.
  • poly-addition salt refers to a salt in which the stoichiometric ratio between the counter-ion and the charged form of the compound is greater than 1 : 1 and is, for example, 2: 1, 3 : 1, 4: 1 and so on, such that the addition salt includes two or more molar equivalents of the counter-ion per one molar equivalent of the compound.
  • prodrug refers to a compound which is converted in the body to an active compound (e.g., a SYNJ2 inhibitor described herein).
  • a prodrug is typically designed to facilitate administration, e.g., by enhancing absorption.
  • a prodrug may comprise, for example, the active compound modified with ester groups, for example, wherein one or more hydroxy groups of the active compound is modified by an acyl (e.g., acetyl) group to form an ester group, and/or wherein one or more carboxylic acid of the active compound is modified by an alkyl (e.g., ethyl) group to form an ester group.
  • each of the compounds described herein, including the salts thereof, can be in a form of a solvate or a hydrate thereof.
  • solvate refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the heterocyclic compounds described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute.
  • hydrate refers to a solvate, as defined hereinabove, where the solvent is water.
  • the present embodiments further encompass any stereoisomers (enantiomers and diastereomers) of the compounds described herein, except in embodiments wherein a specific stereoisomer is explicitly required, as well as any isomorph thereof.
  • alkyl refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., " 1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms.
  • the alkyl group may be substituted or unsubstituted.
  • the substituent group can be, for example, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, and amino, as these terms are defined herein.
  • a "cycloalkyl” group refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system.
  • examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.
  • a cycloalkyl group may be substituted or unsubstituted.
  • the substituent group can be, for example, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, and amino, as these terms are defined herein.
  • alkenyl refers to an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon double bond.
  • alkynyl refers to an alkyl group which consists of at least two carbon atoms and at least one carbon-carbon triple bond.
  • aryl group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be substituted or unsubstituted.
  • the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, and amino, as these terms are defined herein.
  • heteroaryl group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • the heteroaryl group may be substituted or unsubstituted.
  • the substituent group can be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, and amino, as these terms are defined herein.
  • heteroalicyclic group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur.
  • the rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system.
  • the heteroalicyclic may be substituted or unsubstituted.
  • the substituted group can be, for example, lone pair electrons, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N- carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfonamido, hydrazine, and amino, as these terms are defined herein.
  • Representative examples are piperidine, piperazine, tetrahydro
  • a "hydroxy” group refers to an -OH group.
  • amine and “amino” refer to either a -NR'R" group, wherein R' and R" are selected from the group consisting of hydrogen, alkyl, cycloalkyl, heteroalicyclic (bonded through a ring carbon), aryl and heteroaryl (bonded through a ring carbon). R' and R" are bound via a carbon atom thereof. Optionally, R' and R" are selected from the group consisting of hydrogen and alkyl comprising 1 to 4 carbon atoms. Optionally, R' and R" are hydrogen.
  • An “alkoxy” group refers to both an -O-alkyl and an -O-cycloalkyl group, as defined herein.
  • aryloxy refers to both an -O-aryl and an -O-heteroaryl group, as defined herein.
  • a “thiohydroxy” or “thiol” group refers to a -SH group.
  • a “thioalkoxy” group refers to both an -S-alkyl group, and an -S-cycloalkyl group, as defined herein.
  • a "thioaryloxy” group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein.
  • a “disulfide” group refers to both a -S-thioalkoxy and a -S-thioaryloxy group.
  • a disulfide bond describes a -S-S- bond.
  • a “carboxylate” or “carboxyl” encompasses both C-carboxy and O-carboxy groups, as defined herein.
  • a “carboxylic acid” group refers to a C-carboxy group in which R' is hydrogen.
  • esters refers to a C-carboxy group wherein R' is not hydrogen.
  • halo refers to fluorine, chlorine, bromine or iodine.
