WO2010118347A2 - O-linked glycan biosynthesis modulators - Google Patents

O-linked glycan biosynthesis modulators Download PDF

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Publication number
WO2010118347A2
WO2010118347A2 PCT/US2010/030574 US2010030574W WO2010118347A2 WO 2010118347 A2 WO2010118347 A2 WO 2010118347A2 US 2010030574 W US2010030574 W US 2010030574W WO 2010118347 A2 WO2010118347 A2 WO 2010118347A2
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Prior art keywords
linked
linked glycan
core
ppgalnac
glycans
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PCT/US2010/030574
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French (fr)
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WO2010118347A3 (en
Inventor
Brett E. Crawford
Jillian R. Brown
Charles A. Glass
Xiaomei Bai
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Zacharon Pharmaceuticals, Inc.
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Publication of WO2010118347A2 publication Critical patent/WO2010118347A2/en
Publication of WO2010118347A3 publication Critical patent/WO2010118347A3/en

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • O-linked glycans are found in mammals and comprise a plurality of saccharide units linked by O-glycosidic linkages.
  • An O-glycoprotein comprises one or more glycans linked to a protein via an oxygen atom, e.g. via a hydroxy group of a Serine, Threonine or Hydroxylysine residue.
  • a process for modifying populations of O-linked glycans comprise contacting cells that produce such O-linked glycans with a modulator or O-linked glycan biosynthesis (e.g., an inhibitor of O-linked glycan biosynthesis).
  • the O-linked glycan biosynthesis modulators are selective for O-linked glycans (e.g., such modulators do not significantly affect the biosynthesis of other glycans, such as N-linked glycans, gangliosides, heparan sulfate, chondroitin sulfate, keratan sulfate, or the like).
  • O-linked glycan biosynthesis modulators selectively modulate the late stage processes of O-linked glycan biosynthesis. Furthermore, provided in certain embodiments herein is a process for modifying the population of O-linked glycans on a core protein or core proteins, comprising contacting a cell that translationally produces at least one core protein having at least one attached O-linked glycan moiety with an effective amount of a selective inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine -modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, a fucosyl transferase, or a combination thereof.
  • a process for modifying the population of O-linked glycans on one or more proteins associated with a cell comprising contacting a cell that produces O-linked glycans with an effective amount of a selective O- linked glycan biosynthesis inhibitor, the selective O-linked glycan biosynthesis inhibitor being active in a mammalian cell.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein is a selective late stage O- linked glycan biosynthesis inhibitor.
  • the selective late stage O-linked glycan biosynthesis inhibitor utilized in any process described herein is a non-carbohydrate O-linked glycan biosynthesis inhibitor. In certain embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein has a molecular weight of less than 700 g/mol.
  • any process described herein reduces the ratio of glycans containing poly-N-acetyllactosamine chains to glycans that do not contain poly-N- acetyllactosamine chains.
  • any process described herein reduces the ratio X: Y of glycans containing poly-N-acetyllactosamine chains to glycans not containing poly-N-acetyllactosamine chains by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, wherein the ratio X:Y changes to 0.9X:Y, 0.8X:Y, 0.7X:Y, 0.6X:Y, 0.5X:Y, 0.4X:Y, 0.3X:Y, 0.2X:Y, 0.1X:Y, respectively.
  • any process described herein reduces the amount of ( ⁇ l-2) fucosylation, ( ⁇ l-3) fucosylation, ( ⁇ l-4) fucosylation, ( ⁇ 2-3) sialylation, ( ⁇ 2-6) sialylation, terminal ( ⁇ l-3) galactosylation, terminal ( ⁇ l-4) galactosylation, 3-O-sulfation, 6-O- sulfation in the cellular population of O-linked glycan.
  • any process described herein reduces the amount of terminal sialylation and/or terminal GIcNAc residues.
  • any process described herein reduces the amount of selectin ligands in the cellular population of O-linked glycan.
  • the selectin ligand is sialyl Lewis X or 6-O-sulfated sialyl Lewis X (sLe x or 6-sulfo-sLe x ).
  • any process described herein reduces the cellular population of Core 1, Core 2, Core 3, and/or Core 4 O-linked glycan structures. In some embodiments, any process described herein reduces the cellular population of Core 1 , and/or Core 2 O-linked glycan structures.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein reduces the ratio of acidic O-linked glycans to neutral O- linked glycans to less than 50% (e.g., if the original ratio is x:(x+y), a reduction to less than 50% would provide a ratio of ⁇ 0.5x:(x+y)), less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, wherein an acidic O-linked glycan contains sialic acid and/or is sulfated, and wherein a neutral O-linked glycan contains no sialic acid and/or is not sulfated.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein inhibits Core 1 ( ⁇ l-3) galctosyl transferase, core 2 ( ⁇ l-6) N- acetylglucosaminyl transferase, core 3 ( ⁇ l-6) N-acetylglucosaminyl transferase, core 2/4 ( ⁇ l-6) N-acetylglucosaminyl transferase, Lewis ( ⁇ l-3/1-4) fucosyl transferase, FucTIII, FucTIV, FucT VII, ST3GalI, ST3GalII, ST3GalIII, ST3GalIV, GlcNAc6ST-l, GlcNAc6ST-2, Gal3ST4.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process herein indirectly inhibits the Core 1 ( ⁇ l-3) galctosyl transferase, Core 2 ( ⁇ l-6) N-acetylglucosaminyl transferase, core 3 ( ⁇ l-6) N- acetylglucosaminyl transferase, Core 2/4 ( ⁇ l-6) N-acetylglucosaminyl transferase, Lewis ( ⁇ l-3/1-4) fucosyl transferase, FucTIII, FucTIV, FucT VII, ST3GalI, ST3GalII, ST3GalIII, ST3GalIV, GlcNAc6ST-l, GlcNAc6ST-2, Gal3ST4.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process herein directly inhibits the Core 1 ( ⁇ l-3) galctosyl transferase, Core 2 ( ⁇ l-6) N-acetylglucosaminyl transferase, Core 3 ( ⁇ l-6) N-acetylglucosaminyl transferase, Core 2/4 ( ⁇ l-6) N- acetylglucosaminyl transferase, Lewis ( ⁇ l-3/1-4) fucosyl transferase, FucTIII, FucTIV, FucT VII, ST3GalI, ST3GalII, ST3GalIII, ST3GalIV, GlcNAc6ST-l, GlcNAc6ST-2, Gal3ST4.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein inhibits ST3GalI, ST3GalII, or ST3GalIII.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process herein indirectly inhibits ST3GalI, ST3GalII, or ST3GalIII.
  • the selective O-linked glycan biosynthesis inhibitor utilized in any process herein directly inhibits the ST3GalI, ST3GalII, or ST3GalIII.
  • the cell contacted by any process described herein is an inflammatory cell or cancer cell. In certain embodiments, the cell contacted by any process described herein is present in an individual diagnosed with or suspected of having rheumatoid arthritis, Crohn's disease, reperfusion injury or having had an organ transplant. In some embodiments, the cell contacted by any process described herein is present in an individual diagnosed with or suspected of having adenocarcinoma. In certain embodiments, the cell contacted by any process described herein is a breast cancer cell, a colon cancer cell, a prostate cancer cell, or a non-small cell lung cancer cell.
  • compositions comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprise Core 1, Core 2, Core 3, and Core 4 O-linked glycans, and wherein less than 20 mol. %, less than 19 mol. %, less than 18mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. % , less than 5 mol. %, less than 2 mol. %, less than 1 mol.
  • % of the Core 1, Core 2, Core 3, and Core 4 O-linked glycans comprise terminal fucosyl groups.
  • a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprise Core 1 and Core 2 O-linked glycans, and wherein less than 50 mol. %, less than 45 mol. %, less than 40 mol. %, less than 35 mol. %, less than 30 mol. %, less than 25 mol. %, less than 20 mol. %, less than 19 mol.
  • O-linked glycans comprise terminal sialyl groups.
  • a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein less than 50 mol. %, less than 45 mol. %, less than 40 mol.
  • the plurality of O-linked glycans comprise terminal sialyl groups.
  • compositions comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprises less than 20 mol. %, less than 19 mol. %, less than 18 mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. %, less than 5 mol. %, less than 2 mol. %, less than 1 mol. % sLe x .
  • compositions comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprises a plurality of sLe x antigens, and wherein less than 20%, less than 19 mol. %, less than 18mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. % , less than 5 mol. %, less than 2 mol. %, less than 1 mol.
  • % of the sLe x antigens are 6-0 sulfated.
  • a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein less than 50 mol. %, less than 45 mol. %, less than 40 mol. %, less than 35 mol. %, less than 30 mol. %, less than 25 mol. %, less than 20 mol. %, less than 15 mol. %, less than 10 mol. %, less than 5 mol. % of glycoproteins bind to- Wheat Germ Agglutinin (WGA).
  • WGA Wheat Germ Agglutinin
  • a process for modifying the structure of an O-linked glycan on a core protein comprising contacting a cell that translationally produces at least one core protein having at least one attached O-linked glycan moiety with an effective amount of a selective inhibitor of O-linked glycan biosynthesis.
  • the selective inhibitor of O-linked glycan biosynthesis is an inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine-modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, a fucosyl transferase, or a combination thereof.
  • the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc- TI l, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGGalNAc-T
  • the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In one embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4. In one embodiment the inhibitor of the core transferase is an inhibitor of a core 1 ⁇ l-3-galactosyltransferase, a core 2 ⁇ l-6-N-acetylglucosaminyl transferase, a core 3 ⁇ l-3-N-acetylglucosaminyl transferase, a core 4 ⁇ l-6-N- acetylglucosaminyl transferase, a core 5 ⁇ l-3-N-acetylgalactosyl transferase, a core 6 ⁇ l-6- N-acetylglucosaminyl transferase, a core 7 ⁇ l-6-N-acetylgalactosyl transfera
  • the inhibitor is an inhibitor of the core 1 ⁇ l-3-galactosyltransferase. In one embodiment the inhibitor is an inhibitor of the core 2 ⁇ l-6-N-acetylglucosaminyl transferase. In one embodiment the inhibitor of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV, ST3Gal VI or a combination thereof. In yet another embodiment the inhibitor of the sialyl transferase inhibits the addition of a sialic acid residue via an ⁇ 2,3 linkage or ⁇ 2-6 linkage. In a further embodiment the core 1 specific chaperone is Cosmc.
  • the polylactosamine extension enzyme is an i-extension enzyme or a ⁇ l-4 galactosyl transferase IV.
  • the i-extension enzyme is iGnT.
  • the inhibitor of the fucosyl transferase is a selective inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof.
  • the inhibitor of the fucosyl transferase inhibits the addition of a fucose residue via an ⁇ 1,2 linkage, an ⁇ 1,3 linkage or an ⁇ l,4 linkage.
  • the inhibitor of the polypeptide GaINAc transferase inhibits the synthesis of the linkage region, the modification of the linkage region, the initiation of 0-linked glycan synthesis, the synthesis of 0-linked glycan, or a combination thereof.
  • the 0-linked glycan is selected from a blood group antigen A, a blood group antigen B, a blood group antigen H, a Lewis antigen Le x , a Lewis antigen sLe x , and a Lewis antigen Le Y .
  • [0020] in one aspect is a process of inhibiting 0-linked glycan function in a cell comprising contacting the cell with an effective amount of a selective modulator of O-linked glycan biosynthesis.
  • the selective modulator of O-linked glycan biosynthesis is an inhibitor of a polypeptide GaINAc transferase, a modulator of core transferase, a modulator of polylactosamine-modifying glycosyl transferase, a modulator of polylactosamine extension enzyme, a modulator of core 1 specific chaperone, a modulator of sialyl transferase, and a modulator of fucosyl transferase.
  • the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc- T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-
  • the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In one embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4. In another embodiment the inhibitor of the core transferase is an inhibitor of a core 1 ⁇ l-3-galactosyltransferase, a core 2 ⁇ l-6-N-acetylglucosaminyl transferase, a core 3 ⁇ l-3-N-acetylglucosaminyl transferase, a core 4 ⁇ l-6-N-acetylglucosaminyl transferase, a core 5 ⁇ l-3-N-acetylgalactosyl transferase, a core 6 ⁇ l-6-N-acetylglucosaminyl transferase, a core 7 ⁇ l-6-N-acetylgalactosyl
  • the inhibitor is an inhibitor of the core 1 ⁇ l-3-galactosyltransferase. In a further embodiment the inhibitor is an inhibitor of the core 2 ⁇ l-6-N-acetylglucosaminyl transferase. In yet a further embodiment the inhibitor of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV or ST3Gal VI or a combination thereof. In one embodiment the inhibitor of the sialyl transferase inhibits the addition of a sialic acid residue via an ⁇ 2,3 linkage or an ⁇ 2-6 linkage. In another embodiment the core 1 specific chaperone is Cosmc.
  • the polylactosamine extension enzyme is an i-extension enzyme or a ⁇ 1-4 galactosyl transferase IV. In a further embodiment the i-extension enzyme is iGnT.
  • the inhibitor of the fucosyl transferase is an inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof. In one embodiment the inhibitor of the fucosyl transferase inhibits the addition of a fucose residue via an ⁇ l,2 linkage, an ⁇ l,3 linkage or an ⁇ l,4 linkage or a combination thereof.
  • the 0-linked glycan function inhibited is an ability to bind an 0-linked glycan binding lectin.
  • the 0-linked glycan binding lectin is a E-selectin, L-selectin or P-selectin.
  • the cell being contacted is a cell in need thereof, a cell present in an individual suffering from a disease or condition mediated by abnormal O- linked glycan biosynthesis and/or the cell itself is a cell with abnormal 0-linked glycan biosynthesis, a cell present in an individual with normal 0-linked glycan biosynthesis and/or the cell itself is a cell with normal 0-linked glycan biosynthesis.
  • the cell being contacted is a cell present in an individual with normal O- linked glycan biosynthesis (e.g., an individual with a predisposition for or suspected of having a disease or condition mediated by 0-linked glycan biosynthesis) and/or the cell itself is a cell with normal 0-linked glycan biosynthesis.
  • an individual with normal O- linked glycan biosynthesis e.g., an individual with a predisposition for or suspected of having a disease or condition mediated by 0-linked glycan biosynthesis
  • the cell itself is a cell with normal 0-linked glycan biosynthesis.
  • Also presented herein is a method of treating cancer comprising administering a therapeutically effective amount of a selective modulator of polypeptide GaINAc transferase, a core transferase, a polylactosamine -modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, and a fucosyl transferase.
  • the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc- T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGGalNAc-T
  • the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In yet another embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4.
  • the selective modulator of the core transferase is an inhibitor of a core 1 ⁇ l-3-galactosyltransferase, a core 2 ⁇ l-6-N-acetylglucosaminyl transferase, a core 3 ⁇ l-3- N-acetylglucosaminyl transferase, a core 4 ⁇ l-6-N-acetylglucosaminyl transferase, a core 5 ⁇ l-3-N-acetylgalactosyl transferase, a core 6 ⁇ l-6-N-acetylglucosaminyl transferase, a core 7 ⁇ l-6-N-acetylgalactosyl transferase, a core 8 ⁇ 1-3 -galactosyl transferase, or a combination thereof.
  • the selective modulator is an inhibitor of the core 1 ⁇ l-3-galactosyltransferase. In one embodiment the selective modulator is an inhibitor of the core 2 ⁇ l-6-N-acetylglucosaminyl transferase. In another embodiment the selective modulator of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV or ST3Gal VI or a combination thereof. In a further embodiment the selective modulator of the sialyl transferase inhibits the addition of a sialic acid residue via an ⁇ 2,3 linkage or an ⁇ 2-6 linkage. In one embodiment the core 1 specific chaperone is Cosmc.
  • the polylactosamine extension enzyme is an i-extension enzyme or a ⁇ l-4 galactosyl transferase IV.
  • the i-extension enzyme is iGnT.
  • the selective modulator of the fucosyl transferase is an inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof.
  • the selective modulator has a molecular weight of less than 1,000 g/mol.
  • the selective modulator has a molecular weight of less than 700 g/mol.
  • a method of treating a lysosomal storage disease comprising administering a therapeutically effective amount of a selective inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine-modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, and a fucosyl transferase.
  • the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc- T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGGalNAc-T
  • the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In a further embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4.
  • the selective modulator of the core transferase is an inhibitor of a core 1 ⁇ l-3- galactosyltransferase, a core 2 ⁇ l-6-N-acetylglucosaminyl transferase, a core 3 ⁇ 1-3 -N- acetylglucosaminyl transferase, a core 4 ⁇ l-6-N-acetylglucosaminyl transferase, a core 5 ⁇ l-3-N-acetylgalactosyl transferase, a core 6 ⁇ l-6-N-acetylglucosaminyl transferase, a core 7 ⁇ l-6-N-acetylgalactosyl transferase, a core 8 ⁇ 1-3 -galactosyl transferase, or a combination thereof.
  • the selective modulator is an inhibitor of the core 1 ⁇ l-3-galactosyltransferase. In yet another embodiment the selective modulator is an inhibitor of the core 2 ⁇ l-6-N-acetylglucosaminyl transferase. In a further embodiment the selective modulator of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV or ST3Gal VI or a combination thereof. In one embodiment the selective modulator of the sialyl transferase inhibits the addition of a sialic acid residue via an ⁇ 2,3 linkage or an ⁇ 2-6 linkage. In yet a further embodiment the core 1 specific chaperone is Cosmc.
  • the polylactosamine extension enzyme is an i-extension enzyme or a ⁇ l-4 galactosyl transferase IV.
  • the i-extension enzyme is iGnT.
  • the selective modulator of the fucosyl transferase is an inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof.
  • a process for modulating 0-linked glycan degradation in a cell comprising contacting the cell with an effective amount of a selective modulator of an endoglycosidase, an exoglycosidase or a combination thereof.
  • the selective modulator of the endoglycosidase or exoglycosidase is a promoter of ⁇ -N-acetylhexoaminidase.
  • the ⁇ -N-acetylhexoaminidase is ⁇ -GlcNAcase or ⁇ -GalNAcase.
  • the selective modulator of the endoglycosidase or exoglycosidase is a promoter of sialidase.
  • the sialidase is neuraminidase.
  • the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a ⁇ -galactosidase. In a further embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a ⁇ -glucoronidase. In one embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a ⁇ -galactosidase. In yet another embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a Cathepsin A.
  • the cell being contacted is a cell in need thereof, a cell present in an individual suffering from a disease or condition mediated by abnormal O- linked glycan biosynthesis and/or the cell itself is a cell with abnormal O-linked glycan biosynthesis, a cell present in an individual with normal O-linked glycan biosynthesis and/or the cell itself is a cell with normal O-linked glycan biosynthesis.
  • the O-linked glycan is a mucin type O-linked glycan.
  • Also provided herein is a process for identifying a compound that modulates O- linked glycan biosynthesis comprising: a.
  • contacting a mammalian cell with the compound b. contacting the mammalian cell and compound combination with a first labeled probe wherein the first labeled probe binds one or more O-linked glycans; c. incubating the mammalian cell, compound, and the first labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; and e. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans.
  • a process for identifying a compound that selectively modulates O-linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound b. contacting the mammalian cell and compound combination with a first labeled probe and a second labeled probe, wherein the first labeled probe binds one or more O-linked glycans and the second labeled probe binds at least one glycan other than O-linked glycans; c. incubating the mammalian cell, compound, the first labeled probe, and the second labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; e.
  • the mammalian cell is a human sLe x positive cancer cell.
  • the labeled probe comprises a biotinyl moiety and the process further comprises tagging the labeled probe with streptavidin-Cy5-PE.
  • the labeled probe comprises a fluorescent label.
  • the first labeled probe is a labeled protein.
  • the labeled protein is a O-linked glycan -specific lectin.
  • the second labeled probe is a labeled lectin.
  • the labeled lectin is a lectin specific for a glycan other than an O-linked glycan.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans, and wherein less than 25 mol %, less than 20 mol %%, less than 15 mol %, less than 10 mol %, less than 5 mol % of the plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans comprise terminal sialyl groups.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans, and wherein less than 25 mol %, less than 20 mol %, less than 15 mol %, less than 10 mol %, less than 5 mol % of the plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans comprise terminal fucosyl groups.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens, and wherein less than 20% of the plurality of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens are 6-0 sulfated.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens, and wherein less than 20% of the plurality of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens are 3-0 sulfated.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of sLe x antigens, and wherein less than 20% of the sLe x antigens are 6-0 sulfated.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Le x antigens, and wherein less than 20% of the Le x antigens are 3-0 sulfated.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of sLe A antigens, and wherein less than 25 mol %, less than 20 mol %, less than 15 mol %, less than 10 mol %, less than 5 mol % of the sLe A antigens are
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Le A antigens, and wherein less than 25 mol %, less than 20 mol %, less than 15 mol %, less than 10 mol %, less than 5 mol % of the Le A antigens are 3-
  • a process for modulating the amount of one or more of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof in or on a cell comprising contacting the cell with an effective amount of a selective modulator of O-linked glycan biosynthesis.
  • an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises one or more of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof, and wherein the amount of one or more of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof is 40 mol % (or 30 mol %, or 20 mol %, or 10 mol %) less than the amount of one or more of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , s s
  • Figure 1 illustrates the Wheat Germ Agglutinin (WGA) binding proteins (lane 3) are greater than the unbound proteins (lane 4) and representing over 50% of the total proteins in normal human serum.
  • WGA Wheat Germ Agglutinin
  • Figure 2 illustrates O-linked sialic acid dependence on Maackia Amurensis Lectin II
  • Figure 3 illustrates the affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II
  • Figure 4 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia
  • Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
  • Figure 5 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia
  • Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
  • Figure 6 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
  • MoI II Maakia Amurensis Lectin II
  • FGF2 fibroblast growth factor 2
  • Figures 7A-7L illustrate selective modulators of O-linked biosynthesis.
  • O-linked glycan synthesis inhibitors are present on e.g., glycoproteins and mucins, and comprise a plurality of saccharides linked by O-glycosidic linkages.
  • O-linked glycoproteins and mucins comprise a plurality of O-linked glycans attached to a core protein via a hydroxy group of a Serine, Threonine or Hydroxylysine residue.
  • an O-linked glycan is an O-linked GalNAc ⁇ - O-Ser/Thr glycan, an O-linked GlcNAc ⁇ l-O-Ser/Thr glycan, an O-linked Gal ⁇ l-O-Lys glycan, an O-linked Man ⁇ l-O-Ser/Thr glycan, an O-linked Glc ⁇ l-O-Ser glycan, an O- linked Fuc ⁇ l-O-Ser/Thr glycan.
  • O-linked glycan synthesis inhibitors modulate or alter the nature (e.g., the character, structure or concentration) of one or more O-linked glycans on a core protein in a cell, tissue, organ or individual.
  • MAL II binds to glycans containing sialic acid residues, e.g., in an ( ⁇ -2,3) linkage.
  • Figure 3 illustrates the affects of O-linked glycan synthesis inhibitors on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II (MAL II) in treated and untreated cells.
  • MAL II Maakia Amurensis Lectin II
  • Figure 3 illustrates the efficacy of O-linked glycan synthesis inhibitors described herein in inhibiting the formation of normal O-linked glycan (particluarly O-linked glycans to which MAL II binds).
  • O-linked glycans are present on proteins that are heavily glycosylated with O-linked glycans, e.g., mucins.
  • a mucin-type O- linked glycan comprises an N-acetylgalactosamine GaINAc ⁇ -linked to a hydroxyl group of a Ser or Thr residue of a core protein e.g. mucin.
  • mucin-type O-linked glycans comprise one or more Serine or Threonine residues in a core protein that are O- linked to a plurality of linkage units.
  • a linkage unit is a Core- GalNAc ⁇ -O-Ser/Thr unit.
  • a linkage unit O-linked to a core protein at a Ser or Thr hydroxy group is of Formula I or II: wherein R is a core group of the linkage unit, e.g., Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, Core 8 or a keratan.
  • a linkage unit is optionally and independently modified (e.g., polymerized, sialylated, fucosylated).
  • a linkage unit is optionally and independently modified (e.g., polymerized, sialylated, fucosylated).
  • the Core group of the linkage unit is a ⁇ l-3 galactose ( ⁇ l-3Gal) unit, i.e. the linkage unit is a Core 1 O-linked glycan (Gal ⁇ l- 3GalNAc ⁇ -O-Ser/Thr).
  • the Core group of the linkage unit is a Core 1 further modified by attachment of a ⁇ l-6 GIcNAc unit, i.e., the linkage unit is a Core 2 O- linked glycan (Gal ⁇ l-3(GlcNAc ⁇ l-6)GalNAc ⁇ -O-Ser/Thr).
  • the Core group of the linkage unit is a ⁇ l-3GlcNAc unit, i.e., the linkage unit is a Core 3 O-linked glycan (GlcNAc ⁇ l-3GalNAc ⁇ -O-Ser/Thr).
  • the linkage unit is a Core 4 O-linked glycan (GlcNAc ⁇ l-(GlcNAc ⁇ l-6) 3GalNAc ⁇ -O-Ser/Thr), Core 5 O-linked glycan (GalNAc ⁇ l-SGalNAc ⁇ -O-Ser/Thr), Core 6 O-linked glycan (GlcNAc ⁇ l- 6GalNAc ⁇ -O-Ser/Thr), Core 7 O-linked glycan (GalNAc ⁇ l- ⁇ GalNAc ⁇ -O-Ser/Thr) O- linked glycan, Core 8 O-linked glycan (Gal ⁇ l-3GalNAc ⁇ -O-Ser/Thr) or a keratan (Gal ⁇ l- 4GlcNAc ⁇ l- Gal ⁇ l-3GalNAc ⁇ -O-Ser/Thr).
  • one or more of the linkage units is further modified.
  • a further modification of a linkage unit is e.g., polymerization of one or more polylactosamine disaccharide units (e.g., Gal ⁇ l-4GlcNAc or Gal ⁇ l -3GIcNAc) or fucosyl units ( ⁇ l-2 to Gal, or ⁇ l-3 or ⁇ l-4 to GIcNAc) to a linkage unit.
  • a further modification of a linkage unit is e.g., polymerization of a terminal fucosyl unit or a terminal sialic acid unit ( ⁇ 2-3 or ⁇ 2-6 to Gal, or ⁇ 2-6 to GIcNAc) to a linkage unit.
  • a further modification of a linkage unit is e.g., polymerization of a terminal fucosyl unit or a terminal sialic acid ( ⁇ 2-3 or ⁇ 2-6 to Gal, or ⁇ 2-6 to GIcNAc) to a polylactosamine-elongated-linkage unit.
  • a terminal fucosyl unit or a terminal sialic acid ⁇ 2-3 or ⁇ 2-6 to Gal, or ⁇ 2-6 to GIcNAc
  • O-linked glycan synthesis inhibitors described herein modulate O-linked glycan biosynthesis, e.g., synthesis of a linkage unit, modification (e.g., polymerization, sialylation, fucosylation, glucuronidation, galactosylation) of a linkage unit, or chaperones or transporters that mediate glycan biosynthesis.
  • modification e.g., polymerization, sialylation, fucosylation, glucuronidation, galactosylation
  • O- linked glycan synthesis inhibitors described herein selectively modulate (e.g., inhibit) synthesis of a linkage unit, modification (e.g., polymerization, sialylation, fucosylation, glucuronidation, galactosylation) of a linkage unit, or chaperones or transporters that mediate glycan biosynthesis.
  • modification e.g., polymerization, sialylation, fucosylation, glucuronidation, galactosylation
  • modulation of O-linked glycan biosynthesis includes modulation of the production of the linkage unit (e.g., a Core-GalNAc ⁇ -O-Ser/Thr unit) that connects a glycan to a core protein.
  • the modulation of the production of the linkage unit includes the promotion and/or inhibition of the initiation of the synthesis of the O-linked glycan.
  • the promotion and/or inhibition of the initiation of the synthesis of the O-linked glycan includes the promotion and/or inhibition of polypeptide GaINAc transferases (ppGalNAc-Ts), e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc- T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20,
  • the promotion and/or inhibition of the initiation of the synthesis of the O-linked glycan includes the promotion and/or inhibition of polypeptide GaINAc transferases, such as, for example, ppGalNAc-T3, ppGalNAc-Tl, ppGalNAc-T4, ppGalNAc-TIO, ppGalNAc-T20, ppGalNAc-T15 ppGalNAc-T5.
  • the modulation of the production of the linkage unit includes the promotion and/or inhibition of the synthesis of the linkage unit.
  • the modulation of the production of the linkage unit includes inhibition of a glycosyl transferase (e.g., galactosyl transferase, fucosyl transferase, mannosyl transferase, amino sugar transferase).
  • a glycosyl transferase e.g., galactosyl transferase, fucosyl transferase, mannosyl transferase, amino sugar transferase.
  • modulation of the production of the linkage unit includes the promotion and/or inhibition of a Core transferase, e.g., Core 1 ⁇ 1-3 -galactosyl transferase (ClGaIT), Core 2 ⁇ l-6-N-acetylglucosamine transferase (C2GLcNAc-T), Core 3 ⁇ 1-3 -N- acetylglucosamine transferase (C3GlcNAc-T), or Core 5 ⁇ l-3-N-acetylgalactosamine transferase (C5 GaINAc-T).
  • a Core transferase e.g., Core 1 ⁇ 1-3 -galactosyl transferase (ClGaIT), Core 2 ⁇ l-6-N-acetylglucosamine transferase (C2GLcNAc-T), Core 3 ⁇ 1-3 -N- acetylglucosamine transferase (C3Glc
  • modulation of further modification of the linkage unit includes promotion and/or inhibition of polymerization.
  • modulation of polymerization of the linkage unit includes promotion and/or inhibition of polymerization an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., ⁇ l-4-galactosyl transferase IV ( ⁇ 4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV).
  • modulation of further modification of the linkage unit includes promotion and/or inhibition of chain termination of glycan polymerization.
  • modulation of chain termination of glycan polymerization includes promotion and/or inhibition of the polymerization of a sialic acid unit, a fucosyl unit, an ⁇ -Gal unit, a ⁇ -GalNAc unit, or a ⁇ -GlcNAc unit to the glycan.
  • modulation of chain termination of glycan polymerization includes promotion and/or inhibition of a fucosyl transferase, (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), or a UDP-glucuronosyltransferase.
  • a fucosyl transferase e.g., FucTVII, FucTIV
  • sialyl transferase e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV
  • UDP-glucuronosyltransferase e.g., UDP-glucuronosyl
  • the modulation of O-linked glycan biosynthesis also includes modulation of regulators and components of the biosynthetic pathway.
  • the modulation of regulators and components of the biosynthetic pathway includes the promotion and/or inhibition of one or more of a chaperone (e.g., Core 1 ⁇ 3-GalT-specific molecular chaperone (Cosmc)), a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter) or a combination thereof.
  • a chaperone e.g., Core 1 ⁇ 3-GalT-specific molecular chaperone (Cosmc)
  • a transporter e.g., UDP-GaI transporter, UDP-GIcNAc transporter
  • the modulation of O-linked glycan biosynthesis includes modulation of degradation of O-linked glycans. In some embodiments, the modulation of degradation of O-linked glycans promotes and/or inhibits recycling of saccharide units used for glycan biosynthesis. In some embodiments, modulation of degradation of O-linked glycans includes modulation of endoglycosidases and/or exoglycosidases. In some embodiments, modulation of endoglycosidases and/or exoglycosidases includes the promotion and/or inhibition of ⁇ -Nacetylhexosaminidase (e.g.
  • sialidase e.g. neuraminidase
  • ⁇ -galactosidase ⁇ -galactosidase
  • ⁇ - glucuronidase ⁇ -galactosidase or Cathepsin A.
  • the modulation of O-linked glycan biosynthesis includes modulation of the biosynthesis of Mucin-type O-linked glycans.
  • modulation of the biosynthesis of a mucin-type O-linked glycan includes modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan.
  • modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan includes promotion and/or inhibition of ⁇ 2-3 sialyltransferases (e.g., ST3Gal IV, ST2Gal VI), and/or ⁇ l-3fucosyltransferases (e.g., Fuc-TIV, Fuc-TVII), or isoforms thereof.
  • ⁇ 2-3 sialyltransferases e.g., ST3Gal IV, ST2Gal VI
  • ⁇ l-3fucosyltransferases e.g., Fuc-TIV, Fuc-TVII
  • modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan includes promotion and/or inhibition of the biosynthesis of binding domains that mediate biological functions, e.g., Lewis X (sLe x : NeuAc ⁇ 2-3Gal ⁇ l- 4(Fuc ⁇ l-3)GlcNAc) domain of mucin-type O-linked glycans.
  • Lewis X sLe x : NeuAc ⁇ 2-3Gal ⁇ l- 4(Fuc ⁇ l-3)GlcNAc
  • modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan includes promotion and/or inhibition of the biosynthesis of polylactosamine extensions including, e.g., blood group antigens such as A, B, H or the like, Lewis antigens such as Le x , sLe x , Le Y or the like, type 1 blood group antigens such as Le a , Le b or the like.
  • blood group antigens such as A, B, H or the like
  • Lewis antigens such as Le x , sLe x , Le Y or the like
  • type 1 blood group antigens such as Le a , Le b or the like.
  • O-linked glycan biosynthesis inhibitors or modulators of O- linked glycan biosynthesis are compounds that modify the nature (e.g., character, structure and/or concentration) of O-linked glycans endogenous to a cellular compartment (including vesicles), cell, tissue, organ or individual when contacted or administered to the cell, tissue, organ or individual. It is to be understood that contacting a cell, tissue, or organ is possible via the administration to an individual within whom such cell, tissue or organ resides.
  • O-linked glycan biosynthesis inhibitors or modulators of O-linked glycan biosynthesis modify the character and/or concentration of O-linked glycan in a targeted type of cell, tissue type or organ.
  • O-linked glycan synthesis inhibitors or modulators of O-linked glycan biosynthesis modify the character and/or concentration of O- linked glycan in a systemic manner.
  • the modulation of O-linked glycan biosynthesis includes promotion and/or inhibition of one or more of ppGalNac-Ts, Cl ⁇ l-3GalT, C2GlcNAc-T, iGnT, ⁇ 4Gal-TIV, ⁇ 2-3 sialyltransferases, ⁇ l-3 fucosyltransferases, enzymes required to produce nucleotide donors (e.g., UDP-GIc transferase), transporters(e.g., UDP- Gal transporter) or chaperones (e.g., cosmc) that mediate O-linked glycan biosynthesis, or a combination thereof.
