Patent Application
20230321186
This application incorporates by reference the Sequence Listing contained in the following eXtensible Markup Language (XML) file being submitted concurrently herewith:
Candida albicans is an opportunistic fungal pathogen that asymptomatically colonizes the mucosal surfaces of most healthy humans, particularly on surfaces of the oral cavity, gastrointestinal tract, and female genital tract. Alterations to the mucus barrier and perturbations in the microbiota can lead to C. albicans overgrowth and infection, causing conditions such as oral thrush, vulvovaginal candidiasis, and life-threatening systemic candidiasis. The scarcity of antifungal drug classes, their limited efficacy, toxicity, and the development of resistance contribute to a high mortality rate of ˜40% in deep-seated candidiasis, highlighting a profound and urgent need for the development of alternative treatments for fungal infections.
The subject matter disclosed herein is based, in part, on the discovery that mucin glycans, when chemically released from their naturally grafted state, as well as being synthetically produced, can be used as novel therapeutic agents against the fungal pathogen, Candida albicans.
In one aspect, the disclosure provides methods of attenuating virulence of a fungus in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of treating a fungal infection in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of treating a biofilm-related infection in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of maintaining a microbiota in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of repairing or restoring a damaged microbiome or microbiota in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of maintaining a mucus barrier in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of attenuating virulence of a fungus, the methods comprising contacting the fungus with a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of inhibiting formation of a fungal biofilm on a surface, the methods comprising contacting the surface with a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of maintaining a mucus barrier on a surface, the methods comprising contacting the surface with a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides compositions comprising a (i.e., one or more) synthetic mucin glycan.
In another aspect, the disclosure provides compositions comprising a mucin glycan, wherein the purity of the mucin glycan is at least about 30%.
In another aspect, the disclosure provides defined mucin glycan compositions comprising one or more mucin glycans desired mucin glycans, wherein at least 80% of total mucin glycans present in the compositions are of the one or more desired mucin glycans or pharmaceutically acceptable salts thereof.
In another aspect, the disclosure provides defined mucin glycan compositions comprising one or more mucin glycans, wherein at least 80% of total mucin glycans present in the compositions are of the one or more mucin glycans or pharmaceutically acceptable salts thereof.
In some embodiments, a fungus comprises Candida albicans.
In some embodiments, a mucin glycan is a synthetic mucin glycan.
In some embodiments, attenuating virulence of a fungus comprises modulating expression of a virulence-associated gene of a fungus, reducing a fungus's surface adhesion, inhibiting a fungus's morphological transition to an invasive or virulent cell type, inhibiting fungal biofilm formation, reducing a fungus's secretion of a hydrolytic enzyme, or a combination thereof.
In some embodiments, a surface comprises a living surface, an inert surface, or both.
The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.
A description of example embodiments follows.
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
When introducing elements disclosed herein, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. Further, the one or more elements may be the same or different.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including.”
As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the terms “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the disclosure, can in some embodiments, be replaced with the term “consisting of,” or “consisting essentially of” to vary scopes of the disclosure.
As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and/or.”
It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.
Compounds described herein include those described generally, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the relevant contents of which are incorporated herein by reference.
Unless specified otherwise within this specification, the nomenclature used in this specification generally follows the examples and rules stated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, which is incorporated by reference herein for its chemical structure names and rules on naming chemical structures. Optionally, a name of a compound may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.).
“Alkyl” refers to a branched or straight-chain, monovalent, hydrocarbon radical having the specified number of carbon atoms. Thus, “(C2-C8)alkyl” refers to a radical having from 2-8 carbon atoms in a branched or linear arrangement. Typically, alkyl is (C1-C25)alkyl, e.g., (C1-C15)alkyl, (C1-C10)alkyl, (C1-C8)alkyl, (C2-C8)alkyl, (C1-C6)alkyl, (C2-C6)alkyl, (C1-C5)alkyl, (C2-C5)alkyl or (C2-C3)alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, 2-methylpentyl, n-hexyl, and the like. In some embodiments, alkyl is optionally substituted, e.g., with one or more substituents described herein.
“Aryl” refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic), aromatic, hydrocarbon ring system having the specified number of ring atoms, and includes aromatic rings fused to non-aromatic rings, as long as one of the fused rings is an aromatic hydrocarbon. Thus, “(C6-C15)aryl” refers to a ring system having from 6-15 ring atoms. Examples of aryl include phenyl, naphthyl and fluorenyl. In some embodiments, aryl (e.g., (C6-C15)aryl) is phenyl, naphthyl or fluorenyl. In some embodiments, aryl is optionally substituted, e.g., with one or more substituents described herein.
“Heteroaryl” refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic), aromatic, hydrocarbon ring system having the specified number of ring atoms, wherein at least one carbon atom in the ring system has been replaced with a heteroatom selected from nitrogen, sulfur and oxygen. Thus, “(C5-C15)heteroaryl” refers to a heteroaromatic ring system having from 5-15 ring atoms consisting of carbon, nitrogen, sulfur and oxygen. “Heteroaryl” includes heteroaromatic rings fused to non-aromatic rings, as long as one of the fused rings is a heteroaromatic hydrocarbon. A heteroaryl can contain 1, 2, 3 or 4 (e.g., 1, 2 or 3) heteroatoms independently selected from nitrogen, sulfur and oxygen. In some embodiments, a heteroaryl contains 1, 2 or 3 heteroatoms, each of which is nitrogen. Typically, heteroaryl is (C5-C20)heteroaryl, e.g., (C5-C15)heteroaryl, (C5-C12)heteroaryl, C5 heteroaryl or C6 heteroaryl. Monocyclic heteroaryls include, but are not limited to, furan, oxazole, thiophene, triazole, triazene, thiadiazole, oxadiazole, imidazole, isothiazole, isoxazole, pyrazole, pyridazine, pyridine, pyrazine, pyrimidine, pyrrole, tetrazole and thiazole. Bicyclic heteroaryls include, but are not limited to, indolizine, indole, isoindole, indazole, benzimidazole, benzofuran, benzothiazole, purine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine and pteridine. In some embodiments, heteroaryl (e.g., (C5-C15)heteroaryl) is pyridinyl, pyrimidinyl or carbazolyl. In some embodiments, heteroaryl is optionally substituted, e.g., with one or more substituents described herein.
“Alkoxy” refers to an alkyl radical attached through an oxygen linking atom, wherein alkyl is as described herein. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, and the like.
“Halogen” and “halo” are used interchangeably herein and each refers to fluorine, chlorine, bromine, or iodine. In some embodiments, halo is fluoro, chloro or bromo. In some embodiments, halo is fluoro.
Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds produced by the replacement of a hydrogen with deuterium or tritium, or of a carbon with a 13C- or 14C-enriched carbon are within the scope of this disclosure. In all provided structures, any hydrogen atom can also be independently selected from deuterium (2H), tritium (3H) and/or fluorine (18F). Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present disclosure.
“Derived from,” as used herein, refers to a chemical structure that is homologous to or structurally similar to a related chemical structure.
The phrase “pharmaceutically acceptable” means that the substance or composition the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids, and suitable inorganic and organic bases.
Examples of pharmaceutically acceptable acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable acid addition salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxy-ethanesulfonate, hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.
Either the mono-, di- or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form.
Salts derived from appropriate bases include salts derived from inorganic bases, such as alkali metal, alkaline earth metal, and ammonium bases, and salts derived from aliphatic, alicyclic or aromatic organic amines, such as methylamine, trimethylamine and picoline, or N+((C1-C4)alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.
Compounds described herein can also exist as “solvates” or “hydrates.” A “hydrate” is a compound that exists in a composition with one or more water molecules. A hydrate can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. A “solvate” is similar to a hydrate, except that a solvent other than water, such as methanol, ethanol, dimethylformamide, diethyl ether, or the like replaces water. Mixtures of such solvates or hydrates can also be prepared. The source of such solvate or hydrate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.
Compounds disclosed herein may exist as stereoisomers. For example, compounds disclosed herein may have asymmetric centers, chiral axes, and chiral planes (e.g., as described in: E. L. Eliel and S. H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, or as individual diastereomers or enantiomers.
Unless otherwise indicated, all possible isomers and mixtures thereof, including optical isomers, rotamers, tautomers and cis- and trans-isomers, are included in the present invention.
When a disclosed compound is depicted by structure without indicating the stereochemistry, and the compound has one chiral center, it is to be understood that the structure encompasses one enantiomer or diastereomer of the compound separated or substantially separated from the corresponding optical isomer(s), a racemic mixture of the compound and mixtures enriched in one enantiomer or diastereomer relative to its corresponding optical isomer(s).
When a disclosed compound is depicted by a structure indicating stereochemistry, and the compound has more than one chiral center, the stereochemistry indicates relative stereochemistry, rather than the absolute configuration of the substituents around the one or more chiral carbon atoms. “R” and “S” are used to indicate the absolute configuration of substituents around one or more chiral carbon atoms.
“Enantiomers” are pairs of stereoisomers that are non-superimposable mirror images of one another, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center.
“Diastereomers” are stereoisomers that are not related as mirror images, most commonly because they contain two or more asymmetrically substituted carbon atoms.
“Racemate” or “racemic mixture,” as used herein, refer to a mixture containing equimolar quantities of two enantiomers of a compound. Such mixtures exhibit no optical activity (i.e., they do not rotate a plane of polarized light).
Methods of obtaining an optical isomer separated or substantially separated from the corresponding optical isomer(s) are known in the art. For example, an optical isomer can be purified from a racemic mixture by well-known chiral separation techniques, such as, but not limited to, normal- and reverse-phase chromatography, and crystallization. An optical isomer can also be prepared by the use of chiral intermediates or catalysts in synthesis. In some cases, compounds having at least some degree of enantiomeric enrichment can be obtained by physical processes, such as selective crystallization of salts or complexes formed with chiral adjuvants.
As used herein, the term “compound of the disclosure” refers to a compound of any structural formula depicted herein (e.g., a compound of structural formula I or a subformula thereof)), as well as isomers, such as stereoisomers (including diastereoisomers, enantiomers and racemates) and tautomers thereof, isotopologues thereof, and inherently formed moieties (e.g., polymorphs and/or solvates, such as hydrates) thereof. When a moiety is present that is capable of forming a salt, then salts are included as well, in particular, pharmaceutically acceptable salts.
“Pharmaceutically acceptable carrier” refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the agent with which it is formulated and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
“Treating” or “treatment,” as used herein, refers to taking steps to deliver a therapy to a subject, such as a mammal, in need thereof (e.g., as by administering to a mammal one or more therapeutic agents). “Treating” or “treatment” includes inhibiting the disease or condition (e.g., as by slowing or stopping its progression or causing regression of the disease or condition), and relieving the symptoms resulting from the disease or condition. The term “treating” or “treatment” refers to the medical management of a subject with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent or cure a disease, pathological condition, or disorder-such as the particular indications exemplified herein. This term includes active treatment (treatment directed to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy). Treatment also includes diminishment of the extent of the disease or condition; preventing spread of the disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
“Administering” or “administration,” as used herein, refers to providing a compound, composition, or pharmaceutically acceptable salt thereof described herein to a subject in need of treatment or prevention.
“A therapeutically effective amount” or “an effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or biological result (e.g., treatment, healing, inhibition or amelioration of physiological response or condition, etc.). Non-limiting examples of desired therapeutic or biological results include disruption of fungal biofilm formation, growth, and/or maintenance, for example, at or proximate to the surface of an implanted medical device. Effective reductions of signs and/or symptoms associated with fungal infection can be determined by one or more suitable means in the art.
The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as disease state, age, sex, and weight of an individual, e.g., a mammal, mode of administration and the ability of a therapeutic, or combination of therapeutics, to elicit a desired response in an individual.