  • a “sulfonamide” or “sulfonamido” group encompasses both S-sulfonamido and N-sulfonamido groups, as defined herein.
  • a “carbamyl” or “carbamate” group encompasses O-carbamyl and N-carbamyl groups.
  • a “thiocarbamyl” or “thiocarbamate” group encompasses O-thiocarbamyl and N-thiocarbamyl groups.
  • amide encompasses both C-amido and N-amido groups.
  • a “nitro” group refers to an -N0 2 group.
  • a “cyano” group refers to a -C ⁇ N group.
  • hydrazine describes a -N(R')-N(R")R" ' group, with each of R', R" and R" ' as defined hereinabove.
  • a “phosphoric acid” is a phosphate group is which each of R is hydrogen.
  • phosphinyl describes a -PR'R" group, with each of R' and R" as defined hereinabove.
  • a method of inhibiting synaptojanin-2 comprising contacting the synaptojanin-2 with an effective amount of a compound described herein as a synaptojanin-2 inhibitor.
  • the method is effected ex vivo, for example, for research. In some embodiments, the method is effected in vivo. In some embodiments, the method is utilized for treating a disease or disorder in which inhibition of synaptojanin-2 is beneficial (e.g., a condition described herein).
  • an effective amount is less than 100 ⁇ . In some embodiments, an effective amount is less than 10 ⁇ . In some embodiments, an effective amount is less than 5 ⁇ . In some embodiments, an effective amount is less than 2.5 ⁇ .
  • an effective amount is at least 100 % of the IC50 of the compound towards SYNJ2. In some embodiments, an effective amount is at least 200 % of the IC50 of the compound towards SYNJ2. In some embodiments, an effective amount is at least 300 % of the IC50 of the compound towards SYNJ2. In some embodiments, an effective amount is at least 500 % of the IC50 of the compound towards SYNJ2. In some embodiments, an effective amount is at least 1000 % of the IC50 of the compound towards SYNJ2.
  • Non-limiting examples of cancers which can be treated according to some embodiments of any of the aspects of the invention include any solid or non-solid cancer and/or cancer metastasis, including, but is not limiting to, tumors of the gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small and/or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors), endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary tract tumors, prostate cancer, prostate adenocarcinoma, renal cancer (e.g., Wilms' tumor type 2
  • the cancer or tumor is a glioma.
  • the cancer or tumor is not a glioma.
  • the cancer (or the cancer metastasis) is EGF-regulated.
  • the cancer is characterized by over- expression or up-regulation of an ErbB receptor molecule such as EGFR or HER2.
  • Mutations that lead to EGFR overexpression (known as upregulation) or overactivity have been associated with a number of cancers, including lung cancer, anal cancers and glioblastoma multiforme. In this latter case a more or less specific mutation of EGFR, called EGFRvIII is often observed. Mutations, amplifications or misregulations of EGFR or family members are implicated in about 30 % of all epithelial cancers.
  • Mutations involving EGFR could lead to its constant activation, which could result in uncontrolled cell division - a predisposition for cancer. Consequently, mutations of EGFR have been identified in several types of cancer, and it is the target of an expanding class of anticancer therapies [Zhang et al., Clin Invest 117, 2051-2058 (2007)].
  • Amplification or over-expression of the ERBB2 gene occurs in approximately 30 % of breast cancers. It is strongly associated with increased disease recurrence and a worse prognosis. Over-expression is also known to occur in ovarian, stomach, and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma.
  • ErbB-1 - adrenocortical cancer biliary cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, lung cancer (non-small cell, squamous cell carcinoma, adenocarcinoma, and large cell lung cancer), pancreatic cancer, salivary gland cancer, diarrhea benign neoplasm, invasive carcinoma, skin disease, ductal carcinoma in situ, paronychia.
  • ErbB-2 - biliary cancer bladder cancer, breast cancer, cholangiocarcinoma, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, ovarian cancer, pancreatic cancer, salivary gland cancer.