  • nucleotide donors e.g., UDP-GIc transferase
  • transporters e.g., UDP- Gal transporter
  • chaperones e.g., cosmc
  • a single O-linked glycan biosynthesis inhibitor promotes Core 2 synthesis while inhibiting sialylation. In some instances, a single O-linked glycan biosynthesis inhibitor promotes sialylation while inhibiting ppGalNac-Ts. In some instances, a single O-linked glycan biosynthesis inhibitor promotes Core 1 synthesis while inhibiting fucosylation. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits one or more ⁇ 2-3sialyl transferases. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits one or more ⁇ l-3fucosyl transferases.
  • an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits iGnT. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits ⁇ 4-Gal-TIV. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits ppGalNAc-Ts e.g., ppGalNAc-T3, ppGalNAc- Tl, ppGalNAc-T4, ppGalNAc-TIO, ppGalNAc-T20, ppGalNAc-T15 ppGalNAc-T5.
  • an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits Cl- ⁇ l-3-GalT. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits C2 GlcNacT. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits ⁇ - Nacetylhexosaminidase (e.g. promotion and/or inhibition of ⁇ GlcNAc and/or ⁇ GalNAc), sialidase (e.g. neuraminidase), ⁇ -galactosidase, ⁇ -glucuronidase, ⁇ -galactosidase or Cathepsin A.
  • ⁇ - Nacetylhexosaminidase e.g. promotion and/or inhibition of ⁇ GlcNAc and/or ⁇ GalNAc
  • sialidase e.g. neuraminidase
  • specificity includes inhibition, modulation or promotion of the indicated type of sialylation (e.g., Core 2 sialylation), fucosylation (e.g., Core 2 fucosylation), saccharide transfer, polymerization, degradation and/or initiation by a ratio of greater than about 10:1, greater than about 9:1, greater than about 8:1, greater than about 7:1, greater than about 6:1, greater than about 5:1, greater than about 4:1, greater than about 3:1, or greater than about 2:1 over the other types of sialylation, fucosylation, saccharide transfer, polymerization, degradation and/or initiation.
  • sialylation e.g., Core 2 sialylation
  • fucosylation e.g., Core 2 fucosylation
  • saccharide transfer e.g., polymerization, degradation and/or initiation by a ratio of greater than about 10:1, greater than about 9:1, greater than about 8:1, greater than about 7:1, greater than about 6:1, greater than about 5:1, greater than about 4:1, greater than about 3:1,
  • specificity includes inhibition, modulation or promotion of sulfation (e.g., 3-0 sulfation, 6-0 sulfation (e.g., 3-0 sulfation, and/or 6-0 sulfation of one or more of the T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof)) by a ratio of greater than about 10:1, greater than about 9:1, greater than about 8:1, greater than about 7:1, greater than about 6:1, greater than about 5:1, greater than about 4:1, greater than about 3 : 1 , or greater than about 2:1 over the other types of sulfation (e.g., 6-0-sulfation).
  • sulfation e.g., 3-0 sulfation, 6-0 sulfation (e.g., 3-0 sulfation, and/or 6-0 sul
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature (e.g. fucosylation or sialylation) of the O-linked glycan compared to endogenous (i.e., untreated O-linked glycan) O-linked glycan in an amount sufficient to alter or disrupt O-linked glycan binding, O-linked glycan signaling, or a combination thereof.
  • the O-linked glycan synthesis inhibitor alters or disrupts the nature of O-linked glycan in a selected tissue type or organ compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in the selected tissue type or organ.
  • the selected tissue is, by way of non- limiting example, brain tissue, liver tissue, kidney tissue, intestinal tissue, skin tissue, or the like.
  • a O-linked glycan synthesis inhibitor as described herein alters or disrupts the nature of O-linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %).
  • the O- linked glycan synthesis inhibitor described herein alters or disrupts the concentration of O- linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %).
  • an O-linked glycan synthesis inhibitor described herein alters or disrupts the synthesis and/or amount (e.g., the amount and/or number of one or more antigens present) of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens of O-linked glycans compared to endogenous O-linked glycans (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%
  • the O- linked glycan synthesis inhibitor described herein alters or disrupts the fucosylation and/or sialylation of O-linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %).
  • the O- linked glycan synthesis inhibitor described herein alters or disrupts the chain length (or O- linked glycan molecular weight) of an O-linked glycan compared to an endogenous O- linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %; as used herein any % includes mol or w/w % unless otherwise indicated).
  • O-linked glycan synthesis inhibitor described herein alters or disrupts, in combination (e.g., the sum of the change in amount of fucosylation and /or sialylation, concentration, and/or chain length), the nature of an O-linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %).
  • a O-linked glycan synthesis inhibitor as described herein alters or disrupts the fucosylation and/or sialylation of the linkage or Core region of an O-linked glycan compared to endogenous O-linked glycan (i.e., an untreated O-linked glycan) in an organism, organ, tissue or cell by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %).
  • a modified, altered or disrupted O-linked glycan contains less than about 20%, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of terminal sialyl and/or fucosyl residues compared to the amounts of terminal sialyl and/or fucosyl residues in an O-glycan that has not been modified, altered or disrupted.
  • a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 6-0 sulfated T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof compared to the amounts of 6-O-sulfation of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof in an O-glycan that has not been modified, altered or disrupted.
  • a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 3-0 sulfated T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof compared to the amounts of 3-O-sulfation of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof in an O-glycan that has not been modified, altered or disrupted.
  • a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 3-0 sulfated Le x structures compared to the amounts of 3-O- sulfation of Le x structures in an O-glycan that has not been modified, altered or disrupted.
  • a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 6-0 sulfated sLe A structures compared to the amounts of 3-O-sulfation of sLe A structures in an O-glycan that has not been modified, altered or disrupted.
  • a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 6-0 sulfated sLe x structures compared to the amounts of 6-O-sulfation of sLe x structures in an O-glycan that has not been modified, altered or disrupted.
  • the comparison between modified, altered or disrupted O- linked glycan compared to endogenous O-linked glycan is based on the average characteristic (e.g., the concentration, fucosylation, sialylation, chain length or molecular weight, combinations thereof, or the like) of the modified, altered or disrupted O-linked glycan. Furthermore, in some embodiments, the comparison between modified, altered or disrupted O-linked glycan is based on a comparison of the fucosylated and/or sialylated domains of the modified O-linked glycan to the fucosylated and/or sialylated domains of endogenous O-linked glycan.
  • the average characteristic e.g., the concentration, fucosylation, sialylation, chain length or molecular weight, combinations thereof, or the like
  • the degree or nature of fucosylation and/or sialylation in the domains that have high fucosylation and/or sialylation in endogenous O- linked glycan are increased or decreased in the modified O-linked glycan.
  • the degree or nature of fucosylation and/or sialylation in the domains that have low fucosylation and/or sialylation in endogenous O-linked glycan have increased fucosylation and/or sialylation in the modified O-linked glycan.
  • domain organization is determined using enzymes that cleave only terminal sialic acid (e.g., ⁇ - sialidases).
  • domain organization is determined using enzymes that cleave fucosyl linkages (e.g., ⁇ -fucosidases).
  • concentration, amount, character, and/or structure of an O-linked glycan is determined in any suitable manner, including those set forth herein.
  • altering includes increasing or decreasing.
  • disrupting includes reducing or inhibiting.
  • an O-linked glycan biosynthesis inhibitor is a Mucin-type O- linked glycan biosynthesis inhibitor.
  • a Mucin-type O-linked glycan biosynthesis inhibitor described herein alters or disrupts the nature of a Mucin-type O- linked glycan such that it alters or disrupts fucosylation or sialylation of the Mucin-type O- linked glycan.
  • the Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding.
  • the Mucin- type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits O-linked glycan binding and signaling.
  • the Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O- linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM).
  • CAM cell adhesion molecule
  • the CAM is an exogenous CAM, e.g., a bacterial lectin.
  • the CAM is and endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an average fucosylation and/or sialylation of less than about 1.2 mol %, less than about 1.1 mol %, less than about 1.0 mol %, less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, or less than about 0.5 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively.
  • the average fucosylation and/or sialylation refers to the number of sialyl and/or fucosyl substituents on each linkage unit component (e.g., on each Core 1 O-linked glycan (Gal ⁇ l-3GalNAc ⁇ -O-Ser/Thr), on each Core 2 O-linked glycan (Gal ⁇ l-3(GlcNAc ⁇ l-6)GalNAc ⁇ -O-Ser/Thr))of an O-linked glycan.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a pig liver cell, pig liver tissue, a pig liver, or a pig results in an average sialylation and/or fucosylation of less than about 1.0 mol %, less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, or less than about 0.5 mol % in the liver cell, liver tissue, the liver, or the liver of the pig, respectively.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a mouse liver cell, mouse liver tissue, a mouse liver, or a mouse results in an average sialylation and/or fucosylation of less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, less than about 0.5 mol %, less than about 0.4 mol %, or less than about 0.3 mol % in the liver cell, liver tissue, the liver, or the liver of the mouse, respectively.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an average terminal sialylation or fucosylation of less than about 1.2 mol %, less than about 1.1 mol %, less than about 1.0 mol %, less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, or less than about 0.5 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively.
  • sialylation includes sialylation of any glycan ⁇ 2-3 or ⁇ 2-6 to Gal or ⁇ 2-6 to GIcNAc.
  • fucosylation includes terminal and non-terminal fucosylation of any glycan ⁇ l-2 to Gal or ⁇ l-3 or ⁇ l-4 to GIcNAc.
  • mol % is the molar percentage of the selected saccharide component compared to the total number of saccharide components in the O-linked glycan present and/or analyzed.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an average terminal sialylation or fucosylation of each linkage unit of less than about 15 mol %, less than about 14 mol %, less than about 12 mol %, less than about 10 mol %, less than about 8 mol %, less than about 7 mol %, less than about 6 mol %, or less than about 5 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an terminal sialylation or fucosylation of each linkage unit of less than about 7 mol %, less than about 6 mol %, less than about 5 mol %, less than about 4 mol %, less than about 3 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively.
  • an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an terminal sialylation or fucosylation of each linkage unit of less than about 0.7 mol %, less than about 0.6 mol %, less than about 0.5 mol %, less than about 0.4 mol %, or less than about 0.3 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively.
  • the amount of O-linked glycan synthesis inhibitor administered is an effective amount.
  • the effective amount is an amount having a minimal lethality.
  • the LD 50 IED 50 is greater than about 1.1, greater than about 1.2, greater than about 1.3, greater than about 1.4, greater than about 1.5, greater than about 2, greater than about 5, greater than about 10, or more.
  • a therapeutically effective amount is about 0.1 mg to about 1O g.
  • an O-linked glycan biosyntheis inhibitor described herein is a selective O-linked glycan synthesis inhibitor.
  • a selective O-linked glycan inhibitor selectively alters or disrupts the nature (e.g., concentration, chain length, average number of sialic acid residues, fucosylation etc.) of an O-linked glycan compared to other glycans.
  • limiting modifications to glycans limits undesirable or toxic side effects.
  • further restrictions to subsets of glycans further restrict side effects and makes identification, isolation and tracking the effects of the inhibitors more reliable. In some instances, this makes dose determination more reliable.
  • targeting early biosynthetic enzymes would eliminate or severely reduce O-linked glycans which could have global effects on protein folding, protein solubility protein processing as well as other functions involving cell adhesion and signal transduction. These effects could be extremely toxic or lethal.
  • targeting late phase enzymes block modifications that involve more specific receptor binding that is involved in certain cellular adhesion and trafficking interactions.
  • provided herein are late phase O-linked biosynthesis inhibitors.
  • the specific interactions involving late pathway enzymes can be controlled more readily and under controlled conditions (appropriate dosing) could have beneficial effects for a number of diseases.
  • late stage refers to a stage in the biosynthetic process after the initial N-acetylgalactosamine is attached to the ser/thr on the protein, wherein subsequent modifications determine whether the glycan is Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, or Core 8.
  • a late stage biosynthesis inhibitor described herein is an inhibitor that acts in the O-linked glycan biosynthetic pathway after the initial N-acetylgalactosamine is attached to the ser/thr on the protein.
  • late phase in the biosynthetic pathway refers to terminal structures typically on extended O-linked glycans preferentially on core-1 and core-2.
  • selective O-linked glycan biosynthesis inhibitors/modulators include agents that selectively inhibit/modulate one or more of the following: a. biosynthesis of glycan (carbohydrate portion of a molecule) not protein, not nucleic acid, not lipid. b. biosynthesis of specific glycans and/or specific glycans linked to serine (Ser) and threonine (Thr) residues on a protein c. biosynthesis of glycans containing glucose (GIu) d. biosynthesis of glycans containing galactose (Gal) e. biosynthesis of glycans containing N-acetylglucosamine (GIcNAc) f.
  • non mucin glycans including ⁇ - linked O-fucose, ⁇ -linked O- xylose, ⁇ -linked 0-mannose, ⁇ -linked O-GlcNAc (N-acetylglucosamine), ⁇ - or ⁇ -linked O-galactose, and ⁇ - or ⁇ -linked O-glucose glycans 1.
  • Modulation of additional saccharide structures associated with O-glycans associated with mucin glycoproteins the additional saccharides optionally bound to terminal ⁇ l-6 GIcNAc transferred by core 2 ⁇ l-6 GIcNAc.
  • Modification by ⁇ l-3 Gal transferred by ⁇ l-3 galactosyltransferase ( ⁇ l- 3GaIT) forms a Gal ⁇ l-3GlcNAc (neo-N-acetyllactosamine) termed Type-1.
  • Optional modulation of glycosyltransferases to further alter such structures may also be obtained utilizing modulators described herein (e.g., with Gal, GIcNAc, GaINAc, Fuc or Sia). kk.
  • Type-2 residues can be further modified to form structures containing serial disaccharides of GlcNAc( ⁇ l-4)Gal bound together by Gal( ⁇ l-3)GlcNAc linkages forming chains called poly-N- acetlyllactosamine chains. These poly-N-acetlyllactosamine chains further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc, GaINAc and Fuc and Sia. 11.
  • biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc, GaINAc and Fuc to form the A,B, and H blood group antigens.
  • biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc, GaINAc and Fuc to form the Lewis blood group antigens.
  • nn the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc
  • Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by one or more ⁇ l-3FucTs and ⁇ l-2FucTs (as distinct from the Lewis ⁇ l-3/ ⁇ l-4FucT) to carry an ⁇ l-3 fucose residue Lewis X (Le x ) and an ⁇ l-3 fucose residue and an ⁇ 1-2 fucose residue Lewis Y (Le ⁇ ).
  • ss. enzymes required to make the glycans necessary for selectin binding which includes fucosyl transferases, sialyltransferases and sulfotransferases.
  • L-selectin binding requires the 6-sulfo derivative of sialylated Le x (6-sulfo-SLe x ) on both core -2 and extended core-1 glycans.
  • This structure requires two key ⁇ l-3 fucosyl transferases FucT-IV and FucT-VII along with four different sulfotransferases including GlcNA6ST-l and GlcNA6ST-2.
  • L- selectin binding is required for lymphocyte homing as well as adhesion of neutrophils, eosinophils and monocytes to nonlymphoid vascular endothelium. It is also thought to be involved in metastatic tumor spread through the bloodstream and lymph. tt.
  • Lewis X Lewis X
  • Lewis a Lewis a
  • enzymes that glycosylate mucins which may influence mucin glycosylation associated with cancer (carcinoma mucins). Mucin glycosylation is known to be altered on carcinomas. Modulators could be used to therapeutically alter mucin glycosylation for the purpose of developing vaccines and antibodies that recognize the altered mucin glycosylation.
  • enzymes would be enzymes that produce the T antigen, the Tn antigen and the Sialyl Tn antigen, enzymes involved in the Lewis antigen structures and their sialylated and sulfated derivatives, enzymes involved in incomplete blood group antigen production and enzymes involved in abnormal sialylation of carcinoma mucins, glycoproteins and glyco lipids. w. biosynthesis and/or modulation of the character of N- and O- linked glycoproteins and glycolipids containing sialic acid residues ⁇ 2-3 linked to terminal galactose residues (in vertebrates) - catalyzed by 6 ⁇ 2-3 sialyltransferases ST3GalI to ST3GalVI.
  • Glycans bearing ⁇ 2-3 linked Sia may contribute to the circulating half lives of plasma glycoprogeins by virtue of "masking" terminal Gal residues that would contribute to the removal of glycoproteins from serum by the asialoglycoprotein receptor.
  • Sia ⁇ 2-3Gal ⁇ 1- 3GalNAc ⁇ Ser/Thr is important for the viability of CD8+ T cells.
  • ⁇ 2-3 linked Sia is recognized by the hemagglutinin in the envelope of influenza viruses from birds and pigs. ww.
  • ⁇ 2-6 sialic acid is a receptor for influenza viral infection in humans.
  • O-GalNAc glycosylation is an important process because many or all mammalian cell types studied to date express ppGalNAcTs.
  • the O-GalNAc glycans of mucous glycoproteins are essential for their ability to hydrate and protect the underlying epithelium.
  • Mucins also trap bacteria via specific receptor sites within the O-glycans of the mucin. Some sugar residues or their modifications can serve as "decoys," thus masking underlying antigens or receptors.
  • O- acetyl groups on the sialic acid residue of the sialyl-Tn antigen prevent recognition by anti- sialyl-Tn antibodies.
  • Gut bacteria often actively remove this decoy.
  • Bacteria can cleave sulfate with sulfatases or terminal sugars with glycosidases.
  • the O-glycans are hydrophilic and usually negatively charged, they promote binding of water and salts and are major contributors to the viscosity and adhesiveness of mucus, which forms a physical barrier between lumen and epithelium. The removal of microbes and particles trapped in mucus is an important physiological process.
  • O-GalNAc glycans especially in the highly glycosylated mucins, have a significant effect on the conformation of the attached protein.
  • underlying peptide epitopes can be variably recognized by antibodies.
  • O-glycosylation of mucins provides almost complete protection from protease degradation, and it is possible that the sparse O-glycosylation of some secreted glycoproteins, such as the single O-GalNAc glycan on interleukin-2, has a similar protective role.
  • O-GalNAc glycans change during lymphocyte activation and are abnormal in leukemic cells where an increase in core 2 and a decrease in core 1 O-glycans are often seen.
  • the ligands for selectin-mediated interactions between endothelial cells and leukocytes are commonly based upon sialyl Lewis x epitopes attached to core 2 O-GalNAc glycans. This type of selectin-glycan interaction is important for the attachment of leukocytes to the capillary endothelium during homing of lymphocytes or the extravasation of leukocytes during the inflammatory response.
  • mucins carry a higher number of unmodified N- acetylgalactosamine residues and shorter 0-GalNAc glycans.
  • Inhibitor- treated cancer cells lose the ability to bind to E-selectin and endothelial cells in vitro.
  • reproductive tissues produce mucins and O-glycosylated glycoproteins that may have important roles in fertilization.
  • Specific terminal O-glycan structures have been shown to form the ligands for sperm-egg interactions in several species.
  • the changes of O-glycans commonly observed in diseases can be due to the actions of cytokines or growth factors that affect cell growth, differentiation, and cell death and alter the expression of glycosyltransferase genes. Although these glycosylation changes may be considered a "side effect" of a pathological condition, they can also significantly contribute to the ultimate pathology and the course of disease.
  • the biosynthesis of O- GaINAc glycans is often abnormal due to either decreased or increased expression and activities of specific glycosyltransferases. An altered cell-surface glycocalyx may then affect the biology and survival of the cancer cell.
  • cell-surface MUCl and mucin-like glycoproteins have roles in cell adhesion.
  • a number of diseases are associated with abnormal mucin gene expression and abnormal mucin carbohydrate structures and properties. These include cancer, inflammatory bowel disease, lung disease, and cystic fibrosis. The expression of underglycosylated MUCl is often increased in individuals with cancer.
  • selectin ligands are transmembrane glycoproteins which present oligosaccharide structures to the selectins. In some instances, transient bond formations between the selectins and their ligands mediate early steps of the adhesion cascade.
  • selectins e.g., P-selectin, L-selectin, E-selectin, recognize glycoproteins and/or glyco lipids containing the tetrasaccharide sialyl Lewis x (sialyl-CD15). In some embodiments, this tetrasaccharide is found on myeloid cells and is composed of sialic acid, galactose, fucose, and N-actyl-galacosamine.
  • PSGL-I P-selectin glycoprotein ligand
  • PSGL-I is a ligand for P- selectin.
  • PSGL-I is a glycoprotein expressed on blood cells and contains the sialyl Lewis x tetrasaccharide.
  • optimal binding of P-selectin to PSGL-I requires that PSGL-I presents sialyl LewisX on a specific amino-terminal core-2 O-glaycan and sulfate esters on specific amino-terminal tyrosine residues.
  • another P-selectin ligand is CD24, which is important for tumor cell binding to P-selectin.
  • L-selectin GIyCAM- l(Glycosylation-Dependent Cell Adhesion Molecule), CD34, and MAdCAM-I (Mucosal Addressin Cell Adhesion Molecule), and PSGL-I.
  • both sulfate and sialic acid in an ⁇ (2,3) linkage are essential to L-selectin ligand activity.
  • candidate molecules include fucosylated, sialyated oligosaccharides found as components of glycoprotein and glycolipid molecules.
  • a selective 0-linked glycan inhibitor selectively alters or disrupts the sialylation and/or fucosylation of O-linked glycans.
  • a selective O-linked glycan inhibitor selectively reduces or inhibits the sialylation and/or fucosylation of mucin-type O-linked glycans.
  • selectivity of an O- linked glycan synthesis inhibitors is beneficial in order to target specific disorders without adversely impacting properly functioning glycan biosynthetic processes.
  • therapeutic methods utilizing selective O-linked glycan synthesis inhibitors have improved toxicity profiles compared to non-selective glycan synthesis inhibitors.
  • selective O-linked glycan synthesis inhibitors modulate (e.g., inhibit or promote) late stage processes (including, e.g., enzyme activity involved in the O-linked glycan preparation/synthetic pathway, enzyme activity involved in the O-linked glycan degradation pathway, other enzyme activity that affects the character of O-linked glycans, or the like) in the O-linked glycan biosynthetic pathway.
  • late stage refers to a stage in the biosynthetic process after the initial N-acetylgalactosamine is attached to the ser/thr on the protein, wherein subsequent modifications determine whether the glycan is Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, or Core 8.
  • a late stage biosynthesis inhibitor described herein is an inhibitor that acts in the O-linked glycan biosynthetic pathway after the initial N-acetylgalactosamine is attached to the ser/thr on the protein.
  • a selective O-linked glycan biosynthesis inhibitor described herein is a selective Core transferase inhibitor.
  • a selective O-linked glycan biosynthesis inhibitor is a selective polymerization (e.g. polylactosamine polymerization) inhibitor.
  • a selective O-linked glycan biosynthesis inhibitor is a selective sialyl transferase inhibitor.
  • a selective O- linked glycan biosynthesis inhibitor is a selective fucosyl transferase inhibitor.
  • a selective O-linked glycan biosynthesis inhibitor is a selective iGnT inhibitor.
  • the selective O-linked glycan synthesis inhibitor selectively affects the biosynthesis of glycans, such as N-linked, O-linked, lipid linked, or the like, but not glycosaminoglycans (GAGs), such as heparan sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and/or hyaluronan.
  • GAGs glycosaminoglycans
  • selective O-linked glycan inhibitors selectively inhibit glycans compared to GAGs by a ratio of greater than 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1 or more.
  • a O-linked biosynthesis modulator (e.g., inhibitor or promoter) described herein is a selective O-linked synthesis modulator (e.g., inhibitor or promoter).
  • a selective O-linked glycan synthesis modulator affects (e.g., inhibits or promotes) the biosynthesis of O-linked glycans compared to the biosynthesis of one or more of N-linked glycans, glycosaminoglycans (GAGs), glycolipids (e.g., glycan portion thereof), or a combination thereof.
  • the selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g., inhibits or promotes) the synthesis of O-linked glycan over N-linked glycans, glycosaminoglycans (GAGs), glycolipids, or a combination thereof by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
  • a selective O-linked glycan biosynthesis modulator described herein selectively affects (e.g., promotes or inhibits) the biosynthesis of a specific type of O-linked glycan (e.g. mucin type O-linked glycan) compared to one or more other type of O-linked glycan (e.g. non-mucin type O-linked glycans).
  • a selective O-linked glycan synthesis inhibitor described herein selectively affects/inhibits/modulates/promotes the biosynthesis of one or more of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 type of O-linked glycans but does not substantially affect/inhibit/modulate/promote the synthesis of one or more other type of O-linked glycans (e.g., one or more of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 type of O-linked glycans).
  • a selective O- linked glycan biosynthesis modulator selectively modulates (e.g., inhibits or promotes) the synthesis of a first specific type of O-linked glycan compared to one or more different types of O-linked glycans by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
  • a selective O-linked glycan synthesis inhibitor selectively alters or disrupts the nature (e.g., concentration, chain length, average number of sialic acid residues, fucosylation etc.) of a O-linked glycan compared to other glycans (e.g., GAGs, N- linked glycans, glycolipids, or the like, or combinations thereof).
  • glycans e.g., GAGs, N- linked glycans, glycolipids, or the like, or combinations thereof.
  • a selective O-linked glycan inhibitor selectively alters or disrupts the sialylation and/or fucosylation of O-linked glycans compared to the sialylation and/or fucosylation of other glycans (e.g., GAGs, N-linked glycans, glycolipids, or the like, or combinations thereof).
  • other glycans e.g., GAGs, N-linked glycans, glycolipids, or the like, or combinations thereof.
  • a selective O-linked glycan synthesis inhibitor selectively inhibits sialylation and/or fucosylation of O-linked glyans compared to one or more different types of glycans (e.g., GAGs, N-linked glycans, glycolipids, or the like, or combinations thereof) by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
  • GAGs e.g., GAGs, N-linked glycans, glycolipids, or the like, or combinations thereof
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the sulfation (e.g., 6-0 sulfation, 3-0 sulfation) of one or more of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens compared to the sulfation (e.g., 6-0 sulfation, 3-0 sulfation) of one or more of other T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens in the absence of an O-glycan synthesis inhibitor.
  • the sulfation e.g., 6-0 sulfation, 3-0 sulfation
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the 6-0 sulfation of sLe x antigens compared to the 6-0 sulfation of sLe x antigens in the absence of an O-glycan synthesis inhibitor.
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g.
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the 3-0 sulfation of Le x antigens compared to the 3-0 sulfation of Le x antigens in the absence of an O-glycan synthesis inhibitor.
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the -O sulfation of Le x antigens compared to the 3-0 sulfation of sLe x antigens in the absence of an O- glycan synthesis inhibitor by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the amount (e.g.
  • a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the amount (e.g. number of antigens in or on a cell) of one or more of T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or a combination thereof in or on a cell compared to the amount (e.g.
  • a selective O-linked glycan synthesis modulator selectively inhibits or promotes a specific or select characteristic of a O-linked glycan, e.g., the amount of O-linked glycan, the glycan-length of the O-linked glycan, the number of sialic acid residues of a O-linked glycan, N-acetylation, O-sulfation, O-acylation of galactose residues, O-acetylation of sialic acid residues, or the like, or combinations thereof while leaving other characteristics of the O-linked glycan unaffected or significantly unaffected.
  • a specific or select characteristic of a O-linked glycan e.g., the amount of O-linked glycan, the glycan-length of the O-linked glycan, the number of sialic acid residues of a O-linked glycan, N-acetylation, O-sulfation, O-acylation of gal
  • an O-linked glycan synthesis inhibitor selectively modulates (e.g., inhibits or promotes) specific types of action that inhibit O-linked glycan function.
  • an O-linked glycan synthesis inhibitor modulates (e.g., inhibits or promotes) sulfation, glycosylation, phosphorylation, sialylation, fucosylation and/or degradation of endogenous O-linked glyans.
  • a selective O-linked glycan synthesis modulator selectively inhibits or promotes a specific or select characteristic of a O-linked glycan compared to one or more of any other specific or select characteristic of O-linked glycans by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
  • Figures 4-6 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
  • MoI II Maakia Amurensis Lectin II
  • FGF2 fibroblast growth factor 2
  • compounds described herein show selective inhibition of O-linked glycans without a reduction in the unrelated glycan - heparan sulfate. This demonstrates that in certain embodiments, inhibitors described herein have O-linked glycan class selectivity.
  • a selective O-linked glycan synthesis modulator selective affects (e.g., inhibits or promotes) the activity of a specific enzyme involved in the biosynthesis of one or more O-linked glycan (e.g., one or more of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc- T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18
  • the selective O-linked glycan biosynthesis modulator selectively modulates (e.g., inhibits or promotes) one or more enzymes involved in the biosynthesis of one or more O-linked glycans compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
  • a selective O-linked glycan biosynthesis inhibitor described herein selectively modulates (e.g., inhibits or promotes) one or more Core transferases compared to one or more different enzymes involved in the biosynthesis of O-linked glycans.
  • a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g., inhibits or promotes) one or more enzymes invloved in polymerization (e.g. polylactosamine polymerization) compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans.
  • a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g.
  • a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g. inhibits or promotes) one or more fucosyl transferases compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans.
  • a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g. inhibits or promotes) iGnT compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans.
  • selective O-linked glycan modulators e.g., inhibitors or promoters
  • modulate e.g., promote or inhibit
  • the biosynthesis of O-linked glycans with a modified (e.g., increased or decreased) ability to bind with or otherwise associate with one or more proteins, one or more core proteins, one or more lectin, one or more selectin, pathogens or the like (e.g. as compared to glycans that are synthesized in the absence of the modification).
  • a selective O-linked glycan synthesis inhibitor described herein selectively modulates (e.g., promotes or inhibits) the biosynthesis of an O-linked glycan comprising antigens (e.g., T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or the like), blood group antigens (e.g., A, B, ABO, H antigens), type I blood group antigens (e.g., Le a , Le b ), selectin ligands, peanut-agglutinin-binding epitope, salivary mucin MG2 or the like.
  • antigens e.g., T, Le x , sLe x , Le Y , sLe Y , Le A , sLe A , Le B , sLe B antigens or the like
  • blood group antigens
  • selective modulation (e.g., promotion or inhibition) of the synthesis of antigens selectively modulates (promotes or inhibits) cell adhesion, angiogenesis, cancer growth and/or metastasis compared to cell adhesion, angiogenesis, cancer growth and/or metastasis in the absence of selective modulation (e.g., promotion or inhibition) of synthesis of antigens.
  • selective O-linked glycan inhibitors described herein selectively modulate (e.g., promote or inhibit) the biosynthesis of O-linked glycans to acheive one or more specific result (e.g., agglutination, binding or the like).
  • the selective O-linked glycan inhibitor described herein selectively modulates (e.g., promotes or inhibits) the biosynthesis of a specific O-glycan to specifically and/or selectively vary, tune, or optimize the stability, solubility, cellular location, expression of, binding of, and/or activity of O-linked glycans and/or O-linked glycanated proteins produced.
  • a selective O- linked glycan biosynthesis inhibitor selectively modulates (e.g., promotes or inhibits) the biosynthesis of one O-linked glycan comprising antigen in a ratio of greater than 1000:1, greater than 500:1, greater than 250:1, greater than 100:1, greater than 50:1, greater than 25:1, greater than 20:1, greater than 10:1, greater than 5:1, greater than 3 : 1 , or greater than 2:1 over one or more other O-linked glycan comprising antigen, (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-0-linked glycan).
  • antigen e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-0-linked glycan.
  • an O-linked glycan biosynthesis inhibitor described herein is a selective O-linked glycan biosynthesis that inhibits any specific transferase described herein over any one or more other transferase involved in the O-linked glycan biosynthetic pathyway (e.g., over all other transferases involved in the O-linked glycan biosynthetic pathway.
  • an O-linked glycan biosynthesis inhibitor described herein is a selective O-linked glycan biosynthesis inhibitor that inhibits any specific transferase described herein as being involved in the O-linked glycan biosynthetic pathyway over any one or more transferase involved in the biosynthetic pathway of a non-0-linked glycan (e.g., N-linked glycan, glycosaminoglycan, ganglioside, or the like).
  • a non-0-linked glycan e.g., N-linked glycan, glycosaminoglycan, ganglioside, or the like.
  • a selective O-linked glycan biosynthesis inhibitor is selective for (i.e., directly or indirectly inhibits the activity of) a specific enzyme (e.g., transferase) in a ratio of greater than 1000:1 over one or more other enzyme (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan).
  • a specific enzyme e.g., transferase
  • another enzyme involved in the O-linked biosynthetic pathway e.g., another enzyme involved in the biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan.
  • a selective O- linked glycan biosynthesis inhibitor is selective for (i.e., directly or indirectly inhibits the activity of) a specific enzyme in a ratio of greater than 500:1 over one or more other enzyme (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan).
  • a selective O-linked glycan biosynthesis inhibitor is selective for (i.e., directly or indirectly inhibits the activity of) a specific enzyme in a ratio of greater than 250:1, greater than 100:1, greater than 50:1, greater than 25:1, greater than 20:1, greater than 10:1, greater than 5:1, greater than 3 : 1 , or greater than 2 : 1 over one or more other enzyme (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan).
  • a selective O-linked glycan biosynthesis inhibitor selectively inhibits an enzymes selectin receptor binding mucin O-linked glycoproteins.
  • a selective O-linked glycan biosynthesis inhibitor described herein inhibits the enzymes that produce, or otherwise inhibits biosynthesis of, glycans that serve as selectin ligands Lewis X and sialyl-Lewis X (Le x and SLe x ).
  • a selective O-linked glycan biosynthesis inhibitor selectively inhibits one or more of ⁇ l,3 fucosyltransferases, ⁇ 2-3 sialyltransferases, ⁇ 2-6 sialyltransferases and/or sulfotransferases including 6-0 sulfotransferases GlcNAc6ST-l and GlcNAc6ST-2.
  • a selective inhibitor of O-linked glycan biosynthesis includes a selective inhibitor of FucT- VII, glycosyltransferases, ⁇ l,3fucosyltransferase, ⁇ 2-3 sialyltransferases, GlcNAc6ST-l, or GlcNAc6ST-2.
  • an O-linked glycoprotein or O-linked glycan that was prepared by modifying the biosynthesis thereof with any selective inhibitor described herein.
  • a O-linked glycan biosynthesis modulator e.g., a selective O-linked glycan synthesis inhibitor
  • a selective O-linked glycan synthesis inhibitor having suitable cell availability and/or bioavailability to significantly effect the in cyto and/or in vivo biosynthesis of a O-linked glycan (e.g., a specific O-linked glycan or component thereof, in certain instances wherein a selective O-linked glycan synthesis modulator is utilized) when the O-linked glycan biosynthesis modulator is administered to a cell or individual, respectively.