An effective amount of an agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art. For example, suitable dosages can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, or from about 0.01 mg/kg to about 1 mg/kg body weight per treatment. Determining the dosage for a particular agent, subject and disease is well within the abilities of one of skill in the art. Preferably, the dosage does not cause or produces minimal adverse side effects.
In one aspect, the disclosure provides methods of attenuating virulence of a fungus, the methods comprising contacting the fungus with a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In some embodiments, a fungus comprises a yeast. In some embodiments, a fungus (e.g., yeast) is capable of biofilm growth. Non-limiting examples of fungi capable of biofilm growth include Candida species (e.g., Candida albicans, Candida glabrate, C. parapsilosis, Candida dubiliensis, and Candida tropicalis), Cryptococcus neoformans (e.g., C. neoformans), Trichosporon species (e.g., Trichosporon asahii), Aspergillus fumigatus (e.g., A. fumigatus) and Histoplasma capsulatum. In some embodiments, a yeast is from the CTG clade (Candida clade). In some embodiments, a fungus comprises Candida albicans.
In some embodiments, a fungus comprises a fungus population (e.g., a community of Candida albicans).
“Virulence,” as used herein, refers to a phenotypic state of a fungus associated with infection that may harm its host, for example, the host's epithelial tissues. In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises modulating (e.g., downregulating) a virulence-associated gene of the fungus, reducing the fungus's surface adhesion (reducing the fungus from attaching to a surface), inhibiting the fungus's morphological transition to an invasive or virulent cell type, inhibiting fungal biofilm formation, reducing the fungus's secretion of a hydrolytic enzyme, or a combination thereof.
In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises downregulating a gene positively correlated with virulence, upregulating a gene negatively correlated with virulence, or both. Non-limiting examples of virulence-associated gene include genes encoding proteins within the pathways of regulating filamentation and/or adhesion (e.g., encoding adhesins, secreted proteases, cytolytic toxins) and interspecies interactions.
In some embodiments, modulating a virulence-associated gene comprises:
In some embodiments, modulating a virulence-associated gene comprises:
In some embodiments, a method disclosed herein reduces expression of a virulence-associated gene by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces expression of a virulence-associated gene by at least about 30%.
In some embodiments, a method increases expression expression of a virulence-associated gene by at least about 20%, for example, by at least about: 50%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold. In some embodiments, a method increases expression expression of a virulence-associated gene by at least about 3-fold.
Expression of a virulence-associated gene can be determined by a person of ordinary skill using methods known in the art, for example, at the RNA level using RNA sequencing (RNA-Seq) or a microarray.
In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises reducing the fungus's surface adhesion. In some embodiments, a method reduces surface adhesion of a fungus (e.g., Candida albicans) by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces surface adhesion of a fungus (e.g., Candida albicans) by at least about 30%.
The term “surface,” as used herein, refers to an area of upon which fungal biofilm may grow. In some embodiments, a surface comprises a living surface, an inert surface, or both. In some embodiments, a surface comprises a living surface (e.g., a non-keratinized epithelial surface of the body). In some embodiments, a surface comprises an inert surface.
Non-limiting examples of living surfaces include skin and epithelium of the gastrointestinal (GI) tract, oral cavity and reproductive tract. Non-limiting examples of inert surfaces include those of implants and in-dwelling devices, for example contact lenses, dentures, prosthetics (e.g., hip prosthesis, joint prosthesis, voice prosthesis), valves (e.g., mechanical heart valves), pacemakers, catheters (e.g., urinary catheters and central venous catheters), cannulae, vascular access devices, intrauterine devices (IUDs), intravenous lines, endotracheal tubes, enteral feeding tubes, drainage tubes (e.g., wound drains), tracheostomies, instruments (e.g., surgical or examination instruments), laboratory benches, and a material that supports cell growth, replication, and/or maintenance. In some embodiments, a surface is a surface of a device in fluid communication with a subject's circulatory system.
In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises inhibiting morphological transition to an invasive or virulent cell type. In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises inhibiting the fungus's morphological transition to a filamentous state (hyphae formation). In some embodiments, a method reduces morphological transition (e.g., hyphae formation) of a fungus (e.g., Candida albicans) by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces morphological transition (e.g., from yeast to hyphal form) of a fungus (e.g., Candida albicans) by at least about 30%.
In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises inhibiting fungal biofilm formation. “Biofilm,” as used herein, refers to a structured community of fungi enclosed in a (e.g., self-produced) polymeric matrix that is adherent to a surface. See, e.g., Desai et al., Fungal biofilms, drug resistance, and recurrent infection, Cold Spring Harb Perspect Med. 4(10):a019729 (2014) for additional information on fungal biofilm, the contents of which are incorporated by reference in their entirety. In some embodiments, a method reduces biofilm formation by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces biofilm formation by at least about 30%.
In some embodiments, attenuating virulence of a fungus (e.g., Candida albicans) comprises reducing the fungus's secretion of a hydrolytic enzyme. In some embodiments, a method reduces secretion of a hydrolytic enzyme by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, a method reduces secretion of a hydrolytic enzyme by at least about 30%.
In another aspect, the disclosure provides methods of inhibiting formation of a fungal biofilm on a surface, the methods comprise contacting the surface with a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof. A surface may comprise any one or more of the surfaces described herein.
In another aspect, the disclosure provides methods of maintaining a mucus barrier on a surface, the methods comprise contacting the surface with a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of attenuating virulence of a fungus in a subject in need thereof, the methods comprise administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of treating a fungal infection in a subject in need thereof, the methods comprise administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In some embodiments, a fungal infection results from a burn, a wound, keratitis, a bioprosthesis or a medical device, e.g., an indwelling medical device. In some embodiments, a fungal infection is in the lung of a subject.
In another aspect, the disclosure provides methods of treating a biofilm-related infection in a subject in need thereof, the methods comprise administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In some embodiments, a biofilm-related infection results from a burn, a wound, keratitis, a bioprosthesis or an indwelling medical device. In some embodiments, a biofilm-related infection is in the lung of a subject.
In another aspect, the disclosure provides methods of maintaining a microbiota in a subject in need thereof, the methods comprise administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
In another aspect, the disclosure provides methods of maintaining a mucus barrier in a subject in need thereof, the methods comprise administering to the subject a therapeutically effective amount of a mucin glycan, tautomer, stereoisomer and/or a pharmaceutically acceptable salt thereof.
As used herein, “subject” includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.) and livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic animals. In some embodiments, a subject is a human. In some embodiments, the subject is male. In some embodiments the subject is female. In some embodiments, the subject (e.g., human) is immunocompromised (e.g., with AIDS, undergoing an anticancer therapy, or undergoing an immunosuppression therapy). In some embodiments, the subject (e.g., human) has an implanted medical device.
“Subject in need thereof,” as used herein, refers to a subject (e.g., a mammalian subject such as a human) diagnosed with or suspected of having a fungal infection, who will be or has been administered a mucin glycan according to a method of the disclosure. “Subject in need thereof” includes those subjects who already have the undesired physiological change or disease as well as those subjects prone to have the physiological change or disease.
In some embodiments, the subject is diagnosed with or is suspected of having a pulmonary disease (e.g., a chronic pulmonary disease), a lung infection, a mucosal infection, a dermal infection, or an infection, for example, caused by a device, e.g., a medical device. In some embodiments, the subject has a wound (e.g., a burn wound). In some embodiments, the subject is diagnosed with or is suspected of having thrush, vaginal yeast infections, diaper rash or hematogenously disseminated candidiasis. In some embodiments, the subject is diagnosed with or is suspected of having hematogenously disseminated candidiasis. In some embodiments, the subject has a chronic fungal infection.
The administration of the compounds (agents, salts, etc.) and compositions may be carried out in any manner, e.g., by parenteral or nonparenteral administration, including by aerosol inhalation, injection, infusions, ingestion, transfusion, implantation or transplantation. For example, the compositions described herein may be administered to a subject trans-arterially, intradermally, subcutaneously, intratumorally, by intramedullar administration, intranodally, intramuscularly, intravenously (e.g., through an IV drip or by intravenous (i.v.) injection), intranasally, intrathecally or intraperitoneally. In some embodiments, the administration is intravenous. In some embodiments, the administration is topical. In some embodiments, the administration is oral. In some embodiments, the administration is by injection, for instance, directly into a tissue, organ, or site of infection. In some embodiments, the administration is ex vivo. In some embodiments, compounds and compositions are administered by routes such as oral, endobronchial, intrathecal, intracisternal, intra-articular, intraperitoneal, ophthalmic (e.g., in an ophthalmic preparation such as eye drops, intraocular injections, ointments), aerosol, irrigant, peritoneal lavage, endobronchial and intrathecal administration. In one embodiment, the subject has a burn wound and the administration is in an ointment.
In some embodiments, a composition is administered topically, orally, intravenously, nasally, ocularly, or transdermally. In some embodiments, a composition is administered topically. In some embodiments, a composition is administered orally. In some embodiments, a composition is administered intravenously. In some embodiments, a composition is administered nasally. In some embodiments, a composition is administered ocularly. In some embodiments, a composition is administered transdermally.
In some embodiments, a composition is provided in a liquid form. In some embodiments, a composition comprises a dose of from about 0.1 g/liter to about 50 g/liter of mucin glycan(s), for example, about: 0.1 g/liter, 0.2 g/liter, 0.3 g/liter, 0.4 g/liter, 0.5 g/liter, 0.6 g/liter, 0.7 g/liter, 0.8 g/liter, 0.9 g/liter, 1 g/liter, 2 g/liter, 3 g/liter, 4 g/liter, 5 g/liter, 6 g/liter, 7 g/liter, 8 g/liter, 9 g/liter, 10 g/liter, 11 g/liter, 12 g/liter, 13 g/liter, 14 g/liter, 15 g/liter, 16 g/liter, 17 g/liter, 18 g/liter, 19 g/liter, 20 g/liter, 21 g/liter, 22 g/liter, 23 g/liter, 24 g/liter, 25 g/liter, 26 g/liter, 27 g/liter, 28 g/liter, 29 g/liter, 30 g/liter, 31 g/liter, 32 g/liter, 33 g/liter, 34 g/liter, 35 g/liter, 36 g/liter, 37 g/liter, 38 g/liter, 39 g/liter, 40 g/liter, 41 g/liter, 42 g/liter, 43 g/liter, 44 g/liter, 45 g/liter, 46 g/liter, 47 g/liter, 48 g/liter, 49 g/liter or 50 g/liter of mucin glycan(s).
In some embodiments, a composition is provided in a dried form.
In some embodiments, the protein component of a composition is less than about 50% by weight, for example, less than about: 25%, 20%, 18%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1%, by weight.
Administration of a compound, composition, or pharmaceutically acceptable salt described herein may be in conjunction with another active ingredient (e.g., an anti-fungal), for example, simultaneously in the same composition, simultaneously in different dosage forms, or sequentially. A compound, composition, or pharmaceutically acceptable salt described herein and another active ingredient may be formulated in a single combination, multiple combinations, or separate compositions.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to increase rate of fungal (e.g., Candida albicans) clearance in a subject, for example, compared to the same subject were it left untreated. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to increase the rate of fungal (e.g., Candida albicans) clearance by at least about 20%, for example, by at least about: 50%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce expression of a virulence-associated gene (e.g., UME6 or HGC1) by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce expression of a virulence-associated gene by at least about 30%.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to increase expression of a virulence-associated gene (e.g., YWP1) by at least about 20%, for example, by at least about: 50%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to increase expression of a virulence-associated gene by at least about 3-fold.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce surface adhesion of a fungus (e.g., Candida albicans) by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce surface adhesion of a fungus (e.g., Candida albicans) by at least about 30%.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce a morphological transition (e.g., hyphae formation) of a fungus (e.g., Candida albicans) by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce a morphological transition (e.g., from yeast to hyphal form) of a fungus (e.g., Candida albicans) by at least about 30%.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce biofilm formation by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75%, 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce biofilm formation by at least about 30%.