  • the cancer is breast or gastric cancer.
  • ErbB-4 - breast cancer viral leukemia, medulloblastoma, lung cancer and mammary tumor.
  • the inhibitor of SYNJ2 is utilized in addition to an inhibitor of a cell surface receptor associated with an onset or progression of cancer.
  • the receptor is an oncogene.
  • receptors which may be targeted according to the present teachings are receptor tyrosine kinases such as those EGFR, PDGFR, VEGFR, FGFR and ErbB-2.
  • Other surface molecules which can be targeted include integrins matrix metalloproteinases (MMPs), dynamin, TKS5 and CD44.
  • MMPs matrix metalloproteinases
  • Inhibitors of cell surface molecules are well known in the art. A non-limiting list of such inhibitors is provided infra.
  • Cetuximab and panitumumab are examples of monoclonal antibody inhibitors. Other monoclonals in clinical development are zalutumumab, nimotuzumab, and matuzumab. The monoclonal antibodies block the extracellular ligand binding domain. With the binding site blocked, signal molecules can no longer attach there and activate the tyrosine kinase.
  • Another method is using small molecules to inhibit the EGFR tyrosine kinase, which is on the cytoplasmic side of the receptor. Without kinase activity, EGFR is unable to activate itself, which is a prerequisite for binding of downstream adaptor proteins. Ostensibly by halting the signaling cascade in cells that rely on this pathway for growth, tumor proliferation and migration is diminished.
  • Gefitinib, erlotinib, and lapatinib are examples of small molecule kinase inhibitors. Other examples include, Iressa and Tarceva directly target the EGFR.
  • FIER2 is the target of the monoclonal antibody trastuzumab (marketed as Herceptin). Trastuzumab is effective only in cancers where FIER2 is over-expressed. Another monoclonal antibody, pertuzumab, which inhibits dimerization of FIER2 and FIER3 receptors, was approved by the FDA for use in combination with trastuzumab in June 2012.
  • NeuVaxTM (Galena Biopharma) is a peptide-based immunotherapy that directs "killer” T cells to target and destroy cancer cells that express FIER2.
  • HER2 The expression of HER2 is regulated by signaling through estrogen receptors. Estradiol and tamoxifen acting through the estrogen receptor down-regulate the expression of FIER2.
  • antibodies which can be used according to the embodiments of the invention include, without limitation, alemtuzumab (a humanized antibody which targets CD62, and is approved for treatment of chronic lymphocytic leukemia); bevacizumab (a humanized antibody which targets vascular endothelial growth factor, and is approved for treatment of colorectal cancer); brentuximab vedotim (a chimeric antibody which targets CD30, and is approved for treatment of Hoodkin lymphoma and anaplastic large-cell lymphoma); cetuximab (a chimeric antibody which targets epidermal growth factor, and is approved for treatment of colorectal cancer); gemtuzumab ozogamicin (a humanized antibody which targets CD33, and is approved for treatment of acute myelogenous leukemia (with calicheamicin)); ibritumomab tiuxetan (a murine antibody which targets CD20, and is approved for treatment of non- Hogkin lymphoma (with y
  • inhibitors of the SYNJ2 described herein and optionally the inhibitor of the cell surface receptor according to any of the aspects of embodiments of the invention described herein can be administered to the subject per se or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the inhibitor of SYNJ2 (and optionally the inhibitor of the cell surface receptor) accountable for the biological effect, as described herein.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (e.g., SYNJ2 inhibitor) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer or metastatic cancer) or prolong the survival of the subject being treated.
  • active ingredients e.g., SYNJ2 inhibitor
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, e.g., Fingl et al. (1975), in "The Pharmacological Basis of Therapeutics", Ch. 1 p. l).
  • Dosage amount and interval may be adjusted individually to provide SYNJ2 inhibitor levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
  • MEC minimum effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro data, e.g., based on results on a SYNJ2 inhibition assay described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed herein.