  • a significant effect is one wherein a measurable effect, a statistically significant effect, and/or a therapeutic effect is provided to the cell or individual.
  • the specific O-linked glycan synthesis modulator is substantially cell permeable (e.g., when in contact with a cell, a significant percentage/amount of the modulator permeates the cell membrane).
  • the O-linked glycan synthesis modulator e.g., promotor or inhibitor
  • has cellular activit e.g., when put in contact with a cell, the modulator significantly (e.g., therapeutically significantly, physiologically significantly, statistically significantly, or the like) affects cellular O-linked glycan synthesis according to any manner described herein.
  • the O- linked glycan biosynthesis modulator provides a statisitically significant effect and/or therapeutic effect in a cell or individual at a non-toxic concentration, a substantially nontoxic concentration, a concentration below LC50, a concentration below LC20, a concentration below LCOl, or the like.
  • O-linked glycan synthesis inhibitors described herein are inhibitors of one or more of ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc- T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNA
  • O-linked glycan biosynthesis inhibitors described herein are small molecule organic compounds.
  • O-linked glycan biosynthesis inhibitors utilized herein are not polypeptides and/or carbohydrates.
  • a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
  • selective inhibitors of O-linked glycan biosynthesis includes any compound of Figures 7A- 7L. Incubating compounds of Figures 7A- 7L in cells were observed to inhibit glycan- WGA binding, but did not demonstrate a significant inhibition of glycan-FGF binding (non-inhibitory against GAG, HS biosynthesis), glycan- PHA binding (non-inhibitory against N-linked glycan biosynthesis), or glycan-CTB binding (non-inhibitory against ganglioside biosynthesis).
  • selective inhibitors of O-linked glycan biosynthesis include, but are not limited to, the following compounds: 5-bromo-2-chloro- ⁇ /-(4-(pyrrolidin-l-yl)phenyl)benzamide (1); 2-(5-methyl- 1 ,3 ,4-thiadiazol-2-ylthio)- 1 -(2,2,4,6,7-pentamethylquinolin- 1 (2H)-yl)ethanone (2); N-(3- methoxy-5,6,7,8-tetrahydronaphthalen-2-yl)acetamide (3); 4-(4-chlorophenoxy)-iV-(2- nitrophenyl)butanamide (4); JV-butyl-4-ethoxy-iV-ethylbenzamide (5); ( ⁇ )-I- (benzo[ ⁇ i]thiazol-2-yl)-4-(l-(2-ethoxyphenylamino)ethylidene)-3-phenyl-
  • O-linked glycan biosynthesis inhibitors described herein are non-carbohydrate small molecule compounds.
  • Carbohydrates tend to be hydrophilic due to the polyhydroxyls and therefore do not diffuse into cells efficiently.
  • carbohydrates have pharmacokinetic and pharmacodynamic properties in animals that are inappropriate for therapeutic drug effects. Further, the hydroxyls are reactive and may make carbohydrates difficult and expensive to synthesize. In certain instances, carbohydrates are not known to cross the blood-brain barrier. In certain instances, noncarbohydrate small molecules are much less likely to be immunogenic or immunoreactive than are carbohydrates.
  • Carbohydrates include polhydroxyaldehydes, polyhydroxyketones and their simple derivatives or larger compounds that can be hydro lyzed into such units. Carbohydrates also include polhydroxyaldehydes, polyhydroxyketones and their simple derivatives that have been modified such that when they enter cells they are reconverted into polhydroxyaldehydes, polyhydroxyketones.
  • Carbohydrates also include sugar mimetics such as imino structures and alkaloids that inhibit glycosidases such as Deoxynojirimycin, Castanospermine, Australine, Deoxymannojirimycin, Kifunensen, Swainsonine and Mannostatin (page 709 of Essentials of Glycobiology second edition 2008 CSHL Press, CSH, New York.)
  • Non carbohydrate small molecules include, e.g., organic compounds containing less than 3 linked hydroxyl groups with a molecular weight of less than 2000 Daltons.
  • Modulators e.g., inhibitors of glycan synthesis include agents that act directly on the relevant biosynthetic enzymes or indirectly on other targets (e.g. protein kinase, phosphatase, transporter, GPCR, ion channel, hormone receptor, protease, etc.) that would alter the structure of the glycans though effects on biosynthetic (anabolic) enzymes or degradative (catabolic) enzymes.
  • targets e.g. protein kinase, phosphatase, transporter, GPCR, ion channel, hormone receptor, protease, etc.
  • the term "subject”, “patient” or “individual” are used interchangeably herein and refer to mammals and non-mammals, e.g., suffering from a disorder described herein.
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • non-mammals include, but are not limited to, birds, fish and the like.
  • the mammal is a human.
  • treat include alleviating, inhibiting or reducing symptoms, reducing or inhibiting severity of, reducing incidence of, prophylactic treatment of, reducing or inhibiting recurrence of, delaying onset of, delaying recurrence of, abating or ameliorating a disease or condition symptoms, ameliorating the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated, and/or the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient.
  • compositions include preventing additional symptoms, preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition and are intended to include prophylaxis.
  • the terms further include achieving a prophylactic benefit.
  • the compositions are optionally administered to a patient at risk of developing a particular disease, to a patient reporting one or more of the physiological symptoms of a disease, or to a patient at risk of reoccurrence of the disease.
  • combination treatments or prevention methods are contemplated, it is not intended that the agents described herein be limited by the particular nature of the combination.
  • the agents described herein are optionally administered in combination as simple mixtures as well as chemical hybrids.
  • An example of the latter is where the agent is covalently linked to a targeting carrier or to an active pharmaceutical.
  • Covalent binding can be accomplished in many ways, such as, though not limited to, the use of a commercially available cross-linking agent.
  • combination treatments are optionally administered separately or concomitantly.
  • the terms “pharmaceutical combination”, “administering an additional therapy”, “administering an additional therapeutic agent” and the like refer to a pharmaceutical therapy resulting from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients.
  • the term “fixed combination” means that at least one of the agents described herein, and at least one co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • non- fixed combination means that at least one of the agents described herein, and at least one co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with variable intervening time limits, wherein such administration provides effective levels of the two or more agents in the body of the patient.
  • the co-agent is administered once or for a period of time, after which the agent is administered once or over a period of time.
  • the co-agent is administered for a period of time, after which, a therapy involving the administration of both the co-agent and the agent are administered.
  • the agent is administered once or over a period of time, after which, the co- agent is administered once or over a period of time.
  • the terms “co-administration”, “administered in combination with” and their grammatical equivalents are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different times.
  • the agents described herein will be co-administered with other agents.
  • These terms encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present.
  • the agents described herein and the other agent(s) are administered in a single composition.
  • the agents described herein and the other agent(s) are admixed in the composition.
  • the terms "effective amount” or “therapeutically effective amount” as used herein, refer to a sufficient amount of at least one agent being administered which achieve a desired result, e.g., to relieve to some extent one or more symptoms of a disease or condition being treated.
  • the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • the result is the alteration of or the disruption of the structure of endogenous O-linked glycans such that the binding ability, signaling ability or combination thereof of the O-linked glycan is inhibited or reduced.
  • an "effective amount” for therapeutic uses is the amount of the composition comprising an agent as set forth herein required to provide a clinically significant decrease in a disease.
  • An appropriate "effective” amount in any individual case is determined using any suitable technique, such as a dose escalation study.
  • the terms “administer,” “administering”, “administration,” and the like, as used herein, refer to the methods that may be used to enable delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration.
  • agents and compositions described herein are administered orally.
  • pharmaceutically acceptable refers to a material that does not abrogate the biological activity or properties of the agents described herein, and is relatively nontoxic (i.e., the toxicity of the material significantly outweighs the benefit of the material).
  • a pharmaceutically acceptable material may be administered to an individual without causing significant undesirable biological effects or significantly interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • carrier refers to relatively nontoxic chemical agents that, in certain instances, facilitate the incorporation of an agent into cells or tissues.
  • “Pharmaceutically acceptable prodrug” as used herein refers to any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of an agent, which, upon administration to a recipient, is capable of providing, either directly or indirectly, an 0-linked glycan modulator agent described herein or a pharmaceutically active metabolite or residue thereof.
  • Particularly favored prodrugs are those that increase the bioavailability of the 0-linked glycan modulator agents described herein when such agents are administered to a patient ⁇ e.g.
  • pharmaceutically acceptable salts described herein include, by way of non- limiting example, a nitrate, chloride, bromide, phosphate, sulfate, acetate, hexafluorophosphate, citrate, gluconate, benzoate, propionate, butyrate, subsalicylate, maleate, laurate, malate, fumarate, succinate, tartrate, amsonate, pamoate, p-tolunenesulfonate, mesylate and the like.
  • pharmaceutically acceptable salts include, by way of non- limiting example, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium or potassium), ammonium salts and the like.
  • a process for modifying the structure of an 0-linked glycan on a core protein comprising contacting a cell that translationally produces at least one core protein having at least one attached O- linked glycan with an effective amount of any 0-linked glycan synthesis inhibitor described herein.
  • the 0-linked glycan synthesis inhibitor is a selective O- linked glycan synthesis inhibitor, e.g., as described herein.
  • the selective 0-linked glycan synthesis inhibitor is a modulator of (e.g., promotes one or more of, or inhibits one or more of a Core transferase (e.g., modulates a C2-GlcNAcT), polymerization (e.g., modulates a polypeptide GaINAc transferase, also known as a ppGalNAc-T), sialylation (e.g., modulates a sialyl transferase), sulfation (e.g., N or O sulfation), acetylation (e.g., N or O acetylation), phosphorylation, fucosylation (e.g., modulates a fucosyl transferase), a transporter (e.g., UDP-GaI transporter), a chaperone (Cosmc), a polylactosamine extension enzyme, or a combination thereof.
  • a modulator of e.g., promotes one or
  • the inhibitor of a glycosyltransferase inhibits the synthesis of the linkage unit, the initiation of linkage unit synthesis, the polymerization of a linkage unit, the sialylation of a linkage unit, the fucosylation of a linkage unit or a combination thereof.
  • 0-linked glycan synthesis inhibitors modulate (e.g., promote or inhibit) one or more of a Core transferase, iGnT, sialyl transferase, a fucosyl transferase, a ppGalNAc transferase, a ⁇ l- 4galactosyltransferase IV, or combinations thereof.
  • an O- linked glycan synthesis inhibitor selectively modulates (e.g., promote or inhibit) one or more of ppGalNAc-T e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc- TlO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, pp
  • an 0-linked glycan synthesis inhibitor selectively modulates ppGalNAc-T3, ppGalNAc-Tl, ppGalNAc-T4, ppGalNAc-TIO, ppGalNAc-T20, ppGalNAc-T15 or ppGalNAc-T5.
  • an O-linked glycan synthesis inhibitor selectively modulates Core 1 ⁇ 1-3 -galactosyl transferase (ClGaIT), Core 2 ⁇ l-6-N-acetylglucosamine transferase (C2GLcNAc-T), Core 3 ⁇ l-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 ⁇ l-6-N-acetylglucosaminyl transferase (C4GlcNAc-T), or Core 5 ⁇ l-3-N- acetylgalactosamine transferase (C5 GaINAc-T).
  • ClGaIT Core 1 ⁇ 1-3 -galactosyl transferase
  • C2GLcNAc-T Core 2 ⁇ l-6-N-acetylglucosamine transferase
  • C3GlcNAc-T Core 3 ⁇ l-3-N-acetylglu
  • an O-linked glycan synthesis inhibitor selectively modulates an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., ⁇ l-4-galactosyl transferase IV( ⁇ 4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 ⁇ 3-GalT-specif ⁇ c molecular chaperone (Cosmc)), a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter).
  • an i-extension enzyme e.g., i
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • any immunomodulatory factor e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM).
  • CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • the cell is present in an individual (e.g., a human) diagnosed with a disorder mediated by O-linked glycan biosynthesis.
  • the disorder mediated by O-linked glycan biosynthesis is a cancer, a tumor, undesired angiogenesis (e.g., cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, or psoriasis), insufficient angiogenesis (e.g., coronary artery disease, stroke, or delayed wound healing), organomegaly (e.g., hepatosplenomegaly), amyloidosis, skeletal abonormalities, odontoid hypoplasia, hydrops fetalis, sialuria, sialidosis, Ehlers-Danlos syndrome, Walker Warburg syndrome, a wound, or the like.
  • undesired angiogenesis e.g., cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, or psoriasis
  • insufficient angiogenesis e.g., coronary artery disease, stroke, or delayed wound healing
  • the disorder mediated by O-linked glycan biosynthesis is an inflamatory disorder (e.g, an acute or chronic inflammatory disorder) including but not limited to Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific auto immunity, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, organ transplant rejection, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, gastrointestinal allergies, including food allergies, pancreatitis, eosinophilia, conjunctivitis, glomerular nephritis, multiple vasculitides, myasthenia gravis, asthma, chronic obstructive pulmonay disease, myocardial infarction, stroke, transplant rejection, reperfusion injury, autoimmune disease (e.g, Ankylosing spondylitis, systemic lupus erythematosus (SLE), or the like) inflam
  • the cell is present in a human diagnosed with an inflammatory disorder. In certain embodiments, the cell is present in an individual (e.g., a human) diagnosed with abnormal angiogenesis and/or undesired angiogenesis. In some embodiments, the cell is present in an individual (e.g., a human) diagnosed with amyloidosis, a spinal cord injury, hypertriglyceridemia, or the like. [00124] In some embodiments, the cell is present in an individual (e.g., human) diagnosed with a carcinoma or adenocarcinoma.
  • the cell is present in an individual diagnosed with brain cancer, pancreatic cancer, myoloma, ovarian cancer, hepatocellular cancer, breast cancer, colon carcinoma, or melanoma.
  • the cell is a brain cancer cell, pancreatic cancer cell, myoloma cell, ovarian cancer cell, hepatocellular cancer cell, breast cancer cell, colon carcinoma cell, renal cell carcinoma, carcinoma of the gut, lung or urogenital tract, or melanoma cell.
  • the cell is present in an individual (e.g., human) diagnosed with an infectious or viral disease including, by way of non-limiting example, herpes, diphtheria, papilloma virus, hepatitis, HIV, coronavirus, or adenovirus.
  • infectious or viral disease including, by way of non-limiting example, herpes, diphtheria, papilloma virus, hepatitis, HIV, coronavirus, or adenovirus.
  • O-linked glycan synthesis inhibitors described herein are small molecule organic compounds. In certain instances, O-linked glycan synthesis inhibitors utilized herein are not polypeptides or carbohydrates.
  • a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
  • the O-linked glycan synthesis inhibitor is a modulator (e.g., inhibitor or promoter) of a Core transferase, iGnT, a sialyl transferase, a fucosyl transferase, a ppGalNAc transferase, a ⁇ l-4galactosyltransferase IV, or combinations thereof.
  • a modulator e.g., inhibitor or promoter
  • the disorder mediated by an O-linked glycan is a cancer, a tumor, undesired angiogenesis (e.g., cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, or psoriasis), insufficient angiogenesis (e.g., coronary artery disease, stroke, or delayed wound healing), organomegaly (e.g., hepatosplenomegaly), amyloidosis, skeletal abonormalities, odontoid hypoplasia, hydrops fetalis, inflammation, sialuria, sialidosis, Ehlers-Danlos syndrome, Walker Warburg syndrome, a wound, or the like.
  • undesired angiogenesis e.g., cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, or psoriasis
  • insufficient angiogenesis e.g., coronary artery disease, stroke, or delayed wound healing
  • provided herein is a method of treating cancer by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein.
  • a method of treating a tumor by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein.
  • a method of treating undesired angiogenesis by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O- linked glycan synthesis inhibitor described herein.
  • a method for treating sialyl transferase deficiency comprising administering to a patient suffering from a disease or condition mediated by sialyl transferase deficiency a therapeutically effective amount of an O-linked glycan synthesis inhibitor.
  • the disease or condition mediated by sialyl transferase deficiency is thrombocytopenia, leukospenia, sialidosis, and sialuria.
  • provided herein is a method of treating a lysosomal storage disease by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein.
  • a method of treating a sialuria, sialidosis and/or inflammation by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein.
  • provided herein is a method of treating cancer by administering to an individual (e.g., human) a therapeutically effective amount of any O- linked glycan synthesis inhibitor described herein.
  • the cancer is, by way of non-limiting example, brain cancer, pancreatic cancer, myoloma, ovarian cancer, hepatocellular cancer, breast cancer, colon carcinoma, renal cell carcinoma, carcinoma of the gut, lung or urogenital tract, or melanoma.
  • a method of treating an infectious or viral disease by administering to an individual (e.g., human) a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein.
  • the infectious or viral disease includes, by way of non-limiting example, herpes, diphtheria, papilloma virus, hepatitis, HIV, coronavirus, or adenovirus.
  • a process of inhibiting O-linked glycan function in a cell comprising contacting the cell with a selective modulator of O- linked glycan biosynthesis including e.g., mucin biosynthesis.
  • O- linked glycan biosynthesis includes, by way of non- limiting example, (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis.
  • the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O- linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In specific embodiments, the modulator of O- linked glycan biosynthesis promotes polymerization of O-linked glycans. [00131] In some embodiments, the modulator of O-linked glycan biosynthesis modulates (e.g., promotes or inhibits) Core transferases.
  • the modulator of O-linked glycan Core transferase inhibits the synthesis of the linkage region suitable for connecting O-linked glycan to a core protein, the initiation of O-linked glycan synthesis, the synthesis of glycan polymers, or a combination thereof.
  • modulators of O-linked glycan biosynthesis modulated e.g., promote or inhibit) one or more of a ppGalNAc-T, e.g., ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc- T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc
  • a method for treating GaINAc transferase deficiency comprising administering to a patient suffering from a disease or condition mediated by GaINAc transferase deficiency a therapeutically effective amount of an O-linked glycan biosynthesis inhibitor.
  • the disease or condition mediated by GaINAc transferase deficiency is tumorous calcinosis.
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g.
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM).
  • CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of a O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan.
  • a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding. In some specific embodiments, the Mucin-type O- linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin- type O-linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
  • a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM).
  • the CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • the selective modulator of O-linked glycan biosynthesis is a small molecule organic compound.
  • selective modulator of O-linked glycan biosynthesis utilized herein is not a polypeptide or a carbohydrate.
  • the small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
  • Provided in certain embodiments herein is a method of treating cancer or neoplasia comprising administering a therapeutically effective amount of an O-linked glycan biosynthesis inhibitor to a patient in need thereof.
  • the O- linked glycan biosynthesis inhibitor reduces or inhibits tumor growth, reduces or inhibits angiogenesis, or a combination thereof.
  • the O-linked glycan biosynthesis inhibitor is a selective modulator of a Core transferase (e.g., inhibits one or more core transferases), modulator of polymerization (e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts), selective modulator of O-linked glycan sialylation (e.g., inhibits or promotes one or more sialyl transferase), selective modulator or O-linked glycan fucosylation (e.g.
  • a Core transferase e.g., inhibits one or more core transferases
  • modulator of polymerization e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts
  • selective modulator of O-linked glycan sialylation e
  • an O-linked glycan synthesis inhibitor alters or reduces the function of O-linked glycans by one or more of the following non- limiting manners: (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis.
  • the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes polymerization of O-linked glycans.
  • the O-linked glycan synthesis inhibitor is a selective
  • modulators of O-linked glycan biosynthesis modulated e.g., promote or inhibit) one or more of ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc- TlO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19,
  • ppGalNAc-Ts e.g., ppGalNAc-Tl
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-IO, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • any immunomodulatory factor e.g. IL-5, IL- 3, IL-IO, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM).
  • CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of an O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan.
  • a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding.
  • the Mucin- type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
  • a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM).
  • the CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • O-linked glycan synthesis inhibitors described herein are small molecule organic compounds.
  • O-linked glycan synthesis utilized herein are not polypeptides or carbohydrates.
  • a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
  • a method of treating a lysosomal storage disease comprising administering a therapeutically effective amount of an O-linked glycan biosynthesis inhibitor to an individual (e.g., a human) in need thereof.
  • the O-linked glycan synthesis is a selective inhibitor of a Core transferase (e.g., inhibits one or more core transferases), modulator of polymerization (e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts), selective modulator of O-linked glycan sialylation (e.g., inhibits or promotes one or more sialyl transferase), selective modulator, O-linked glycan fucosylation (e.g. inhibits or promotes one or more fucosyl transferases), or selective modulator of O-linked glycan degradation (e.g. inhibits or promotes one or more of sialidases, glycosidases, fucosidases).
  • a Core transferase e.g., inhibits one or more core transferases
  • modulator of polymerization e.g., inhibits or promotes one or more of iGnT or ppGl
  • an O-linked glycan synthesis inhibitor alters or reduces the function of O-linked glycans by one or more of the following non- limiting manners: (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis.
  • the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes polymerization of O-linked glycans. [00146] In some embodiments, the O-linked glycan synthesis inhibitor is a selective
  • the selective O-linked glycan synthesis inhibitor is a modulator of (e.g., promotes one or more of, or inhibits one or more of) ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc- T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18,
  • ppGalNAc-Ts e.g., promotes one or more of, or inhibit
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • any immunomodulatory factor e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM).
  • CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of an O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan.
  • a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding.
  • the Mucin- type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
  • a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM).
  • the CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • O-linked glycan synthesis inhibitors described herein are small molecule organic compounds.
  • O-linked glycan synthesis inhibitors utilized herein are not polypeptides or carbohydrates.
  • a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
  • a method of reducing the mean or median sialylation or fucosylation of a mucin in (or endogenous to) an individual comprising administering a therapeutically effective amount of a O-linked glycan synthesis inhibitor to a patient in need thereof.
  • the method of reducing the mean or median sialylation or fucosylation of a mucin in (or endogenous to) an individual is suitable for treating cancer or the symptoms thereof.
  • the O-linked glycan synthesis is a selective inhibitor of a Core transferase (e.g., inhibits one or more core transferases), modulator of polymerization (e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts), selective modulator of O-linked glycan sialylation (e.g., inhibits or promotes one or more sialyl transferase), selective modulator or O-linked glycan fucosylation (e.g. inhibits or promotes one or more fucosyl transferases).
  • the cancer is by way of example, a carcinoma, or an adenocarcinoma.
  • the adenocarcinoma is by way of example, a bladder cancer, a breast cancer, a cervical cancer, a colon cancer, an esophagus cancer, a lung cancer, a non- small cell lung cancer, a pancreas cancer, a prostate cancer, a stomach cancer, a urachal cancer, a vaginal cancer.
  • an O-linked glycan synthesis inhibitor alters or reduces the function of O-linked glycans by one or more of the following non- limiting manners: (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis.
  • the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In other embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O-linked glycans. In some embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In further embodiments, the modulator of O-linked glycan biosynthesis promotes polymerization of O-linked glycans. In other embodiments, the modulator of O-linked glycan biosynthesis inhibits degradation of O-linked glycans. In further embodiments, the modulator of O-linked glycan biosynthesis promotes degradation of O-linked glycans.
  • the O-linked glycan synthesis inhibitor is a selective
  • the selective O-linked glycan synthesis inhibitor is a modulator of (e.g., promotes one or more of, or inhibits one or more of) ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc- T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18,
  • ppGalNAc-Ts e.g., promotes one or more of, or inhibit
  • ⁇ -Nacetylhexosaminidase e.g. ⁇ GlcNAc and/or ⁇ GalNAc
  • sialidase e.g. neuraminidase
  • ⁇ -galactosidase ⁇ -glucuronidase
  • ⁇ - galactosidase ⁇ - galactosidase or Cathepsin A
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-IO, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • any immunomodulatory factor e.g. IL-5, IL- 3, IL-IO, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)
  • an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM).
  • CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of an O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan.
  • a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding.
  • the Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O- linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
  • a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor.
  • the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM).
  • CAM cell adhesion molecule
  • the CAM is an exogenous CAM, e.g., bacterial lectins.
  • the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
  • O-linked glycan synthesis inhibitors described herein are small molecule organic compounds.
  • O-linked glycan synthesis inhibitors utilized herein are not polypeptides or carbohydrates.
  • a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
  • processes for identifying inhibitors of the biosynthesis of mucin-type O-linked glycans or for identifying genes involved in (including selective inhibitors or modulators of) the biosynthesis of mucin-type O-linked glycans are also provided herein.
  • processes for identifying modulators of enzymes involved in the biosynthesis of mucin-type O-linked glycans are also provided herein.
  • a cell-based high throughput process for identifying and/or screening for (1) O-linked glycan biosynthesis inhibitors; (2) genes involved in (including selective regulators of) the biosynthesis of mucin-type O-linked glycans; (3) O- linked glycan biosynthesis modulators; or (4) combinations thereof.
  • a library of small-molecule chemical compounds (including oligopeptides and oligonucleotides) is screened; in other embodiments, a library of siRNA is screened; in other embodiments, both types of libraries are simultaneously or sequentially screened.
  • the siRNA library is enzymatically generated; or rationally synthesized; or randomly generated; or a combination thereof.
  • protocols for screening siRNA libraries in high-throughpout genetic screens is found in the Journal of Cancer Molecules: 1(1), 19-24, 2005.
  • a process for identifying a compound that modulates O-linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound in combination with a labeled probe that binds one or more O-linked glycans; b. incubating the mammalian cell, compound and labeled probe; c. collecting the labeled probe that is bound to one or more O-linked glycans; and d. detecting or measuring the amount of the labeled probe bound to one or more O-linked glycans.
  • a process for identifying a compound that selectively modulates O-linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound b. contacting the mammalian cell and compound combination with a first labeled probe and a second labeled probe, wherein the first labeled probe binds one or more O-linked glycans and the second labeled probe binds at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; c.
  • glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • the mammalian cell, compound, the first labeled probe, and the second labeled probe incubating the mammalian cell, compound, the first labeled probe, and the second labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; e. collecting the second labeled probe that is bound to at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; f. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans; and g.
  • glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • at least one glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • a process for identifying compounds that selectively modulate O-linked glycans biosynthesis comprising: a. contacting a first mammalian cell with the compound b. contacting the first mammalian cell and compound combination with a first labeled probe, wherein the first labeled probe binds one or more O-linked glycans; c. incubating the first mammalian cell, compound, the first labeled probe, and the second labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; e. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans; f.
  • contacting a second mammalian cell with the compound wherein the second mammalian cell is of the same type as the first mammalian cell; g. contacting the second mammalian cell and compound combination with a second labeled probe, wherein the second labeled probe binds at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; h.
  • glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • the second labeled probe that is bound to at least one glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • at least one glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • detecting or measuring the amount of the second labeled probe bound to at least one glycan e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like
  • a process for identifying a compound that modulates O-linked glycan biosynthesis comprising: a. collecting O-linked glycans from a first mammalian cell of a selected type, wherein the O-linked glycan comprises a plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 and/or Core 8 O-linked glycans; b. cleaving the O-linked glycans into a plurality of monosaccharide, disaccharide or oligosaccharide component parts; c. detecting or measuring the amount of one of more of the monosaccharide, disaccharide or oligosaccharide component parts; d.
  • a second mammalian cell of the selected type with the compound; e. collecting O-linked glycans from the second mammalian cell of a selected type; f. cleaving the O-linked glycans into a plurality of monosaccharide, disaccharide or oligosaccharide component parts; g. detecting or measuring the amounts of one of more of the monosaccharide, disaccharide or oligosaccharide component parts; h. comparing: i. the amounts of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 and/or Core 8 O-linked glycans or one or more component parts thereof produced by the first and second mammalian cells; ii.
  • monosaccharide, disaccharide or oligosaccharide component parts characteristic of O-linked glycans comprise terminal sialic acid residues in mucin-type O-linked glycans. In some embodiments, monosaccharide, disaccharide or oligosaccharide component parts characteristic of O-linked glycans comprise terminal fucosyl residues in mucin-type O-linked glycans.
  • monosaccharide, disaccharide or oligosaccharide component parts characteristic of O-linked glycans comprise 3-0 sulfated monosaccharide, disaccharide or oligosaccharide component parts in sLe x O-linked glycan structures.
  • the amount of terminal sialylation in O-glycans collected from a first mammalian cell is compared with the amount of terminal sialylation in O-glycans collected from a second mammalian cell.
  • the amount of terminal fucosylation in O-glycans collected from a first mammalian cell is compared with the amount of terminal fucosylation in O-glycans collected from a second mammalian cell.
  • incubating the mixture of the compound with the at least one cell expressing at least one O-linked glycan is performed for a predetermined time. In one embodiment, incubation is for a period of about 12 hours. In another embodiment, incubating the mixture is for a period of about 18 hours. In another embodiment, about 24 hours. In yet another embodiment, about 36 hours. In a further embodiment, 48 hours. In another embodiment, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days.
  • the process(es) described herein are useful for high- throughput analysis of a O-linked glycan biosynthesis inhibitor or a positive or negative regulatory gene for O-linked glycan biosynthesis.
  • the amounts of O-linked glycan and/or monsaccharides, disaccharides or oligosaccharides characteristic of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 and/or Core 8 O-linked glycans are measured with an analytical device.
  • the analytical device is a fluorimeter.
  • the analytical device is a fluorescent plate reader.
  • fluorescence is measured at an excitation of 450 - 600 nm and emission of 600 - 700 nm.
  • the detecting or measuring process is developed using a robotic pipettor.
  • the inhibitor of O-linked glycan biosynthesis is an inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine- modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, and a fucosyl transferase, or a combination thereof.
  • the inhibitor of O-linked glycan biosynthesis is an inhibitor of O- linked mucin-type glycosylation.
  • the process further comprises comparing the amount of first labeled probe bound to O-linked glycans to the amount of the second labeled probe bound to at least one glycan other than O-linked glycans (e.g., to determine a ratio of the amount of first labeled probe bound to the amount of second labeled probe bound under substantially similar conditions).
  • a label utilized in any process described herein is any suitable label such as, by way of non- limiting example, a fluorescent label, a dye, a radiolabel, or the like.
  • the labeled probe comprises a biotinyl moiety and the process further comprises tagging the labeled probe with streptavidin-Cy5-PE.
  • the first probe is any O-linked glycan binding protein, e.g., a selectin.
  • the selectin is, by way of non- limiting example, P-selectin, L- selectin or E-selectin.
  • the amount of bound labeled probes are detected in any suitable manner, e.g., with a fluorimeter, a radiation detector, or the like.
  • the first and second probes are labeled in a manner so as to be independently detectable.
  • the first and second probes are contacted to the cells separately (i.e., to different cells of the same type) and independently analyzed.
  • the at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans is, by way of non- limiting example, chondroitin sulfate, gangliosides, N-linked glycans, heparan sulfate or the like.
  • a third labeled probe that binds at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) not bound by the first or second labeled probe is also utilized.
  • Second and additional labeled probes include any labeled compound or labeled lectin suitable (e.g., a labeled compound or lectin that binds a ganglioside, a GAG, a non-sulfated GAG, an N-linked glycan, an N-linked glycan, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratin sulfate, and/or hyaluronan).
  • labeled compound or labeled lectin suitable e.g., a labeled compound or lectin that binds a ganglioside, a GAG, a non-sulfated GAG, an N-linked glycan, an N-linked glycan, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratin sulfate, and/or h
  • labeled probes included labeled forms of one or more of, by way of non- limiting example, Wheat Germ Agglutinin (WGA) from Triticum vulgaris (as a probe for binding N-linked and O- linked glycans with terminal GIcNAc residues and clustered sialic acid residues); Phaseolus Vulgaris Aggutinin (PHA) from Phaseolus vulgaris (as a probe for binding N-linked glycans); Cholera Toxin B-subunit (CTB) from Vibrio cholera (as a probe for binding sialic acid modified glycolipids); Concanavalin A (ConA) from Canavalia ensiformis (as a probe for binding mannose residues in N-linked glycans); and/or Jacalin from Artocarpus integrifolia (as a probe for binding O-linked glycans).
  • WGA Wheat Germ Agglutinin
  • PHA Phaseolus
  • WGA Wheat Germ Agglutinin
  • PHA Phaseolus Vulgaris Aggutinin
  • CTB Cholera Toxin B-subunit
  • the mammalian cell e.g., human cell
  • the mammalian cell is selected from any suitable mammalian cell.
  • the mammalian cell is, by way of non-limiting example, a human cancer cell (e.g., human cervical cancer cell (HeLa)), a human ovarian cancer cell (SKOV), a human lung cancer cell (Hal8), a human meduloblastoma cancer cell (DAOY), a Chinese Hamster Ovary (CHO) cell, an adenocarcinoma cell, a melanoma cell, or a human primary cell.
  • a human cancer cell e.g., human cervical cancer cell (HeLa)
  • SKOV human ovarian cancer cell
  • Hal8 human lung cancer cell
  • DAOY human meduloblastoma cancer cell
  • CHO Chinese Hamster Ovary
  • the cell includes a plurality (e.g., 2, 3 , 4 or all) of a human cancer cell (e.g., human cervical cancer cell (HeLa)), a human ovarian cancer cell (SKOV), a human lung cancer cell (Hal8), a human meduloblastoma cancer cell (DAOY), and/or a Chinese Hamster Ovary (CHO) cell.
  • a human cancer cell e.g., human cervical cancer cell (HeLa)
  • SKOV human ovarian cancer cell
  • Hal8 human lung cancer cell
  • DAOY human meduloblastoma cancer cell
  • CHO Chinese Hamster Ovary
  • Contact with such cells optionally occurs in parallel, concurrently, or sequentially.
  • contact with multiple cells identifies inhibitors (e.g., selective O-linked glycan synthesis inhibitors) that inhibit O- linked glycan biosynthesis in multiple cell lines.
  • utilization of a plurality of cell lines allows the elimination or minimization of false positives in
  • any process described herein comprises contacting the compound to a first cell (type), contacting the compound to a second cell (type), and, optionally, contacting the compound to additional cells (types), and repeating the process described for each of the first, second and any additional cell types utilized (e.g., to determine if a O-linked glycan inhibitor is selective for multiple cell lines or to determine which types of cell lines that the O-linked glycan inhibitor selectively targets).
  • the process further comprises comparing the amount of labeled probe (or the amount of first, second or any additional labeled probe) that is bound in each type of cell (e.g., to determine selectively of inhibiting O-linked glycan biosynthesis compared to the biosynthesis of other types of glycans).
  • comparing the amount of labeled probe (or the amount of first, second or any additional labeled probe) that is bound in each type of cell e.g., to determine selectively of inhibiting O-linked glycan biosynthesis compared to the biosynthesis of other types of glycans.