In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce secretion of a hydrolytic enzyme by at least about 10%, for example, by at least about: 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%, or by about: 10-99%, 15-99%, 15-95%, 20-95%, 20-90%, 25-90%, 25-85%, 30-85%, 30-80%, 35-80%, 35-75% 40-75%, 40-70%, 45-70%, 45-65%, 50-65% or 50-60%. In some embodiments, an amount (e.g., a therapeutically effective amount) of mucin glycan or pharmaceutically acceptable salt thereof is sufficient to reduce secretion of a hydrolytic enzyme by at least about 30%.
In another aspect, the disclosure provides defined or semi-defined mucin glycan compositions, comprising one or more desired mucin glycans or pharmaceutically acceptable salts thereof, wherein at least 80% of total mucin glycans present in the compositions are of the one or more desired mucin glycans or pharmaceutically acceptable salts thereof.
A defined, or semi-defined mucin glycan composition comprises at least 1 desired mucin glycan or pharmaceutically acceptable salt thereof, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more desired mucin glycans or pharmaceutically acceptable salts or 1-20, 1-18, 1-15, 1-12, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 2-5, 2-4, 2-3, 3-20, 3-18, 3-15, 3-12, 3-10, 3-8, 3-5, 3-4, 4-20, 4-18, 4-15, 4-12, 4-10, 4-8, 4-5, 5-20, 5-18, 5-15, 5-12, 5-10, 5-8, 8-20, 8-18, 8-15, 8-12 or 8-10 desired mucin glycans or pharmaceutically acceptable salts thereof.
In some embodiments, a defined, or semi-defined mucin glycan composition comprises at least 2 desired mucin glycans or pharmaceutically acceptable salts thereof at a desired ratio. In some embodiments, a desired ratio of a first versus a second desired mucin glycan or pharmaceutically acceptable salt thereof is about: 1:1, 3:4, 2:3, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10 or 1:15.
In some embodiments, a mucin glycan composition is defined. In other embodiments, a mucin glycan composition is semi-defined.
In some embodiments, at least 85%, 90%, 95%, 99%, 99.5% or 99.9% of the total mucin glycans or pharmaceutically acceptable salts thereof present in a composition are of the one or more desired mucin glycans or pharmaceutically acceptable salts thereof. In some embodiments, about 80-99.9% of the total mucin glycans or pharmaceutically acceptable salts thereof present in a composition are of the one or more desired mucin glycans or pharmaceutically acceptable salts thereof, for example, about: 85-99.9%, 85-99.5%, 85-99%, 85-98%, 85-95%, 88-99.9%, 88-99.5%, 88-99%, 88-98%, 88-95%, 90-99.9%, 90-99.5%, 90-99%, 90-98%, 90-95%, 92-99.9%, 92-99.5%, 92-99%, 92-98%, 92-95%, 95-99.9%, 95-99.5%, 95-99% or 95-98% of the total mucin glycans present in the composition are of the one or more desired mucin glycans or pharmaceutically acceptable salts thereof.
In some embodiments, a defined, or semi-defined mucin glycan composition is incorporated into a formulation for therapeutic administration (e.g., a pharmaceutical composition). In some embodiments, a pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or diluents. In some embodiments, a pharmaceutical composition further comprises one or more additional therapeutics, i.e., therapeutic agents (e.g., an antifungal). Pharmaceutical compositions may be formulated into preparations in, fore example, solid, semi-solid, liquid or gaseous forms, such as capsules, gels, granules, microspheres, ointments, powders, solutions, drops, and tablets. Defined, or semi-defined mucin glycan compositions may be formulated for various routes of administration, for example, oral formulations, intravenous formulations, or in the form of a douche. In some embodiments, a defined, or semi-defined mucin glycan composition is formulated into an ointment.
In some embodiments, a defined, or semi-defined mucin glycan composition is incorporated into a coating (e.g., a film) for any one or more of the surfaces described herein (e.g., to inhibit formation of a fungal biofilm). In some embodiments, a coating (e.g., a single- of multi-layer film) is biocompatible.
In another aspect, the disclosure provides compositions comprising a synthetic mucin glycan.
In another aspect, the disclosure provides compositions comprising a mucin glycan, wherein the purity of the mucin glycan is at least about 30%. In some embodiments, the purity of the mucin glycan is at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99.5%. In some embodiments, the purity of the mucin glycan is at least about 50%. In some embodiments, the purity of the mucin glycan is at least about 99.5%. In some embodiments, the composition comprises at least about 100 mg of a mucin glycan having a purity of at least about 99.5%.
Mucins are heavily O-glycosylated glycoproteins that are found in mucous secretions (secreted mucins) and on the cell surface (membrane-bound (transmembrane) mucins). Secreted mucins include gel-forming mucins and non-gel-forming (soluble) mucins.
Mucin genes are expressed in a tissue- and/or region-specific fashion, for example, in the airway, digestive system, reproductive system, and different regions of the gastrointestinal tract. About 20 different mucin genes have been cloned, including gel-forming mucin genes such as MUC2, MUC5AC, MUC5B, MUC6 and MUC19; soluble mucin genes such as MUC7, MUC8, MUC9 and MUC20; and transmembrane mucin genes such as MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC13, MUC15 and MUC21.
Non-limiting examples of mucin genes include human MUC1 (e.g., GenBank: AAA60019.1, UniProtKB/Swiss-Prot: P15941.3, Gene ID 4582), porcine MUC1 (e.g., NCBI: XP_020945387.1), human MUC2 (e.g., GenBank: AAB95295.1, Gene ID 4583), porcine MUC2 (e.g., NCBI: XP_020938243.1), human MUC5AC (e.g., GenBank: ABV02582.1, UniProtKB/Swiss-Prot: P98088.4, Gene ID 4586), porcine MUC5AC (e.g., NCBI: XP_020938242.1), human MUC5B (e.g., UniProtKB/Swiss-Prot: Q9HC84.3, Gene ID 727897), porcine MUC5B (e.g., NCBI: XP_020938146.1), human MUC6 (e.g., GenBank: AZL49144.1) and porcine MUC6 (e.g., NCBI: XP_020938133.1).
Additional non-limiting examples of mucin genes include MUC2 (e.g., HomoloGene 130504, 131905, 132025, or 133451), MUC5AC (e.g., UniGene IDs 3881294, 1370646, 1774723, 1133368, 441382, and 5878683, HomoloGene 130646; Gene ID 100170143; and reference sequences AAC48526, AAD19833, and AAD19832), MUC5B (e.g., HomoloGene 124413), MUC6 (e.g., HomoloGene 18768), MUC19 (bovine submaxillary mucin (BSM), e.g., Gene ID 100140959; HomoloGene 130967; and reference protein sequence XP_0035861 12.1).
A mucin protein comprises an amino region and/or a carboxy region that are cysteine-rich and a central region enriched in serine and/or threonine residues. Native mucin glycans are typically built upon an N-acetylgalactosamine that is O-linked via its C-1 hydroxyl to serine or threonine residues of a mucin protein. The monosaccharide unit or series of monosaccharide units that, in a native mucin glycan, would be O-linked via the C-1 hydroxyl of the monosaccharide unit or first monosaccharide unit in the series of monosaccharide units, respectively, to a serine or threonine residue of the mucin protein is also referred to herein as the “glycan core” or “mucin glycan core.”
As used herein, the term “mucin glycan” refers to a compound comprising (e.g., consisting of) a glycan, or a portion thereof, found on a native mucin. “Mucin glycans” can be natural (e.g., derived from purification) or synthetic. In some embodiments, the mucin glycan is a synthetic mucin glycan.
In some embodiments, a mucin glycan is purified from a non-human animal, for example, a domesticated mammal such as a porcine, a bovine. In some embodiments, a mucin glycan is gut-derived. In some embodiments, a mucin glycan is prepared from cell culture (e.g., of a recombinant cell line or a non-recombinant cell line).
In some embodiments, a mucin glycan is released from a mucin clycoprotein, for example, by enzymatic degradation, or a chemical release (reductive or non-reductive).
It has been found that native glycans exhibit certain common cores, such as those described herein as Cores 1-8. Thus, in some embodiments, a mucin glycan comprises a glycan core. In some embodiments, the glycan core comprises GalNAc, e.g., as the sole monosaccharide in the core or as the first monosaccharide unit in a series of monosaccharide units that make up the core. In some embodiments, the glycan core comprises (e.g., consists of) two or more monosaccharide units linked via O-glycosidic linkages (e.g., GalNAc and one or more additional monosaccharide units linked via O-glycosidic linkages).
It will be understood that one or more monosaccharide units of a mucin glycan compound and/or glycan core described herein can be optionally substituted (e.g., unsubstituted; substituted) by one or more (e.g., from one to ten, from one to five, from one to three) independently selected substituents in accordance with this disclosure as, for example, when a monosaccharide in a glycan core is substituted with an additional monosaccharide unit via an O-glycosidic linkage. Typically, when a monosaccharide unit of a glycan core is substituted with an additional monosaccharide unit, the hydrogen atom of a hydroxyl group of the monosaccharide of the glycan core being substituted is replaced with the substituent, such as the additional monosaccharide unit.
It will also be understood that the mucin glycan compound, in addition to comprising a glycan may contain further, non-saccharide substituents. Thus, for example, one or more oxygen atoms of the monosaccharide hydroxyls in the glycan may be independently substituted, as by replacing the hydrogen atom of a monosaccharide hydroxyl with a substituent, such as alkyl (e.g., methyl, ethyl, n-propyl, isopropyl), aryl (e.g., phenyl, biaryl), —C(O)H or —C(O)alkyl (e.g., acetyl). See, for example, Compounds 3 and 6 described herein, which are substituted with methyl at the 1-position of the galactopyranoside. In some embodiments, the oxygen atom of the hydroxyl at the anomeric/C-1 position of the monosaccharide unit or first monosaccharide unit in the series of monosaccharide units of the glycan core is substituted (e.g., with alkyl (e.g., methyl, ethyl, n-propyl, isopropyl), aryl (e.g., phenyl, biaryl), —C(O)H or —C(O)alkyl (e.g., acetyl)). In some embodiments, the oxygen atom of the hydroxyl at the anomeric/C-1 position of the GalNAc residue of Cores 1-8 is substituted (e.g., with alkyl (e.g., methyl, ethyl, n-propyl, isopropyl), aryl (e.g., phenyl, biaryl), —C(O)H or —C(O)alkyl (e.g., acetyl)). In further embodiments, the oxygen atoms(s) of the remaining hydroxyls in the glycan or glycan core are not substituted with a non-saccharide substituent. In further embodiments, the oxygen atom(s) of the remaining hydroxyls in the glycan or glycan core are not substituted.
Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
When a group is substituted herein, the substituted group can have a suitable substituent at each substitutable position of the group and, when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent can be the same or different at every position.
Suitable substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkyl, alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidino, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, cycloalkyl, heterocyclyl, aryl, biaryl or heteroaryl. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. For instance, substituent(s) of a substituted alkyl may include substituted and unsubstituted forms of hydroxyl, amino, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate) and carbonyls (including ketones, aldehydes, carboxylates, and esters), and the like.
It will also be appreciated by a person of ordinary skill in the art, that a mucin glycan having hydroxyl at the anomeric/C-1 position of the GalNAc residue of Cores 1-8 may equilibrate into various different forms, for example, in aqueous solutions, such as aqueous formulations and in the body. The process by which equilibration occurs is known as glycan mutarotation/tautomerization. The tautomers (e.g., ring-chain tautomers, including both the cyclic (such as, furanose and pyranose) tautomeric forms and the linear or open-chain tautomeric forms) as well as the isomers (e.g., anomers, including both the alpha- and beta-anomers) resulting from glycan mutarotation/tautomerization are within the scope of the present disclosure. Thus, in some embodiments of any of the structural formulas herein, the structural formula includes tautomers (e.g., ring-chain tautomers, including both the cyclic (such as, furanose and pyranose) tautomeric forms and the linear or open-chain tautomeric forms) and isomers (e.g., anomers) thereof, in particular, those resulting from glycan mutarotation/tautomerization.