  • a kit for the treatment of cancer or prevention of cancer metastasis comprising a packaging material packaging a compound described herein as a SYNJ2 inhibitor and an inhibitor of a cell surface receptor associated with an onset or progression of cancer, as described herein.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases "ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • mice Female CB-17 SCID mice (Harlan Laboratories, Haslett, MI; 15 per group) were implanted in the fat pad with MDA-MB-231 cells (1.4 xlO 6 cells/mouse). Two and six weeks post implantation, mice were anesthetized, tumor sizes were measured and metastases in lymph nodes were visualized using a fluorescent binocular. For lung metastases, mice were sacrificed, lungs were removed, washed, and images were acquired using a fluorescent binocular. Two-sided Fischer's exact test was used for analysis of lymph node metastasis. Tumor growth measurements used the Exact-sig [2x1 -tailed]) Mann-Whitney test.
  • Anti-EGFR for western blot analysis was from Alexis (Lausen, Switzerland).
  • Anti Ras- GAP and anti-AKT antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Anti-EEAl, anti-Rab5, anti-Rab4, and anti Racl were from BD Transduction Laboratories (Franklin Lakes, NJ).
  • Anti-SYNJ2 mAb was from Abnova (Taipei, Taiwan).
  • the following secondary antibodies were used: goat anti-mouse IgG and goat anti-rabbit IgG antibodies conjugated to Horseradish peroxidase (HRP) were purchased from Jackson ImmunoRe search Laboratories (Bar Harbor, Maine).
  • Texas-red transferrin, goat anti mouse Alexa-488, Alexa-555 and Alexa-647 secondary antibodies were from Invitrogen (Carlsbad, CA).
  • siRNA control was from "Thermo scientific Dharmacon” cat. D-001810-10- 05; siRNA sequence against SYNJ2 is GGACAGCACUGCAGGUGUU; all shRNA were from SIGMA Israel: shRNA control- cat. SHC002; shRNA sequences against SYNJ2 used is
  • MCF10A cells were grown in DMEM:F12 (1 : 1) medium supplemented with antibiotics, insulin (10 ⁇ g/mL), cholera toxin (0.1 ⁇ g/mL), hydrocortisone (0.5 ⁇ g/mL), heat-inactivated horse serum (5% vol/vol), and EGF (10 ng/mL).
  • Human mammary MDA-MB-231 cells were grown in RPMI-1640 (Gibco BRL; Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (Gibco), lmM sodium pyruvate and a penicillin-streptomycin mixture (100 unit/ml; 0.1 mg/ml; Beit Haemek, Israel).
  • the MDA-MB-231 -RFP stable cell-line was a kind gift from Prof. Hadasa Degani (The Weizmann Institute of Science, Israel). Plasmid transfections were performed using Fugen-HD according to the manufacture's guidelines (Roche, Mannheim, Germany). Alternatively, for transient mRNA knockdown experiments using siRNA oligonucleotides, cells were transfected with Oligofectamine (Invitrogen). Lentiviral vectors and virus production:
  • Non-targeted shRNA hairpins (control) and hairpins directed against human SYNJ2 were produced in HEK-293T cells following the manufacture's guidelines (Sigma).
  • Target cells were infected with shRNA-encoding lentiviruses supplemented with polybrene (8 ⁇ g/mL), and cultured in the presence of puromycin (2 ⁇ g/mL) for 4 days.
  • Stable gene-specific delivery of human SYNJ2 was performed using the ViraPower lentiviral expression system (Invitrogen), following the manufacture's guidelines.
  • 3D image stacks were acquired every 70-300 ms along the Z-axis by varying the position of the piezo electrically controlled stage (step size: 0.1-0.4 ⁇ ).
  • live cell fluorescence microscopy was carried out using the DeltaVision system (Applied Precision, Issaqua, WA) and images were processed using the prism software.