  • selectivity of a compound that modulates O-linked glycan biosynthesis is determined by utilizing a similar process as described for determining whether or not the compound modulates O-linked glycan biosynthesis, e.g., by: a. contacting a mammalian cell with the compound in combination with a labeled probe that binds one or more non-O-linked glycan (e.g., GAG or other class of glycan); b. incubating the mammalian cell, compound and labeled probe; c. collecting the labeled probe that is bound to non- O-linked glycan (e.g., GAG or other class of glycan); and d. detecting or measuring the amount of labeled probe bound to non-O-linked glycan (e.g., GAG or other class of glycan).
  • a labeled probe that binds one or more non-O-linked glycan e.g., GAG or other class of g
  • this process is repeated for any number of non- O- linked glycans (e.g., GAG or other class of glycan).
  • the non- O- linked glycans are, by way of non- limiting example, chondroitin sulfate, heparan sulfate, N- linked glycans, gangliosides, or the like.
  • the mammalian cell is selected from any suitable mammalian cell.
  • the mammalian cell is, by way of non- limiting example, a human cancer cell (e.g., human cervical cancer cell (HeLa)) a human ovarian cancer cell (SKOV), a human lung cancer cell (Hal8), a human meduloblastoma cancer cell (DAOY) or a human primary cell.
  • the process is repeated utilizing one or more additional cell types.
  • the results (e.g., of (c), and/or (d)) from the one or more additional cell types are compared to each other and the results (e.g., of (c), and/or (d)) from the first cell type.
  • the O-linked glycans and/or the modified O-linked glycans are cleaved in any suitable manner.
  • the O-linked glycans and/or the modified O-linked glycans are cleaved using a suitable enzyme such as a protease that is selective for highly sialylated, highly substituted mucins, or in any other suitable chemical manner.
  • a suitable enzyme such as a protease that is selective for highly sialylated, highly substituted mucins, or in any other suitable chemical manner.
  • the amount of one of more of monosaccharide, disaccharide or oligosaccharide units present in the cell and/or the characteristic of the O- linked glycans in a cell are determined in any suitable manner.
  • the amount of sialic acid units present and/or the amount of O-sulfation (e.g., 3-O-sulfation) of the glucosylamine groups, or a combination thereof is determined utilizing a carbozole assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), capillary elecrophoresis, gel electrophoseis, mass spectrum (MS) analysis, HPLC electrospray ionization tandem mass spectrometry, nuclear magnetic resonance (NMR) analysis, or the like.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • MS mass spectrum
  • HPLC electrospray ionization tandem mass spectrometry nuclear magnetic resonance (NMR) analysis, or the like.
  • the process described is a process for identifying compounds that selectively modulate O-linked glycan biosynthesis.
  • the process also comprises collecting one or more non-O-linked glycan (e.g., a sulfated glycan, such as chondroitin sulfate, N-linked glycans, or the like) from the cell, both without incubation with the compound and with incubation with the compound; cleaving each of such non-O-linked glycans; measuring the character of each of such non- O-linked glycan; and comparing the character of the non-O-linked glycan that was not incubated with the character of the non-O-linked glycan that was incubated.
  • a non-O-linked glycan e.g., a sulfated glycan, such as chondroitin sulfate, N-linked glycans, or the like
  • the character includes, by way of non-limiting example, the chain length of the non-O-linked glycan, the amount of sulfation of the non-O-linked glycan, the location of sulfation of the non-O-linked glycan, the structure of the non-O-linked glycan, the composition of the non-O-linked glycan, or the like.
  • glycosaminoglycans N-linked glycans, O-linked glycans and lipid linked glycans can be determined using any suitable method, including, by way of non- limiting example, monosaccharide compositional analysis, capillary electrophoresis, gel electrophoresis, gel filtration, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrum (MS) analysis, HPLC electrospray ionization tandem mass spectrometry, nuclear magnetic resonance (NMR) analysis, or the like.
  • HPLC high performance liquid chromatography
  • TLC thin layer chromatography
  • MS mass spectrum
  • HPLC electrospray ionization tandem mass spectrometry nuclear magnetic resonance (NMR) analysis, or the like.
  • At least one therapeutic compound described herein i.e., any O-linked glycan inhibitor described herein
  • another therapeutic agent i.e., any O-linked glycan inhibitor described herein
  • one of the side effects experienced by a patient upon receiving one of the O-linked glycan inhibitors described herein is nausea
  • an anti- nausea agent in combination with the initial therapeutic agent.
  • the therapeutic effectiveness of one of the O-linked glycan inhibitors described herein is enhanced by administration of an adjuvant (i.e., by itself the adjuvant has minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced).
  • the benefit experienced by a patient is increased by administering one of O-linked glycan inhibitors described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit.
  • another therapeutic agent which also includes a therapeutic regimen
  • the overall benefit experienced by the patient is in some embodiments additive of the two therapeutic agents or in other embodiments, the patient experiences a synergistic benefit.
  • the particular choice of compounds depends upon the diagnosis of the attending physicians and their judgment of the condition of the patient and the appropriate treatment protocol.
  • the compounds are optionally administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the patient, and the actual choice of compounds used.
  • the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol is based on an evaluation of the disease being treated and the condition of the patient.
  • therapeutically-effective dosages vary when the drugs are used in treatment combinations.
  • Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.
  • dosages of the co-administered compounds vary depending on the type of co-drug employed, on the specific drug employed, on the disease or condition being treated and so forth.
  • the compound provided herein is optionally administered either simultaneously with the biologically active agent(s), or sequentially. In certain instances, if administered sequentially, the attending physician will decide on the appropriate sequence of therapeutic compound described herein in combination with the additional therapeutic agent.
  • the multiple therapeutic agents are optionally administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). In certain instances, one of the therapeutic agents is optionally given in multiple doses. In other instances, both are optionally given as multiple doses. If not simultaneous, the timing between the multiple doses is any suitable timing, e.g, from more than zero weeks to less than four weeks.
  • the additional therapeutic agent is utilized to achieve remission (partial or complete) of a cancer, whereupon the therapeutic agent described herein (e.g., any O-linked glycan) is subsequently administered.
  • the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations are also envisioned (including two or more therapeutic compounds described herein).
  • a dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought is modified in accordance with a variety of factors. These factors include the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject.
  • the dosage regimen actually employed varies and deviates from the dosage regimens set forth herein.
  • the pharmaceutical agents which make up the combination therapy disclosed herein are provided in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration.
  • the pharmaceutical agents that make up the combination therapy are administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step administration.
  • two-step administration regimen calls for sequential administration of the active agents or spaced-apart administration of the separate active agents.
  • the time period between the multiple administration steps varies, by way of non-limiting example, from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent.
  • the O-linked glycan inhibitors described herein also are optionally used in combination with procedures that provide additional or synergistic benefit to the patient.
  • patients are expected to find therapeutic and/or prophylactic benefit in the methods described herein, wherein pharmaceutical composition of a compound disclosed herein and /or combinations with other therapeutics are combined with genetic testing to determine whether that individual is a carrier of a gene or gene mutation that is known to be correlated with certain diseases or conditions.
  • the O-linked glycan inhibitors described herein and combination therapies are administered before, during or after the occurrence of a disease or condition. Timing of administering the composition containing an O-linked glycan inhibitor is optionally varied to suit the needs of the individual treated.
  • the O-linked glycan inhibitors are used as a prophylactic and are administered continuously to subjects with a propensity to develop conditions or diseases in order to prevent the occurrence of the disease or condition.
  • the compounds and compositions are administered to a subject during or as soon as possible after the onset of the symptoms.
  • the administration of the O-linked glycan inhibitors are optionally initiated within the first 48 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms.
  • the initial administration is achieved by any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof.
  • the compound should be administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months.
  • the length of treatment is optionally varied for each subject based on known criteria.
  • the compound or a formulation containing the compound is administered for at least 2 weeks, between about 1 month to about 5 years, or from about 1 month to about 3 years.
  • therapeutic agents are combined with or utilized in combination with one or more of the following therapeutic agents in any combination: immunosuppressants or anti-cancer therapies (e.g., radiation, surgery or anti-cancer agents).
  • one or more of the anti-cancer agents are proapoptotic agents.
  • anti-cancer agents include, by way of non- limiting example: gossypol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec®), geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, or PD184352, TaxolTM, also referred to as "paclitaxel”, is an anti-cancer drug which acts by enhancing and stabilizing microtubule formation, and analogs of Taxol
  • anti-cancer agents include inhibitors of mitogen- activated protein kinase signaling, e.g., U0126, PD98059, PD184352, PD0325901, ARRY- 142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies (e.g., rituxan).
  • mitogen- activated protein kinase signaling e.g., U0126, PD98059, PD184352, PD0325901, ARRY- 142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002
  • Syk inhibitors e.g., mTOR inhibitors
  • mTOR inhibitors e.g., rituxan
  • anti-cancer agents include Adriamycin, Dactinomycin, Bleomycin,
  • Vinblastine Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefmgol; chlorambucil; cirolemycin;
  • anti-cancer agents include: 20-epi-l, 25 dihydroxyvitamin D3; 5- ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara- CDP-DL-PTBA
  • anticancer agents that include alkylating agents, antimetabolites, natural products, or hormones, e.g., nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, ete.), or triazenes (decarbazine, etc.).
  • nitrogen mustards e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g., carmustine, lomusitne, ete.
  • triazenes decarbazine, etc.
  • antimetabolites include but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin).
  • folic acid analog e.g., methotrexate
  • pyrimidine analogs e.g., Cytarabine
  • purine analogs e.g., mercaptopurine, thioguanine, pentostatin.
  • natural products include but are not limited to vinca alkaloids
  • antibiotics e.g., daunorubicin, doxorubicin, bleomycin
  • enzymes e.g., L-asparaginase
  • biological response modifiers e.g., interferon alpha
  • alkylating agents include, but are not limited to, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin, etc.), or triazenes (decarbazine, ete.).
  • nitrogen mustards e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, etc.
  • ethylenimine and methylmelamines e.g., hexamethlymelamine, thiotepa
  • alkyl sulfonates e
  • antimetabolites include, but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.
  • folic acid analog e.g., methotrexate
  • pyrimidine analogs e.g., fluorouracil, floxouridine, Cytarabine
  • purine analogs e.g., mercaptopurine, thioguanine, pentostatin.
  • hormones and antagonists include, but are not limited to, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), gonadotropin releasing hormone analog (e.g., leuprolide).
  • adrenocorticosteroids e.g., prednisone
  • progestins e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate
  • estrogens e.g., diethlystilbestrol
  • platinum coordination complexes e.g., cisplatin, carboblatin
  • anthracenedione e.g., mitoxantrone
  • substituted urea e.g., hydroxyurea
  • methyl hydrazine derivative e.g., procarbazine
  • adrenocortical suppressant e.g., mitotane, aminoglutethimide
  • provided herein is a method of treating lymphoma comprising administering a therapeutically effective amount of a compound described herein in combination with an antibody to CD20 and/or a CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy.
  • a method of treating leukemia comprising administering a therapeutically effective amount of a compound described herein in combination with ATRA, methotrexate, cyclophosphamide and the like.
  • compositions are formulated in a conventional manner using one or more physiologically acceptable carriers including, e.g., excipients and auxiliaries which facilitate processing of the active compounds into preparations which are suitable for pharmaceutical use.
  • physiologically acceptable carriers including, e.g., excipients and auxiliaries which facilitate processing of the active compounds into preparations which are suitable for pharmaceutical use.
  • proper formulation is dependent upon the route of administration chosen.
  • a summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.
  • a pharmaceutical composition refers to a mixture of an O- linked glycan inhibitor described herein, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the 0-linked glycan inhibitor to an individual or cell.
  • therapeutically effective amounts of O- linked glycan inhibitors described herein are administered in a pharmaceutical composition to an individual having a disease, disorder, or condition to be treated.
  • the individual is a human.
  • the 0-linked glycan inhibitors described herein are either utilized singly or in combination with one or more additional therapeutic agents.
  • the pharmaceutical formulations described herein are administered to an individual in any manner, including one or more of multiple administration routes, such as, by way of non- limiting example, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes.
  • oral parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes.
  • compositions described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.
  • Pharmaceutical compositions including a compound described herein are optionally manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • a pharmaceutical compositions described herein includes one or more O-linked glycan inhibitors described herein, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form.
  • the compounds described herein are utilized as an JV-oxide or in a crystalline or amorphous form (i.e., a polymorph).
  • an active metabolite or prodrug of a compound described herein is utilized.
  • a compound described herein exists as tautomers. All tautomers are included within the scope of the compounds presented herein.
  • a compound described herein exists in an unsolvated or solvated form, wherein solvated forms comprise any pharmaceutically acceptable solvent, e.g., water, ethanol, and the like.
  • solvated forms of the O-linked glycan inhibitors presented herein are also considered to be disclosed herein.
  • a "carrier” includes, in some embodiments, a pharmaceutically acceptable excipient and is selected on the basis of compatibility with O-linked glycan inhibitors disclosed herein, and the release profile properties of the desired dosage form.
  • Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.
  • the pharmaceutical compositions described herein is formulated as a dosage form.
  • a dosage form comprising an O-linked glycan inhibitor described herein, suitable for administration to an individual.
  • suitable dosage forms include, by way of non-limiting example, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.
  • aqueous oral dispersions liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.
  • the pharmaceutical solid dosage forms described herein optionally include an additional therapeutic compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof.
  • a compatible carrier such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of the O-linked glycan inhibitor.
  • an O-linked glycan inhibitor described herein is in the form of a particle and some or all of the particles of the compound are coated. In certain embodiments, some or all of the particles of an O- linked glycan inhibitor described herein are microencapsulated. In some embodiment, the particles of the O-linked glycan inhibitor described herein are not microencapsulated and are uncoated.
  • the pharmaceutical composition described herein is in unit dosage forms suitable for single administration of precise dosages.
  • the formulation is divided into unit doses containing appropriate quantities of one or more therapeutic compound.
  • the unit dosage is in the form of a package containing discrete quantities of the formulation.
  • Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • Aqueous suspension compositions are optionally packaged in single-dose non-reclosable containers.
  • multiple-dose re-closeable containers are used.
  • multiple dose containers comprise a preservative in the composition.
  • formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative.
  • unit dosage form which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative.
  • Cells are grown in the presence of GalNAc ⁇ -O-benzyl for two days, then analyzed by flow cytometry using fluorescently labeled versions of WGA. Quantification of the inhibition of binding is carried out using streptavidin-Cy5-PE. LS 180 shows reduced binding of MAH and CSLEX-I after GalNAc ⁇ -O-benzyl treatment. This indicates that
  • WGA lectins primarily bind O-linked structures on this cell line.
  • Figure 1 see Hongsachart et al, Glycoproteomic analysis ofWGA-bound glycoprotein biomarkers in sera from patients with lung adenocarcinoma, Electrophoresis
  • O-linked glycan synthesis inhibitors are tested on at least three independent occasions, in duplicate over a dose range.
  • O-linked glycan specificity is then determined by probing with lectins and/or proteins that bind to other glycan classes (chondroitin sulfate, Heparan sulfate, N-linked, etc.).
  • Free glycans are obtained from the peptide backbone by hydrazino lysis which involves reacting with hydrazine (hydrazinolysis), acetylating with acetic anhydride / sodium bicarbonate, acidification and purification of the free glycans.
  • Preferential release of O-linked glycans is selected by altering the conditions (time and temperature) of the hydrazinolysis reaction.
  • the released glycans are labeled e.g. with the fluorescent tag, 2-aminobenzamide (2-AB) by reductive amination.
  • Glycan structures can then be analyzed by HPLC (e.g.
  • composition and sequence of the glycans can be further analyzed by digestion at specific monosaccharide residues with one or a combination of specific glycosidases e.g. sialic acid (A. ureafaciens sialidase), galactose (S. pneumoniae ⁇ -galactosidase), fucose (bovine epididymis ⁇ -fucosidase), N- acetylhexosamine (jackbean ⁇ -N-acetylhexosaminidase), N-acetylglucosamine (S.
  • sialic acid A. ureafaciens sialidase
  • galactose S. pneumoniae ⁇ -galactosidase
  • fucose bovine epididymis ⁇ -fucosidase
  • N- acetylhexosamine jackbean ⁇ -N-acetylhexosaminidase
  • Unlabeled glycans are analyzed by mass spectrometry (MS). In addition, sialic acid residues are esterified. Neutral (digested as outlined above) and sialic acid methyl ester containing oligosaccharides are analyzed by MS including MALDI MS on an instrument externally calibrated with a mixture of dextran oligomers [00218] Another method for analyzing glycans on glycoproteins involves removing
  • N-linked glycans from the polypeptide with the enzyme Peptide: N-Glycosidase F, also known as PNGase F.
  • Peptide also known as PNGase F.
  • cell or tissue material is extracted with detergent. Then it is reduced, carboxymethylated, digested with trypsin and the glycopeptides purified by reverse phase Cl 8 column chromatography.
  • N-linked glycans are released from the peptides with PNGase F while the O-linked glycan containing peptides are repurified by reverse phase C 18 column chromatography.
  • O-linked glycans are then released from the peptides by reductive elimination (NaH4 and NaOH), desalted and cleaned up with a reverse phase C18 column chromatography (C 18 Sep-Pak cartridge).
  • the purified O-linked glycans are permethylated and can be analyzed by various techniques including matrix-assisted laser desorption ionization "time-of-flight" (MALDI-TOF) and collisionally activated dissociation electrospray tandem mass spectrometry (CAD-ES- MS/MS).
  • MALDI-TOF matrix-assisted laser desorption ionization
  • CAD-ES- MS/MS collisionally activated dissociation electrospray tandem mass spectrometry
  • linkage analyses the permethylated glycans are hydrolyzed, reduced, acetylated and analyzed by gas chromatography mass spectrometry (GC-MS).
  • GC-MS gas chromatography mass spectrometry
  • LS 180 cells were treated with a carbohydrate-based inhibitor of O-linked sialylation, GalNAc ⁇ -O-benzyl, and with sialidase (2 hr at 37 0 C). As seen in Figure 2, LS 180 cells show reduced binding to Maackia Amurensis Lectin II (MAH) after treatment with GalNAc ⁇ -O-benzyl. LS 180 cells treated with sialidase also showed reduced binding to biotinylated MAH (identified with PE-Cy5 Streptavidin), which verifies that the lectin binds primarily to sialic acid structures in O-linked glycans.
  • MAH Maackia Amurensis Lectin II
  • LS 180 cells treated with sialidase also showed reduced binding to biotinylated MAH (identified with PE-Cy5 Streptavidin), which verifies that the lectin binds primarily to sialic acid structures in O-linked glycans.
  • CHO cells make only core 1 type oligosaccharides, since they lack the Core 2 N-acetylglucosamine transferase (C2GlcNAc-T). Thus, the use of this cell line to screen for inhibitors that block the formation of sialylated core 1 is contemplated herein.
  • C2GlcNAc-T Core 2 N-acetylglucosamine transferase
  • CHO-lecl cells are stably transfected with human C2GlcNAc-T. 36% of O-linked glycans produced by CHO cells transfected with C2GlcNAc-T consist of Core 2 structures.
  • Core 2 glycans are further modified with O-linked sialic acid by endogenous ⁇ 2,3 sialic acid transferases.
  • endogenous ⁇ 2,3 sialic acid transferases In order to induce the expression of the sLe x structure, cells with human ⁇ l,3 fucosyltransferase VII (Fuc-TVII) are also transfected.
  • Efficient transfection and expression of the transferases are monitored by flow cytometry using fluorescent lectins.
  • Individual clonal lines are isolated based on increased WGA and MAH II binding and transfected with Fuc-TVII to determine the level of CSLEX-I binding.
  • CHO lines having the desired characteristics are then tested with the methods as described above.
  • Figure 3 illustrates the affects of O-linked inhibitor compounds on the ability of Maakia Amurensis Lectin II (MaI II) to bind to treated and untreated U937 cells.
  • Other compounds listed in Figures 7A- 7L demonstrated similar activity.
  • EXAMPLE 5
  • MAL II binds to glycans containing sialic acid residues, e.g., in an ( ⁇ -2,3) linkage.
  • FGF2 is specific for another class of glycans (heparan sulfate).
  • Cultured DAOY cells were treated with and without the test compounds. After 2 days of growth the cells were released with 5 mM EDTA. Parallel cultures were then probed with either MAL II or FGF2 for 1 hour on ice. After washing to remove unbound lectin, bound lectins were detected with streptavidin-Cy5-PE. After washing to remove the unbound streptavidin-Cy5- PE the bound probes were quantified (separately) using flow cytometry.
  • FIG. 4-6 illustrate the specificity of the indicated O-linked inhibitors by probing with Maakia Amurensis Lectin II (MaI II) and with another lectin, fibroblast growth factor 2 (FGF2). These compounds show selective inhibition of O-linked glycans without a reduction in the unrelated glycan - heparan sulfate. This demonstrates that these inhibitors have glycan class selectivity.
  • Other compounds listed in Figures 7A- 7K demonstrated similar activity.
  • EXAMPLE 6 Method of Treatment [00228] Human Clinical Trial of the Safety and/or Efficacy of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) therapy.
  • selective O-linked glycan biosynthesis inhibitor e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof
  • Study Design This will be a Phase I, single-center, open-label, randomized dose escalation study followed by a Phase II study in cancer patients with a cancer that can be biopsied (e.g., adenocarcinoma). Patients should not have had exposure to an O-linked glycan biosynthesis inhibitor prior to the study entry. Patients must not have received treatment for their cancer within 2 weeks of beginning the trial. Treatments include the use of chemotherapy, hematopoietic growth factors, and biologic therapy such as monoclonal antibodies. The exception is the use of hydroxyurea for patients with WBC > 30 x 103/ ⁇ L. This duration of time appears adequate for wash out due to the relatively short-acting nature of most anti-leukemia agents.
  • Phase I Patients receive (e.g., intravenous, oral, ip, or the like) selective O- linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) daily for 5 consecutive days or 7 days a week. Doses of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A- 7L, or a pharmaceutically acceptable salt thereof) may be held or modified for toxicity based on assessments as outlined below.
  • selective O-linked glycan biosynthesis inhibitor e.g., a compound of Figures 7A- 7L, or a pharmaceutically acceptable salt thereof
  • Treatment repeats every 28 days in the absence of unacceptable toxicity.
  • Cohorts of 3-6 patients receive escalating doses of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) until the maximum tolerated dose (MTD) for the selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) is determined.
  • the MTD is defined as the dose preceding that at which 2 of 3 or 2 of 6 patients experience dose-limiting toxicity.
  • Blood Sampling Serial blood is drawn by direct vein puncture before and after administration of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof).
  • Venous blood samples (5 mL) for determination of serum concentrations are obtained at about 10 minutes prior to dosing and at approximately the following times after dosing: days 1, 2, 3, 4, 5, 6, 7, and 14. Each serum sample is divided into two aliquots. All serum samples are stored at -2O 0 C. Serum samples are shipped on dry ice.
  • Pharmacokinetics Patients undergo plasma/serum sample collection for pharmacokinetic evaluation before beginning treatment and at days 1, 2, 3, 4, 5, 6, 7, and 14. Pharmacokinetic parameters are calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetics parameters are determined: peak serum concentration (C max ); time to peak serum concentration (t max ); area under the concentration- time curve (AUC) from time zero to the last blood sampling time (AUCo-72) calculated with the use of the linear trapezoidal rule; and terminal elimination half- life (ti/2), computed from the elimination rate constant. The elimination rate constant is estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot.
  • Patient response is assessed via imaging with X-ray, CT scans, and MRI, and imaging is performed prior to beginning the study and at the end of the first cycle, with additional imaging performed every four weeks or at the end of subsequent cycles. Imaging modalities are chosen based upon the cancer type and feasibility/availability, and the same imaging modality is utilized for similar cancer types as well as throughout each patient's study course. Response rates are determined using the RECIST criteria. (Therasse et al, J. Natl. Cancer Inst.

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Abstract

Provided herein are O-linked glycan inhibitors, including modulators of polypeptide GalNAc transferase, core transferase, polylactosamine-modifying glycosyl transferase, polylactosamine extension enzyme, core 1 specific chaperone, sialyl transferase, fucosyl transferase, and O-linked glycan sulfotransferases.

Description

O-LINKED GLYCAN BIOSYNTHESIS MODULATORS
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 61/168,527, filed 10 April 2009, and U.S. Provisional Application No. 61/290,350, filed 28 December 2009, which applications are incorporated herein by reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] Certain inventions described herein were made with the support of the United States government under Contract 1 R21 NS053595 by the National Institutes of Health.
BACKGROUND OF THE INVENTION
[0003] O-linked glycans are found in mammals and comprise a plurality of saccharide units linked by O-glycosidic linkages. An O-glycoprotein comprises one or more glycans linked to a protein via an oxygen atom, e.g. via a hydroxy group of a Serine, Threonine or Hydroxylysine residue.
SUMMARY OF THE INVENTION
[0004] Provided in certain embodiments, herein is a process for modifying populations of O-linked glycans. In some embodiments, such processes comprise contacting cells that produce such O-linked glycans with a modulator or O-linked glycan biosynthesis (e.g., an inhibitor of O-linked glycan biosynthesis). In specific embodiments, the O-linked glycan biosynthesis modulators are selective for O-linked glycans (e.g., such modulators do not significantly affect the biosynthesis of other glycans, such as N-linked glycans, gangliosides, heparan sulfate, chondroitin sulfate, keratan sulfate, or the like). In some embodiments, O-linked glycan biosynthesis modulators selectively modulate the late stage processes of O-linked glycan biosynthesis. Furthermore, provided in certain embodiments herein is a process for modifying the population of O-linked glycans on a core protein or core proteins, comprising contacting a cell that translationally produces at least one core protein having at least one attached O-linked glycan moiety with an effective amount of a selective inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine -modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, a fucosyl transferase, or a combination thereof.
. 1 . WSGR Ref. 36670-704.601 [0005] Provided in some embodiments herein is a process for modifying the population of O-linked glycans on one or more proteins associated with a cell, the process comprising contacting a cell that produces O-linked glycans with an effective amount of a selective O- linked glycan biosynthesis inhibitor, the selective O-linked glycan biosynthesis inhibitor being active in a mammalian cell. In certain embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein is a selective late stage O- linked glycan biosynthesis inhibitor. In some embodiments, the selective late stage O-linked glycan biosynthesis inhibitor utilized in any process described herein is a non-carbohydrate O-linked glycan biosynthesis inhibitor. In certain embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein has a molecular weight of less than 700 g/mol.
[0006] In some embodiments, any process described herein reduces the ratio of glycans containing poly-N-acetyllactosamine chains to glycans that do not contain poly-N- acetyllactosamine chains. In certain embodiments, any process described herein reduces the ratio X: Y of glycans containing poly-N-acetyllactosamine chains to glycans not containing poly-N-acetyllactosamine chains by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, wherein the ratio X:Y changes to 0.9X:Y, 0.8X:Y, 0.7X:Y, 0.6X:Y, 0.5X:Y, 0.4X:Y, 0.3X:Y, 0.2X:Y, 0.1X:Y, respectively.
[0007] In certain embodiments, any process described herein reduces the amount of (αl-2) fucosylation, (αl-3) fucosylation, (αl-4) fucosylation, (α2-3) sialylation, (α2-6) sialylation, terminal (βl-3) galactosylation, terminal (βl-4) galactosylation, 3-O-sulfation, 6-O- sulfation in the cellular population of O-linked glycan. In some embodiments, any process described herein reduces the amount of terminal sialylation and/or terminal GIcNAc residues. In certain embodiments, any process described herein reduces the amount of selectin ligands in the cellular population of O-linked glycan. In specific embodiments, the selectin ligand is sialyl Lewis X or 6-O-sulfated sialyl Lewis X (sLex or 6-sulfo-sLex). In certain embodiments, any process described herein reduces the cellular population of Core 1, Core 2, Core 3, and/or Core 4 O-linked glycan structures. In some embodiments, any process described herein reduces the cellular population of Core 1 , and/or Core 2 O-linked glycan structures.
[0008] In certain embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein reduces the ratio of acidic O-linked glycans to neutral O- linked glycans to less than 50% (e.g., if the original ratio is x:(x+y), a reduction to less than 50% would provide a ratio of <0.5x:(x+y)), less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, wherein an acidic O-linked glycan contains sialic acid and/or is sulfated, and wherein a neutral O-linked glycan contains no sialic acid and/or is not sulfated. [0009] In some embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein inhibits Core 1 (βl-3) galctosyl transferase, core 2 (βl-6) N- acetylglucosaminyl transferase, core 3 (βl-6) N-acetylglucosaminyl transferase, core 2/4 (βl-6) N-acetylglucosaminyl transferase, Lewis (αl-3/1-4) fucosyl transferase, FucTIII, FucTIV, FucT VII, ST3GalI, ST3GalII, ST3GalIII, ST3GalIV, GlcNAc6ST-l, GlcNAc6ST-2, Gal3ST4. In specific embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process herein indirectly inhibits the Core 1 (βl-3) galctosyl transferase, Core 2 (βl-6) N-acetylglucosaminyl transferase, core 3 (βl-6) N- acetylglucosaminyl transferase, Core 2/4 (βl-6) N-acetylglucosaminyl transferase, Lewis (αl-3/1-4) fucosyl transferase, FucTIII, FucTIV, FucT VII, ST3GalI, ST3GalII, ST3GalIII, ST3GalIV, GlcNAc6ST-l, GlcNAc6ST-2, Gal3ST4. In another specific embodiment, the selective O-linked glycan biosynthesis inhibitor utilized in any process herein directly inhibits the Core 1 (βl-3) galctosyl transferase, Core 2 (βl-6) N-acetylglucosaminyl transferase, Core 3 (βl-6) N-acetylglucosaminyl transferase, Core 2/4 (βl-6) N- acetylglucosaminyl transferase, Lewis (αl-3/1-4) fucosyl transferase, FucTIII, FucTIV, FucT VII, ST3GalI, ST3GalII, ST3GalIII, ST3GalIV, GlcNAc6ST-l, GlcNAc6ST-2, Gal3ST4. In certain embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process described herein inhibits ST3GalI, ST3GalII, or ST3GalIII. In specific embodiments, the selective O-linked glycan biosynthesis inhibitor utilized in any process herein indirectly inhibits ST3GalI, ST3GalII, or ST3GalIII. In another specific embodiment, the selective O-linked glycan biosynthesis inhibitor utilized in any process herein directly inhibits the ST3GalI, ST3GalII, or ST3GalIII.
[0010] In some embodiments, the cell contacted by any process described herein is an inflammatory cell or cancer cell. In certain embodiments, the cell contacted by any process described herein is present in an individual diagnosed with or suspected of having rheumatoid arthritis, Crohn's disease, reperfusion injury or having had an organ transplant. In some embodiments, the cell contacted by any process described herein is present in an individual diagnosed with or suspected of having adenocarcinoma. In certain embodiments, the cell contacted by any process described herein is a breast cancer cell, a colon cancer cell, a prostate cancer cell, or a non-small cell lung cancer cell. [0011] In on aspect, disclosed herein is a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprise Core 1, Core 2, Core 3, and Core 4 O-linked glycans, and wherein less than 20 mol. %, less than 19 mol. %, less than 18mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. % , less than 5 mol. %, less than 2 mol. %, less than 1 mol. % of the Core 1, Core 2, Core 3, and Core 4 O-linked glycans comprise terminal fucosyl groups. [0012] In one aspect, disclosed herein is a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprise Core 1 and Core 2 O-linked glycans, and wherein less than 50 mol. %, less than 45 mol. %, less than 40 mol. %, less than 35 mol. %, less than 30 mol. %, less than 25 mol. %, less than 20 mol. %, less than 19 mol. %, less than 18 mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. % , less than 5 mol. %, less than 2 mol. %, less than 1 mol. % of the Core 1 and Core 2, O-linked glycans comprise terminal sialyl groups. [0013] In some embodiments, disclosed herein is a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein less than 50 mol. %, less than 45 mol. %, less than 40 mol. %, less than 35 mol. %, less than 30 mol. %, less than 25 mol. %, less than 20 mol. %, less than 15 mol. %, less than 10 mol. %, less than 5 mol. % of the plurality of O-linked glycans comprise terminal sialyl groups.
[0014] In some embodiments, disclosed herein is a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprises less than 20 mol. %, less than 19 mol. %, less than 18 mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. %, less than 5 mol. %, less than 2 mol. %, less than 1 mol. % sLex.
[0015] In certain embodiments, disclosed herein is a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein the plurality of O-linked glycans comprises a plurality of sLex antigens, and wherein less than 20%, less than 19 mol. %, less than 18mol. % , less than 17 mol. % , less than 16 mol. % , less than 15 mol. % , less than 10 mol. % , less than 5 mol. %, less than 2 mol. %, less than 1 mol. % of the sLex antigens are 6-0 sulfated. [0016] In some embodiments, disclosed herein is a composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein less than 50 mol. %, less than 45 mol. %, less than 40 mol. %, less than 35 mol. %, less than 30 mol. %, less than 25 mol. %, less than 20 mol. %, less than 15 mol. %, less than 10 mol. %, less than 5 mol. % of glycoproteins bind to- Wheat Germ Agglutinin (WGA).
[0017] In one aspect is a process for modifying the structure of an O-linked glycan on a core protein, the process comprising contacting a cell that translationally produces at least one core protein having at least one attached O-linked glycan moiety with an effective amount of a selective inhibitor of O-linked glycan biosynthesis.
[0018] In one embodiment is a process for modifying the structure of an O-linked glycan on a core protein, wherein the selective inhibitor of O-linked glycan biosynthesis is an inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine-modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, a fucosyl transferase, or a combination thereof.
[0019] In another embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc- TI l, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-T24, or a combination thereof. In one embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In one embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4. In one embodiment the inhibitor of the core transferase is an inhibitor of a core 1 βl-3-galactosyltransferase, a core 2 βl-6-N-acetylglucosaminyl transferase, a core 3 βl-3-N-acetylglucosaminyl transferase, a core 4 βl-6-N- acetylglucosaminyl transferase, a core 5 αl-3-N-acetylgalactosyl transferase, a core 6 βl-6- N-acetylglucosaminyl transferase, a core 7 αl-6-N-acetylgalactosyl transferase, a core 8 α 1-3 -galactosyl transferase, or a combination thereof. In one embodiment the inhibitor is an inhibitor of the core 1 βl-3-galactosyltransferase. In one embodiment the inhibitor is an inhibitor of the core 2 βl-6-N-acetylglucosaminyl transferase. In one embodiment the inhibitor of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV, ST3Gal VI or a combination thereof. In yet another embodiment the inhibitor of the sialyl transferase inhibits the addition of a sialic acid residue via an α2,3 linkage or α2-6 linkage. In a further embodiment the core 1 specific chaperone is Cosmc. In yet a further embodiment the polylactosamine extension enzyme is an i-extension enzyme or a βl-4 galactosyl transferase IV. In one embodiment the i-extension enzyme is iGnT. In another embodiment the inhibitor of the fucosyl transferase is a selective inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof. In yet another embodiment the inhibitor of the fucosyl transferase inhibits the addition of a fucose residue via an α 1,2 linkage, an α 1,3 linkage or an αl,4 linkage. In a further embodiment the inhibitor of the polypeptide GaINAc transferase inhibits the synthesis of the linkage region, the modification of the linkage region, the initiation of 0-linked glycan synthesis, the synthesis of 0-linked glycan, or a combination thereof. In yet a further embodiment the 0-linked glycan is selected from a blood group antigen A, a blood group antigen B, a blood group antigen H, a Lewis antigen Lex, a Lewis antigen sLex, and a Lewis antigen LeY.