In some embodiments, a mucin glycan is a primate (e.g., human) mucin glycan. In some embodiments, a mucin glycan is a non-primate (e.g., porcine, bovine, or mouse) mucin glycan.
In some embodiments, a native mucin is expressed in the airway, the digestive system, the reproductive system, or a combination thereof. In some embodiments, a native mucin is expressed in the digestive system. In some embodiments, a mucin glycan comprises a gastric mucin glycan, a salivary gland mucin glycan, an intestinal mucin glycan, or a combination thereof.
In some embodiments, a mucin glycan is a secreted mucin glycan (e.g., of the digestive system). In some embodiments, a mucin glycan is a gel-forming mucin glycan (e.g., of the digestive system).
In some embodiments, a mucin glycan comprises an oligosaccharide of from about 2 to about 10 monosaccharide subunits in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 monosaccharide subunits in length. In some embodiments, a mucin glycan comprises an oligosaccharide of from about 2 to about 6 monosaccharide subunits in length. In some embodiments, a mucin glycan comprises an oligosaccharide of from about 4 to about 6 monosaccharide subunits in length. In some embodiments, a mucin glycan comprises an oligosaccharide of from about 6 to about 8 monosaccharide subunits in length. In some embodiments, a mucin glycan comprises an oligosaccharide of from about 6 to about 10 monosaccharide subunits in length. In some embodiments, a mucin glycan comprises an oligosaccharide of from about 8 to about 10 monosaccharide subunits in length.
Non-limiting examples of linear and/or branched oligosaccharides include galactose, N-acetylgalactosamine, fucose, glucose and N-acetylglucosamine, with heterogenous linkages within any one glycan, e.g., a mixture of α2-3, α2-6, α1-2, α1-3, α1-4, β1-3, β1-4, β1-6, etc. The numbering is shown with respect to D-glucopyranose, where “α” refers to the configuration of a cyclic sugar where the oxygen on the anomeric carbon is on the opposite face of the ring relative to the substituent on the other carbon flanking the ring oxygen and “β” refers to the configuration of a cyclic sugar where the oxygen on the anomeric carbon is on the same face of the ring as the substituent on the other carbon flanking the ring oxygen.
In some embodiments, a mucin glycan comprises one or more N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), mannose (Man), fucose (Fuc), N-acetylneuraminic acid (Neu5Ac), galactose (Gal) or a combination thereof. In some embodiments, a mucin glycan comprises one or more N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), N-acetylneuraminic acid (Neu5Ac), galactose (Gal) or a combination thereof. In some embodiments, a mucin glycan further comprises sialic acid.
In some embodiments, a mucin glycan is unsulfated. In other embodiments, a mucin glycan is sulfated.
In some embodiments, a mucin glycan comprises a glycan core of the following structural formula:
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan structure selected from the group consisting of 1a, 2, 3a, 4, 9c, 15b, 16b, 18b, 5, 6, 7 and 25b of
In some embodiments, a mucin glycan (e.g., Core 1 mucin glycan) further comprises fucose, sialic acid, or a combination thereof. In some embodiments, a mucin glycan comprises the following structural formula:
or a tautomer or stereoisomer thereof, or
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan core of the following structural formula:
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan structure selected from the group consisting of 8a, 9a, 9b, 10a, 11a, 12a, 12b, 13, 14a, 15a, 16a, 17, 18a, 19, 20a, 21a, 21b, 22, 23a, 24a, 24b, 25a, 26a, 27, 28, 29, 30, 31, 32, 33a, 34a, 35, 36, 37, 38a, 38b, 39, 40, 41, 42, 43, 44, 45, 46, 47a, 48, 49a, 50, 51, 52, 53a and 54 of
In some embodiments, a mucin glycan (e.g., Core 2 mucin glycan) further comprises fucose, galactose, or a combination thereof. In some embodiments, a mucin glycan comprises the following structural formula:
or a tautomer or stereoisomer thereof or
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan core of the following structural formula:
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan structure selected from the group consisting of 8b, 10b, 20b, 26b and 55 of
In some embodiments, a mucin glycan comprises a glycan core of the following structural formula:
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan structure selected from the group consisting of 34b and 56 of
In some embodiments, a mucin glycan comprises a glycan core of the following structural formula:
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan structure selected from the group consisting of 1b, 3b, 11b, 14b, 57, 8c, 23b, 23c, 24c, 33b, 58, 47b, 49b and 53b of
In some embodiments, a mucin glycan comprises a glycan core of one of the following structural formula:
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises a glycan structure of 5 of
In some embodiments, a mucin glycan comprises a glycan core of one of the following structural formula:
or a tautomer or stereoisomer thereof,
or a tautomer or stereoisomer thereof,
or a tautomer or stereoisomer thereof, or
or a tautomer or stereoisomer thereof.
In some embodiments, a mucin glycan comprises (e.g., consists of) a MUC2 glycan, a MUC5AC glycan, a MUC5B glycan, or a combination thereof. In some embodiments, a mucin glycan comprises a porcine MUC2 glycan, a porcine MUC5AC glycan, a human MUC5B glycan, or a combination thereof.
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (HexNAc)1 (e.g., GlyTouCan Accession: G57321FI); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G73318SN, G33986KK); (Hex)1 (HexNAc)1 (NeuGc)1 (e.g., G64527IJ); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 (e.g., G47180UC); (Hex)2 (HexNAc)1 (Deoxyhexose)2 (e.g., G68200GL); (Hex)1 (HexNAc)1 (NeuAc)2 (e.g., G01614ZM); (Hex)2 (HexNAc)1 (Deoxyhexose)3 (e.g., G82961CS); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 (e.g., G49549VN, G95742RK); (Hex)1 (HexNAc)1 (NeuAc)1 (NeuGc)1 (e.g., G49527BY); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G75749JP, G40270LS); (Hex)3 (HexNAc)2 (Deoxyhexose)4 (e.g., G93469SN, G15747RC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)2 (HexNAc)2 (e.g., G64973KT); (Hex)1 (HexNAc)3 (e.g., G68893BQ, G23438NR); (Hex)1 (HexNAc)2 (Deoxyhexose)2 (e.g., G89748NG, G09520ZQ); (Hex)1 (HexNAc)2 (NeuAc)1 (e.g., G85608AG, G64844ET); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)1 (HexNAc)2 (NeuGc)1 (e.g., G60426XC); (Hex)3 (HexNAc)2 (e.g., G16404NW, G25323VU); (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G28878FC); (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW); (Hex)1 (HexNAc)4 (e.g., G59229NY); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)2 (HexNAc)2 (NeuAc)1 (e.g., G46748BU); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (e.g., G15849KC, G81911LP, G41486OC); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)3 (HexNAc)3 (e.g., G86537AD, G89585FG); (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ); (Hex)2 (HexNAc)2 (Deoxyhexose)3 (e.g., G01532FF); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G77740PR, G59155GF); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)2 (HexNAc)3 (Deoxyhexose)2 (e.g., G68384KC, G39326PP); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); (Hex)2 (HexNAc)4 (Deoxyhexose)1 (e.g., G23700TV); (Hex)3 (HexNAc)4 (e.g., G94517VF, G57672ST); (Hex)3 (HexNAc)2 (Deoxyhexose)3 (e.g., G70416EY, G13012GZ); (Hex)2 (HexNAc)5 (e.g., G23048PE); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G74607VK, G81461IK); (Hex)3 (HexNAc)3 (Deoxyhexose)2 (e.g., G35949CT, G71094KR, G08426KY); (Hex)2 (HexNAc)4 (Deoxyhexose)2 (e.g., G90965BZ); (Hex)3 (HexNAc)4 (Deoxyhexose)1 (e.g., G02990AF, G29956GF); (Hex)3 (HexNAc)3 (Deoxyhexose)3 (e.g., G23021IW); (Hex)4 (HexNAc)3 (Deoxyhexose)2 (e.g., G68308CM); (Hex)3 (HexNAc)4 (Deoxyhexose)2 (e.g., G93333OF); (Hex)4 (HexNAc)4 (Deoxyhexose)1 (e.g., G94768NG); (Hex)2 (HexNAc)5 (Deoxyhexose)2 (e.g., G25612EW); (Hex)3 (HexNAc)3 (Deoxyhexose)4 (e.g., G84853WN); (Hex)5 (HexNAc)6 (Deoxyhexose)3 (e.g., G65612SS); (Hex)3 (HexNAc)5 (Deoxyhexose)1 (e.g., G05252QE); (Hex)7 (HexNAc)6 (Deoxyhexose)1 (e.g., G61898SS); (Hex)3 (HexNAc)6 (e.g., G62609ZF); (Hex)3 (HexNAc)4 (Deoxyhexose)3 (e.g., G59787TQ); (Hex)4 (HexNAc)4 (Deoxyhexose)2 (e.g., G98518WL); (Hex)6 (HexNAc)7 (Deoxyhexose)2 (e.g., G29852ZH); (Hex)4 (HexNAc)5 (Deoxyhexose)1 (e.g., G92547QZ); (Hex)3 (HexNAc)4 (Deoxyhexose)4 (e.g., G89469SP); (Hex)5 (HexNAc)4 (Deoxyhexose)2 (e.g., G21630AC); (Hex)4 (HexNAc)5 (Deoxyhexose)2 (e.g., G82251ZP, G18603ZQ); (Hex)5 (HexNAc)5 (Deoxyhexose)1 (e.g., G25957KN); (Hex)4 (HexNAc)6 (Deoxyhexose)1 (e.g., G32752FJ); (Hex)6 (HexNAc)5 (Deoxyhexose)1 (e.g., G37901JE, G84713IO); (Hex)4 (HexNAc)6 (Deoxyhexose)2 (e.g., G99804SJ); (Hex)4 (HexNAc)5 (Deoxyhexose)4 (e.g., G11381FO); (Hex)5 (HexNAc)5 (Deoxyhexose)3 (e.g., G90829NZ); (Hex)6 (HexNAc)5 (Deoxyhexose)2 (e.g., G70999YJ, G02681FY); (Hex)5 (HexNAc)6 (Deoxyhexose)2 (e.g., G44467ZE); (Hex)3 (HexNAc)5 (Deoxyhexose)2 (e.g., G28921PH); (Hex)6 (HexNAc)5 (Deoxyhexose)3 (e.g., G18501TC); (Hex)4 (HexNAc)4 (Deoxyhexose)3 (e.g., G66166BF); (HexNAc)2 (e.g., G00041MO, G00057MO); (HexNAc)2 (NeuAc)1 (e.g., G63334FZ); (HexNAc)2 (NeuGc)1 (e.g., G09441IP); (Hex)3 (HexNAc)5 (e.g., G00505CR); (Hex)2 (HexNAc)5 (Deoxyhexose)1 (e.g., G09396HG); (Hex)2 (HexNAc)1 (e.g., G28052FT); (Hex)2 (HexNAc)1 (NeuAc)1 (e.g., G59126YU); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); and (Hex)1 (NeuGc)1 (e.g., G38557KR, G59867EM).
“Hex” refers to hexose, a monosaccharide with six carbon atoms, C6H12O6. “NAC” refers to N-acetylcysteine. “NeuAc” or “Neu5Ac” refers to N-acetylneuraminic acid. “NeuGc” or “Neu5Gc” refers to N-glycolylneuraminic acid. “HexNAc” refers to N-acetylhexosamine. “Deoxyhexose” refers to any deoxysugar derived from a hexose.