  • EGF Human recombinant EGF was labeled with IODOGEN as follows: EGF (5 ⁇ g) was mixed in an Iodogen-coated tube (1 mg of reagent) with Na 125 I (lmCi). Following 15 minutes of incubation at 23°C, albumin was added to a final concentration of 0.1 mg/ml, and the mixture was separated on an Excellulose GF-5 column.
  • MDA-MB-231 cells were seeded in triplicates for each time point in 24-well plates, with an additional well plated for control. 48 hours later, cells were starved for 4 hours and stimulated with EGF (2 ng/ml) at 37°C for the indicated time intervals. Subsequently, they were placed on ice, rinsed once with binding buffer (DME medium, albumin 1 %, Hepes 20 mM, pH 7.5), and subjected to mild acid/salt wash (0.2 M Na Acetate buffer pH 4.5, 0.5 M NaCl) to remove surface-bound EGF. Thereafter, cells were incubated with a radiolabeled EGF for 1.5 hours at 4°C and rinsed with binding buffer.
  • binding buffer DME medium, albumin 1 %, Hepes 20 mM, pH 7.5
  • mild acid/salt wash 0.2 M Na Acetate buffer pH 4.5, 0.5 M NaCl
  • control well was incubated with a radiolabelled EGF and an excess of unlabelled EGF. Finally, cells were lysed with 1M NaOH, and radioactivity was determined using a ⁇ -counter. Data represent the percentage of receptors on the cell surface relative to time 0.
  • Cells (2 x 10 4 /well) were seeded in triplicates in 24-well plates, with an additional well plated for control. Thereafter, cells were incubated with a radiolabelled EGF for 1.5 hours at 4°C and rinsed with binding buffer. The control well was incubated with a radiolabelled EGF and an excess of unlabelled EGF. Finally, cells were lysed in 1M NaOH solution and radioactivity was determined. Data represent the percentage of receptors on the cell surface relative to control cells.
  • Membranes were blocked in TBST buffer (0.02 M Tris-HCl (pH 7.5), 0.15 M NaCl and 0.05% Tween 20) containing 10% low-fat milk, blotted with a primary antibody for 1 hour, washed with TBST and incubated for 30 minutes with a secondary antibody conjugated to HRP.
  • TBST buffer 0.02 M Tris-HCl (pH 7.5), 0.15 M NaCl and 0.05% Tween 20
  • Wound healing assays were performed according to manufacturer's protocol (ibidi GmbH, Germany). Briefly, MCF10A cells were trypsinized, re-suspended in EGF-deprived medium (7.0 x 10 5 cells/mL) and 70 ⁇ plated into each well, resulting in a confluent layer within 24 hours. Thereafter, Culture-Inserts were removed by using sterile tweezers and cells were allowed to migrate for 2 hours.
  • MDA-MB-231 cells were pre-incubated for 30 minutes at 37°C with Alexa Fluor 488-transferrin (25 ⁇ g/ml in serum-free medium) or for 10 minutes with Alexa Fluor 488-EGF (40 ng/mL).
  • Surface-bound ligands were detached by incubation for 30 minute at 4°C in an acidic buffer (150mM NaCl, ImM MgCl 2 , 0.125mM CaCl 2 , 0.1M glycine), prior to transfer to 37°C for the indicated time intervals, to allow for recycling of the internalized ligands.
  • Cells were analyzed either by imaging or by FACS.
  • a construct encoding two TAPP1-PH domains was cloned in tandem (Figure 18) into pET28 plasmid containing an N-terminal Flag tag and C-terminal 6xHis tag and expressed in E. coli BL21(DE3) following induction with 200 ⁇ IPTG.
  • the bacteria was grown at 15 °C and then lysed with a cell disrupter. Cell debris was removed by centrifugation and the protein was captured on a Ni column (HisPrep FF 16/10, GE Healthcare) equilibrated with 50 mM Tris pH 8, 0.5 M NaCl and 20 mM imidazole. The protein was eluted in the same buffer containing 0.5 M Imidazole.