[0020] In one aspect is a process of inhibiting 0-linked glycan function in a cell comprising contacting the cell with an effective amount of a selective modulator of O-linked glycan biosynthesis.
[0021] In yet another embodiment the selective modulator of O-linked glycan biosynthesis is an inhibitor of a polypeptide GaINAc transferase, a modulator of core transferase, a modulator of polylactosamine-modifying glycosyl transferase, a modulator of polylactosamine extension enzyme, a modulator of core 1 specific chaperone, a modulator of sialyl transferase, and a modulator of fucosyl transferase. In a further embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc- T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-T24, or a combination thereof. In yet a further embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In one embodiment the inhibitor of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4. In another embodiment the inhibitor of the core transferase is an inhibitor of a core 1 βl-3-galactosyltransferase, a core 2 βl-6-N-acetylglucosaminyl transferase, a core 3 βl-3-N-acetylglucosaminyl transferase, a core 4 βl-6-N-acetylglucosaminyl transferase, a core 5 αl-3-N-acetylgalactosyl transferase, a core 6 βl-6-N-acetylglucosaminyl transferase, a core 7 αl-6-N-acetylgalactosyl transferase, a core 8 α 1-3 -galactosyl transferase, or a combination thereof. In yet another embodiment the inhibitor is an inhibitor of the core 1 βl-3-galactosyltransferase. In a further embodiment the inhibitor is an inhibitor of the core 2 βl-6-N-acetylglucosaminyl transferase. In yet a further embodiment the inhibitor of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV or ST3Gal VI or a combination thereof. In one embodiment the inhibitor of the sialyl transferase inhibits the addition of a sialic acid residue via an α2,3 linkage or an α2-6 linkage. In another embodiment the core 1 specific chaperone is Cosmc. In yet another embodiment the polylactosamine extension enzyme is an i-extension enzyme or a β 1-4 galactosyl transferase IV. In a further embodiment the i-extension enzyme is iGnT. In yet a further embodiment the inhibitor of the fucosyl transferase is an inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof. In one embodiment the inhibitor of the fucosyl transferase inhibits the addition of a fucose residue via an αl,2 linkage, an αl,3 linkage or an αl,4 linkage or a combination thereof. In another embodiment the 0-linked glycan function inhibited is an ability to bind an 0-linked glycan binding lectin. In a further embodiment the 0-linked glycan binding lectin is a E-selectin, L-selectin or P-selectin.
[0022] In yet a further embodiment the cell being contacted is a cell in need thereof, a cell present in an individual suffering from a disease or condition mediated by abnormal O- linked glycan biosynthesis and/or the cell itself is a cell with abnormal 0-linked glycan biosynthesis, a cell present in an individual with normal 0-linked glycan biosynthesis and/or the cell itself is a cell with normal 0-linked glycan biosynthesis. In some embodiments, the cell being contacted is a cell present in an individual with normal O- linked glycan biosynthesis (e.g., an individual with a predisposition for or suspected of having a disease or condition mediated by 0-linked glycan biosynthesis) and/or the cell itself is a cell with normal 0-linked glycan biosynthesis.
[0023] Also presented herein is a method of treating cancer comprising administering a therapeutically effective amount of a selective modulator of polypeptide GaINAc transferase, a core transferase, a polylactosamine -modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, and a fucosyl transferase. [0024] In one embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc- T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-T24, or a combination thereof. In another embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In yet another embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4. In a further embodiment the selective modulator of the core transferase is an inhibitor of a core 1 βl-3-galactosyltransferase, a core 2 βl-6-N-acetylglucosaminyl transferase, a core 3 βl-3- N-acetylglucosaminyl transferase, a core 4 βl-6-N-acetylglucosaminyl transferase, a core 5 αl-3-N-acetylgalactosyl transferase, a core 6 βl-6-N-acetylglucosaminyl transferase, a core 7 αl-6-N-acetylgalactosyl transferase, a core 8 α 1-3 -galactosyl transferase, or a combination thereof. In yet a further embodiment the selective modulator is an inhibitor of the core 1 βl-3-galactosyltransferase. In one embodiment the selective modulator is an inhibitor of the core 2 βl-6-N-acetylglucosaminyl transferase. In another embodiment the selective modulator of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV or ST3Gal VI or a combination thereof. In a further embodiment the selective modulator of the sialyl transferase inhibits the addition of a sialic acid residue via an α2,3 linkage or an α2-6 linkage. In one embodiment the core 1 specific chaperone is Cosmc. In yet a further embodiment the polylactosamine extension enzyme is an i-extension enzyme or a βl-4 galactosyl transferase IV. In one embodiment the i-extension enzyme is iGnT. In one embodiment the selective modulator of the fucosyl transferase is an inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof. In another embodiment the selective modulator has a molecular weight of less than 1,000 g/mol. In yet another embodiment the selective modulator has a molecular weight of less than 700 g/mol. [0025] Presented herein is a method of treating a lysosomal storage disease comprising administering a therapeutically effective amount of a selective inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine-modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, and a fucosyl transferase. [0026] In one embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc- T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-T24, or a combination thereof. In one embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T3. In a further embodiment the selective modulator of the polypeptide GaINAc transferase is an inhibitor of ppGalNAc-T4. In yet a further embodiment the selective modulator of the core transferase is an inhibitor of a core 1 βl-3- galactosyltransferase, a core 2 βl-6-N-acetylglucosaminyl transferase, a core 3 β 1-3 -N- acetylglucosaminyl transferase, a core 4 βl-6-N-acetylglucosaminyl transferase, a core 5 αl-3-N-acetylgalactosyl transferase, a core 6 βl-6-N-acetylglucosaminyl transferase, a core 7 αl-6-N-acetylgalactosyl transferase, a core 8 α 1-3 -galactosyl transferase, or a combination thereof. In another embodiment the selective modulator is an inhibitor of the core 1 βl-3-galactosyltransferase. In yet another embodiment the selective modulator is an inhibitor of the core 2 βl-6-N-acetylglucosaminyl transferase. In a further embodiment the selective modulator of the sialyl transferase is an inhibitor of ST6Gal I, ST3Gal IV or ST3Gal VI or a combination thereof. In one embodiment the selective modulator of the sialyl transferase inhibits the addition of a sialic acid residue via an α2,3 linkage or an α2-6 linkage. In yet a further embodiment the core 1 specific chaperone is Cosmc. In another embodiment the polylactosamine extension enzyme is an i-extension enzyme or a βl-4 galactosyl transferase IV. In a further embodiment the i-extension enzyme is iGnT. In one embodiment the selective modulator of the fucosyl transferase is an inhibitor of fucosyl transferase VII or fucosyl transferase IV or a combination thereof. [0027] In one aspect is a process for modulating 0-linked glycan degradation in a cell comprising contacting the cell with an effective amount of a selective modulator of an endoglycosidase, an exoglycosidase or a combination thereof. [0028] In one embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of β-N-acetylhexoaminidase. In yet another embodiment the β-N-acetylhexoaminidase is β-GlcNAcase or β-GalNAcase. In a further embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of sialidase. In one embodiment the sialidase is neuraminidase. In another embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a β-galactosidase. In a further embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a β-glucoronidase. In one embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a α-galactosidase. In yet another embodiment the selective modulator of the endoglycosidase or exoglycosidase is a promoter of a Cathepsin A.
[0029] In some embodiments, the cell being contacted is a cell in need thereof, a cell present in an individual suffering from a disease or condition mediated by abnormal O- linked glycan biosynthesis and/or the cell itself is a cell with abnormal O-linked glycan biosynthesis, a cell present in an individual with normal O-linked glycan biosynthesis and/or the cell itself is a cell with normal O-linked glycan biosynthesis. [0030] In yet a further embodiment the O-linked glycan is a mucin type O-linked glycan. [0031] Also provided herein is a process for identifying a compound that modulates O- linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound b. contacting the mammalian cell and compound combination with a first labeled probe wherein the first labeled probe binds one or more O-linked glycans; c. incubating the mammalian cell, compound, and the first labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; and e. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans.
[0032] Further provided herein is a process for identifying a compound that selectively modulates O-linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound b. contacting the mammalian cell and compound combination with a first labeled probe and a second labeled probe, wherein the first labeled probe binds one or more O-linked glycans and the second labeled probe binds at least one glycan other than O-linked glycans; c. incubating the mammalian cell, compound, the first labeled probe, and the second labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; e. collecting the second labeled probe that is bound to at least one glycan other than O-linked glycans; f. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans; and g. detecting or measuring the amount of the second labeled probe bound to at least one glycan other than O-linked glycans.
[0033] In some embodiments, the mammalian cell is a human sLex positive cancer cell. In some embodiments, the labeled probe comprises a biotinyl moiety and the process further comprises tagging the labeled probe with streptavidin-Cy5-PE. In some embodiments, the labeled probe comprises a fluorescent label. In some embodiments, the first labeled probe is a labeled protein. In some embodiments, the labeled protein is a O-linked glycan -specific lectin. In some embodiments, the second labeled probe is a labeled lectin. In some embodiments, the labeled lectin is a lectin specific for a glycan other than an O-linked glycan.
[0034] Provided, in some embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans, and wherein less than 25 mol %, less than 20 mol %%, less than 15 mol %, less than 10 mol %, less than 5 mol % of the plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans comprise terminal sialyl groups. [0035] Provided, in some embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans, and wherein less than 25 mol %, less than 20 mol %, less than 15 mol %, less than 10 mol %, less than 5 mol % of the plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 O-linked glycans comprise terminal fucosyl groups.
[0036] Provided, in some embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens, and wherein less than 20% of the plurality of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens are 6-0 sulfated.
[0037] Provided, in some embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens, and wherein less than 20% of the plurality of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens are 3-0 sulfated.
[0038] Provided, in certain embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of sLex antigens, and wherein less than 20% of the sLex antigens are 6-0 sulfated.
[0039] Provided, in certain embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of Lex antigens, and wherein less than 20% of the Lex antigens are 3-0 sulfated.
[0040] Provided in certain embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of sLeA antigens, and wherein less than 25 mol %, less than 20 mol %, less than 15 mol %, less than 10 mol %, less than 5 mol % of the sLeA antigens are
6-0 sulfated.
[0041] Provided in certain embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises a plurality of LeA antigens, and wherein less than 25 mol %, less than 20 mol %, less than 15 mol %, less than 10 mol %, less than 5 mol % of the LeA antigens are 3-
O sulfated.
[0042] Provided, in certain embodiments, is a process for modulating the amount of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in or on a cell comprising contacting the cell with an effective amount of a selective modulator of O-linked glycan biosynthesis.
[0043] Provided, in certain embodiments, is an O-linked proteoglycan comprising a core protein covalently linked to at least one O-linked glycan, wherein the at least one O-linked glycan comprises one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof, and wherein the amount of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof is 40 mol % (or 30 mol %, or 20 mol %, or 10 mol %) less than the amount of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof, in an endogenous O-linked proteoglycan.
[0044] Other objects and features of the methods, compositions and uses described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The features disclosed herein are set forth with particularity in the appended claims.
A better understanding of the features and advantages will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the embodiments are utilized, and the accompanying drawings of which:
[0046] Figure 1 illustrates the Wheat Germ Agglutinin (WGA) binding proteins (lane 3) are greater than the unbound proteins (lane 4) and representing over 50% of the total proteins in normal human serum.
[0047] Figure 2 illustrates O-linked sialic acid dependence on Maackia Amurensis Lectin II
(MAH). Untreated LS 180 cells bind MAH while treatment with GalNAca-O-benzyl, a carbohydrate-based inhibitor, reduced MAH binding.
[0048] Figure 3 illustrates the affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II
(MaI II) in treated and untreated cells.
[0049] Figure 4 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia
Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
[0050] Figure 5 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia
Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
[0051] Figure 6 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells.
[0052] Figures 7A-7L illustrate selective modulators of O-linked biosynthesis.
DETAILED DESCRIPTION OF THE INVENTION
O-linked glycan Synthesis Inhibitors
[0053] Provided in certain embodiments, herein are O-linked glycan synthesis inhibitors. O- linked glycans are present on e.g., glycoproteins and mucins, and comprise a plurality of saccharides linked by O-glycosidic linkages. O-linked glycoproteins and mucins comprise a plurality of O-linked glycans attached to a core protein via a hydroxy group of a Serine, Threonine or Hydroxylysine residue. There is broad variability in the saccharide units that comprise a glycan, e.g, in various instances, an O-linked glycan is an O-linked GalNAcα- O-Ser/Thr glycan, an O-linked GlcNAcβl-O-Ser/Thr glycan, an O-linked Galβl-O-Lys glycan, an O-linked Manαl-O-Ser/Thr glycan, an O-linked Glcβl-O-Ser glycan, an O- linked Fucαl-O-Ser/Thr glycan. In general, O-linked glycan synthesis inhibitors modulate or alter the nature (e.g., the character, structure or concentration) of one or more O-linked glycans on a core protein in a cell, tissue, organ or individual.
[0054] MAL II binds to glycans containing sialic acid residues, e.g., in an (α-2,3) linkage. Figure 3 illustrates the affects of O-linked glycan synthesis inhibitors on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II (MAL II) in treated and untreated cells. Thus, Figure 3 illustrates the efficacy of O-linked glycan synthesis inhibitors described herein in inhibiting the formation of normal O-linked glycan (particluarly O-linked glycans to which MAL II binds). [0055] In certain instances, O-linked glycans are present on proteins that are heavily glycosylated with O-linked glycans, e.g., mucins. In certain instances a mucin-type O- linked glycan comprises an N-acetylgalactosamine GaINAc α-linked to a hydroxyl group of a Ser or Thr residue of a core protein e.g. mucin. In certain instances, mucin-type O-linked glycans comprise one or more Serine or Threonine residues in a core protein that are O- linked to a plurality of linkage units. In certain instances, a linkage unit is a Core- GalNAcα-O-Ser/Thr unit. In certain instances, a linkage unit O-linked to a core protein at a Ser or Thr hydroxy group is of Formula I or II:
Figure imgf000017_0001
wherein R is a core group of the linkage unit, e.g., Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, Core 8 or a keratan.
[0056] In certain instances a linkage unit is optionally and independently modified (e.g., polymerized, sialylated, fucosylated). Within the 0-linked glycans, there is broad variability with respect to the length, location, degree of polymerization, branching, fucosylation and/or sialylation. In various instances, the Core group of the linkage unit is a βl-3 galactose (βl-3Gal) unit, i.e. the linkage unit is a Core 1 O-linked glycan (Galβl- 3GalNAcα-O-Ser/Thr). In certain instances the Core group of the linkage unit is a Core 1 further modified by attachment of a βl-6 GIcNAc unit, i.e., the linkage unit is a Core 2 O- linked glycan (Galβl-3(GlcNAcβl-6)GalNAcα-O-Ser/Thr). In certain instances, the Core group of the linkage unit is a βl-3GlcNAc unit, i.e., the linkage unit is a Core 3 O-linked glycan (GlcNAcβl-3GalNAcα-O-Ser/Thr). In some instances, the linkage unit is a Core 4 O-linked glycan (GlcNAcβl-(GlcNAcβl-6) 3GalNAcα-O-Ser/Thr), Core 5 O-linked glycan (GalNAcαl-SGalNAcα-O-Ser/Thr), Core 6 O-linked glycan (GlcNAcβl- 6GalNAcα-O-Ser/Thr), Core 7 O-linked glycan (GalNAcαl-όGalNAcα-O-Ser/Thr) O- linked glycan, Core 8 O-linked glycan (Galαl-3GalNAcα-O-Ser/Thr) or a keratan (Galβl- 4GlcNAcβl- Galβl-3GalNAcα-O-Ser/Thr).
[0057] In certain instances, one or more of the linkage units, is further modified. In certain instances a further modification of a linkage unit is e.g., polymerization of one or more polylactosamine disaccharide units (e.g., Galβl-4GlcNAc or Galβl -3GIcNAc) or fucosyl units (αl-2 to Gal, or αl-3 or αl-4 to GIcNAc) to a linkage unit. In certain instances, a further modification of a linkage unit is e.g., polymerization of a terminal fucosyl unit or a terminal sialic acid unit (α2-3 or α2-6 to Gal, or α2-6 to GIcNAc) to a linkage unit. In certain instances, a further modification of a linkage unit is e.g., polymerization of a terminal fucosyl unit or a terminal sialic acid (α2-3 or α2-6 to Gal, or α2-6 to GIcNAc) to a polylactosamine-elongated-linkage unit. There is broad variability with respect to degree of polymerization, fucosylation and/or sialylation within glycan chains present on a core protein. [0058] In some instances, O-linked glycan synthesis inhibitors described herein modulate O-linked glycan biosynthesis, e.g., synthesis of a linkage unit, modification (e.g., polymerization, sialylation, fucosylation, glucuronidation, galactosylation) of a linkage unit, or chaperones or transporters that mediate glycan biosynthesis. In some embodiments, O- linked glycan synthesis inhibitors described herein selectively modulate (e.g., inhibit) synthesis of a linkage unit, modification (e.g., polymerization, sialylation, fucosylation, glucuronidation, galactosylation) of a linkage unit, or chaperones or transporters that mediate glycan biosynthesis.
[0059] In some instances, modulation of O-linked glycan biosynthesis includes modulation of the production of the linkage unit (e.g., a Core-GalNAcα-O-Ser/Thr unit) that connects a glycan to a core protein. In some embodiments, the modulation of the production of the linkage unit includes the promotion and/or inhibition of the initiation of the synthesis of the O-linked glycan. In some embodiments, the promotion and/or inhibition of the initiation of the synthesis of the O-linked glycan includes the promotion and/or inhibition of polypeptide GaINAc transferases (ppGalNAc-Ts), e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc- T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-T24, or a combination thereof. In other embodiments, the promotion and/or inhibition of the initiation of the synthesis of the O-linked glycan includes the promotion and/or inhibition of polypeptide GaINAc transferases, such as, for example, ppGalNAc-T3, ppGalNAc-Tl, ppGalNAc-T4, ppGalNAc-TIO, ppGalNAc-T20, ppGalNAc-T15 ppGalNAc-T5. In some embodiments, the modulation of the production of the linkage unit includes the promotion and/or inhibition of the synthesis of the linkage unit. In some embodiments, the modulation of the production of the linkage unit includes inhibition of a glycosyl transferase (e.g., galactosyl transferase, fucosyl transferase, mannosyl transferase, amino sugar transferase). In some instances, modulation of the production of the linkage unit includes the promotion and/or inhibition of a Core transferase, e.g., Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N-acetylglucosamine transferase (C2GLcNAc-T), Core 3 β 1-3 -N- acetylglucosamine transferase (C3GlcNAc-T), or Core 5 αl-3-N-acetylgalactosamine transferase (C5 GaINAc-T). [0060] In some embodiments, modulation of O-linked glycan biosynthesis includes modulation of further modification of the linkage unit. In some instances, modulation of further modification of the linkage unit includes promotion and/or inhibition of polymerization. In some instances, modulation of polymerization of the linkage unit includes promotion and/or inhibition of polymerization an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV (β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV). In some instances, modulation of further modification of the linkage unit includes promotion and/or inhibition of chain termination of glycan polymerization. In some embodiments, modulation of chain termination of glycan polymerization includes promotion and/or inhibition of the polymerization of a sialic acid unit, a fucosyl unit, an α-Gal unit, a β-GalNAc unit, or a β-GlcNAc unit to the glycan. In some embodiments, modulation of chain termination of glycan polymerization includes promotion and/or inhibition of a fucosyl transferase, (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), or a UDP-glucuronosyltransferase.
[0061] The modulation of O-linked glycan biosynthesis also includes modulation of regulators and components of the biosynthetic pathway. In some embodiments, the modulation of regulators and components of the biosynthetic pathway includes the promotion and/or inhibition of one or more of a chaperone (e.g., Core 1 β3-GalT-specific molecular chaperone (Cosmc)), a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter) or a combination thereof.
[0062] In certain embodiments, the modulation of O-linked glycan biosynthesis includes modulation of degradation of O-linked glycans. In some embodiments, the modulation of degradation of O-linked glycans promotes and/or inhibits recycling of saccharide units used for glycan biosynthesis. In some embodiments, modulation of degradation of O-linked glycans includes modulation of endoglycosidases and/or exoglycosidases. In some embodiments, modulation of endoglycosidases and/or exoglycosidases includes the promotion and/or inhibition of β-Nacetylhexosaminidase (e.g. promotion and/or inhibition of βGlcNAc and/or βGalNAc), sialidase (e.g. neuraminidase), β-galactosidase, β- glucuronidase, α-galactosidase or Cathepsin A.
[0063] In certain embodiments, the modulation of O-linked glycan biosynthesis includes modulation of the biosynthesis of Mucin-type O-linked glycans. In other embodiments, modulation of the biosynthesis of a mucin-type O-linked glycan includes modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan. In some embodiments, modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan includes promotion and/or inhibition of α2-3 sialyltransferases (e.g., ST3Gal IV, ST2Gal VI), and/or αl-3fucosyltransferases (e.g., Fuc-TIV, Fuc-TVII), or isoforms thereof. [0064] In some embodiments, modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan includes promotion and/or inhibition of the biosynthesis of binding domains that mediate biological functions, e.g., Lewis X (sLex: NeuAcα2-3Galβl- 4(Fucαl-3)GlcNAc) domain of mucin-type O-linked glycans. In certain embodiments, modulation of fucosylation and/or sialylation of a mucin-type O-linked glycan includes promotion and/or inhibition of the biosynthesis of polylactosamine extensions including, e.g., blood group antigens such as A, B, H or the like, Lewis antigens such as Lex, sLex, LeY or the like, type 1 blood group antigens such as Lea, Leb or the like. [0065] In certain embodiments, O-linked glycan biosynthesis inhibitors or modulators of O- linked glycan biosynthesis are compounds that modify the nature (e.g., character, structure and/or concentration) of O-linked glycans endogenous to a cellular compartment (including vesicles), cell, tissue, organ or individual when contacted or administered to the cell, tissue, organ or individual. It is to be understood that contacting a cell, tissue, or organ is possible via the administration to an individual within whom such cell, tissue or organ resides. In certain instances, O-linked glycan biosynthesis inhibitors or modulators of O-linked glycan biosynthesis modify the character and/or concentration of O-linked glycan in a targeted type of cell, tissue type or organ. In other instances, O-linked glycan synthesis inhibitors or modulators of O-linked glycan biosynthesis modify the character and/or concentration of O- linked glycan in a systemic manner.
[0066] In some instances, the modulation of O-linked glycan biosynthesis includes promotion and/or inhibition of one or more of ppGalNac-Ts, Cl βl-3GalT, C2GlcNAc-T, iGnT, β4Gal-TIV, α2-3 sialyltransferases, αl-3 fucosyltransferases, enzymes required to produce nucleotide donors (e.g., UDP-GIc transferase), transporters(e.g., UDP- Gal transporter) or chaperones (e.g., cosmc) that mediate O-linked glycan biosynthesis, or a combination thereof. In some instances, a single O-linked glycan biosynthesis inhibitor promotes Core 2 synthesis while inhibiting sialylation. In some instances, a single O-linked glycan biosynthesis inhibitor promotes sialylation while inhibiting ppGalNac-Ts. In some instances, a single O-linked glycan biosynthesis inhibitor promotes Core 1 synthesis while inhibiting fucosylation. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits one or more α2-3sialyl transferases. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits one or more αl-3fucosyl transferases. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits iGnT. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits β4-Gal-TIV. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits ppGalNAc-Ts e.g., ppGalNAc-T3, ppGalNAc- Tl, ppGalNAc-T4, ppGalNAc-TIO, ppGalNAc-T20, ppGalNAc-T15 ppGalNAc-T5. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits Cl-βl-3-GalT. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits C2 GlcNacT. In certain instances, an O-linked glycan biosynthesis inhibitor specifically modulates, promotes or inhibits β- Nacetylhexosaminidase (e.g. promotion and/or inhibition of βGlcNAc and/or βGalNAc), sialidase (e.g. neuraminidase), β-galactosidase, β-glucuronidase, α-galactosidase or Cathepsin A. In certain instances, specificity includes inhibition, modulation or promotion of the indicated type of sialylation (e.g., Core 2 sialylation), fucosylation (e.g., Core 2 fucosylation), saccharide transfer, polymerization, degradation and/or initiation by a ratio of greater than about 10:1, greater than about 9:1, greater than about 8:1, greater than about 7:1, greater than about 6:1, greater than about 5:1, greater than about 4:1, greater than about 3:1, or greater than about 2:1 over the other types of sialylation, fucosylation, saccharide transfer, polymerization, degradation and/or initiation. In certain instances, specificity includes inhibition, modulation or promotion of sulfation (e.g., 3-0 sulfation, 6-0 sulfation (e.g., 3-0 sulfation, and/or 6-0 sulfation of one or more of the T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof)) by a ratio of greater than about 10:1, greater than about 9:1, greater than about 8:1, greater than about 7:1, greater than about 6:1, greater than about 5:1, greater than about 4:1, greater than about 3 : 1 , or greater than about 2:1 over the other types of sulfation (e.g., 6-0-sulfation). [0067] In certain embodiments, an O-linked glycan synthesis inhibitor (in some embodiments, used interchangeably herein with a modulator of O-linked glycan biosynthesis) alters or disrupts the nature (e.g. fucosylation or sialylation) of the O-linked glycan compared to endogenous (i.e., untreated O-linked glycan) O-linked glycan in an amount sufficient to alter or disrupt O-linked glycan binding, O-linked glycan signaling, or a combination thereof. In some embodiments, the O-linked glycan synthesis inhibitor alters or disrupts the nature of O-linked glycan in a selected tissue type or organ compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in the selected tissue type or organ. In some embodiments, the selected tissue is, by way of non- limiting example, brain tissue, liver tissue, kidney tissue, intestinal tissue, skin tissue, or the like. In some embodiments, a O-linked glycan synthesis inhibitor as described herein alters or disrupts the nature of O-linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %). In certain embodiments, the O- linked glycan synthesis inhibitor described herein alters or disrupts the concentration of O- linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %). In certain embodiments, an O-linked glycan synthesis inhibitor described herein alters or disrupts the synthesis and/or amount (e.g., the amount and/or number of one or more antigens present) of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens of O-linked glycans compared to endogenous O-linked glycans (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %). In certain embodiments, the O- linked glycan synthesis inhibitor described herein alters or disrupts the fucosylation and/or sialylation of O-linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %). In certain embodiments, the O- linked glycan synthesis inhibitor described herein alters or disrupts the chain length (or O- linked glycan molecular weight) of an O-linked glycan compared to an endogenous O- linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %; as used herein any % includes mol or w/w % unless otherwise indicated). In certain embodiments, O-linked glycan synthesis inhibitor described herein alters or disrupts, in combination (e.g., the sum of the change in amount of fucosylation and /or sialylation, concentration, and/or chain length), the nature of an O-linked glycan compared to endogenous O-linked glycan (i.e., O-linked glycan not treated with an O-linked glycan synthesis inhibitor) in a cell, tissue, organ, or individual by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %). In certain embodiments, a O-linked glycan synthesis inhibitor as described herein alters or disrupts the fucosylation and/or sialylation of the linkage or Core region of an O-linked glycan compared to endogenous O-linked glycan (i.e., an untreated O-linked glycan) in an organism, organ, tissue or cell by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or more (mol or w/w %). As used herein, endogenous O-linked glycan is described as O-linked glycan present in the absence of treatment or contact with an O-linked glycan synthesis inhibitor. [0068] In some embodiments, a modified, altered or disrupted O-linked glycan contains less than about 20%, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of terminal sialyl and/or fucosyl residues compared to the amounts of terminal sialyl and/or fucosyl residues in an O-glycan that has not been modified, altered or disrupted. In some embodiments, a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 6-0 sulfated T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof compared to the amounts of 6-O-sulfation of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in an O-glycan that has not been modified, altered or disrupted. In some embodiments, a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 3-0 sulfated T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof compared to the amounts of 3-O-sulfation of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in an O-glycan that has not been modified, altered or disrupted. In some embodiments, a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 3-0 sulfated Lex structures compared to the amounts of 3-O- sulfation of Lex structures in an O-glycan that has not been modified, altered or disrupted. In some embodiments, a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 6-0 sulfated sLeA structures compared to the amounts of 3-O-sulfation of sLeA structures in an O-glycan that has not been modified, altered or disrupted. In some embodiments, a modified, altered or disrupted O-linked glycan contains less than about 20 mol %, less than about 30 mol %, less than about 40 mol %, less than about 50 mol %, less than about 60 mol %, less than about 70 mol % or less than about 80 mol % of 6-0 sulfated sLex structures compared to the amounts of 6-O-sulfation of sLex structures in an O-glycan that has not been modified, altered or disrupted. [0069] In some embodiments, the comparison between modified, altered or disrupted O- linked glycan compared to endogenous O-linked glycan is based on the average characteristic (e.g., the concentration, fucosylation, sialylation, chain length or molecular weight, combinations thereof, or the like) of the modified, altered or disrupted O-linked glycan. Furthermore, in some embodiments, the comparison between modified, altered or disrupted O-linked glycan is based on a comparison of the fucosylated and/or sialylated domains of the modified O-linked glycan to the fucosylated and/or sialylated domains of endogenous O-linked glycan. In some instances, the degree or nature of fucosylation and/or sialylation in the domains that have high fucosylation and/or sialylation in endogenous O- linked glycan are increased or decreased in the modified O-linked glycan. Similarly, in certain instances, the degree or nature of fucosylation and/or sialylation in the domains that have low fucosylation and/or sialylation in endogenous O-linked glycan have increased fucosylation and/or sialylation in the modified O-linked glycan. In some instances, domain organization is determined using enzymes that cleave only terminal sialic acid (e.g., α- sialidases). In some instances, domain organization is determined using enzymes that cleave fucosyl linkages (e.g., α-fucosidases). In other embodiments, the concentration, amount, character, and/or structure of an O-linked glycan is determined in any suitable manner, including those set forth herein. As used herein, altering includes increasing or decreasing. Furthermore, as used herein, disrupting includes reducing or inhibiting. [0070] In some embodiments, an O-linked glycan biosynthesis inhibitor is a Mucin-type O- linked glycan biosynthesis inhibitor. In other embodiments, a Mucin-type O-linked glycan biosynthesis inhibitor described herein alters or disrupts the nature of a Mucin-type O- linked glycan such that it alters or disrupts fucosylation or sialylation of the Mucin-type O- linked glycan. In some specific embodiments, the Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding. In some embodiments, the Mucin- type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits O-linked glycan binding and signaling. [0071] In some instances, the Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O- linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM). In certain embodiments, the CAM is an exogenous CAM, e.g., a bacterial lectin. In certain embodiments, the CAM is and endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
[0072] In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an average fucosylation and/or sialylation of less than about 1.2 mol %, less than about 1.1 mol %, less than about 1.0 mol %, less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, or less than about 0.5 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively. As used herein, the average fucosylation and/or sialylation refers to the number of sialyl and/or fucosyl substituents on each linkage unit component (e.g., on each Core 1 O-linked glycan (Galβl-3GalNAcα-O-Ser/Thr), on each Core 2 O-linked glycan (Galβl-3(GlcNAcβl-6)GalNAcα-O-Ser/Thr))of an O-linked glycan. In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a pig liver cell, pig liver tissue, a pig liver, or a pig results in an average sialylation and/or fucosylation of less than about 1.0 mol %, less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, or less than about 0.5 mol % in the liver cell, liver tissue, the liver, or the liver of the pig, respectively. In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a mouse liver cell, mouse liver tissue, a mouse liver, or a mouse results in an average sialylation and/or fucosylation of less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, less than about 0.5 mol %, less than about 0.4 mol %, or less than about 0.3 mol % in the liver cell, liver tissue, the liver, or the liver of the mouse, respectively.
[0073] In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an average terminal sialylation or fucosylation of less than about 1.2 mol %, less than about 1.1 mol %, less than about 1.0 mol %, less than about 0.9 mol %, less than about 0.8 mol %, less than about 0.7 mol %, less than about 0.6 mol %, or less than about 0.5 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively. As used herein, unless otherwise indicated, sialylation includes sialylation of any glycan α2-3 or α2-6 to Gal or α2-6 to GIcNAc. As used herein, unless otherwise indicated, fucosylation includes terminal and non-terminal fucosylation of any glycan αl-2 to Gal or αl-3 or αl-4 to GIcNAc. Furthermore, as used herein, mol % is the molar percentage of the selected saccharide component compared to the total number of saccharide components in the O-linked glycan present and/or analyzed. In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an average terminal sialylation or fucosylation of each linkage unit of less than about 15 mol %, less than about 14 mol %, less than about 12 mol %, less than about 10 mol %, less than about 8 mol %, less than about 7 mol %, less than about 6 mol %, or less than about 5 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively. In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an terminal sialylation or fucosylation of each linkage unit of less than about 7 mol %, less than about 6 mol %, less than about 5 mol %, less than about 4 mol %, less than about 3 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively. In some embodiments, an O-linked glycan biosynthesis inhibitor is an agent that when contacted or administered to a human liver cell, a human liver tissue, a human liver, or a human results in an terminal sialylation or fucosylation of each linkage unit of less than about 0.7 mol %, less than about 0.6 mol %, less than about 0.5 mol %, less than about 0.4 mol %, or less than about 0.3 mol % in the liver cell, liver tissue, the liver, or the liver of the human, respectively.