In some embodiments, a mucin glycan comprises a porcine MUC2 glycan.
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (NeuGc)1 (e.g., G38557KR, G59867EM); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)1 (HexNAc)1 (NeuGc)1 (e.g., G64527IJ); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (HexNAc)2 (e.g., G00041MO, G00057MO); (HexNAc)1 (e.g., G57321FI); (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G28878FC); (Hex)2 (HexNAc)2 (NeuAc)1 (e.g., G46748BU); (Hex)2 (HexNAc)2 (e.g., G64973KT); (HexNAc)2 (NeuAc)1 (e.g., G63334FZ); (Hex)2 (HexNAc)1 (e.g., G28052FT); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)1 (HexNAc)3 (e.g., G68893BQ, G23438NR); (HexNAc)2 (NeuGc)1 (e.g., G09441IP); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)1 (HexNAc)2 (NeuAc)1 (e.g., G85608AG, G64844ET); (Hex)2 (HexNAc)3 (Deoxyhexose)2 (e.g., G68384KC, G39326PP); (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW); (Hex)1 (HexNAc)1 (NeuAc)2 (e.g., G01614ZM); (Hex)1 (HexNAc)2 (NeuGc)1 (e.g., G60426XC); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 (e.g., G47180UC); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G75749JP, G40270LS); (Hex)4 (HexNAc)5 (Deoxyhexose)1 (e.g., G92547QZ); (Hex)3 (HexNAc)4 (Deoxyhexose)1 (e.g., G02990AF, G29956GF); (Hex)2 (HexNAc)4 (Deoxyhexose)2 (e.g., G90965BZ); (Hex)3 (HexNAc)5 (Deoxyhexose)1 (e.g., G05252QE); (Hex)2 (HexNAc)1 (NeuAc)1 (e.g., G59126YU); (Hex)3 (HexNAc)4 (e.g., G94517VF, G57672ST); (Hex)4 (HexNAc)4 (Deoxyhexose)1 (e.g., G94768NG); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)1 (HexNAc)1 (NeuAc)1 (NeuGc)1 (e.g., G49527BY); (Hex)2 (HexNAc)4 (Deoxyhexose)1 (e.g., G23700TV); (Hex)3 (HexNAc)5 (e.g., G00505CR); (Hex)4 (HexNAc)5 (Deoxyhexose)2 (e.g., G82251ZP, G18603ZQ); (Hex)5 (HexNAc)5 (Deoxyhexose)1 (e.g., G25957KN); (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); (Hex)4 (HexNAc)6 (Deoxyhexose)1 (e.g., G32752FJ); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (e.g., G15849KC, G81911LP, G41486OC); (Hex)2 (HexNAc)1 (Deoxyhexose)2 (e.g., G68200GL); (Hex)4 (HexNAc)6 (Deoxyhexose)2 (e.g., G99804SJ); (Hex)3 (HexNAc)2 (e.g., G16404NW, G25323VU); (Hex)5 (HexNAc)6 (Deoxyhexose)2 (e.g., G44467ZE); and (Hex)3 (HexNAc)3 (e.g., G86537AD, G89585FG).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (NeuGc)1 (e.g., G38557KR, G59867EM); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)1 (HexNAc)1 (NeuGc)1 (e.g., G64527IJ); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (HexNAc)2 (e.g., G00041MO, G00057MO); (HexNAc)1 (e.g., G57321FI); (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G28878FC); (Hex)2 (HexNAc)2 (NeuAc)1 (e.g., G46748BU); (Hex)2 (HexNAc)2 (e.g., G64973KT); (HexNAc)2 (NeuAc)1 (e.g., G63334FZ); and (Hex)2 (HexNAc)1 (e.g., G28052FT).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (NeuGc)1 (e.g., G38557KR, G59867EM); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); and (Hex)1 (HexNAc)1 (NeuGc)1 (e.g., G64527IJ).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); and (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC).
In some embodiments, a mucin glycan comprises a porcine MUC5AC glycan.
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW); (Hex)2 (HexNAc)2 (e.g., G64973KT); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); (Hex)3 (HexNAc)4 (e.g., G94517VF, G57672ST); (Hex)2 (HexNAc)4 (Deoxyhexose)1 (e.g., G23700TV); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G73318SN, G33986KK); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)1 (HexNAc)3 (e.g., G68893BQ, G23438NR); (Hex)3 (HexNAc)4 (Deoxyhexose)1 (e.g., G02990AF, G29956GF); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)3 (HexNAc)4 (Deoxyhexose)2 (e.g., G93333OF); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (e.g., G15849KC, G81911LP, G41486OC); (Hex)2 (HexNAc)1 (e.g., G28052FT); (Hex)3 (HexNAc)3 (Deoxyhexose)2 (e.g., G35949CT, G71094KR, G08426KY); (Hex)3 (HexNAc)3 (e.g., G86537AD, G89585FG); (Hex)1 (HexNAc)2 (NeuAc)1 (e.g., G85608AG, G64844ET); (Hex)3 (HexNAc)5 (e.g., G00505CR); (HexNAc)1 (e.g., G57321FI); (Hex)1 (HexNAc)4 (e.g., G59229NY); (Hex)4 (HexNAc)3 (Deoxyhexose)2 (e.g., G68308CM); (Hex)3 (HexNAc)5 (Deoxyhexose)1 (e.g., G05252QE); (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G28878FC); (Hex)2 (HexNAc)1 (NeuAc)1 (e.g., G59126YU); (Hex)4 (HexNAc)4 (Deoxyhexose)1 (e.g., G94768NG); (Hex)2 (HexNAc)2 (NeuAc)1 (e.g., G46748BU); (Hex)3 (HexNAc)2 (e.g., G16404NW, G25323VU); (Hex)2 (HexNAc)3 (Deoxyhexose)2 (e.g., G68384KC, G39326PP); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 (e.g., G47180UC); (Hex)4 (HexNAc)4 (Deoxyhexose)2 (e.g., G98518WL); (Hex)2 (HexNAc)4 (Deoxyhexose)2 (e.g., G90965BZ); (Hex)2 (HexNAc)5 (e.g., G23048PE); (Hex)4 (HexNAc)5 (Deoxyhexose)2 (e.g., G82251ZP, G18603ZQ); (Hex)3 (HexNAc)6 (e.g., G62609ZF); (Hex)2 (HexNAc)1 (Deoxyhexose)2 (e.g., G68200GL); (Hex)4 (HexNAc)4 (Deoxyhexose)3 (e.g., G66166BF); (Hex)4 (HexNAc)5 (Deoxyhexose)1 (e.g., G92547QZ); (Hex)3 (HexNAc)5 (Deoxyhexose)2 (e.g., G28921PH); (Hex)2 (HexNAc)5 (Deoxyhexose)1 (e.g., G09396HG); (Hex)6 (HexNAc)5 (Deoxyhexose)3 (e.g., G18501TC); (Hex)4 (HexNAc)6 (Deoxyhexose)1 (e.g., G32752FJ); (Hex)5 (HexNAc)6 (Deoxyhexose)3 (e.g., G65612SS); (Hex)7 (HexNAc)6 (Deoxyhexose)1 (e.g., G61898SS); (Hex)5 (HexNAc)4 (Deoxyhexose)2 (e.g., G21630AC); (Hex)5 (HexNAc)5 (Deoxyhexose)1 (e.g., G25957KN); (Hex)6 (HexNAc)5 (Deoxyhexose)1 (e.g., G37901JE, G84713IO); (Hex)6 (HexNAc)7 (Deoxyhexose)2 (e.g., G29852ZH); (Hex)4 (HexNAc)6 (Deoxyhexose)2 (e.g., G99804SJ); (Hex)5 (HexNAc)5 (Deoxyhexose)3 (e.g., G90829NZ); (Hex)6 (HexNAc)5 (Deoxyhexose)2 (e.g., G70999YJ, G02681FY); and (Hex)5 (HexNAc)6 (Deoxyhexose)2 (e.g., G44467ZE).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW); (Hex)2 (HexNAc)2 (e.g., G64973KT); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); (Hex)3 (HexNAc)4 (e.g., G94517VF, G57672ST); (Hex)2 (HexNAc)4 (Deoxyhexose)1 (e.g., G23700TV); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G73318SN, G33986KK); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)1 (HexNAc)3 (e.g., G68893BQ, G23438NR); (Hex)3 (HexNAc)4 (Deoxyhexose)1 (e.g., G02990AF, G29956GF); and (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW); (Hex)2 (HexNAc)2 (e.g., G64973KT); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); and (Hex)3 (HexNAc)4 (e.g., G94517VF, G57672ST).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); and (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ).
In some embodiments, a mucin glycan comprises a human MUC5B glycan.