  • TAPP1-PH domain Fractions containing the TAPP1-PH domain were injected into a size exclusion column (Hiload_26/60_Superdex 75, GE Healthcare) equilibrated with buffer containing 50 mM Tris pH 8 and 100 mM NaCl.
  • the pooled peak containing TAPPl-PH domain was diluted three fold with 20 mM sodium phosphate buffer pH 7.2 and loaded onto a cation exchange column (HiTrap_SP_FF_5ml, GE Healthcare) equilibrated with the same phosphate buffer.
  • the pure protein was eluted from the column with a linear gradient of the phosphate buffer containing 1 M NaCl (TAPPl-PH domain elutes at 200 mM NaCl).
  • the fractions containing the pure TAPPl-PH domain as evaluated by SDS- PAGE were pooled together and protein concentration was determined by Bradford reagent and ⁇ 28 ⁇ (extinction coefficient of 20,520) quantization. The protein was divided into aliquots, flash frozen with liquid nitrogen and stored at -80 °C.
  • Stabilizing SOP lipid mix (x50) was prepared in a glass tube by adding 100 ⁇ of SOPS (Avanti Inc., 50 mg / ml in chloroform) and 50 ⁇ Cholesterol (Sigma Aldrich, 10 mg / ml in chloroform). The mix was air-dried using gentle nitrogen steam to evaporate the chloroform.
  • the evaporated lipid mix was then re-suspended in 10 ml of 0.25 mg / ml Ci 2 E 8 (Avanti Inc.) by 1 minute vortex at room temperature.
  • a reaction mix comprising PBS, DTT, MgC12 (all from Sigma Aldrich), SOP lipid mix (x50), full length purified SYNJ2 (OriGene, cat no. TP315160) and PI(3,4,5)P3 (Echelon Bioscience, cat no. P-3908), with or without a tested compound.
  • PI(3,4,5)P3 was added, the reaction mix was incubated in 33 °C for 8 minutes to allow production of PI(3,4)P2 by SYNJ2 5 '-phosphatase activity.
  • MCF10A Human mammary epithelial cells exhibit strong migratory and invasive phenotypes when cultured with EGF family ligands ( Figures 1A and IB), but treatment with serum is insufficient to propel cell motility.
  • the EGFR-induced motile phenotype associates with transcriptional up-regulation of 425 genes [Amit et al., Nat Genet 39, 503-512 (2007)].
  • SYNJ2 a GFP fusion; SYNJ2-OX, Figure IE.
  • SYNJ2-OX cells displayed a pro-migratory phenotype characterized by membrane ruffling (Figure 2C), along with enhanced basal and EGF-induced migratory and invasive capacities ( Figures 2D and 2C).
  • siRNAs small interfering RNAs
  • Figure 1G small interfering RNAs
  • the highly metastatic MDA-MB-231 breast cancer Red fluorescent protein (RFP) expressing cells were used to generate subclones overexpressing either SYNJ2 or LacZ (control), as well as sub-clones expressing shControl or SYNJ2-specific hairpins (shSYNJ2; Figure 3A).
  • Enhanced expression of SYNJ2 conferred an elongated morphology in 2D cultures (Figure 3B) and extensive invasive arms, when cells were grown in 3D cultures ( Figure 4A).
  • SYNJ2 knockdown abrogated invasive patterns ( Figure 4B).
  • re-expression of the mutant failed to restore the invasive capacity ( Figure 4D), indicating that the phosphatase activity of SYNJ2 is essential for the invasive phenotype.
  • Time-lapse images of MDA-MB-231 cells expressing GFP-SYNJ2 (Figure 3E), as well as immunofluorescence using anti-SYNJ2 antibodies ( Figure 3F), reflected two major patterns of SYNJ2 distribution: small peripheral assemblies, which localized to the leading edge (black arrowheads in Figure 3E), and a second population of larger assemblies, which were located closer to the cell centre (blue arrowheads).