[0074] In certain embodiments, the amount of O-linked glycan synthesis inhibitor administered is an effective amount. In further embodiments, the effective amount is an amount having a minimal lethality. In more specific embodiments, the LD50IED50 is greater than about 1.1, greater than about 1.2, greater than about 1.3, greater than about 1.4, greater than about 1.5, greater than about 2, greater than about 5, greater than about 10, or more. In some embodiments, a therapeutically effective amount is about 0.1 mg to about 1O g. Selectivity
[0075] In some embodiments, an O-linked glycan biosyntheis inhibitor described herein is a selective O-linked glycan synthesis inhibitor. In some embodiments, a selective O-linked glycan inhibitor selectively alters or disrupts the nature (e.g., concentration, chain length, average number of sialic acid residues, fucosylation etc.) of an O-linked glycan compared to other glycans. In certain instances, limiting modifications to glycans limits undesirable or toxic side effects. In some instances, further restrictions to subsets of glycans, further restrict side effects and makes identification, isolation and tracking the effects of the inhibitors more reliable. In some instances, this makes dose determination more reliable. [0076] In certain instances, targeting early biosynthetic enzymes would eliminate or severely reduce O-linked glycans which could have global effects on protein folding, protein solubility protein processing as well as other functions involving cell adhesion and signal transduction. These effects could be extremely toxic or lethal. In some instances, however, targeting late phase enzymes block modifications that involve more specific receptor binding that is involved in certain cellular adhesion and trafficking interactions. Thus, in certain embodiments, provided herein are late phase O-linked biosynthesis inhibitors. In some instances, the specific interactions involving late pathway enzymes can be controlled more readily and under controlled conditions (appropriate dosing) could have beneficial effects for a number of diseases. In certain embodiments, late stage refers to a stage in the biosynthetic process after the initial N-acetylgalactosamine is attached to the ser/thr on the protein, wherein subsequent modifications determine whether the glycan is Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, or Core 8. In some embodiments, a late stage biosynthesis inhibitor described herein is an inhibitor that acts in the O-linked glycan biosynthetic pathway after the initial N-acetylgalactosamine is attached to the ser/thr on the protein. In some instances, late phase in the biosynthetic pathway refers to terminal structures typically on extended O-linked glycans preferentially on core-1 and core-2. [0077] In embodiments, selective O-linked glycan biosynthesis inhibitors/modulators include agents that selectively inhibit/modulate one or more of the following: a. biosynthesis of glycan (carbohydrate portion of a molecule) not protein, not nucleic acid, not lipid. b. biosynthesis of specific glycans and/or specific glycans linked to serine (Ser) and threonine (Thr) residues on a protein c. biosynthesis of glycans containing glucose (GIu) d. biosynthesis of glycans containing galactose (Gal) e. biosynthesis of glycans containing N-acetylglucosamine (GIcNAc) f. biosynthesis of glycans containing N-acetylgalatosamine (GaINAc) g. biosynthesis of glycans containing mannose (Man) h. biosynthesis of glycans containing xylose (XyI) i. biosynthesis of glycans containing fucose (Fuc) j. biosynthesis of glycans containing sialic acid (Sia) k. biosynthesis of non mucin glycans including α- linked O-fucose, β-linked O- xylose, α-linked 0-mannose, β-linked O-GlcNAc (N-acetylglucosamine), α- or β-linked O-galactose, and α- or β-linked O-glucose glycans 1. biosynthesis of glycans linked to glycoproteins via α-linked N- acetylgalactosamine (GaINAc) residue to the -OH of serine or threonine by an O-glycosidic bond; the structures named Mucin O-glycans or 0-GalNAc glycans. m. biosynthesis of mucin O-glycans (e.g., selectively modulate 0-GalNAc glycans of mucous glycoproteins, biosynthesis of sialyl Lewisx epitopes
(e.g., for modulating selection-binding properties of their cell surface as a mechanism to invade tissues)) n. biosynthesis of Tn antigen GalNAcαSer/Thr o. biosynthesis of Sialyl-Tn antigen Siaα2-6GalNAcαSer/Thr p. biosynthesis of Core 1 or T antigen Galβl-3GalNAcαSer/Thr q. biosynthesis of Core 2 GlcNAcβl-6(Galβl-
3)GalNAcαSer/Thr r. biosynthesis of Core 3 GlcNAcβl-3GalNAcαSer/Thr s. biosynthesis of Core 4 GlcNAcβl-6(GlcNAcβl-
3)GalNAcαSer/Thr t. biosynthesis of Core 5 GalNAcαl-3GalNAcαSer/Thr u. biosynthesis of Core 6 GlcNAcβl-6GalNAcαSer/Thr v. biosynthesis of Core 7 GalNAcαl-6GalNAcαSer/Thr w. biosynthesis of Core 8 Galαl-3GalNAcαSer/Thr x. Glycosyltransferases solely involved in the assembly of Mucin 0-linked glycoproteins y. Polypeptide N-acetylgalactosaminyltransferase ppGalNAcT-1 to -24 z. Core 1 βl-3 galactosyltransferase ClGaIT-I or T synthase aa. Core 2 βl-6 N-acetylglucosaminyltransferase C2GnT-l, C2GnT-3 bb. Core 3 βl-3 N-acetylglucosaminyltransferase C3 GnT-I cc. Core 2/4 βl-6 N-acetylglucosaminyltransferase C2GnT-2 dd. Elongation βl-3 N-acetylglucosaminyltransferase elongation β3 GnT-I to -8 ee. Core 1 α2-3 sialyltransferase ST3Gal I, ST3Gal
IV ff. α2-6 sialyltransferase ST6GalNAc I, II,
III or IV gg. Core 1 3-O-sulfotransferase Gal3ST4 hh. Secretor gene αl-2 fucosyltransferase FucT-I, FucT-II ii. Modulation of extension of initial N-acetylgalactosamine with sugars including galactose, N-acetylglucosamine, fucose or sialic acid but not mannose, glucose or xylose. Certain modifications of Mucin O-linked glycans include O-acetylation of sialic acid and O-sulfation of galactose and N-acetylglucosamine jj. Modulation of additional saccharide structures associated with O-glycans associated with mucin glycoproteins, the additional saccharides optionally bound to terminal βl-6 GIcNAc transferred by core 2 βl-6 GIcNAc. Modification by βl-3 Gal transferred by βl-3 galactosyltransferase (βl- 3GaIT) forms a Gal βl-3GlcNAc (neo-N-acetyllactosamine) termed Type-1. Optional modulation of glycosyltransferases to further alter such structures may also be obtained utilizing modulators described herein (e.g., with Gal, GIcNAc, GaINAc, Fuc or Sia). kk. biosynthesis and/or modulation of the character of O-glycans associated with mucin glycoproteins with additional saccharide structures preferentially bound to terminal βl-6 GIcNAc transferred by core 2 βl-6 GIcNAc. Modification of terminal βl-6 GIcNAc by βl-4Gal transferred by β 1-4 galactosyltransferase (βl -4GaIT) forms a Gal βl -4GIcNAc (N- acetyllactosamine) termed Type-2. Type-2 residues can be further modified to form structures containing serial disaccharides of GlcNAc(β l-4)Gal bound together by Gal(β l-3)GlcNAc linkages forming chains called poly-N- acetlyllactosamine chains. These poly-N-acetlyllactosamine chains further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc, GaINAc and Fuc and Sia. 11. biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc, GaINAc and Fuc to form the A,B, and H blood group antigens. mm. biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by glycosyltransferases to from structures containing Gal, GIcNAc, GaINAc and Fuc to form the Lewis blood group antigens. nn. biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by the Lewis αl-3/ αl-4FucT to carry an α 1-3 fucose residue termed Lewis a (Lea). oo. biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by the Lewis αl-3/ αl-4FucT to carry an αl-3 fucose residue and an αl-4 fucose reside termed Lewis a (Leb). pp. biosynthesis and/or modulation of the character of Type 1 and Type 2 structures containing N-acetyllactosamine and neo-N-acetyollactosamine units further acted upon by one or more αl-3FucTs and αl-2FucTs (as distinct from the Lewis αl-3/ αl-4FucT) to carry an αl-3 fucose residue Lewis X (Lex) and an αl-3 fucose residue and an α 1-2 fucose residue Lewis Y (Leγ). qq. biosynthesis and/or modulation of the character of Forms of the Lea determinants that are sialylated and or sulfated members of the Lewis blood group antigen family that have important functions in selectin-dependent leukocyte and tumor cell adhesion processes. rr. biosynthesis and/or modulation of the character of the sialylated and/or sulfated derivatives of Lex which function as a selectin ligand on glycoproteins and glycolipids of leukocytes and tumor cells. ss. enzymes required to make the glycans necessary for selectin binding which includes fucosyl transferases, sialyltransferases and sulfotransferases. For example L-selectin binding requires the 6-sulfo derivative of sialylated Lex (6-sulfo-SLex) on both core -2 and extended core-1 glycans. This structure requires two key αl-3 fucosyl transferases FucT-IV and FucT-VII along with four different sulfotransferases including GlcNA6ST-l and GlcNA6ST-2. L- selectin binding is required for lymphocyte homing as well as adhesion of neutrophils, eosinophils and monocytes to nonlymphoid vascular endothelium. It is also thought to be involved in metastatic tumor spread through the bloodstream and lymph. tt. enzymes necessary for the production of Lewis X (Lex ) and Lewis a (Lea ) antigens and their sialylated and/or sulfated derivative that are required for P- selectin, L-selectin and E-selectin binding that correlate with tumor progression, metastatic spread and poor prognosis in humans. uu. enzymes that glycosylate mucins which may influence mucin glycosylation associated with cancer (carcinoma mucins). Mucin glycosylation is known to be altered on carcinomas. Modulators could be used to therapeutically alter mucin glycosylation for the purpose of developing vaccines and antibodies that recognize the altered mucin glycosylation. Examples of enzymes would be enzymes that produce the T antigen, the Tn antigen and the Sialyl Tn antigen, enzymes involved in the Lewis antigen structures and their sialylated and sulfated derivatives, enzymes involved in incomplete blood group antigen production and enzymes involved in abnormal sialylation of carcinoma mucins, glycoproteins and glyco lipids. w. biosynthesis and/or modulation of the character of N- and O- linked glycoproteins and glycolipids containing sialic acid residues α2-3 linked to terminal galactose residues (in vertebrates) - catalyzed by 6 α2-3 sialyltransferases ST3GalI to ST3GalVI. Glycans bearing α2-3 linked Sia may contribute to the circulating half lives of plasma glycoprogeins by virtue of "masking" terminal Gal residues that would contribute to the removal of glycoproteins from serum by the asialoglycoprotein receptor. Siaα2-3Galβ 1- 3GalNAcαSer/Thr is important for the viability of CD8+ T cells. α2-3 linked Sia is recognized by the hemagglutinin in the envelope of influenza viruses from birds and pigs. ww. biosynthesis and/or modulation of the character of N- and O- linked glycoproteins and glycolipids containing sialic acid residues α2-6 linked to terminal Gal residues, terminal or subterminal GaINAc residues(ST6Gal-I, STόGal-II and ST6GalNAc-I - ST6GalNAc-IV) or on internal GaINAc (ST6GalNAc-III). α2-6 sialic acid is a receptor for influenza viral infection in humans.
[0078] It is noted that function O-GalNAc glycosylation is an important process because many or all mammalian cell types studied to date express ppGalNAcTs. In the secreted mucins of the respiratory, gastrointestinal, and genitourinary tracts, as well as those of the eyes, the O-GalNAc glycans of mucous glycoproteins are essential for their ability to hydrate and protect the underlying epithelium. Mucins also trap bacteria via specific receptor sites within the O-glycans of the mucin. Some sugar residues or their modifications can serve as "decoys," thus masking underlying antigens or receptors. For example, O- acetyl groups on the sialic acid residue of the sialyl-Tn antigen prevent recognition by anti- sialyl-Tn antibodies. Gut bacteria often actively remove this decoy. Bacteria can cleave sulfate with sulfatases or terminal sugars with glycosidases. Because the O-glycans are hydrophilic and usually negatively charged, they promote binding of water and salts and are major contributors to the viscosity and adhesiveness of mucus, which forms a physical barrier between lumen and epithelium. The removal of microbes and particles trapped in mucus is an important physiological process. However, in diseases such as cystic fibrosis, the abnormally high viscosity of the mucus leads to obstruction and life-threatening tissue malfunction). Moreover, in certain instances, O-GalNAc glycans, especially in the highly glycosylated mucins, have a significant effect on the conformation of the attached protein. Depending on the size and bulkiness of O-glycans, underlying peptide epitopes can be variably recognized by antibodies. O-glycosylation of mucins provides almost complete protection from protease degradation, and it is possible that the sparse O-glycosylation of some secreted glycoproteins, such as the single O-GalNAc glycan on interleukin-2, has a similar protective role. In some instances, O-GalNAc glycans change during lymphocyte activation and are abnormal in leukemic cells where an increase in core 2 and a decrease in core 1 O-glycans are often seen. The ligands for selectin-mediated interactions between endothelial cells and leukocytes are commonly based upon sialyl Lewisx epitopes attached to core 2 O-GalNAc glycans. This type of selectin-glycan interaction is important for the attachment of leukocytes to the capillary endothelium during homing of lymphocytes or the extravasation of leukocytes during the inflammatory response. Removal of core 2 O- GaINAc glycans by eliminating the C2GnT-l gene in mice results in a severe deficiency in the immune system, particularly in the selectin-binding capability of leukocytes. In certain instances, cancer cells express sialyl Lewisx epitopes and may thus use the selectin-binding properties of their cell surface as a mechanism to invade tissues. The role of O-glycans in this process has been studied in cell lines treated with the O-glycan extension inhibitor benzyl-α-GalNAc. This compound acts as a competitive substrate for the synthesis of core 1, core 2, core 3, and core 4 O-glycans in cells and thus causes a reduction in the synthesis of complex 0-GalNAc glycans. As a consequence, mucins carry a higher number of unmodified N- acetylgalactosamine residues and shorter 0-GalNAc glycans. Inhibitor- treated cancer cells lose the ability to bind to E-selectin and endothelial cells in vitro. In some instances, reproductive tissues produce mucins and O-glycosylated glycoproteins that may have important roles in fertilization. Specific terminal O-glycan structures have been shown to form the ligands for sperm-egg interactions in several species. In certain instances, the changes of O-glycans commonly observed in diseases can be due to the actions of cytokines or growth factors that affect cell growth, differentiation, and cell death and alter the expression of glycosyltransferase genes. Although these glycosylation changes may be considered a "side effect" of a pathological condition, they can also significantly contribute to the ultimate pathology and the course of disease. In cancer, the biosynthesis of O- GaINAc glycans is often abnormal due to either decreased or increased expression and activities of specific glycosyltransferases. An altered cell-surface glycocalyx may then affect the biology and survival of the cancer cell. In some instances, cell-surface MUCl and mucin-like glycoproteins have roles in cell adhesion. A number of diseases are associated with abnormal mucin gene expression and abnormal mucin carbohydrate structures and properties. These include cancer, inflammatory bowel disease, lung disease, and cystic fibrosis. The expression of underglycosylated MUCl is often increased in individuals with cancer.
[0079] In certain embodiments, selectin ligands are transmembrane glycoproteins which present oligosaccharide structures to the selectins. In some instances, transient bond formations between the selectins and their ligands mediate early steps of the adhesion cascade. In certain instances, selectins, e.g., P-selectin, L-selectin, E-selectin, recognize glycoproteins and/or glyco lipids containing the tetrasaccharide sialyl Lewisx (sialyl-CD15). In some embodiments, this tetrasaccharide is found on myeloid cells and is composed of sialic acid, galactose, fucose, and N-actyl-galacosamine.
[0080] In certain instances, PSGL-I (P-selectin glycoprotein ligand) is a ligand for P- selectin. In some instances, PSGL-I is a glycoprotein expressed on blood cells and contains the sialyl Lewisx tetrasaccharide. In certain instances, optimal binding of P-selectin to PSGL-I requires that PSGL-I presents sialyl LewisX on a specific amino-terminal core-2 O-glaycan and sulfate esters on specific amino-terminal tyrosine residues. In certain embodiments, another P-selectin ligand is CD24, which is important for tumor cell binding to P-selectin. In some instances, four possible ligands have been identified for L-selectin: GIyCAM- l(Glycosylation-Dependent Cell Adhesion Molecule), CD34, and MAdCAM-I (Mucosal Addressin Cell Adhesion Molecule), and PSGL-I. In some instances, both sulfate and sialic acid in an α(2,3) linkage are essential to L-selectin ligand activity. Although specific ligands for E-selectin are not yet known, in certain instances, candidate molecules include fucosylated, sialyated oligosaccharides found as components of glycoprotein and glycolipid molecules.
[0081] In some embodiments, a selective 0-linked glycan inhibitor selectively alters or disrupts the sialylation and/or fucosylation of O-linked glycans. In some embodiments, a selective O-linked glycan inhibitor selectively reduces or inhibits the sialylation and/or fucosylation of mucin-type O-linked glycans. In certain embodiments, selectivity of an O- linked glycan synthesis inhibitors is beneficial in order to target specific disorders without adversely impacting properly functioning glycan biosynthetic processes. In some embodiments, therapeutic methods utilizing selective O-linked glycan synthesis inhibitors have improved toxicity profiles compared to non-selective glycan synthesis inhibitors. In some embodiments, selective O-linked glycan synthesis inhibitors modulate (e.g., inhibit or promote) late stage processes (including, e.g., enzyme activity involved in the O-linked glycan preparation/synthetic pathway, enzyme activity involved in the O-linked glycan degradation pathway, other enzyme activity that affects the character of O-linked glycans, or the like) in the O-linked glycan biosynthetic pathway. In certain embodiments, late stage refers to a stage in the biosynthetic process after the initial N-acetylgalactosamine is attached to the ser/thr on the protein, wherein subsequent modifications determine whether the glycan is Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7, or Core 8. In some embodiments, a late stage biosynthesis inhibitor described herein is an inhibitor that acts in the O-linked glycan biosynthetic pathway after the initial N-acetylgalactosamine is attached to the ser/thr on the protein.
[0082] In some embodiments, a selective O-linked glycan biosynthesis inhibitor described herein is a selective Core transferase inhibitor. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor is a selective polymerization (e.g. polylactosamine polymerization) inhibitor. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor is a selective sialyl transferase inhibitor. In certain embodiments, a selective O- linked glycan biosynthesis inhibitor is a selective fucosyl transferase inhibitor. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor is a selective iGnT inhibitor.
[0083] In certain embodiments, the selective O-linked glycan synthesis inhibitor selectively affects the biosynthesis of glycans, such as N-linked, O-linked, lipid linked, or the like, but not glycosaminoglycans (GAGs), such as heparan sulfate, chondroitin sulfate, dermatan sulfate, keratin sulfate, and/or hyaluronan. In certain embodiments, selective O-linked glycan inhibitors selectively inhibit glycans compared to GAGs by a ratio of greater than 2:1, 3:1, 4:1, 5:1, 6:1, 8:1, 10:1 or more.
[0084] In some embodiments, a O-linked biosynthesis modulator (e.g., inhibitor or promoter) described herein is a selective O-linked synthesis modulator (e.g., inhibitor or promoter). In some embodiments, a selective O-linked glycan synthesis modulator affects (e.g., inhibits or promotes) the biosynthesis of O-linked glycans compared to the biosynthesis of one or more of N-linked glycans, glycosaminoglycans (GAGs), glycolipids (e.g., glycan portion thereof), or a combination thereof. In various embodiments, the selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g., inhibits or promotes) the synthesis of O-linked glycan over N-linked glycans, glycosaminoglycans (GAGs), glycolipids, or a combination thereof by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more. [0085] In certain embodiments, a selective O-linked glycan biosynthesis modulator described herein selectively affects (e.g., promotes or inhibits) the biosynthesis of a specific type of O-linked glycan (e.g. mucin type O-linked glycan) compared to one or more other type of O-linked glycan (e.g. non-mucin type O-linked glycans).
[0086] In certain embodiments, a selective O-linked glycan synthesis inhibitor described herein selectively affects/inhibits/modulates/promotes the biosynthesis of one or more of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 type of O-linked glycans but does not substantially affect/inhibit/modulate/promote the synthesis of one or more other type of O-linked glycans (e.g., one or more of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 or Core 8 type of O-linked glycans). In certain embodiments, a selective O- linked glycan biosynthesis modulator (e.g., inhibitor or promoter) selectively modulates (e.g., inhibits or promotes) the synthesis of a first specific type of O-linked glycan compared to one or more different types of O-linked glycans by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more. [0087] In some embodiments, a selective O-linked glycan synthesis inhibitor selectively alters or disrupts the nature (e.g., concentration, chain length, average number of sialic acid residues, fucosylation etc.) of a O-linked glycan compared to other glycans (e.g., GAGs, N- linked glycans, glycolipids, or the like, or combinations thereof). In specific embodiments, a selective O-linked glycan inhibitor selectively alters or disrupts the sialylation and/or fucosylation of O-linked glycans compared to the sialylation and/or fucosylation of other glycans (e.g., GAGs, N-linked glycans, glycolipids, or the like, or combinations thereof). In certain embodiments, a selective O-linked glycan synthesis inhibitor selectively inhibits sialylation and/or fucosylation of O-linked glyans compared to one or more different types of glycans (e.g., GAGs, N-linked glycans, glycolipids, or the like, or combinations thereof) by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
[0088] In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the sulfation (e.g., 6-0 sulfation, 3-0 sulfation) of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens compared to the sulfation (e.g., 6-0 sulfation, 3-0 sulfation) of one or more of other T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens in the absence of an O-glycan synthesis inhibitor. In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the 6-0 sulfation of sLex antigens compared to the 6-0 sulfation of sLex antigens in the absence of an O-glycan synthesis inhibitor. In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the 6-0 sulfation of sLex antigens compared to the 6-0 sulfation of sLex antigens in the absence of an O-glycan synthesis inhibitor by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more. In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the 3-0 sulfation of Lex antigens compared to the 3-0 sulfation of Lex antigens in the absence of an O-glycan synthesis inhibitor. In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the -O sulfation of Lex antigens compared to the 3-0 sulfation of sLex antigens in the absence of an O- glycan synthesis inhibitor by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more. [0089] In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the amount (e.g. number of antigens in or on a cell) of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in or on a cell compared to the amount (e.g. number of antigens in or on a cell) of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in or on a cell in the absence of an O-glycan synthesis inhibitor. In some embodiments, a selective O-linked glycan synthesis inhibitor selectively modulates (e.g. promotes or inhibits) the amount (e.g. number of antigens in or on a cell) of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in or on a cell compared to the amount (e.g. number of antigens in or on a cell) of one or more of T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof in or on a cell in the absence of an O-glycan synthesis inhibitor by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
[0090] In some embodiments, a selective O-linked glycan synthesis modulator selectively inhibits or promotes a specific or select characteristic of a O-linked glycan, e.g., the amount of O-linked glycan, the glycan-length of the O-linked glycan, the number of sialic acid residues of a O-linked glycan, N-acetylation, O-sulfation, O-acylation of galactose residues, O-acetylation of sialic acid residues, or the like, or combinations thereof while leaving other characteristics of the O-linked glycan unaffected or significantly unaffected. In certain embodiments, an O-linked glycan synthesis inhibitor selectively modulates (e.g., inhibits or promotes) specific types of action that inhibit O-linked glycan function. For example, in some embodiments, an O-linked glycan synthesis inhibitor modulates (e.g., inhibits or promotes) sulfation, glycosylation, phosphorylation, sialylation, fucosylation and/or degradation of endogenous O-linked glyans. In some embodiments, a selective O-linked glycan synthesis modulator selectively inhibits or promotes a specific or select characteristic of a O-linked glycan compared to one or more of any other specific or select characteristic of O-linked glycans by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
[0091] Figures 4-6 illustrate the specificity of affects of O-linked inhibitor compounds on the biosynthesis of O-linked glycans, by demonstrating the binding ability of Maakia Amurensis Lectin II (MaI II) in treated cells compared to the binding ability of fibroblast growth factor 2 (FGF2) in treated cells. Thus, in some embodiments, compounds described herein show selective inhibition of O-linked glycans without a reduction in the unrelated glycan - heparan sulfate. This demonstrates that in certain embodiments, inhibitors described herein have O-linked glycan class selectivity.
[0092] In certain embodiments, a selective O-linked glycan synthesis modulator (e.g., inhibitor or promoter) selective affects (e.g., inhibits or promotes) the activity of a specific enzyme involved in the biosynthesis of one or more O-linked glycan (e.g., one or more of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc- T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N-acetylglucosamine transferase (C2GlcNAc-T), Core 3 βl-3-N- acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc-T), Core 5 αl -3 -N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CoGIcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 β3-GalT-specific molecular chaperone (Cosmc)), or a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter) while not affecting or not significantly affecting the activity of one or more of any other enzyme involved in the biosynthesis of one or more O-linked glycans (e.g., one or more of ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc- T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N-acetylglucosamine transferase (C2GlcNAc-T), Core 3 βl-3-N-acetylglucosamine transferase (CSGIcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc- T), Core 5 α 1-3 -N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CoGIcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, STβGall, STβGalll, ST6GalIII, STβGallV), a chaperone (e.g., Core 1 β3-GalT-specifϊc molecular chaperone (Cosmc)), or a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter), any other enzyme described herein as being involved in the biosynthesis of a O-linked glycan, or a combination thereof, and/or any enzyme involved in the biosynthesis of other glycans (e.g., GAGs, glycolipids, or the like, or combinations thereof). In certain embodiments, the selective O-linked glycan biosynthesis modulator (e.g., inhibitor or promoter) selectively modulates (e.g., inhibits or promotes) one or more enzymes involved in the biosynthesis of one or more O-linked glycans compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans by a ratio of greater than about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 8:1, about 10:1 or more.
[0093] In some embodiments, a selective O-linked glycan biosynthesis inhibitor described herein selectively modulates (e.g., inhibits or promotes) one or more Core transferases compared to one or more different enzymes involved in the biosynthesis of O-linked glycans. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g., inhibits or promotes) one or more enzymes invloved in polymerization (e.g. polylactosamine polymerization) compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g. inhibits or promotes) one or more sialyl transferases compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g. inhibits or promotes) one or more fucosyl transferases compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans. In certain embodiments, a selective O-linked glycan biosynthesis inhibitor selectively modulates (e.g. inhibits or promotes) iGnT compared to one or more different enzymes involved in the biosynthesis of one or more O-linked glycans.
[0094] In certain embodiments, selective O-linked glycan modulators (e.g., inhibitors or promoters) described herein modulate (e.g., promote or inhibit) the biosynthesis of O-linked glycans with a modified (e.g., increased or decreased) ability to bind with or otherwise associate with one or more proteins, one or more core proteins, one or more lectin, one or more selectin, pathogens or the like (e.g. as compared to glycans that are synthesized in the absence of the modification). In specific embodiments, a selective O-linked glycan synthesis inhibitor described herein selectively modulates (e.g., promotes or inhibits) the biosynthesis of an O-linked glycan comprising antigens (e.g., T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or the like), blood group antigens (e.g., A, B, ABO, H antigens), type I blood group antigens (e.g., Lea, Leb), selectin ligands, peanut-agglutinin-binding epitope, salivary mucin MG2 or the like. In some embodiments, selective modulation (e.g., promotion or inhibition) of the synthesis of antigens (e.g., T, Lex, sLex, LeY, sLeY, LeA, sLeA, LeB, sLeB antigens or the like) selectively modulates (promotes or inhibits) cell adhesion, angiogenesis, cancer growth and/or metastasis compared to cell adhesion, angiogenesis, cancer growth and/or metastasis in the absence of selective modulation (e.g., promotion or inhibition) of synthesis of antigens. In some embodiments, selective O-linked glycan inhibitors described herein selectively modulate (e.g., promote or inhibit) the biosynthesis of O-linked glycans to acheive one or more specific result (e.g., agglutination, binding or the like). In specific embodiments, the selective O-linked glycan inhibitor described herein selectively modulates (e.g., promotes or inhibits) the biosynthesis of a specific O-glycan to specifically and/or selectively vary, tune, or optimize the stability, solubility, cellular location, expression of, binding of, and/or activity of O-linked glycans and/or O-linked glycanated proteins produced. In various embodiments, a selective O- linked glycan biosynthesis inhibitor selectively modulates (e.g., promotes or inhibits) the biosynthesis of one O-linked glycan comprising antigen in a ratio of greater than 1000:1, greater than 500:1, greater than 250:1, greater than 100:1, greater than 50:1, greater than 25:1, greater than 20:1, greater than 10:1, greater than 5:1, greater than 3 : 1 , or greater than 2:1 over one or more other O-linked glycan comprising antigen, (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-0-linked glycan).
[0095] In some embodiments, an O-linked glycan biosynthesis inhibitor described herein is a selective O-linked glycan biosynthesis that inhibits any specific transferase described herein over any one or more other transferase involved in the O-linked glycan biosynthetic pathyway (e.g., over all other transferases involved in the O-linked glycan biosynthetic pathway. In certain embodiments, an O-linked glycan biosynthesis inhibitor described herein is a selective O-linked glycan biosynthesis inhibitor that inhibits any specific transferase described herein as being involved in the O-linked glycan biosynthetic pathyway over any one or more transferase involved in the biosynthetic pathway of a non-0-linked glycan (e.g., N-linked glycan, glycosaminoglycan, ganglioside, or the like). [0096] In certain embodiments, a selective O-linked glycan biosynthesis inhibitor is selective for (i.e., directly or indirectly inhibits the activity of) a specific enzyme (e.g., transferase) in a ratio of greater than 1000:1 over one or more other enzyme (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan). In specific embodiments, a selective O- linked glycan biosynthesis inhibitor is selective for (i.e., directly or indirectly inhibits the activity of) a specific enzyme in a ratio of greater than 500:1 over one or more other enzyme (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan). In specific embodiments, a selective O-linked glycan biosynthesis inhibitor is selective for (i.e., directly or indirectly inhibits the activity of) a specific enzyme in a ratio of greater than 250:1, greater than 100:1, greater than 50:1, greater than 25:1, greater than 20:1, greater than 10:1, greater than 5:1, greater than 3 : 1 , or greater than 2 : 1 over one or more other enzyme (e.g., another enzyme involved in the O-linked biosynthetic pathway, and/or another enzyme involved in the biosynthetic pathway of a non-O-linked glycan). [0097] In certain embodiments, a selective O-linked glycan biosynthesis inhibitor selectively inhibits an enzymes selectin receptor binding mucin O-linked glycoproteins. In specific embodiments, a selective O-linked glycan biosynthesis inhibitor described herein inhibits the enzymes that produce, or otherwise inhibits biosynthesis of, glycans that serve as selectin ligands Lewis X and sialyl-Lewis X (Lex and SLex). In certain embodiments, a selective O-linked glycan biosynthesis inhibitor selectively inhibits one or more of αl,3 fucosyltransferases, α2-3 sialyltransferases, α2-6 sialyltransferases and/or sulfotransferases including 6-0 sulfotransferases GlcNAc6ST-l and GlcNAc6ST-2. In certain embodiments, a selective inhibitor of O-linked glycan biosynthesis includes a selective inhibitor of FucT- VII, glycosyltransferases, αl,3fucosyltransferase, α2-3 sialyltransferases, GlcNAc6ST-l, or GlcNAc6ST-2.
[0098] Moreover, in certain embodiments, provided herein is an O-linked glycoprotein or O-linked glycan that was prepared by modifying the biosynthesis thereof with any selective inhibitor described herein.
[0099] In some embodiments, provided herein is a O-linked glycan biosynthesis modulator (e.g., a selective O-linked glycan synthesis inhibitor) having suitable cell availability and/or bioavailability to significantly effect the in cyto and/or in vivo biosynthesis of a O-linked glycan (e.g., a specific O-linked glycan or component thereof, in certain instances wherein a selective O-linked glycan synthesis modulator is utilized) when the O-linked glycan biosynthesis modulator is administered to a cell or individual, respectively. In certain instances, a significant effect is one wherein a measurable effect, a statistically significant effect, and/or a therapeutic effect is provided to the cell or individual. In certain specific embodiments, the specific O-linked glycan synthesis modulator is substantially cell permeable (e.g., when in contact with a cell, a significant percentage/amount of the modulator permeates the cell membrane). In some embodiments, the O-linked glycan synthesis modulator (e.g., promotor or inhibitor) has cellular activit (e.g., when put in contact with a cell, the modulator significantly (e.g., therapeutically significantly, physiologically significantly, statistically significantly, or the like) affects cellular O-linked glycan synthesis according to any manner described herein. In some embodiments, the O- linked glycan biosynthesis modulator provides a statisitically significant effect and/or therapeutic effect in a cell or individual at a non-toxic concentration, a substantially nontoxic concentration, a concentration below LC50, a concentration below LC20, a concentration below LCOl, or the like.
Compounds
[00100] In certain embodiments, O-linked glycan synthesis inhibitors described herein are inhibitors of one or more of ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc- T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N- acetylglucosamine transferase (C2GlcNAc-T), Core 3 βl-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc- T), Core 5 αl-3-N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CoGIcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 β3-GalT-specific molecular chaperone (Cosmc)), or a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter). [00101] In certain embodiments, O-linked glycan biosynthesis inhibitors described herein are small molecule organic compounds. Thus, in certain instances, O-linked glycan biosynthesis inhibitors utilized herein are not polypeptides and/or carbohydrates. In some embodiments, in certain embodiments, a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
[00102] In some embodiments, selective inhibitors of O-linked glycan biosynthesis includes any compound of Figures 7A- 7L. Incubating compounds of Figures 7A- 7L in cells were observed to inhibit glycan- WGA binding, but did not demonstrate a significant inhibition of glycan-FGF binding (non-inhibitory against GAG, HS biosynthesis), glycan- PHA binding (non-inhibitory against N-linked glycan biosynthesis), or glycan-CTB binding (non-inhibitory against ganglioside biosynthesis). In certain embodiment, selective inhibitors of O-linked glycan biosynthesis include, but are not limited to, the following compounds: 5-bromo-2-chloro-Λ/-(4-(pyrrolidin-l-yl)phenyl)benzamide (1); 2-(5-methyl- 1 ,3 ,4-thiadiazol-2-ylthio)- 1 -(2,2,4,6,7-pentamethylquinolin- 1 (2H)-yl)ethanone (2); N-(3- methoxy-5,6,7,8-tetrahydronaphthalen-2-yl)acetamide (3); 4-(4-chlorophenoxy)-iV-(2- nitrophenyl)butanamide (4); JV-butyl-4-ethoxy-iV-ethylbenzamide (5); (^)-I- (benzo[<i]thiazol-2-yl)-4-(l-(2-ethoxyphenylamino)ethylidene)-3-phenyl-lH-pyrazol-5(4H)- one (6); Λ/-(2-(3-fluorophenyl)benzo[J]oxazol-5-yl)pentanamide (7); l-(4-(4-nitro-3- (phenethylamino)phenyl)piperazin- 1 -yl)propan- 1 -one (8); 5-ethoxy-2-(2- phenoxyethylthio)-lH-benzo[<i]imidazole (9); ethyl 4-isopropoxyphenylcarbamate (10); butyl 6-methoxybenzo[<i]thiazol-2-ylcarbamate (11); N-(3-bromophenyl)-4-(4- chlorophenoxy)butanamide (12).