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)3 (HexNAc)3 (Deoxyhexose)2 (e.g., G35949CT, G71094KR, G08426KY); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)2 (Deoxyhexose)2 (e.g., G89748NG, G09520ZQ); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G73318SN, G33986KK); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)2 (HexNAc)3 (Deoxyhexose)2 (e.g., G68384KC, G39326PP); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (e.g., G15849KC, G81911LP, G41486OC); (Hex)2 (HexNAc)1 (e.g., G28052FT); (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G28878FC); (Hex)3 (HexNAc)4 (Deoxyhexose)3 (e.g., G59787TQ); (Hex)2 (HexNAc)1 (Deoxyhexose)2 (e.g., G68200GL); (Hex)2 (HexNAc)2 (NeuAc)1 (e.g., G46748BU); (Hex)3 (HexNAc)2 (Deoxyhexose)3 (e.g., G70416EY, G13012GZ); (Hex)2 (HexNAc)4 (Deoxyhexose)2 (e.g., G90965BZ); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); (Hex)3 (HexNAc)3 (Deoxyhexose)3 (e.g., G23021IW); (Hex)2 (HexNAc)2 (e.g., G64973KT); (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW); (Hex)2 (HexNAc)2 (Deoxyhexose)3 (e.g., G01532FF); (Hex)3 (HexNAc)4 (Deoxyhexose)4 (e.g., G89469SP); (Hex)3 (HexNAc)2 (e.g., G16404NW, G25323VU); (Hex)3 (HexNAc)4 (Deoxyhexose)1 (e.g., G02990AF, G29956GF); (HexNAc)2 (NeuAc)1 (e.g., G63334FZ); (Hex)3 (HexNAc)4 (Deoxyhexose)2 (e.g., G93333OF); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G75749JP, G40270LS); (Hex)3 (HexNAc)2 (Deoxyhexose)4 (e.g., G93469SN, G15747RC); (Hex)1 (HexNAc)2 (NeuAc)1 (e.g., G85608AG, G64844ET); (Hex)1 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 (e.g., G47180UC); (Hex)4 (HexNAc)3 (Deoxyhexose)2 (e.g., G68308CM); (Hex)4 (HexNAc)4 (Deoxyhexose)2 (e.g., G98518WL); (HexNAc)2 (e.g., G00041MO, G00057MO); (Hex)4 (HexNAc)4 (Deoxyhexose)3 (e.g., G66166BF); (Hex)2 (HexNAc)4 (Deoxyhexose)1 (e.g., G23700TV); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G74607VK, G81461IK); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (NeuAc)1 (e.g., G49549VN, G95742RK); (Hex)3 (HexNAc)3 (Deoxyhexose)4 (e.g., G84853WN); (Hex)5 (HexNAc)4 (Deoxyhexose)2 (e.g., G21630AC); (HexNAc)1 (e.g., G57321FI); (Hex)1 (HexNAc)3 (e.g., G68893BQ, G23438NR); (Hex)2 (HexNAc)1 (Deoxyhexose)3 (e.g., G82961CS); (Hex)2 (HexNAc)5 (Deoxyhexose)2 (e.g., G25612EW); (Hex)4 (HexNAc)4 (Deoxyhexose)1 (e.g., G94768NG); (Hex)4 (HexNAc)5 (Deoxyhexose)4 (e.g., G11381FO); (Hex)4 (HexNAc)5 (Deoxyhexose)2 (e.g., G82251ZP, G18603ZQ); (Hex)5 (HexNAc)5 (Deoxyhexose)1 (e.g., G25957KN); (Hex)2 (HexNAc)4 (e.g., G34764BK, G12074QJ); (Hex)4 (HexNAc)5 (Deoxyhexose)1 (e.g., G92547QZ); (Hex)1 (NeuGc)1 (e.g., G38557KR, G59867EM); (Hex)5 (HexNAc)6 (Deoxyhexose)2 (e.g., G44467ZE); (Hex)6 (HexNAc)5 (Deoxyhexose)2 (e.g., G70999YJ, G02681FY); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (NeuAc)1 (e.g., G77740PR, G59155GF); (Hex)5 (HexNAc)6 (Deoxyhexose)3 (e.g., G65612SS); (Hex)3 (HexNAc)3 (e.g., G86537AD, G89585FG); (Hex)3 (HexNAc)5 (Deoxyhexose)1 (e.g., G05252QE); (Hex)5 (HexNAc)5 (Deoxyhexose)3 (e.g., G90829NZ); (Hex)2 (HexNAc)1 (NeuAc)1 (e.g., G59126YU); (Hex)7 (HexNAc)6 (Deoxyhexose)1 (e.g., G61898SS); and (Hex)3 (HexNAc)4 (e.g., G94517VF, G57672ST).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)3 (HexNAc)3 (Deoxyhexose)2 (e.g., G35949CT, G71094KR, G08426KY); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)2 (Deoxyhexose)2 (e.g., G89748NG, G09520ZQ); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G73318SN, G33986KK); (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G91459EI, G26724PP, G52132CU); (Hex)2 (HexNAc)3 (Deoxyhexose)2 (e.g., G68384KC, G39326PP); (Hex)3 (HexNAc)2 (Deoxyhexose)1 (e.g., G15849KC, G81911LP, G41486OC); (Hex)2 (HexNAc)1 (e.g., G28052FT); (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G28878FC); (Hex)3 (HexNAc)4 (Deoxyhexose)3 (e.g., G59787TQ); (Hex)2 (HexNAc)1 (Deoxyhexose)2 (e.g., G68200GL); (Hex)2 (HexNAc)2 (NeuAc)1 (e.g., G46748BU); (Hex)3 (HexNAc)2 (Deoxyhexose)3 (e.g., G70416EY, G13012GZ); (Hex)2 (HexNAc)4 (Deoxyhexose)2 (e.g., G90965BZ); (Hex)3 (HexNAc)3 (Deoxyhexose)1 (e.g., G66741QE, G16458JH); (Hex)3 (HexNAc)3 (Deoxyhexose)3 (e.g., G23021IW); (Hex)2 (HexNAc)2 (e.g., G64973KT); and (Hex)2 (HexNAc)3 (e.g., G96915PP, G13483MW).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); (Hex)1 (Deoxyhexose)1 (e.g., G00068MO); (Hex)3 (HexNAc)3 (Deoxyhexose)2 (e.g., G35949CT, G71094KR, G08426KY); (Hex)2 (HexNAc)2 (Deoxyhexose)2 (e.g., G23119MJ); (Hex)1 (HexNAc)1 (NeuAc)1 (e.g., G65562ZE); (Hex)3 (HexNAc)2 (Deoxyhexose)2 (e.g., G20310DM, G78177LC); (Hex)1 (HexNAc)2 (e.g., G00033MO, G61730RY, G56868BH); (Hex)1 (HexNAc)2 (Deoxyhexose)2 (e.g., G89748NG, G09520ZQ); (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G94514IB, G61216ZY); and (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G73318SN, G33986KK).
In some embodiments, a mucin glycan comprises a formula selected from the group consisting of (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G94435QH); (Hex)1 (HexNAc)1 (e.g., G76355TG, G85856KC); (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G32426JY, G74353FF); (Hex)1 (NeuAc)1 (e.g., G30207PZ, G63069TR); and (Hex)1 (Deoxyhexose)1 (e.g., G00068MO).
In some embodiments, a mucin glycan (e.g., MUC2, MUC5B or MUC5AC glycan) is a sulfated mucin glycan.
In some embodiments, a sulfated mucin glycan comprises a formula selected from the group consisting of S1 (Hex)1 (HexNAc)1 (e.g., G10634LC); S1 (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G08671QK); S1 (Hex)1 (HexNAc)2 (e.g., G32406CO); S1 (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G24803MV); S1 (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G10520JC); S1 (Hex)1 (HexNAc)2 (NeuAc)1 (e.g., G96888OD); S1 (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G24803MV); S1 (Hex)1 (HexNAc)2 (NeuGc)1 (e.g., G60426XC); S1 (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G72091WB, G60644GY); and S1 (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G35891PL).
In some embodiments, a sulfated mucin glycan comprises a sulfated MUC2 glycan, a sulfated MUC5AC glycan, a sulfated MUC5B glycan, or a combination thereof.
In some embodiments, a sulfated MUC2 glycan comprises a formula selected from the group consisting of S1 (Hex)1 (HexNAc)2 (e.g., G32406CO); S1 (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G10520JC); S1 (Hex)1 (HexNAc)2 (NeuAc)1 (e.g., G96888OD); S1 (Hex)1 (HexNAc)3 (Deoxyhexose)1 (e.g., G72091WB, G60644GY); and S1 (Hex)1 (HexNAc)2 (NeuGc)1 (e.g., G60426XC).
In some embodiments, a sulfated MUC5AC glycan comprises a formula selected from the group consisting of S1 (Hex)1 (HexNAc)2 (Deoxyhexose)1 (e.g., G10520JC); S1 (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G24803MV); S1 (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G24803MV); S1 (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G35891PL); and S1 (Hex)1 (HexNAc)2 (e.g., G32406CO).
In some embodiments, a sulfated MUC5B glycan comprises a formula selected from the group consisting of S1 (Hex)2 (HexNAc)1 (Deoxyhexose)1 (e.g., G24803MV); S1 (Hex)1 (HexNAc)1 (Deoxyhexose)1 (e.g., G08671QK); S1 (Hex)1 (HexNAc)1 (e.g., G10634LC); S1 (Hex)2 (HexNAc)2 (Deoxyhexose)1 (e.g., G24803MV); and S1 (Hex)2 (HexNAc)3 (Deoxyhexose)1 (e.g., G35891PL).
In some embodiments, a mucin glycan comprises a synthesized (e.g., chemically synthesized) mucin glycan, a purified (e.g., native) mucin glycan, or a combination thereof. In some embodiments, a mucin glycan comprises a synthesized mucin glycan. Methods for chemically synthesizing mucin glycans are described herein; methods for purifying mucin glycan are described herein and are known in the art.
Concentrations of a mucin glycan in compositions or methods disclosed herein can vary, for example, depending on the desired use. In some embodiments, a mucin glycan concentration is about: 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9% (w/v) in a solution. In some embodiments, a mucin glycan concentration in a solution is within physiological concentration ranges of the mucin glycan.
Headings used in this application are for convenience only and do not affect the interpretation of this application.
Preferred features of each of the aspects provided by the disclosure are applicable to all of the other aspects of the disclosure mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g., elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the disclosure, including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed disclosure piecemeal without departing from the disclosure. For example, for materials that are disclosed, while specific reference of each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements A-D is disclosed, then, even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application, including elements of a composition of matter and steps of method of making or using the compositions.
or a tautomer or stereoisomer thereof.
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Candida albicans is an opportunistic fungal pathogen that asymptomatically colonizes the mucosal surfaces of most healthy humans1,2. Alterations to the mucus barrier and microbiota can lead to C. albicans overgrowth and infection, causing conditions such as oral thrush, vulvovaginal candidiasis, and life-threatening systemic candidiasis1,3. The scarcity of antifungal drug classes, their limited efficacy, toxicity, and the development of resistance4 contribute to a mortality rate of ˜40% in deep-seated candidiasis3, highlighting an urgent need for alternative treatments to fungal infections.
Targeting pathogenic mechanisms rather than growth represents an attractive approach for developing novel antimicrobial agents. The infection of diverse host niches is supported by a wide range of C. albicans virulence and fitness attributes, including the morphological yeast-to-hyphal transition (filamentation), adhesin expression, biofilm formation, and the secretion of hydrolytic enzymes that damage the underlying epithelium5. The yeast-to-hyphal transition is a major virulence factor5 and is integral for robust biofilms, which are intrinsically resistant to treatment, posing a significant clinical challenge6. Strikingly, despite its potential for pathogenicity, C. albicans is accommodated in healthy mucus7, suggesting that mucus may underpin novel strategies for preventing C. albicans virulence.
Mucus is a complex viscoelastic secretion that coats all non-keratinized epithelial surfaces in the body that are exposed to and communicate with the external environment8. Much of the microbiota is housed in the mucus layer, serving as a protective barrier and microbial niche8,9. Mucins are the main structural component of mucus and play an integral role in attenuating virulence traits in various cross-kingdom pathogens, including C. albicans2,10. Mucin exposure suppresses C. albicans virulence phenotypes, including the formation of host-cell-penetrating hyphae10. However, the mechanisms through which mucins attenuate virulence in C. albicans remain unknown, impeding their application for therapeutic intervention.
To close this gap, the mechanism and biochemical motifs of mucins that suppress C. albicans virulence gene expression and phenotypes were characterized. By isolating and characterizing mucin-derived glycans across major mucosal surfaces, it was determined that mucin glycans repress C. albicans virulence traits including filamentation, adhesion, and biofilm formation and alter fungal-bacterial dynamics. It was identified that specific Core-1- and Core-2-modified glycan structures within the mucin polymer suppress filamentation and downregulate filamentation-associated genes in C. albicans. These results elucidate the mechanisms by which healthy mucins attenuate C. albicans pathogenicity, suggesting therapeutic candidates for treating C. albicans infection without disrupting the microbiota (and potential evolution of antifungal resistance) that normally accompanies the killing of cells.
C. albicans Strains and Media
Strains were maintained on yeast extract peptone dextrose (YPD) agar (2% Bacto peptone, 2% glucose, 1% yeast extract, 2% agar) and grown at 30° C. Single colonies were inoculated into YPD broth and grown with shaking overnight at 30° C. prior to each experiment. Experiments were performed with Gibco RPMI 1640 medium (Life Technologies, Carlsbad, CA; #31800-089) buffered with 165 mM 3-(N-morpholino)propanesulfonic acid (MOPS) and supplemented with 0.2% NaHCO3 and 2% glucose; YPD medium with 10% fetal bovine serum; GlcNAc medium (0.5% N-acetylglucosamine, 0.5% peptone, 0.3% KH2PO4); Spider medium (1% nutrient broth, 1% D-mannitol, 2 g K2HPO4, 50 mg/mL arginine, 10 mg/mL histidine, and 50 mg/mL tryptophan); or Lee's medium45. Growth curves were performed in Synthetic Defined (SD)+0.004% (w/v) L-Arginine+0.0025% (w/v) L-Leucine media with 2% glucose.
The C. albicans reference strains used in this study were SC5314 and HGFP3. Strain HGFP3 was constructed by inserting the GFP gene next to the promoter of HWP1, a gene encoding a hyphal cell wall protein, in SC5314; this strain was provided by E. Mylonakis (Massachusetts General Hospital, Boston, MA) with the permission of P. Sundstrom. Homozygous deletion strains were obtained from the transcriptional factor deletion collection and were provided by the Fungal Genome Stock Center (www.fgsc.net). The following C. albicans strains used for pathway analyses were gifts from Paul Kauman (University of Massachusetts, Medical School): AV55 (ura3::λimm434/ura3::λimm 434; LEU2::pCK1-efg1-T206E::URA3); DH409 (ura3::λimm434/ura3; ras1-G13V); and CDH72-1 (ura3/ura3 cph1Δ::hisG/cph1Δ::hisG; ADH1prCPH1).