  • small peripheral assemblies which localized to the leading edge
  • blue arrowheads blue arrowheads
  • SYNJ2 rapidly assembled at the base of emerging lamellipodia, underneath the forming leading edge ( Figures 3E, 3F).
  • GFP-SYNJ2 MDA-MB-231 subclone (GFP-SYNJ2 cells) was generated and analyzed for the formation and consumption of GFP-SYNJ2 puncta. These were classified into kinetically distinct sub-populations: dynamic puncta that localized to ruffling membranes and puncta localized to discrete regions proximal to the cell centre ( Figure 5A). Notably, GFP-SYNJ2 puncta showed minimal overlap with assemblies marked by RFP-Clathrin light chain A ( Figure 5A) or RFP-Caveolin 1 ( Figure 6A), suggesting minor localization to Clathrin-coated pits or to caveolae.
  • Intracellular trapping of EGFR bears functional consequences: in line with their well- characterized chemotactic function [Mouneimne et al., Curr Biol 16, 2193-2205 (2006); van Rheenen et al., Cell Biology 179, 1247-1259 (2007)], EGFRs localized to the leading edge of mammary cells, but EGFRs of shSYNJ2 cells lost their polarized distribution and accumulated in large, actin-decorated vesicles (Figure 8C).
  • the endocytic system maintains several distinct compartments, which are defined by specific phosphoinositides (PI) [Gruenberg & Stenmark, Nat Rev Mol Cell Biol 5, 317-323 (2004)], and the present analyses uncovered strong dependency on SYNJ2.
  • PI phosphoinositides
  • TKS5 was observed, a PI(3,4)P 2 and a binder of Cortactin that serves as a signpost of invadopodia [Courtneidge et al., Cold Spring Harb Symp Quant Biol 70, 16 -171 (2005)].
  • endogenous TKS5 localized to multiple ventral sites of matrix degradation in control MDA-MB-231 cells, but almost no active sites were found in siSYNJ2 cells, and TKS5 lost its ventral location (Figure 6E; X-Y and Z panels).
  • SYNJ2 appears necessary at a step preceding TKS5 engagement, consistent with sequential action of PI3K [Yamaguchi et al., Cell Biology 193, 1275- 1288 (2011)] and SYNJ2, which respectively generate PI(3,4,5)P 3 and then PI(3,4)P 2 , to anchor TKS5 at sites of EGFR-induced activation of PI3K.
  • transcript levels of SYNJ2 were analyzed in the NCI-60 panel of 60 human cancer lines. In line with contribution to motile phenotypes, it was found that high transcript levels of SYNJ2 associate with mesenchymal phenotypes.
  • a set of 331 paraffin-embedded samples of breast carcinomas NJ2 were immunostained (Figure 16A). Importantly, expression intensity of SYNJ2 was positively associated with prognostically unfavorable subtypes defined by HER2 overexpression (/? ⁇ 0.001) and/or lack of estrogen receptor (/? ⁇ 0.001).
  • a fluorescence polarization competitive assay was used, based on the principle that when excited by polarized light, fluorescent molecules bound to a larger element (e.g., a protein) exhibit more polarized fluorescence than do free, rapidly rotating fluorescent molecules in solution. Addition of detector molecule (e.g., a binding protein) that binds a fluorescent probe therefore increases the polarization readings for fluorescent measurements of a solution (see Figure 17A).
  • detector molecule e.g., a binding protein
  • This assay was used to screen libraries of test compounds obtained from AnalytiCon and ChemBridge.
  • the present inventors measured, in the presence of different test compounds, the enzymatic de-phosphoryiation by SYNJ2 or SYNJl of the 5' position of PI(3,4,5,)P3 to produce PI(3,4)P2.
  • the solution containing the PI(3,4)P2 products was mixed with a mixture of PI(3,4)P2 binding protein (detector) and a fluorescent PI(3,4)P2 (probe).