[00103] In certain embodiments, O-linked glycan biosynthesis inhibitors described herein are non-carbohydrate small molecule compounds. Carbohydrates tend to be hydrophilic due to the polyhydroxyls and therefore do not diffuse into cells efficiently. In some instances, carbohydrates have pharmacokinetic and pharmacodynamic properties in animals that are inappropriate for therapeutic drug effects. Further, the hydroxyls are reactive and may make carbohydrates difficult and expensive to synthesize. In certain instances, carbohydrates are not known to cross the blood-brain barrier. In certain instances, noncarbohydrate small molecules are much less likely to be immunogenic or immunoreactive than are carbohydrates. [00104] Carbohydrates include polhydroxyaldehydes, polyhydroxyketones and their simple derivatives or larger compounds that can be hydro lyzed into such units. Carbohydrates also include polhydroxyaldehydes, polyhydroxyketones and their simple derivatives that have been modified such that when they enter cells they are reconverted into polhydroxyaldehydes, polyhydroxyketones. Carbohydrates also include sugar mimetics such as imino structures and alkaloids that inhibit glycosidases such as Deoxynojirimycin, Castanospermine, Australine, Deoxymannojirimycin, Kifunensen, Swainsonine and Mannostatin (page 709 of Essentials of Glycobiology second edition 2008 CSHL Press, CSH, New York.) Non carbohydrate small molecules include, e.g., organic compounds containing less than 3 linked hydroxyl groups with a molecular weight of less than 2000 Daltons.
[00105] Modulators (e.g., inhibitors) of glycan synthesis include agents that act directly on the relevant biosynthetic enzymes or indirectly on other targets (e.g. protein kinase, phosphatase, transporter, GPCR, ion channel, hormone receptor, protease, etc.) that would alter the structure of the glycans though effects on biosynthetic (anabolic) enzymes or degradative (catabolic) enzymes.
General Definitions
[00106] The term "subject", "patient" or "individual" are used interchangeably herein and refer to mammals and non-mammals, e.g., suffering from a disorder described herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.
[00107] The terms "treat," "treating" or "treatment," and other grammatical equivalents as used herein, include alleviating, inhibiting or reducing symptoms, reducing or inhibiting severity of, reducing incidence of, prophylactic treatment of, reducing or inhibiting recurrence of, delaying onset of, delaying recurrence of, abating or ameliorating a disease or condition symptoms, ameliorating the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms further include achieving a therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated, and/or the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient.
[00108] The terms "prevent," "preventing" or "prevention," and other grammatical equivalents as used herein, include preventing additional symptoms, preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition and are intended to include prophylaxis. The terms further include achieving a prophylactic benefit. For prophylactic benefit, the compositions are optionally administered to a patient at risk of developing a particular disease, to a patient reporting one or more of the physiological symptoms of a disease, or to a patient at risk of reoccurrence of the disease.
[00109] Where combination treatments or prevention methods are contemplated, it is not intended that the agents described herein be limited by the particular nature of the combination. For example, the agents described herein are optionally administered in combination as simple mixtures as well as chemical hybrids. An example of the latter is where the agent is covalently linked to a targeting carrier or to an active pharmaceutical. Covalent binding can be accomplished in many ways, such as, though not limited to, the use of a commercially available cross-linking agent. Furthermore, combination treatments are optionally administered separately or concomitantly.
[00110] As used herein, the terms "pharmaceutical combination", "administering an additional therapy", "administering an additional therapeutic agent" and the like refer to a pharmaceutical therapy resulting from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that at least one of the agents described herein, and at least one co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non- fixed combination" means that at least one of the agents described herein, and at least one co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with variable intervening time limits, wherein such administration provides effective levels of the two or more agents in the body of the patient. In some instances, the co-agent is administered once or for a period of time, after which the agent is administered once or over a period of time. In other instances, the co-agent is administered for a period of time, after which, a therapy involving the administration of both the co-agent and the agent are administered. In still other embodiments, the agent is administered once or over a period of time, after which, the co- agent is administered once or over a period of time. These also apply to cocktail therapies, e.g. the administration of three or more active ingredients.
[00111] As used herein, the terms "co-administration", "administered in combination with" and their grammatical equivalents are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different times. In some embodiments the agents described herein will be co-administered with other agents. These terms encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. They include simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition in which both agents are present. Thus, in some embodiments, the agents described herein and the other agent(s) are administered in a single composition. In some embodiments, the agents described herein and the other agent(s) are admixed in the composition.
[00112] The terms "effective amount" or "therapeutically effective amount" as used herein, refer to a sufficient amount of at least one agent being administered which achieve a desired result, e.g., to relieve to some extent one or more symptoms of a disease or condition being treated. In certain instances, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In specific instances, the result is the alteration of or the disruption of the structure of endogenous O-linked glycans such that the binding ability, signaling ability or combination thereof of the O-linked glycan is inhibited or reduced. In certain instances, an "effective amount" for therapeutic uses is the amount of the composition comprising an agent as set forth herein required to provide a clinically significant decrease in a disease. An appropriate "effective" amount in any individual case is determined using any suitable technique, such as a dose escalation study. [00113] The terms "administer," "administering", "administration," and the like, as used herein, refer to the methods that may be used to enable delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. In some embodiments, those familiar with administration techniques that are employed with the agents and methods described herein, e.g., as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In certain embodiments, the agents and compositions described herein are administered orally.
[00114] The term "pharmaceutically acceptable" as used herein, refers to a material that does not abrogate the biological activity or properties of the agents described herein, and is relatively nontoxic (i.e., the toxicity of the material significantly outweighs the benefit of the material). In some instances, a pharmaceutically acceptable material may be administered to an individual without causing significant undesirable biological effects or significantly interacting in a deleterious manner with any of the components of the composition in which it is contained.
[00115] The term "carrier" as used herein, refers to relatively nontoxic chemical agents that, in certain instances, facilitate the incorporation of an agent into cells or tissues. [00116] "Pharmaceutically acceptable prodrug" as used herein, refers to any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of an agent, which, upon administration to a recipient, is capable of providing, either directly or indirectly, an 0-linked glycan modulator agent described herein or a pharmaceutically active metabolite or residue thereof. Particularly favored prodrugs are those that increase the bioavailability of the 0-linked glycan modulator agents described herein when such agents are administered to a patient {e.g. , by allowing an orally administered agent to be more readily absorbed into blood) or which enhance delivery of the parent agent to a biological compartment (e.g., the brain or lymphatic system). In various embodiments, pharmaceutically acceptable salts described herein include, by way of non- limiting example, a nitrate, chloride, bromide, phosphate, sulfate, acetate, hexafluorophosphate, citrate, gluconate, benzoate, propionate, butyrate, subsalicylate, maleate, laurate, malate, fumarate, succinate, tartrate, amsonate, pamoate, p-tolunenesulfonate, mesylate and the like. Furthermore, pharmaceutically acceptable salts include, by way of non- limiting example, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium or potassium), ammonium salts and the like. Methods
[00117] Provided in certain embodiments herein is a process for modifying the structure of an 0-linked glycan on a core protein (e.g. a mucin), comprising contacting a cell that translationally produces at least one core protein having at least one attached O- linked glycan with an effective amount of any 0-linked glycan synthesis inhibitor described herein. In some embodiments, the 0-linked glycan synthesis inhibitor is a selective O- linked glycan synthesis inhibitor, e.g., as described herein. In some embodiments, the selective 0-linked glycan synthesis inhibitor is a modulator of (e.g., promotes one or more of, or inhibits one or more of a Core transferase (e.g., modulates a C2-GlcNAcT), polymerization (e.g., modulates a polypeptide GaINAc transferase, also known as a ppGalNAc-T), sialylation (e.g., modulates a sialyl transferase), sulfation (e.g., N or O sulfation), acetylation (e.g., N or O acetylation), phosphorylation, fucosylation (e.g., modulates a fucosyl transferase), a transporter (e.g., UDP-GaI transporter), a chaperone (Cosmc), a polylactosamine extension enzyme, or a combination thereof. [00118] In some embodiments, the 0-linked glycan synthesis inhibitor modulates
(e.g., promote or inhibit) a glycosyltransferase. In some embodiments, the inhibitor of a glycosyltransferase inhibits the synthesis of the linkage unit, the initiation of linkage unit synthesis, the polymerization of a linkage unit, the sialylation of a linkage unit, the fucosylation of a linkage unit or a combination thereof. In some embodiments, 0-linked glycan synthesis inhibitors modulate (e.g., promote or inhibit) one or more of a Core transferase, iGnT, sialyl transferase, a fucosyl transferase, a ppGalNAc transferase, a βl- 4galactosyltransferase IV, or combinations thereof. In more specific embodiments an O- linked glycan synthesis inhibitor selectively modulates (e.g., promote or inhibit) one or more of ppGalNAc-T e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc- TlO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and ppGalNAc-T24. In another embodiment, an 0-linked glycan synthesis inhibitor selectively modulates ppGalNAc-T3, ppGalNAc-Tl, ppGalNAc-T4, ppGalNAc-TIO, ppGalNAc-T20, ppGalNAc-T15 or ppGalNAc-T5.
[00119] In a further embodiment, an O-linked glycan synthesis inhibitor selectively modulates Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N-acetylglucosamine transferase (C2GLcNAc-T), Core 3 βl-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc-T), or Core 5 αl-3-N- acetylgalactosamine transferase (C5 GaINAc-T).
[00120] In yet a further embodiment, an O-linked glycan synthesis inhibitor selectively modulates an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 β3-GalT-specifϊc molecular chaperone (Cosmc)), a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter).
[00121] In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin. [00122] In certain embodiments, the cell is present in an individual (e.g., a human) diagnosed with a disorder mediated by O-linked glycan biosynthesis. In certain instances, the disorder mediated by O-linked glycan biosynthesis is a cancer, a tumor, undesired angiogenesis (e.g., cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, or psoriasis), insufficient angiogenesis (e.g., coronary artery disease, stroke, or delayed wound healing), organomegaly (e.g., hepatosplenomegaly), amyloidosis, skeletal abonormalities, odontoid hypoplasia, hydrops fetalis, sialuria, sialidosis, Ehlers-Danlos syndrome, Walker Warburg syndrome, a wound, or the like. In certain instances, the disorder mediated by O-linked glycan biosynthesis is an inflamatory disorder (e.g, an acute or chronic inflammatory disorder) including but not limited to Crohn's disease, reactive arthritis, including Lyme disease, insulin-dependent diabetes, organ-specific auto immunity, Hashimoto's thyroiditis and Grave's disease, contact dermatitis, psoriasis, organ transplant rejection, graft rejection, graft versus host disease, sarcoidosis, atopic conditions, gastrointestinal allergies, including food allergies, pancreatitis, eosinophilia, conjunctivitis, glomerular nephritis, multiple vasculitides, myasthenia gravis, asthma, chronic obstructive pulmonay disease, myocardial infarction, stroke, transplant rejection, reperfusion injury, autoimmune disease (e.g, Ankylosing spondylitis, systemic lupus erythematosus (SLE), or the like) inflamatory bowel disease, psoriasis, arthritis (including, e.g., rheumatoid arthritis), allergic rhinitis, berillium disease, bronchiectasis, bronchitis, bronchopneumonia, cystic fibrosis, diphtheria, dyspnea, emphysema, allergic bronchopulmonary aspergillosis, pneumonia, acute pulmonary edema, pertussis, pharyngitis, atelectasis, Wegener's granulomatosis, Legionnaires disease, pleurisy, rheumatic fever, sinusitis or the like. [00123] In some embodiments, the cell is present in a human diagnosed with cancer.
In some embodiments, the cell is present in a human diagnosed with an inflammatory disorder. In certain embodiments, the cell is present in an individual (e.g., a human) diagnosed with abnormal angiogenesis and/or undesired angiogenesis. In some embodiments, the cell is present in an individual (e.g., a human) diagnosed with amyloidosis, a spinal cord injury, hypertriglyceridemia, or the like. [00124] In some embodiments, the cell is present in an individual (e.g., human) diagnosed with a carcinoma or adenocarcinoma. In some embodiments, the cell is present in an individual diagnosed with brain cancer, pancreatic cancer, myoloma, ovarian cancer, hepatocellular cancer, breast cancer, colon carcinoma, or melanoma. In certain embodiments, the cell is a brain cancer cell, pancreatic cancer cell, myoloma cell, ovarian cancer cell, hepatocellular cancer cell, breast cancer cell, colon carcinoma cell, renal cell carcinoma, carcinoma of the gut, lung or urogenital tract, or melanoma cell. [00125] In some embodiments, the cell is present in an individual (e.g., human) diagnosed with an infectious or viral disease including, by way of non-limiting example, herpes, diphtheria, papilloma virus, hepatitis, HIV, coronavirus, or adenovirus. [00126] In certain embodiments, O-linked glycan synthesis inhibitors described herein are small molecule organic compounds. In certain instances, O-linked glycan synthesis inhibitors utilized herein are not polypeptides or carbohydrates. In some embodiments, a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
[00127] In certain embodiments, provided herein is a method of treating a disorder mediated by O-linked glycan synthesis by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein. In specific embodiments, the O-linked glycan synthesis inhibitor is a modulator (e.g., inhibitor or promoter) of a Core transferase, iGnT, a sialyl transferase, a fucosyl transferase, a ppGalNAc transferase, a βl-4galactosyltransferase IV, or combinations thereof. In certain instances, the disorder mediated by an O-linked glycan is a cancer, a tumor, undesired angiogenesis (e.g., cancer, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, or psoriasis), insufficient angiogenesis (e.g., coronary artery disease, stroke, or delayed wound healing), organomegaly (e.g., hepatosplenomegaly), amyloidosis, skeletal abonormalities, odontoid hypoplasia, hydrops fetalis, inflammation, sialuria, sialidosis, Ehlers-Danlos syndrome, Walker Warburg syndrome, a wound, or the like. In some embodiments, provided herein is a method of treating cancer by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein. In some embodiments, provided herein is a method of treating a tumor by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein. In some embodiments, provided herein is a method of treating undesired angiogenesis by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O- linked glycan synthesis inhibitor described herein.
[00128] In other embodiments, provided herein are methods for treating sialyl transferase deficiency, the method comprising administering to a patient suffering from a disease or condition mediated by sialyl transferase deficiency a therapeutically effective amount of an O-linked glycan synthesis inhibitor. In one embodiment, the disease or condition mediated by sialyl transferase deficiency is thrombocytopenia, leukospenia, sialidosis, and sialuria. [00129] In some embodiments, provided herein is a method of treating a lysosomal storage disease by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein. In some embodiments, provided herein is a method of treating a sialuria, sialidosis and/or inflammation by administering to an individual (e.g., a human) in need thereof a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein. In some embodiments, provided herein is a method of treating cancer by administering to an individual (e.g., human) a therapeutically effective amount of any O- linked glycan synthesis inhibitor described herein. In some embodiments, the cancer is, by way of non- limiting example, brain cancer, pancreatic cancer, myoloma, ovarian cancer, hepatocellular cancer, breast cancer, colon carcinoma, renal cell carcinoma, carcinoma of the gut, lung or urogenital tract, or melanoma. In some embodiments, provided herein is a method of treating an infectious or viral disease by administering to an individual (e.g., human) a therapeutically effective amount of any O-linked glycan synthesis inhibitor described herein. In some embodiments, the infectious or viral disease includes, by way of non-limiting example, herpes, diphtheria, papilloma virus, hepatitis, HIV, coronavirus, or adenovirus.
[00130] Provided in certain embodiments herein is a process of inhibiting O-linked glycan function in a cell comprising contacting the cell with a selective modulator of O- linked glycan biosynthesis including e.g., mucin biosynthesis. In various embodiments, O- linked glycan biosynthesis, as used herein, includes, by way of non- limiting example, (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O- linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In specific embodiments, the modulator of O- linked glycan biosynthesis promotes polymerization of O-linked glycans. [00131] In some embodiments, the modulator of O-linked glycan biosynthesis modulates (e.g., promotes or inhibits) Core transferases. In some embodiments, the modulator of O-linked glycan Core transferase inhibits the synthesis of the linkage region suitable for connecting O-linked glycan to a core protein, the initiation of O-linked glycan synthesis, the synthesis of glycan polymers, or a combination thereof. In some embodiments, modulators of O-linked glycan biosynthesis modulated (e.g., promote or inhibit) one or more of a ppGalNAc-T, e.g., ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc- T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N-acetylglucosamine transferase (C2GlcNAc-T), Core 3 βl-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc- T), Core 5 αl-3-N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CoGIcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 β3-GalT-specific molecular chaperone (Cosmc)), or a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter) or a combination thereof.
[00132] In another embodiment, provided herein is a method for treating GaINAc transferase deficiency, the method comprising administering to a patient suffering from a disease or condition mediated by GaINAc transferase deficiency a therapeutically effective amount of an O-linked glycan biosynthesis inhibitor. In one embodiment, the disease or condition mediated by GaINAc transferase deficiency is tumorous calcinosis. [00133] In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin. [00134] In certain embodiments, an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of a O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan. In some specific embodiments, a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding. In some specific embodiments, the Mucin-type O- linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin- type O-linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
[00135] In some instances, a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
[00136] In certain embodiments, the selective modulator of O-linked glycan biosynthesis is a small molecule organic compound. In certain instances, selective modulator of O-linked glycan biosynthesis utilized herein is not a polypeptide or a carbohydrate. In certain embodiments, the small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol. [00137] Provided in certain embodiments herein is a method of treating cancer or neoplasia comprising administering a therapeutically effective amount of an O-linked glycan biosynthesis inhibitor to a patient in need thereof. In some embodiments, the O- linked glycan biosynthesis inhibitor reduces or inhibits tumor growth, reduces or inhibits angiogenesis, or a combination thereof. In certain embodiments, the O-linked glycan biosynthesis inhibitor is a selective modulator of a Core transferase (e.g., inhibits one or more core transferases), modulator of polymerization (e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts), selective modulator of O-linked glycan sialylation (e.g., inhibits or promotes one or more sialyl transferase), selective modulator or O-linked glycan fucosylation (e.g. inhibits or promotes one or more fucosyl transferases). [00138] In various embodiments, an O-linked glycan synthesis inhibitor alters or reduces the function of O-linked glycans by one or more of the following non- limiting manners: (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes polymerization of O-linked glycans.
[00139] In some embodiments, the O-linked glycan synthesis inhibitor is a selective
O-linked glycan synthesis inhibitor, e.g., as described herein. In some embodiments, modulators of O-linked glycan biosynthesis modulated (e.g., promote or inhibit) one or more of ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc-T8, ppGalNAc-T9, ppGalNAc- TlO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 βl-3- galactosyl transferase (ClGaIT), Core 2 βl-6-N-acetylglucosamine transferase (C2GlcNAc- T), Core 3 βl-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N- acetylglucosaminyl transferase (C4GlcNAc-T), Core 5 αl-3-N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CoGIcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, STβGall, STβGalll, ST6GalIII, STβGallV), a chaperone (e.g., Core 1 β3-GalT-specifϊc molecular chaperone (Cosmc)), or a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter) or a combination thereof.
[00140] In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-IO, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin. [00141] In certain embodiments, an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of an O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan. In some specific embodiments, a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding. In some specific embodiments, the Mucin- type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
[00142] In some instances, a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
[00143] In certain embodiments, O-linked glycan synthesis inhibitors described herein are small molecule organic compounds. In certain instances, O-linked glycan synthesis utilized herein are not polypeptides or carbohydrates. In some embodiments, a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
[00144] Provided in some embodiments herein is a method of treating a lysosomal storage disease comprising administering a therapeutically effective amount of an O-linked glycan biosynthesis inhibitor to an individual (e.g., a human) in need thereof. In certain embodiments, the O-linked glycan synthesis is a selective inhibitor of a Core transferase (e.g., inhibits one or more core transferases), modulator of polymerization (e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts), selective modulator of O-linked glycan sialylation (e.g., inhibits or promotes one or more sialyl transferase), selective modulator, O-linked glycan fucosylation (e.g. inhibits or promotes one or more fucosyl transferases), or selective modulator of O-linked glycan degradation (e.g. inhibits or promotes one or more of sialidases, glycosidases, fucosidases).
[00145] In various embodiments, an O-linked glycan synthesis inhibitor alters or reduces the function of O-linked glycans by one or more of the following non- limiting manners: (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In specific embodiments, the modulator of O-linked glycan biosynthesis promotes polymerization of O-linked glycans. [00146] In some embodiments, the O-linked glycan synthesis inhibitor is a selective
O-linked glycan synthesis inhibitor, e.g., as described herein. In some embodiments, the selective O-linked glycan synthesis inhibitor is a modulator of (e.g., promotes one or more of, or inhibits one or more of) ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc- T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N- acetylglucosamine transferase (C2GlcNAc-T), Core 3 βl-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc- T), Core 5 αl-3-N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CoGIcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 β3-GalT-specific molecular chaperone (Cosmc)), or a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter) or a combination thereof.
[00147] In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-10, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin. [00148] In certain embodiments, an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of an O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan. In some specific embodiments, a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding. In some specific embodiments, the Mucin- type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling.
[00149] In some instances, a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
[00150] In certain embodiments, O-linked glycan synthesis inhibitors described herein are small molecule organic compounds. In certain instances, O-linked glycan synthesis inhibitors utilized herein are not polypeptides or carbohydrates. In some embodiments, a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol.
[00151] Provided in certain embodiments herein is a method of reducing the mean or median sialylation or fucosylation of a mucin in (or endogenous to) an individual comprising administering a therapeutically effective amount of a O-linked glycan synthesis inhibitor to a patient in need thereof. In certain embodiments, the method of reducing the mean or median sialylation or fucosylation of a mucin in (or endogenous to) an individual is suitable for treating cancer or the symptoms thereof. In certain embodiments, the O-linked glycan synthesis is a selective inhibitor of a Core transferase (e.g., inhibits one or more core transferases), modulator of polymerization (e.g., inhibits or promotes one or more of iGnT or ppGlcNAc-Ts), selective modulator of O-linked glycan sialylation (e.g., inhibits or promotes one or more sialyl transferase), selective modulator or O-linked glycan fucosylation (e.g. inhibits or promotes one or more fucosyl transferases). In specific embodiments, the cancer is by way of example, a carcinoma, or an adenocarcinoma. In other specific embodiments, the adenocarcinoma is by way of example, a bladder cancer, a breast cancer, a cervical cancer, a colon cancer, an esophagus cancer, a lung cancer, a non- small cell lung cancer, a pancreas cancer, a prostate cancer, a stomach cancer, a urachal cancer, a vaginal cancer.
[00152] In various embodiments, an O-linked glycan synthesis inhibitor alters or reduces the function of O-linked glycans by one or more of the following non- limiting manners: (1) inhibition of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis; and/or (2) promotion of (a) a Core transferase; (b) polymerization; (c) fucosylation; (d) sialylation (e) degradation and/or (f) chaperones and/or transporter that mediate O-linked glycan synthesis. In some embodiments, the modulator of O-linked glycan biosynthesis inhibits sialylation and/or fucosylation of O-linked glycans. In other embodiments, the modulator of O-linked glycan biosynthesis promotes sialylation and/or fucosylation of O-linked glycans. In some embodiments, the modulator of O-linked glycan biosynthesis inhibits polymerization of O-linked glycans. In further embodiments, the modulator of O-linked glycan biosynthesis promotes polymerization of O-linked glycans. In other embodiments, the modulator of O-linked glycan biosynthesis inhibits degradation of O-linked glycans. In further embodiments, the modulator of O-linked glycan biosynthesis promotes degradation of O-linked glycans.
[00153] In some embodiments, the O-linked glycan synthesis inhibitor is a selective
O-linked glycan synthesis inhibitor, e.g., as described herein. In some embodiments, the selective O-linked glycan synthesis inhibitor is a modulator of (e.g., promotes one or more of, or inhibits one or more of) ppGalNAc-Ts e.g., ppGalNAc-Tl, ppGalNAc-T2, ppGalNAc-T3, ppGalNAc-T4, ppGalNAc-T5, ppGalNAc-T6, ppGalNAc-T7, ppGalNAc- T8, ppGalNAc-T9, ppGalNAc-TIO, ppGalNAc-Tl 1, ppGalNAc-T12, ppGalNAc-T13, ppGalNAc-T14, ppGalNAc-T15, ppGalNAc-T16, ppGalNAc-T17, ppGalNAc-T18, ppGalNAc-T19, ppGalNAc-T20, ppGalNAc-T21, ppGalNAc-T22, ppGalNAc-T23, and/or ppGalNAc-T24; Core 1 β 1-3 -galactosyl transferase (ClGaIT), Core 2 βl-6-N- acetylglucosamine transferase (C2GlcNAc-T), Core 3 βl-3-N-acetylglucosamine transferase (C3GlcNAc-T), a Core 4 βl-6-N-acetylglucosaminyl transferase (C4GlcNAc- T), Core 5 αl-3-N-acetylgalactosamine transferase (C5GalNAc-T), a Core 6 tranferase (CβGlcNAc-T), a Core 7 transferase (C7GalNAc-T), an i-extension enzyme, (e.g., iGnT), a polylactosamine extension enzyme, (e.g., βl-4-galactosyl transferase IV(β4Gal-TIV)) or a fucosyl transferase (e.g., FucTVII, FucTIV), a sialyl transferase (e.g., ST3GalI, ST3GalII, ST3GalIV, ST3GalVI, ST6GalI, ST6GalII, ST6GalIII, ST6GalIV), a chaperone (e.g., Core 1 β3-GalT-specific molecular chaperone (Cosmc)), a transporter (e.g., UDP-GaI transporter, UDP-GIcNAc transporter), a glycosidase (e.g. β-Nacetylhexosaminidase (e.g. βGlcNAc and/or βGalNAc), a sialidase (e.g. neuraminidase), β-galactosidase, β-glucuronidase, α- galactosidase or Cathepsin A) or a combination thereof.
[00154] In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any immunomodulatory factor (e.g. IL-5, IL- 3, IL-IO, galectin, Granulocyte -macrophage colony-stimulating factor (GM-CSF)) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, an O-linked glycan synthesis inhibitor alters or disrupts the nature of the O-linked glycan (e.g., sialylation, fucosylation) such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of an O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non-limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin. [00155] In certain embodiments, an effective amount of a Mucin type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of an O-linked glycan such that it alters or disrupts fucosylation or sialylation of a Mucin type O-linked glycan. In some embodiments, a Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits Mucin-type O-linked glycan binding. In some embodiments, the Mucin-type O-linked glycan synthesis inhibitor described herein alters or disrupts the nature of the Mucin-type O- linked glycan such that it inhibits Mucin type O-linked glycan binding and signaling. [00156] In some instances, a Mucin-type O-linked glycan synthesis inhibitor alters or disrupts the nature of the Mucin-type O-linked glycan such that it inhibits the binding, signaling, or a combination thereof of any lectin (including polypeptides) subject to Mucin type O-linked glycan binding, signaling or a combination thereof, compared to binding in the absence of a Mucin-type O-linked glycan synthesis inhibitor. In some instances, the polypeptide is, by way of non- limiting example, a cell adhesion molecule (CAM). In certain embodiments the CAM is an exogenous CAM, e.g., bacterial lectins. In certain embodiments, the CAM is an endogenous CAM and includes, by way of non- limiting examples, E-selectin, L-selectin or P-selectin.
[00157] In certain embodiments, O-linked glycan synthesis inhibitors described herein are small molecule organic compounds. In certain instances, O-linked glycan synthesis inhibitors utilized herein are not polypeptides or carbohydrates. In some embodiments, a small molecule organic compound has a molecular weight of less than about 2,000 g/mol, less than about 1,500 g/mol, less than about 1,000 g/mol, less than 700 g/mol, or less than about 500 g/mol. Screening Processes
[00158] Provided herein are processes for identifying inhibitors of the biosynthesis of mucin-type O-linked glycans or for identifying genes involved in (including selective inhibitors or modulators of) the biosynthesis of mucin-type O-linked glycans. Also provided herein are processes for identifying modulators of enzymes involved in the biosynthesis of mucin-type O-linked glycans.
[00159] In one embodiment is a cell-based high throughput process for identifying and/or screening for (1) O-linked glycan biosynthesis inhibitors; (2) genes involved in (including selective regulators of) the biosynthesis of mucin-type O-linked glycans; (3) O- linked glycan biosynthesis modulators; or (4) combinations thereof. In one embodiment, a library of small-molecule chemical compounds (including oligopeptides and oligonucleotides) is screened; in other embodiments, a library of siRNA is screened; in other embodiments, both types of libraries are simultaneously or sequentially screened. [00160] In certain embodiments, the siRNA library is enzymatically generated; or rationally synthesized; or randomly generated; or a combination thereof. Non-limiting examples of protocols for screening siRNA libraries in high-throughpout genetic screens is found in the Journal of Cancer Molecules: 1(1), 19-24, 2005.
[00161] Provided in some embodiments is a process for identifying a compound that modulates O-linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound in combination with a labeled probe that binds one or more O-linked glycans; b. incubating the mammalian cell, compound and labeled probe; c. collecting the labeled probe that is bound to one or more O-linked glycans; and d. detecting or measuring the amount of the labeled probe bound to one or more O-linked glycans.
[00162] In more specific embodiments, provided herein is a process for identifying a compound that selectively modulates O-linked glycan biosynthesis comprising: a. contacting a mammalian cell with the compound b. contacting the mammalian cell and compound combination with a first labeled probe and a second labeled probe, wherein the first labeled probe binds one or more O-linked glycans and the second labeled probe binds at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; c. incubating the mammalian cell, compound, the first labeled probe, and the second labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; e. collecting the second labeled probe that is bound to at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; f. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans; and g. detecting or measuring the amount of the second labeled probe bound to at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans.
[00163] Similarly, in some embodiments provided herein is a process for identifying compounds that selectively modulate O-linked glycans biosynthesis comprising: a. contacting a first mammalian cell with the compound b. contacting the first mammalian cell and compound combination with a first labeled probe, wherein the first labeled probe binds one or more O-linked glycans; c. incubating the first mammalian cell, compound, the first labeled probe, and the second labeled probe; d. collecting the first labeled probe that is bound to one or more O-linked glycans; e. detecting or measuring the amount of first labeled probe bound to one or more O-linked glycans; f. contacting a second mammalian cell with the compound, wherein the second mammalian cell is of the same type as the first mammalian cell; g. contacting the second mammalian cell and compound combination with a second labeled probe, wherein the second labeled probe binds at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; h. collecting the second labeled probe that is bound to at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans; and i. detecting or measuring the amount of the second labeled probe bound to at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans.
[00164] In some embodiments, provided herein is a process for identifying a compound that modulates O-linked glycan biosynthesis comprising: a. collecting O-linked glycans from a first mammalian cell of a selected type, wherein the O-linked glycan comprises a plurality of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 and/or Core 8 O-linked glycans; b. cleaving the O-linked glycans into a plurality of monosaccharide, disaccharide or oligosaccharide component parts; c. detecting or measuring the amount of one of more of the monosaccharide, disaccharide or oligosaccharide component parts; d. contacting and incubating a second mammalian cell of the selected type with the compound; e. collecting O-linked glycans from the second mammalian cell of a selected type; f. cleaving the O-linked glycans into a plurality of monosaccharide, disaccharide or oligosaccharide component parts; g. detecting or measuring the amounts of one of more of the monosaccharide, disaccharide or oligosaccharide component parts; h. comparing: i. the amounts of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 and/or Core 8 O-linked glycans or one or more component parts thereof produced by the first and second mammalian cells; ii. the amounts of sialyl residues present in the O-linked glycans or one or more of the monosaccharide, disaccharide or oligosaccharide component parts thereof; iii. the amounts of 3-0 sulfation present in the O-linked glycans and/or one or more of the T, Lex, sLex, Leγ, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof, or one or more of the monosaccharide, disaccharide or oligosaccharide component parts therof; iv. the amounts of 6-0 sulfation present in the O-linked glycans and/or one or more of the T, Lex, sLex, Leγ, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof, one or more of the monosaccharide, disaccharide or oligosaccharide component parts therof; v. the amounts (e.g., number and/or concentration) of one or more of the T, Lex, sLex, Leγ, sLeY, LeA, sLeA, LeB, sLeB antigens or a combination thereof, or one or more monosaccharide, disaccharide or oligosaccharide component parts therof; vi. the amounts of fucosyl residues present in the O-linked glycans or one or more of the monosaccharide, disaccharide or oligosaccharide component parts thereof; or vii. a combination thereof.
[00165] In some embodiments, monosaccharide, disaccharide or oligosaccharide component parts characteristic of O-linked glycans comprise terminal sialic acid residues in mucin-type O-linked glycans. In some embodiments, monosaccharide, disaccharide or oligosaccharide component parts characteristic of O-linked glycans comprise terminal fucosyl residues in mucin-type O-linked glycans. In some embodiments, monosaccharide, disaccharide or oligosaccharide component parts characteristic of O-linked glycans comprise 3-0 sulfated monosaccharide, disaccharide or oligosaccharide component parts in sLex O-linked glycan structures. In some embodiments, the amount of terminal sialylation in O-glycans collected from a first mammalian cell is compared with the amount of terminal sialylation in O-glycans collected from a second mammalian cell. In some embodiments, the amount of terminal fucosylation in O-glycans collected from a first mammalian cell is compared with the amount of terminal fucosylation in O-glycans collected from a second mammalian cell.
[00166] In some embodiments, incubating the mixture of the compound with the at least one cell expressing at least one O-linked glycan is performed for a predetermined time. In one embodiment, incubation is for a period of about 12 hours. In another embodiment, incubating the mixture is for a period of about 18 hours. In another embodiment, about 24 hours. In yet another embodiment, about 36 hours. In a further embodiment, 48 hours. In another embodiment, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days.
[00167] In one embodiment, the process(es) described herein are useful for high- throughput analysis of a O-linked glycan biosynthesis inhibitor or a positive or negative regulatory gene for O-linked glycan biosynthesis. In certain embodiments, the amounts of O-linked glycan and/or monsaccharides, disaccharides or oligosaccharides characteristic of Core 1, Core 2, Core 3, Core 4, Core 5, Core 6, Core 7 and/or Core 8 O-linked glycans are measured with an analytical device. In some embodiments, the analytical device is a fluorimeter. In some embodiments, the analytical device is a fluorescent plate reader. In some embodiments, fluorescence is measured at an excitation of 450 - 600 nm and emission of 600 - 700 nm. In some embodiments, the detecting or measuring process is developed using a robotic pipettor.