Collection of Human Saliva
Submandibular saliva was collected from healthy human volunteers using a custom vacuum pump, pooled, centrifuged at 2,500×g for 5 min, and phenylmethylsulfonylfluoride (1 mM) was added.
Mucin Purification
This study used native porcine gastric mucins (MUC5AC), porcine intestinal mucins (MUC2), and human salivary mucins (MUC5B), which differ from industrially purified mucins in their rheological properties and bioactivities10,46. Native mucins were purified as described previously10,18. In brief, mucus was scraped from fresh pig stomachs and intestines and solubilized in sodium chloride. Insoluble material was removed via ultracentrifugation at 190,000×g for 1 h at 4° C. (Beckman 50.2 Ti rotor with polycarbonate bottles). Submandibular saliva was collected from human volunteers as described above using a custom vacuum pump, pooled, centrifuged, and protease inhibitors were added10. Mucins were purified using size-exclusion chromatography on separate Sepharose CL-2B columns. Mucin fractions were then desalted, concentrated, and lyophilized for storage at −80° C. Lyophilized mucins were reconstituted by shaking them gently at 4° C. overnight in the desired medium.
Mass spectrometry is routinely used to monitor the composition of purified mucin extracts. This type of analysis has shown that mucin extracts purified from porcine stomach mucus, for example, are composed predominantly of MUC5AC, with small quantities of MUC2, MUC5B, and MUC6, as well as histones, actin, and albumin47.
Isolation of Mucin Oligosaccharides
Non-reductive alkaline β-elimination ammonolysis was applied to dissociate non-reduced glycans from mucins as described previously18,48. Purified mucins were dissolved in ammonium hydroxide saturated with ammonium carbonate and incubated at 60° C. for 40 h to release oligosaccharide glycosylamines and partially deglycosylated mucins. Volatile salts were removed using repeated centrifugal evaporation and the oligosaccharide glycosylamines were separated from residual deglycosylated mucins via centrifugal filtration through 3-5 kDa molecular weight cut-off membranes (Amicon, Miami, FL; Ultracel) in accordance with the manufacturer's instructions. The resulting oligosaccharide glycosylamines were converted to reducing oligosaccharide hemiacetals via treatment with boric acid. Residual boric acid was removed via repeated centrifugal evaporation from methanol. Oligosaccharides were further purified using solid-phase extraction using Hypercarb mini-columns (ThermoFisher, Waltham, MA) and residual solvents were removed through centrifugal evaporation.
Analysis of Mucin O-Glycan Profiles
Glycans released from MUC2, MUC5B, and MUC5AC were permethylated and analyzed by nanospray ionization tandem mass spectrometry (NSI-MS) following direct infusion into a linear/orbital hybrid ion trap instrument (Orbitrap-LTQ Discovery, ThermoFisher) operated in positive ion mode for non-sulfated glycans or in negative mode for the detection of sulfated glycans. The permethylated O-glycans were dissolved in 1 mM sodium hydroxide in methanol/water (1:1) for infusion at a syringe flow rate of 0.60 μl/min and capillary temperature set to 210° C.49. For fragmentation by collision-induced dissociation (CID) in MS/MS and MSn, a normalized collision energy of 35-40% was applied. Detection and relative quantification of the prevalence of individual glycans was accomplished using the total ion mapping (TIM) functionality of the Xcalibur software package version 2.0 (ThermoFisher) as previously described49. For TIM, the m/z range from 600 to 2000 was automatically scanned in successive 2.8 mass unit windows with a window-to-window overlap of 0.8 mass units, which allowed the naturally occurring isotopes of each glycan species to be summed into a single response, thereby increasing detection sensitivity. Most glycan components were identified as singly, doubly, and/or triply charged, sodiated species (M+Na) in positive mode or as singly or doubly charged (M−H) species in negative mode. Charge states for each glycan were deconvoluted manually and summed for quantification. Structural representations of mucin glycans were based on topologic features detected upon CID fragmentation and knowledge of O-glycan biosynthetic pathways. Approximately 33% of the m/z values reported here were associated with 2 or 3 isomeric glycan structures. NSI-MS/MS and MSn were used as needed to assign isomeric heterogeneity at each of these m/z values. For purposes of representing and comparing the heterogeneity of the glycan profile associated with each mucin, the signal intensity associated with an m/z value comprised of more than one glycan was assigned to the most abundant glycan structure among the isomers. Graphic representations of glycan monosaccharide residues are consistent with the Symbol Nomenclature For Glycans (SNFG) as adopted by the glycomics and glycobiology communities. Glycomics data and metadata were obtained and are presented in accordance with MIRAGE standards and the Athens Guidelines50. GlyTouCan accessions were retrieved from the GlyTouCan repository through GlyGen for glycan instances in which accessions already existed. If new accessions were required for glycans not previously placed in the repository, the desired structural representations were generated in GlycoGlyph and submitted directly to GlyTouCan for registration51. All raw mass spectrometric data related to mucin glycan profiles were deposited at GlycoPost52. Heatmap and other data analysis was performed on extracted signal intensities using Prism GraphPad and Excel software.
Filamentation Assay
Hyphal growth of C. albicans was induced by diluting cells to OD600=0.05 into prewarmed hyphae-inducing medium as indicated and incubating at 37° C. (200 rpm) in a glass-bottom, 96-well plate. Cells were grown in hyphae-inducing medium for several hours, as described in BRIEF DESCRIPTION OF THE DRAWINGS. Images were acquired with a confocal laser scanning microscope (Zeiss, Oberkochen, Germany; LSM 800) equipped with a ×63/1.4 NA oil-immersion or a 25× objective. Images were analyzed using Zeiss ZEN v.2.1. Representative micrographs are shown.
RNA Extraction
For the extraction of the RNA of C. albicans grown in the presence or absence of mucins, 1 mL of RPMI or 0.5% w/v MUC2, MUC5AC, or MUC5B in RPMI was inoculated with 10 μL of an overnight culture of strain SC5314 and incubated in a culture tube at 37° C. with shaking (180 rpm) for 8 h. Total RNA was extracted using the Epicentre MasterPure Yeast RNA Purification Kit and treated with Sigma-Aldrich (St. Louis, MO) AMPD1 amplification-grade DNase I.
For RNA extraction from C. albicans grown in the presence or absence of mucin glycans, 100 μL of RPMI or 0.1% w/v MUC5AC glycans in RPMI were inoculated with a 1:50 dilution of an overnight culture of strain SC5314 and incubated at 37° C. for the time indicated. Total RNA was extracted with the MasterPure RNA Purification Kit (Lucigen, Middleton, WI) and residual DNA was removed using the Turbo DNA-free kit (Ambion, Austin, TX). The integrity of the total RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). rRNA was removed using the Ribo-Zero® rRNA Removal Kit (Yeast; Epicentre).
RNA Sequencing
For RNA sequencing of C. albicans grown in mucins, poly(A) RNA was isolated from total RNA via two rounds of purification. Samples were run on a MiSeq with a paired-end protocol and read lengths of 150 bp.
For RNA sequencing of C. albicans grown in mucin glycans, Illumina RNA-seq was used. Libraries were produced using the KAPA RNA HyperPrep kit (Kapa Biosystems, Wilmington, MA) and sequenced using the Illumina HiSeq platform with a single-end protocol and read lengths of 40 or 50 nucleotides.
RT-qPCR Analysis
A list of the primers used in this study is provided in Table 1. qPCR with reverse transcription (RT-qPCR) was performed using a two-step method. First-strand cDNA was synthesized from total RNA using the ProtoScript IV First Strand cDNA Synthesis kit (New England Biolabs, Ipswich, MA). The cDNA was used as template for RT-qPCR using a SYBR PowerUp Master Mix kit (Applied Biosystems, Life Technologies) on a Roche LightCycler 480 real-time PCR system. The ACT1 and 18S rRNA genes were used as endogenous controls as specified. The elimination of contaminating DNA was confirmed using qPCR amplification of ACT1 and 18S rRNA genes on control samples that did not have reverse transcriptase added during cDNA synthesis. Changes in gene expression were calculated based on mean change in qPCR cycle threshold (ΔCt) using the ΔΔCt method (fold change=2−ΔΔCt−ΔΔCt).
Analysis of RNA Sequencing Data
For mucins RNA sequencing experiments, reads were mapped to the C. albicans SC5314 haplotype A genome, version A22-s05-m05-r03, retrieved from the Candida Genome Database (www.candidagenome.org) using Rsubread v1.28.153. Read summarization was performed on the gene level using featureCounts54 using annotations from a modified version of C. albicans SC5314 haplotype A, which only contained protein coding genes. Multimapping read pairs, read pairs mapping across more than one gene, and read pairs in which ends mapped to different chromosomes were removed from downstream analyses. Genes that had <10 counts per million in at least two samples were discarded. Remaining gene counts were normalized using trimmed mean of M-values55. Differential expression analysis was performed using limma v3.34.956 with voom-transformed read counts57. Genes were considered differentially expressed when P<0.05 after the false-discovery rate was controlled using Benjamini-Hochberg correction.
For mucin glycans RNA sequencing experiments, reads were mapped to the C. albicans SC5314 haplotype A genome, version A22-s05-m05-r03, retrieved from the Candida Genome Database (www.candidagenome.org) using the Galaxy Server58. Read summarization was performed on the gene level with annotations from a modified version of C. albicans SC5314 haplotype A. Multimapping read pairs, read pairs mapping across more than one gene, and read pairs in which ends mapped to different chromosomes were removed from downstream analyses. Differential expression analysis was performed using DESEQ259. Genes were considered differentially expressed when P<0.05 after the false-discovery rate was controlled using Benjamini-Hochberg correction.
Functional category (pathway) assignments were obtained from Candida Genome Database Gene Ontology annotations and assessed using PANTHER60. Over-representation of biological pathways in mucin was assessed using one-sided Fisher's exact test followed by a Benjamini-Hochberg procedure for multiple corrections, for differentially expressed genes from n=3 replicates. Enrichment of pathways in MUC5AC glycans was determined based on mean log2-transformed fold changes from n=3 replicates and calculated with the two-sided Mann-Whitney U-test followed by a Benjamini-Hochberg procedure for multiple corrections. Heat maps and scatter plots of gene expression data were constructed using GraphPad Prism.
Murine Wound C. albicans Protocol
Female, 8-week old, SKH-1 mice were anesthetized with isoflurane and given buprenorphine (0.05 mg/kg) before wounding with a 6-mm punch biopsy to generate two identical full-thickness dermal wounds on the dorsal side of the mouse. Wounds were kept covered with an occlusive dressing (Opsite Flexifix) throughout the duration of the experiment. After a 24 h recovery period, wounds were inoculated with 30 μl volume of PBS containing 108 SC513 Enol-mCherry yeast. Topical treatments of 30 μl of 0.5% MUC2, or PBS were administered to each wound on Day 1, 3, and 5 post-infection. Wounds were gently washed with 500 ul of sterile PBS and bandages changed prior to each treatment. Wound fluorescence was imaged daily with IVIS Lumina II optical imaging system to assess fungal burden. At Day 5 and Day 7, wound biopsy specimens were collected and CFUs were calculated per gram of tissue.
The mice used in this experiment were housed at 72° F. at 30% humidity with a 12-hour light/dark cycle.