  • the detector protein used was the purified PH-domain of Tappl that selectively binds PI(3,4)P2.
  • the assay was performed using different concentrations of each test compound, and IC50 values were calculated from the data for different concentrations of test compound.
  • the IC50 for inhibition of SYNJl was higher (at least 15.9 ⁇ ) than that of the IC50 for inhibition of SYNJ2, indicating that the compounds selectively inhibited SYNJ2.
  • Compound 18 exhibited an IC50 of 1.0 ⁇ for inhibition of SYNJl . Binding of Compound 18 to SYNJ2 was confirmed by microscale thermophoresis (data not shown).
  • the effect of Compounds 3, 8, 12, 14, 15, 16 and 18 on cell invasion was determined using an invasion assay performed using MDA-MB-231 breast cancer cells and BioCoat Matrigel Chambers, as described in Example 1.
  • the compounds were assayed at a concentration of 10 ⁇ , except for Compound 18, which was toxic to the cells at a concentration of 10 ⁇ was assayed at a concentration of 1 or 2 ⁇ .
  • the effect of Compound 18 on cell migration was further determined using a migration assay performed on MCF IOA cells in a Boy den chamber.
  • the MCF IOA cells were assayed for migration or invasion in the presence of various concentrations of Compound 18 (and 10 ng/ml EGF). Cells that reached the filter bottom after 18 hours were stained and quantified.
  • Compound 18 reduced cell migration in a dose-dependent manner, exhibiting an EC50 of about 0.8 ⁇ .
  • mice 12 (also referred to herein as " P-360” or " P-000360”) was tested in vivo.
  • MDA-MB- 231 breast cancer cells expressing GFP (green fluorescent protein) were implanted (2.5xl0 6 per mouse) in the fat pad of 20 SCID mice (aged 6 weeks old). After tumors reached a volume of approximately 3x3x3 mm 3 , mice were randomized into two groups, and Compound 12 (5 mg/kg) or carrier (DMSO) were injected twice a week for 6 weeks. Tumor mass was then determined.
  • GFP green fluorescent protein
  • Compound 2 (also referred to herein as “NP-3195" or “NP-003195”) on tumor mass was tested in vivo, as described hereinabove for Compound 12.
  • the effect of Compound 2 on lung metastasis was also tested in vivo, according to procedures such as described in Example 1. As shown in Figure 22, Compound 2 modestly reduced attenuated tumor growth in comparison to the control group.
  • SYNJ2 inhibitors can be effective at inhibiting tumor growth and/or metastasis in vivo.
  • MDA-MB-231 cells were immunoblotted with antibodies against EGFR (endothelial growth factor receptor) treated with 0.78 ⁇ of Compound 18 (pyrvinium) or with carrier (DMSO). For comparison, the same immunoblotting was performed on cells subjected to SYNJ2 knockdown and control knockdown.
  • EGFR endothelial growth factor receptor
  • DMSO carrier
  • SYNJ2 inhibitors affect cells, at least in part, by causing less EGFR to be present on the surface of cells, thereby reducing their ability to bind EGF (endothelial growth factor).

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Abstract

L'invention concerne des inhibiteurs de synaptojanine-2 ainsi que des nouveaux procédés et des nouvelles utilisations les employant pour prévenir une métastase de tumeur, traiter un cancer ou inhiber synaptojanine-2. Les composés décrits ici comprennent la chlorhexidine et le pyrvinium, le composé répondant à la formule : (I) et des composés caractérisés par la formule générale : X-L-[(Y)i-(Z)j]-(L-X)k et/ou par la formule générale : (II) dans lesquelles L, X, Y, Z, D, E, i, j et k sont définis ici.
EP14827537.3A 2013-11-28 2014-11-27 Inhibiteurs de synaptojanine-2 et leurs utilisations Withdrawn EP3074044A2 (fr)

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