[00168] In one embodiment the inhibitor of O-linked glycan biosynthesis is an inhibitor of a polypeptide GaINAc transferase, a core transferase, a polylactosamine- modifying glycosyl transferase, a polylactosamine extension enzyme, a core 1 specific chaperone, a sialyl transferase, and a fucosyl transferase, or a combination thereof. In another embodiment the inhibitor of O-linked glycan biosynthesis is an inhibitor of O- linked mucin-type glycosylation.
[00169] In some embodiments, the process further comprises comparing the amount of first labeled probe bound to O-linked glycans to the amount of the second labeled probe bound to at least one glycan other than O-linked glycans (e.g., to determine a ratio of the amount of first labeled probe bound to the amount of second labeled probe bound under substantially similar conditions).
[00170] In certain embodiments, a label utilized in any process described herein is any suitable label such as, by way of non- limiting example, a fluorescent label, a dye, a radiolabel, or the like. In some embodiments, the labeled probe comprises a biotinyl moiety and the process further comprises tagging the labeled probe with streptavidin-Cy5-PE. In certain embodiments, the first probe is any O-linked glycan binding protein, e.g., a selectin. In specific embodiments, the selectin is, by way of non- limiting example, P-selectin, L- selectin or E-selectin. In various embodiments, the amount of bound labeled probes are detected in any suitable manner, e.g., with a fluorimeter, a radiation detector, or the like. [00171] In certain embodiments, the first and second probes are labeled in a manner so as to be independently detectable. In some embodiments, the first and second probes are contacted to the cells separately (i.e., to different cells of the same type) and independently analyzed. In some embodiments, the at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) other than O-linked glycans is, by way of non- limiting example, chondroitin sulfate, gangliosides, N-linked glycans, heparan sulfate or the like. Furthermore, in some embodiments, a third labeled probe that binds at least one glycan (e.g., a GAG, a sulfated GAG, an N-linked glycan, or the like) not bound by the first or second labeled probe is also utilized. Additional labeled probes are also optionally utilized. [00172] Second and additional labeled probes include any labeled compound or labeled lectin suitable (e.g., a labeled compound or lectin that binds a ganglioside, a GAG, a non-sulfated GAG, an N-linked glycan, an N-linked glycan, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratin sulfate, and/or hyaluronan). In some embodiments, labeled probes included labeled forms of one or more of, by way of non- limiting example, Wheat Germ Agglutinin (WGA) from Triticum vulgaris (as a probe for binding N-linked and O- linked glycans with terminal GIcNAc residues and clustered sialic acid residues); Phaseolus Vulgaris Aggutinin (PHA) from Phaseolus vulgaris (as a probe for binding N-linked glycans); Cholera Toxin B-subunit (CTB) from Vibrio cholera (as a probe for binding sialic acid modified glycolipids); Concanavalin A (ConA) from Canavalia ensiformis (as a probe for binding mannose residues in N-linked glycans); and/or Jacalin from Artocarpus integrifolia (as a probe for binding O-linked glycans). In specific embodiments, labeled forms of each of Wheat Germ Agglutinin (WGA) from Triticum vulgaris (as a probe for binding N-linked and O-linked glycans with terminal GIcNAc residues and clustered sialic acid residues); Phaseolus Vulgaris Aggutinin (PHA) from Phaseolus vulgaris (as a probe for binding N-linked glycans); and Cholera Toxin B-subunit (CTB) from Vibrio cholera (as a probe for binding sialic acid modified glycolipids) are utilized.
[00173] Contact with first, second and additional labeled probes occurs in parallel, concurrently, or sequentially. In certain embodiments, contact the compounds and multiple probes allows identification of selective O-linked glycan inhibitors. [00174] In some embodiments, the mammalian cell (e.g., human cell) is selected from any suitable mammalian cell. In specific embodiments, the mammalian cell is, by way of non- limiting example, a human cancer cell (e.g., human cervical cancer cell (HeLa)), a human ovarian cancer cell (SKOV), a human lung cancer cell (Hal8), a human meduloblastoma cancer cell (DAOY), a Chinese Hamster Ovary (CHO) cell, an adenocarcinoma cell, a melanoma cell, or a human primary cell. In certain embodiments, included herein are processes wherein the cell includes a plurality (e.g., 2, 3 , 4 or all) of a human cancer cell (e.g., human cervical cancer cell (HeLa)), a human ovarian cancer cell (SKOV), a human lung cancer cell (Hal8), a human meduloblastoma cancer cell (DAOY), and/or a Chinese Hamster Ovary (CHO) cell. Contact with such cells optionally occurs in parallel, concurrently, or sequentially. In certain embodiments, contact with multiple cells identifies inhibitors (e.g., selective O-linked glycan synthesis inhibitors) that inhibit O- linked glycan biosynthesis in multiple cell lines. In some instances, utilization of a plurality of cell lines allows the elimination or minimization of false positives in identifying O-linked glycan inhibitors.
[00175] Thus, in some embodiments, any process described herein comprises contacting the compound to a first cell (type), contacting the compound to a second cell (type), and, optionally, contacting the compound to additional cells (types), and repeating the process described for each of the first, second and any additional cell types utilized (e.g., to determine if a O-linked glycan inhibitor is selective for multiple cell lines or to determine which types of cell lines that the O-linked glycan inhibitor selectively targets). Furthermore, in such embodiments, the process further comprises comparing the amount of labeled probe (or the amount of first, second or any additional labeled probe) that is bound in each type of cell (e.g., to determine selectively of inhibiting O-linked glycan biosynthesis compared to the biosynthesis of other types of glycans). [00176] In some embodiments, once a compound that modulates O-linked glycan biosynthesis is determined by the process described, a similar process is optionally utilized to determine whether or not the compound selectively modulates O-linked glycan biosynthesis. Specifically, selectivity of a compound that modulates O-linked glycan biosynthesis is determined by utilizing a similar process as described for determining whether or not the compound modulates O-linked glycan biosynthesis, e.g., by: a. contacting a mammalian cell with the compound in combination with a labeled probe that binds one or more non-O-linked glycan (e.g., GAG or other class of glycan); b. incubating the mammalian cell, compound and labeled probe; c. collecting the labeled probe that is bound to non- O-linked glycan (e.g., GAG or other class of glycan); and d. detecting or measuring the amount of labeled probe bound to non-O-linked glycan (e.g., GAG or other class of glycan).
[00177] In various embodiments, this process is repeated for any number of non- O- linked glycans (e.g., GAG or other class of glycan). In some embodiments, the non- O- linked glycans are, by way of non- limiting example, chondroitin sulfate, heparan sulfate, N- linked glycans, gangliosides, or the like.
[00178] In some embodiments, the mammalian cell (e.g., human cell) is selected from any suitable mammalian cell. In specific embodiments, the mammalian cell is, by way of non- limiting example, a human cancer cell (e.g., human cervical cancer cell (HeLa)) a human ovarian cancer cell (SKOV), a human lung cancer cell (Hal8), a human meduloblastoma cancer cell (DAOY) or a human primary cell. Furthermore, in some embodiments, the process is repeated utilizing one or more additional cell types. In certain embodiments, the results (e.g., of (c), and/or (d)) from the one or more additional cell types (e.g., a second, third, fourth, fifth or the like cell types) are compared to each other and the results (e.g., of (c), and/or (d)) from the first cell type.
[00179] In certain embodiments, the O-linked glycans and/or the modified O-linked glycans are cleaved in any suitable manner. In some embodiments, the O-linked glycans and/or the modified O-linked glycans are cleaved using a suitable enzyme such as a protease that is selective for highly sialylated, highly substituted mucins, or in any other suitable chemical manner. [00180] In some embodiments, the amount of one of more of monosaccharide, disaccharide or oligosaccharide units present in the cell and/or the characteristic of the O- linked glycans in a cell are determined in any suitable manner. For example, in some embodiments, the amount of sialic acid units present and/or the amount of O-sulfation (e.g., 3-O-sulfation) of the glucosylamine groups, or a combination thereof is determined utilizing a carbozole assay, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), capillary elecrophoresis, gel electrophoseis, mass spectrum (MS) analysis, HPLC electrospray ionization tandem mass spectrometry, nuclear magnetic resonance (NMR) analysis, or the like.
[00181] Moreover, in certain embodiments, the process described is a process for identifying compounds that selectively modulate O-linked glycan biosynthesis. In such embodiments, the process also comprises collecting one or more non-O-linked glycan (e.g., a sulfated glycan, such as chondroitin sulfate, N-linked glycans, or the like) from the cell, both without incubation with the compound and with incubation with the compound; cleaving each of such non-O-linked glycans; measuring the character of each of such non- O-linked glycan; and comparing the character of the non-O-linked glycan that was not incubated with the character of the non-O-linked glycan that was incubated. In certain embodiments, the character includes, by way of non-limiting example, the chain length of the non-O-linked glycan, the amount of sulfation of the non-O-linked glycan, the location of sulfation of the non-O-linked glycan, the structure of the non-O-linked glycan, the composition of the non-O-linked glycan, or the like. The structure of glycosaminoglycans, N-linked glycans, O-linked glycans and lipid linked glycans can be determined using any suitable method, including, by way of non- limiting example, monosaccharide compositional analysis, capillary electrophoresis, gel electrophoresis, gel filtration, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrum (MS) analysis, HPLC electrospray ionization tandem mass spectrometry, nuclear magnetic resonance (NMR) analysis, or the like. Combinations
[00182] In certain instances, it is appropriate to administer at least one therapeutic compound described herein (i.e., any O-linked glycan inhibitor described herein) in combination with another therapeutic agent. By way of example only, if one of the side effects experienced by a patient upon receiving one of the O-linked glycan inhibitors described herein is nausea, then it is appropriate in certain instances to administer an anti- nausea agent in combination with the initial therapeutic agent. Or, by way of example only, the therapeutic effectiveness of one of the O-linked glycan inhibitors described herein is enhanced by administration of an adjuvant (i.e., by itself the adjuvant has minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit experienced by a patient is increased by administering one of O-linked glycan inhibitors described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit. In any case, regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient is in some embodiments additive of the two therapeutic agents or in other embodiments, the patient experiences a synergistic benefit.
[00183] In some embodiments, the particular choice of compounds depends upon the diagnosis of the attending physicians and their judgment of the condition of the patient and the appropriate treatment protocol. The compounds are optionally administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the patient, and the actual choice of compounds used. In certain instances, the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is based on an evaluation of the disease being treated and the condition of the patient. [00184] In some embodiments, therapeutically-effective dosages vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.
[00185] In some embodiments of the combination therapies described herein, dosages of the co-administered compounds vary depending on the type of co-drug employed, on the specific drug employed, on the disease or condition being treated and so forth. In addition, when co-administered with one or more biologically active agents, the compound provided herein is optionally administered either simultaneously with the biologically active agent(s), or sequentially. In certain instances, if administered sequentially, the attending physician will decide on the appropriate sequence of therapeutic compound described herein in combination with the additional therapeutic agent.
[00186] The multiple therapeutic agents (at least one of which is an O-linked glycan inhibitor described herein) are optionally administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). In certain instances, one of the therapeutic agents is optionally given in multiple doses. In other instances, both are optionally given as multiple doses. If not simultaneous, the timing between the multiple doses is any suitable timing, e.g, from more than zero weeks to less than four weeks. In some embodiments, the additional therapeutic agent is utilized to achieve remission (partial or complete) of a cancer, whereupon the therapeutic agent described herein (e.g., any O-linked glycan) is subsequently administered. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations are also envisioned (including two or more therapeutic compounds described herein). [00187] In certain embodiments, a dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought, is modified in accordance with a variety of factors. These factors include the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, in various embodiments, the dosage regimen actually employed varies and deviates from the dosage regimens set forth herein. [00188] In some embodiments, the pharmaceutical agents which make up the combination therapy disclosed herein are provided in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. In certain embodiments, the pharmaceutical agents that make up the combination therapy are administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step administration. In some embodiments, two-step administration regimen calls for sequential administration of the active agents or spaced-apart administration of the separate active agents. In certain embodiments, the time period between the multiple administration steps varies, by way of non-limiting example, from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent. [00189] In addition, the O-linked glycan inhibitors described herein also are optionally used in combination with procedures that provide additional or synergistic benefit to the patient. By way of example only, patients are expected to find therapeutic and/or prophylactic benefit in the methods described herein, wherein pharmaceutical composition of a compound disclosed herein and /or combinations with other therapeutics are combined with genetic testing to determine whether that individual is a carrier of a gene or gene mutation that is known to be correlated with certain diseases or conditions. [00190] In various embodiments, the O-linked glycan inhibitors described herein and combination therapies are administered before, during or after the occurrence of a disease or condition. Timing of administering the composition containing an O-linked glycan inhibitor is optionally varied to suit the needs of the individual treated. Thus, in certain embodiments, the O-linked glycan inhibitors are used as a prophylactic and are administered continuously to subjects with a propensity to develop conditions or diseases in order to prevent the occurrence of the disease or condition. In some embodiments, the compounds and compositions are administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the O-linked glycan inhibitors are optionally initiated within the first 48 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration is achieved by any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof. In some embodiments, the compound should be administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. The length of treatment is optionally varied for each subject based on known criteria. In exemplary embodiments, the compound or a formulation containing the compound is administered for at least 2 weeks, between about 1 month to about 5 years, or from about 1 month to about 3 years. [00191] In certain embodiments, therapeutic agents are combined with or utilized in combination with one or more of the following therapeutic agents in any combination: immunosuppressants or anti-cancer therapies (e.g., radiation, surgery or anti-cancer agents). [00192] In some embodiments, one or more of the anti-cancer agents are proapoptotic agents. Examples of anti-cancer agents include, by way of non- limiting example: gossypol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec®), geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, or PD184352, Taxol™, also referred to as "paclitaxel", is an anti-cancer drug which acts by enhancing and stabilizing microtubule formation, and analogs of Taxol™, such as Taxotere™. Compounds that have the basic taxane skeleton as a common structure feature, have also been shown to have the ability to arrest cells in the G2-M phases due to stabilized microtubules and may be useful for treating cancer in combination with the compounds described herein. [00193] Further examples of anti-cancer agents include inhibitors of mitogen- activated protein kinase signaling, e.g., U0126, PD98059, PD184352, PD0325901, ARRY- 142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies (e.g., rituxan).
[00194] Other anti-cancer agents include Adriamycin, Dactinomycin, Bleomycin,
Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefmgol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin Il (including recombinant interleukin II, or rlL2), interferon alpha-2a; interferon alpha-2b; interferon α- nl; interferon α-n3; interferon beta-la; interferon gamma-1 b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfϊmer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safϊngol; safϊngol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfm; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.
[00195] Other anti-cancer agents include: 20-epi-l, 25 dihydroxyvitamin D3; 5- ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara- CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9- dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N- acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfϊmer sodium; porfϊromycin; prednisone; propyl bis- acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safϊngol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofϊran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfϊn; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[00196] Yet other anticancer agents that include alkylating agents, antimetabolites, natural products, or hormones, e.g., nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, ete.), or triazenes (decarbazine, etc.). Examples of antimetabolites include but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin). [00197] Examples of natural products include but are not limited to vinca alkaloids
(e.g., vinblastin, vincristine), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), or biological response modifiers (e.g., interferon alpha).
[00198] Examples of alkylating agents include, but are not limited to, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan, etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin, etc.), or triazenes (decarbazine, ete.). Examples of antimetabolites include, but are not limited to folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin. [00199] Examples of hormones and antagonists include, but are not limited to, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), gonadotropin releasing hormone analog (e.g., leuprolide). Other agents that can be used in the methods and compositions described herein for the treatment or prevention of cancer include platinum coordination complexes (e.g., cisplatin, carboblatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide). [00200] In some embodiments, provided herein is a method of treating lymphoma comprising administering a therapeutically effective amount of a compound described herein in combination with an antibody to CD20 and/or a CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) therapy. In certain embodiments, provided herein is a method of treating leukemia comprising administering a therapeutically effective amount of a compound described herein in combination with ATRA, methotrexate, cyclophosphamide and the like. Pharmaceutical Compositions
[00201] In certain embodiments, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers including, e.g., excipients and auxiliaries which facilitate processing of the active compounds into preparations which are suitable for pharmaceutical use. In certain embodiments, proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999). [00202] A pharmaceutical composition, as used herein, refers to a mixture of an O- linked glycan inhibitor described herein, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. In certain instances, the pharmaceutical composition facilitates administration of the 0-linked glycan inhibitor to an individual or cell. In certain embodiments of practicing the methods of treatment or use provided herein, therapeutically effective amounts of O- linked glycan inhibitors described herein are administered in a pharmaceutical composition to an individual having a disease, disorder, or condition to be treated. In specific embodiments, the individual is a human. As discussed herein, the 0-linked glycan inhibitors described herein are either utilized singly or in combination with one or more additional therapeutic agents.
[00203] In certain embodiments, the pharmaceutical formulations described herein are administered to an individual in any manner, including one or more of multiple administration routes, such as, by way of non- limiting example, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. [00204] Pharmaceutical compositions including a compound described herein are optionally manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes. [00205] In certain embodiments, a pharmaceutical compositions described herein includes one or more O-linked glycan inhibitors described herein, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In some embodiments, the compounds described herein are utilized as an JV-oxide or in a crystalline or amorphous form (i.e., a polymorph). In certain embodiments, an active metabolite or prodrug of a compound described herein is utilized. In some situations, a compound described herein exists as tautomers. All tautomers are included within the scope of the compounds presented herein. In certain embodiments, a compound described herein exists in an unsolvated or solvated form, wherein solvated forms comprise any pharmaceutically acceptable solvent, e.g., water, ethanol, and the like. The solvated forms of the O-linked glycan inhibitors presented herein are also considered to be disclosed herein. [00206] A "carrier" includes, in some embodiments, a pharmaceutically acceptable excipient and is selected on the basis of compatibility with O-linked glycan inhibitors disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999). [00207] Moreover, in certain embodiments, the pharmaceutical compositions described herein is formulated as a dosage form. As such, in some embodiments, provided herein is a dosage form comprising an O-linked glycan inhibitor described herein, suitable for administration to an individual. In certain embodiments, suitable dosage forms include, by way of non-limiting example, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.
[00208] The pharmaceutical solid dosage forms described herein optionally include an additional therapeutic compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof. In some aspects, using coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of the O-linked glycan inhibitor. In one embodiment, an O-linked glycan inhibitor described herein is in the form of a particle and some or all of the particles of the compound are coated. In certain embodiments, some or all of the particles of an O- linked glycan inhibitor described herein are microencapsulated. In some embodiment, the particles of the O-linked glycan inhibitor described herein are not microencapsulated and are uncoated.
[00209] In certain embodiments, the pharmaceutical composition described herein is in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more therapeutic compound. In some embodiments, the unit dosage is in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions are optionally packaged in single-dose non-reclosable containers. In some embodiments, multiple-dose re-closeable containers are used. In certain instances, multiple dose containers comprise a preservative in the composition. By way of example only, formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative. [00210] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. The starting materials and reagents used for the processes, methods, and compositions described herein are synthesized or are obtained from commercial sources, such as, but not limited to, Sigma-Aldrich, Acros Organics, Fluka, and Fischer Scientific.
EXAMPLES EXAMPLE 1:
Primary assay
[00211] The impact of an O-linked glycan synthesis inhibitor on the ability of a protein (e.g., Wheat Germ Agglutinin (WGA)) to bind to O-linked glycans in a mammalian cell line is tested by incubating cells in the presence of an O-linked glycan synthesis modulator. WGA is applied to a panel of cell lines at a concentration range covering from about 100 pg/ml to about 100 ug/ml in 3-fold increments.
[00212] Cells are grown in the presence of GalNAcα-O-benzyl for two days, then analyzed by flow cytometry using fluorescently labeled versions of WGA. Quantification of the inhibition of binding is carried out using streptavidin-Cy5-PE. LS 180 shows reduced binding of MAH and CSLEX-I after GalNAcα-O-benzyl treatment. This indicates that
WGA lectins primarily bind O-linked structures on this cell line.
[00213] Figure 1 (see Hongsachart et al, Glycoproteomic analysis ofWGA-bound glycoprotein biomarkers in sera from patients with lung adenocarcinoma, Electrophoresis
(2009), 30, 1206-1220) demonstrates that the WGA binding proteins (lane 3) are greater than the unbound proteins (lane 4). This indicates that the WGA bound glycoproteins represent over 50% of the total proteins in a normal human serum.
[00214] The O-linked glycan synthesis inhibitors are tested on at least three independent occasions, in duplicate over a dose range.
Secondary assay
[00215] O-linked glycan specificity is then determined by probing with lectins and/or proteins that bind to other glycan classes (chondroitin sulfate, Heparan sulfate, N-linked, etc.).
Determination of composition of O-linked glycans [00216] Free glycans are obtained from the peptide backbone by hydrazino lysis which involves reacting with hydrazine (hydrazinolysis), acetylating with acetic anhydride / sodium bicarbonate, acidification and purification of the free glycans. Preferential release of O-linked glycans is selected by altering the conditions (time and temperature) of the hydrazinolysis reaction. Typically, the released glycans are labeled e.g. with the fluorescent tag, 2-aminobenzamide (2-AB) by reductive amination. Glycan structures can then be analyzed by HPLC (e.g. Glycoprep N column, Oxford Glycosciences) using a buffer gradient and fluorescence detection. The composition and sequence of the glycans can be further analyzed by digestion at specific monosaccharide residues with one or a combination of specific glycosidases e.g. sialic acid (A. ureafaciens sialidase), galactose (S. pneumoniae β-galactosidase), fucose (bovine epididymis α-fucosidase), N- acetylhexosamine (jackbean β-N-acetylhexosaminidase), N-acetylglucosamine (S. pneumoniae N-acetyl-β-D-glucosaminidase), mannose (jackbean α-mannnosidase) and internal galactose (B. fragilis endo-β-galactosidase). Following digestion the glycans are reanalyzed by HPLC.
[00217] Unlabeled glycans are analyzed by mass spectrometry (MS). In addition, sialic acid residues are esterified. Neutral (digested as outlined above) and sialic acid methyl ester containing oligosaccharides are analyzed by MS including MALDI MS on an instrument externally calibrated with a mixture of dextran oligomers [00218] Another method for analyzing glycans on glycoproteins involves removing
N-linked glycans from the polypeptide with the enzyme Peptide: N-Glycosidase F, also known as PNGase F. In the procedure, cell or tissue material is extracted with detergent. Then it is reduced, carboxymethylated, digested with trypsin and the glycopeptides purified by reverse phase Cl 8 column chromatography. N-linked glycans are released from the peptides with PNGase F while the O-linked glycan containing peptides are repurified by reverse phase C 18 column chromatography. O-linked glycans are then released from the peptides by reductive elimination (NaH4 and NaOH), desalted and cleaned up with a reverse phase C18 column chromatography (C 18 Sep-Pak cartridge). For MS analysis, the purified O-linked glycans are permethylated and can be analyzed by various techniques including matrix-assisted laser desorption ionization "time-of-flight" (MALDI-TOF) and collisionally activated dissociation electrospray tandem mass spectrometry (CAD-ES- MS/MS). For linkage analyses the permethylated glycans are hydrolyzed, reduced, acetylated and analyzed by gas chromatography mass spectrometry (GC-MS). EXAMPLE 2:
[00219] LS 180 cells were treated with a carbohydrate-based inhibitor of O-linked sialylation, GalNAcα-O-benzyl, and with sialidase (2 hr at 37 0C). As seen in Figure 2, LS 180 cells show reduced binding to Maackia Amurensis Lectin II (MAH) after treatment with GalNAcα-O-benzyl. LS 180 cells treated with sialidase also showed reduced binding to biotinylated MAH (identified with PE-Cy5 Streptavidin), which verifies that the lectin binds primarily to sialic acid structures in O-linked glycans. EXAMPLE 3:
[00220] The generation of a CHO cell line that presents sialic acid only on O-linked glycans is described herein. A mutant CHO cell line is available that lacks complex sialylated N-linked glycans due to a mutation in GIcNAc transferase I (CHO-lecl, ATCC CRL- 1735) Reduction of glyco lipids is eliminated using the glycolipid inhibitor, PDMP (20 uM) if needed. The combination of the genetic lesion in CHO-lecl and pharmacological inhibition of glycolipid biosynthesis should result in a cell line that presents sialic acid only on O-linked glycans. CHO cells make only core 1 type oligosaccharides, since they lack the Core 2 N-acetylglucosamine transferase (C2GlcNAc-T). Thus, the use of this cell line to screen for inhibitors that block the formation of sialylated core 1 is contemplated herein. [00221] In order to produce a CHO-lecl cell line that binds all of the lectins described herein, CHO-lecl cells are stably transfected with human C2GlcNAc-T. 36% of O-linked glycans produced by CHO cells transfected with C2GlcNAc-T consist of Core 2 structures. The majority of these Core 2 glycans are further modified with O-linked sialic acid by endogenous α2,3 sialic acid transferases. In order to induce the expression of the sLex structure, cells with human αl,3 fucosyltransferase VII (Fuc-TVII) are also transfected.
[00222] Efficient transfection and expression of the transferases are monitored by flow cytometry using fluorescent lectins. Individual clonal lines are isolated based on increased WGA and MAH II binding and transfected with Fuc-TVII to determine the level of CSLEX-I binding. CHO lines having the desired characteristics are then tested with the methods as described above.
[00223] While preferred embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only. Various alternatives to the embodiments described herein are optionally employed in practicing the inventions. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. EXAMPLE 4:
[00224] Suspension U937 cells were treated with and without test compounds. After
2 days of growth the cells were probed with MaI II for 1 hour on ice (MAL II binds to glycans containing sialic acid, e.g., in an (α-2,3) linkage). After washing to remove unbound MaI II, MaI II was detected with streptavidin-Cy5-PE. After washing to remove the unbound streptavidin-Cy5-PE the bound probe was quantified using flow cytometry. Compound doses are in uM. The Y-axis shows the % of binding relative to control cells. Control cells were treated with vehicle only. The test compounds were tested on at least 3 independent occasions in duplicate over a dose range.
[00225] Figure 3 illustrates the affects of O-linked inhibitor compounds on the ability of Maakia Amurensis Lectin II (MaI II) to bind to treated and untreated U937 cells. Other compounds listed in Figures 7A- 7L demonstrated similar activity. EXAMPLE 5:
[00226] MAL II binds to glycans containing sialic acid residues, e.g., in an (α-2,3) linkage. FGF2 is specific for another class of glycans (heparan sulfate). Cultured DAOY cells were treated with and without the test compounds. After 2 days of growth the cells were released with 5 mM EDTA. Parallel cultures were then probed with either MAL II or FGF2 for 1 hour on ice. After washing to remove unbound lectin, bound lectins were detected with streptavidin-Cy5-PE. After washing to remove the unbound streptavidin-Cy5- PE the bound probes were quantified (separately) using flow cytometry. Compound doses are in uM. The Y-axis shows the % binding relative to the untreated cells. The test compounds were tested on at least 3 independent occasions in duplicate over a dose range. [00227] Figures 4-6 illustrate the specificity of the indicated O-linked inhibitors by probing with Maakia Amurensis Lectin II (MaI II) and with another lectin, fibroblast growth factor 2 (FGF2). These compounds show selective inhibition of O-linked glycans without a reduction in the unrelated glycan - heparan sulfate. This demonstrates that these inhibitors have glycan class selectivity. Other compounds listed in Figures 7A- 7K demonstrated similar activity. EXAMPLE 6: Method of Treatment [00228] Human Clinical Trial of the Safety and/or Efficacy of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) therapy.
[00229] Objective: To determine the safety, pharmacokinetics, and efficacy of administered selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 36A-36I, or a pharmaceutically acceptable salt thereof).
[00230] Study Design: This will be a Phase I, single-center, open-label, randomized dose escalation study followed by a Phase II study in cancer patients with a cancer that can be biopsied (e.g., adenocarcinoma). Patients should not have had exposure to an O-linked glycan biosynthesis inhibitor prior to the study entry. Patients must not have received treatment for their cancer within 2 weeks of beginning the trial. Treatments include the use of chemotherapy, hematopoietic growth factors, and biologic therapy such as monoclonal antibodies. The exception is the use of hydroxyurea for patients with WBC > 30 x 103/μL. This duration of time appears adequate for wash out due to the relatively short-acting nature of most anti-leukemia agents. Patients must have recovered from all toxicities (to grade 0 or 1) associated with previous treatment. All subjects are evaluated for safety and all blood collections for pharmacokinetic analysis are collected as scheduled. All studies are performed with institutional ethics committee approval and patient consent. [00231] Phase I: Patients receive (e.g., intravenous, oral, ip, or the like) selective O- linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) daily for 5 consecutive days or 7 days a week. Doses of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A- 7L, or a pharmaceutically acceptable salt thereof) may be held or modified for toxicity based on assessments as outlined below. Treatment repeats every 28 days in the absence of unacceptable toxicity. Cohorts of 3-6 patients receive escalating doses of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) until the maximum tolerated dose (MTD) for the selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) is determined. The MTD is defined as the dose preceding that at which 2 of 3 or 2 of 6 patients experience dose-limiting toxicity. Dose limiting toxicities are determined in any suitable manner, e.g., according to the definitions and standards set by the National Cancer Institute (NCI) Common Terminology for Adverse Events (CTCAE) Version 3.0 (August 9, 2006). [00232] Phase II: Patients receive selective O-linked glycan biosynthesis inhibitor
(e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof) as in phase I at the MTD determined in phase I. Treatment repeats every 6 weeks for 2-6 courses in the absence of disease progression or unacceptable toxicity. After completion of 2 courses of study therapy, patients who achieve a complete or partial response may receive an additional 4 courses. Patients who maintain stable disease for more than 2 months after completion of 6 courses of study therapy may receive an additional 6 courses at the time of disease progression, provided they meet original eligibility criteria. [00233] Blood Sampling: Serial blood is drawn by direct vein puncture before and after administration of selective O-linked glycan biosynthesis inhibitor (e.g., a compound of Figures 7A-7L, or a pharmaceutically acceptable salt thereof). Venous blood samples (5 mL) for determination of serum concentrations are obtained at about 10 minutes prior to dosing and at approximately the following times after dosing: days 1, 2, 3, 4, 5, 6, 7, and 14. Each serum sample is divided into two aliquots. All serum samples are stored at -2O0C. Serum samples are shipped on dry ice.
[00234] Pharmacokinetics : Patients undergo plasma/serum sample collection for pharmacokinetic evaluation before beginning treatment and at days 1, 2, 3, 4, 5, 6, 7, and 14. Pharmacokinetic parameters are calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetics parameters are determined: peak serum concentration (Cmax); time to peak serum concentration (tmax); area under the concentration- time curve (AUC) from time zero to the last blood sampling time (AUCo-72) calculated with the use of the linear trapezoidal rule; and terminal elimination half- life (ti/2), computed from the elimination rate constant. The elimination rate constant is estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot. The mean, standard deviation (SD), and coefficient of variation (CV) of the pharmacokinetic parameters are calculated for each treatment. The ratio of the parameter means (preserved formulation/non-preserved formulation) is calculated. [00235] Patient Response: Patient response is assessed via imaging with X-ray, CT scans, and MRI, and imaging is performed prior to beginning the study and at the end of the first cycle, with additional imaging performed every four weeks or at the end of subsequent cycles. Imaging modalities are chosen based upon the cancer type and feasibility/availability, and the same imaging modality is utilized for similar cancer types as well as throughout each patient's study course. Response rates are determined using the RECIST criteria. (Therasse et al, J. Natl. Cancer Inst. 2000 Feb 2; 92(3):205-16; http://ctep.cancer.gov/forms/TherasseRECISTINCI.pdf). Patients also undergo cancer/tumor biopsy to assess changes in progenitor cancer cell phenotype and clonogenic growth by flow cytometry, Western blotting, and IHC, and for changes in cytogenetics by FISH or TaqMan PCR for specific chromosomal translocations. After completion of study treatment, patients are followed periodically for 4 weeks.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A process for modifying the population of O-linked glycans on one or more proteins associated with a cell, the process comprising contacting a cell that produces O-linked glycans with an effective amount of a selective late stage O-linked glycan biosynthesis inhibitor, the selective late stage O-linked glycan biosynthesis inhibitor being active in a mammalian cell.
2. The process of claim 1, wherein the selective late stage O-linked glycan biosynthesis inhibitor is a non-carbohydrate O-linked glycan biosynthesis inhibitor.
3. The process of any of claims 1-2, wherein the selective O-linked glycan biosynthesis inhibitor has a molecular weight of less than 700 g/mol.
4. The process of any of claims 1-3, wherein the process reduces the ratio of glycans containing poly-N-acetyllactosamine chains to glycans that do not contain poly-N- acetyllactosamine chains.
5. The process of any of claims 1-3, wherein the process reduces the amount of terminal sialylation and/or terminal GIcNAc residues.
6. The process of any of claims 1-3, wherein the process reduces the amount of selectin ligands in the cellular population of O-linked glycan.
7. The process of any of claims 1-3, wherein the process reduces the cellular population of Core 1 and/or Core 2 O-linked glycan structures.
8. The process of any of claims 1-3, wherein the selective O-linked glycan biosynthesis inhibitor reduces the ratio of acidic O-linked glycans to neutral O-linked glycans to less than 50%.
9. The process of any of claims 1-3, wherein the selective O-linked glycan biosynthesis inhibitor inhibits ST3GalI, ST3GalII, or ST3GalIII.
10. The process of claim 9, wherein the selective O-linked glycan biosynthesis inhibitor indirectly inhibits ST3GalI, ST3GalII, or ST3GalIII.
11. The process of claim 9, wherein the selective O-linked glycan biosynthesis inhibitor directly inhibits ST3GalI, ST3GalII, or ST3GalIII.
12. The process of any of claims 1-11, wherein the cell is an inflammatory cell or cancer cell.
13. The process of any of claims 1-12, wherein the cell is present in an individual diagnosed with or suspected of having rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, reperfusion injury, or having had an organ transplant.
14. The process of any of claims 1-12, wherein the cell is present in an individual diagnosed with or suspected of having adenocarcinoma.
15. A composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein less than 50 mol. % of the plurality of O-linked glycans comprise terminal sialyl groups.
16. A composition comprising a plurality of glycoproteins, the glycoproteins comprising a plurality of O-linked glycans covalently linked to a core protein, wherein less than 50 mol. % of the glycoproteins bind to WGA.
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