Polystyrene Attachment Assay
Strain HGFP3 was pregrown overnight in YPD medium at 30° C., diluted to OD600=0.1, added into prewarmed RPMI medium in a polystyrene 96-well plate, and incubated at 37° C. for the time indicated in the figure legend. Medium was decanted and plates were washed three times with phosphate-buffered saline. Images were acquired with a confocal laser scanning microscope (LSM 800) equipped with a ×20/1.4 NA objective. The excitation wavelength for GFP was 488 nm. Four images were recorded for each well and for at least three independent wells. Images were analyzed in Fiji as follows: each image was converted to 8-bit and the contrast was enhanced (0.4% saturated pixels), then thresholded to create a binary image. Each image was analyzed using the Analyze Particles tool to measure the surface area covered by cells as described previously10. The mean surface area measurements of the images for each condition were calculated.
Biofilm Formation Assays and Visualization
In vitro biofilm growth assays were carried out in RPMI medium by growing the biofilm directly on a 96-well polystyrene plate. Briefly, strain SC5314 was grown overnight in YPD at 30° C., washed twice with phosphate-buffered saline, then diluted to OD600=0.5 in 100 μL of RPMI in a 96-well polystyrene plate. The inoculated plate was incubated at 37° C. for 90 min to facilitate attachment of yeast cells to the surface. Nonadherent cells were washed twice with phosphate-buffered saline, and samples were subsequently submerged in fresh RPMI. Biofilms were grown for 24 h at 37° C. For CFU enumeration, the medium containing the planktonic cells was removed and plated on YPD plates. Biofilms were resuspended with phosphate-buffered saline, disrupted by pipetting, serially diluted in phosphate-buffered saline, and plated on YPD plates. Biofilms and planktonic cells were imaged using a Zeiss wide-field fluorescence microscope.
Coculture Viability Assays
An overnight SC5314 culture grown in YPD was diluted 1:100 into RPMI in a 96-well plate (MatTek, Ashland, MA) with or without 0.1% MUC5AC glycans and grown for 4 h with shaking at 37° C. A control well without C. albicans was included. Concurrently, 2 ml of LB were inoculated with 40 l Pseudomonas aeruginosa strain PA14 and grown for 4 h with shaking at 37° C. RPMI was then removed from C. albicans and replaced with 200 μl SLB. P. aeruginosa was added to a final OD600=0.25. At 0 h, 24 h, 48 h, and 72 h, the contents of the wells were homogenized and a 5-μl aliquot was serially diluted in phosphate-buffered saline. Dilutions were plated on YPD agar+Gm30+Tet60 (to select for C. albicans) and Cetrimide agar (to select for P. aeruginosa) and incubated overnight at 30° C. and 37° C., respectively. Colonies were counted after incubation.
Confocal Imaging of Coculture
Images were acquired with a confocal laser scanning microscope (LSM 800) equipped with a ×63/1.4 NA oil-immersion objective. Images were analyzed using Zeiss ZEN v.2.1. C. albicans was stained with 20 μg/ml calcofluor white. The excitation and emission wavelengths for calcofluor white were 365 nm and 445 nm, respectively; the excitation and emission wavelengths for mCherry were 587 nm and 610 nm, respectively.
Glycan Synthesis and Analysis
All commercial reagents were used as supplied unless otherwise stated, and solvents were dried and distilled using standard techniques. Thin layer chromatography was performed on silica-coated glass plates (TLC Silica Gel 60 F254; Merck, Rahway, NJ) with detection via fluorescence, charring with 5% H2SO4(aq), or staining with a ceric ammonium molybdate solution. Organic solutions were concentrated and/or evaporated to dry under vacuum in a water bath (<50° C.). Molecular sieves were dried at 400° C. under vacuum for 20-30 min prior to use. Amberlite IR-120H resin was washed extensively with MeOH and dried under vacuum prior to use. Medium-pressure liquid chromatography was performed using a CombiFlash Companion equipped with RediSep normal-phase flash columns, and solvent gradients refer to sloped gradients with concentrations reported as % v/v. Nuclear magnetic resonance spectra were recorded on a Bruker Avance DMX-500 (500 MHz) spectrometer, and assignments achieved with the assistance of 2D gCOSY, 2D gTOCSY, 2D gHSQC, and 2D gHMBC; chemical shifts are expressed in ppm and referenced to either Si(CH3)4 (for CDCl3), residual CHD2OD (for CD3OD), or a MeOH internal standard (for D2O). Low-resolution electron-spray ionization mass spectrometry was performed with a Waters micromass ZQ. High-resolution mass spectrometry was performed with an Agilent 1100 LC equipped with a photodiode array detector, and a Micromass QTOF I equipped with a 4 GHz digital-time converter. Optical rotation was determined in a 10-cm cell at 20° C. using a Perkin-Elmer Model 341 polarimeter. High-performance liquid chromatography was performed with an Agilent 1100 LC equipped with an Atlantis T3 (3 mm, 2.1×100 mm) C18 column and ELSD detection.
Statistical Analysis
Unless noted otherwise, experiments were performed with at least three biological replicates consisting of at least three technical replicates, and results are presented as mean SEM. Microscopy images depicted are representative and similar results were observed in different fields of view across at minimum three independent biological replicates. Raw data are available as Source Data. MUC2, MUC5AC, and MUC5B and their associated glycans were tested from several purification batches with consistent results.
Data and Materials Availability
High-throughput sequencing data presented were deposited in the Gene Expression Omnibus (GEO) under accession number GSE197249 (
Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast to hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear, despite their potential for therapeutic interventions. Here, it was determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. Greater than 100 mucin O-glycans from three major mucosal surfaces were isolated and catalogued, and it was established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation, and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, three structures (Core 1, Core 1+fucose, and Core 2+galactose) were identified to be sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, Examples 2-6 identify mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.
While previous studies identified mucin polymers as candidates for managing C. albicans virulence in vitro10, it is unclear whether mucin activity persists in native and complex mucus or in the context of an intact immune system, which may indicate whether mucins are viable candidates for therapeutic intervention on mucosal surfaces. Here, mucus from three distinct sources (
One candidate for mediating this function is mucin polymers, which suppress C. albicans adhesion in vitro10. To determine whether mucins are the dominant adhesion-suppressing factor in mucus, high-molecular-weight components were removed from porcine intestinal mucus using a centrifugal filter with a 100-kDa cutoff. The filtrate was less effective in preventing adhesion than whole mucus (
To clarify how mucins regulate C. albicans physiology, RNA sequencing was performed on cells grown in RPMI medium with or without (0.5% w/v) MUC2 (intestinal mucin), MUC5B (salivary mucin), or MUC5AC (gastric/respiratory mucin), representing mucins secreted on mucosal surfaces abundantly colonized by C. albicans. Each mucin type elicited a specific gene-expression profile (
The 262 downregulated genes include, among others, ADH2, ALG1, ALG5, AMS1, AOX1, AOX2, ASM3, ATO1, ATO2, AYR2, BEM1, BMT1, BMT3, BTA1, BUD14, CCT6, CDC3, CDC4, CHS2, CHS3, CHS7, CHS8, CHT2, CIP1, CLA4, CLG1, CSH1, CSP2, CTF1, CTR1, CUP1, DEF1, DFI1, DPP3, ECE1, ERF1, EXG2, FAA4, FAV1, FAV2, FDH1, FRE30, FRE7, FRP3, GAC1, GAD1, GAP4, GCD2, GDT1, GLG2, GLX3, GRE2, GUT1, HAC1, HEX3, HGC1, HGT1, HGT2, HRT1, HSP21, HSP30, HTS1, HWP1, HYR1, IDH2, IFD6, IHD1, IHD2, ISU1, KEX2, KRE1, KRS1, LAC1, LYS143, MAC1, MAL31, MDM34, MDN1, MED7, MET10, MET14, MET3, MEU1, MKC1, MKK2, MNN12, MRV8, MSI3, NAM7, NIP1, PBR1, PCL1, PGA11, PGA13, PGA32, PGA54, PGI1, PHO112, PHO86, PHO89, PIN3, PRN1, PRN2, PRN3, PRN4, PST1, PTP3, RBT7, RFG1, RHB1, RHO2, RLM1, RPS42, RTA4, SAL6, SAP2, SAP5, SIR2, SKO1, SMF12, SMI1B, SOD3, SPO7, STB3, TEN1, THI13, TIF, UAP1, UGA1, UME6, WOR3, WSC1, YHB5, YKE2, YMC2, YOX1, YVC1, ZCF27, ZDS1, and ZFU2.
The 343 upregulated genes include, among others, ACO2, AFP99, AGE1, ALT1, AMO1, AMO2, APE3, ARO8, ARP4, ASN1, ATF1, ATM1, AXL1, BAT21, BAT22, BUB1, BUL1, CAN1, CAR1, CAR2, CAT8, CBF1, CDC14, CDC20, CDC21, CDC5, CHS1, CHT3, CLB4, CMK1, CRD2, CTP1, CWH8, DAG7, DAK2, DAO1, DAO2, DOA1, DOT1, DQD1, DSE1, DUR1,2, EBP7, ECM15, EMP46, ENG1, ERG1, ERG11, ERG13, ERG24, ERG251, ERG26, ERG27, ERG3, ERG5, ERG6, ERG9, FBA1, FCY21, FGR23, FGR29, FGR41, FGR50, FLO9, FMA1, GAL1, GAP1, GAP2, GAT1, GCN4, GCS1, GDH3, GIS2, GLN1, GLO1, GLT1, GOR1, GPD1, GPM2, GYP1, HEM13, HEM14, HEM3, HGT7, HIS1, HIS4, HIS5, HIS7, HMS1, HNM1, HOF1, HOM2, HOM6, HOS3, HRK1, HRQ2, HTA3, HYU1, IDP1, IFE2, IFG3, IFR2, ILV1, ILV3, ILV6, INT1, KIP1, KIP2, KTR4, LAP4, LCB4, LEU1, LEU2, LEU4, LIG1, LIP8, LYS1, LYS12, LYS144, LYS2, LYS22, LYS4, LYS5, LYS9, MBP1, MDM10, MED17, MEP1, MEP2, MIH1, MIS11, MNN22, MPH1, NCP1, NIT3, NPR1, NRG1, NUF2, NUP, OPT3, OPT4, PCL5, PCL7, PDE2, PGA38, PGA45, PGA48, PHO87, PIR1, POS5, PPS1, PRO1, PRO3, PTR22, PUT1, PUT2, QDR1, RAD32, RAD54, RBE1, RBT1, RHD1, RHD3, RIA1, RNH35, RNR1, RNR22, ROD1, RPD31, SCT1, SCW11, SEO1, SHE9, SIM1, SIZ1, SMC2, SMC4, SNF5, SNO1, SNZ1, SSN6, STP1, SUR2, SUT1, SWE1, SWI4, TFA1, TGL99, THI20, THR1, THR4, TNA1, TOA2, TOS4, TPO3, TRP4, TRP5, TYE7, UBP13, UPC2, VPS4, XKS1, YCG1, YCS4, YHB4, YWP1, and ZCF16.
Filamentation- and adhesion-regulating pathways were enriched in the shared downregulated genes. Notably, all three mucins caused the downregulation of various virulence-associated genes (
The roles of mucins were investigated in the context of an intact immune system (
Mucin glycans can regulate host-microbe interactions: they serve as nutrients15, microbial binding sites8, and signaling molecules18,19. To determine whether mucin glycans mediate virulence suppression, glycans were isolated via non-reductive, alkaline β-elimination, which preserved the structural heterogeneity of glycan chains, yielding a library of glycans released from MUC5AC. The released glycans were analyzed as permethylated derivatives using nanospray-ionization multi-dimensional mass spectrometry (NSI-MSn, ThermoFisher Orbitrap Discovery) to characterize structural topology features beyond simple monosaccharide composition (
To determine whether the mucin glycan pool can replicate mucin-induced virulence suppression, RNA sequencing of C. albicans in medium with or without 0.1% w/v MUC5AC glycans was performed. A pooled library of MUC5AC glycans triggered global gene expression changes, with 233 and 308 genes significantly upregulated and downregulated, respectively, compared with cells grown in medium alone (P<0.05). Similar to intact mucins, MUC5AC glycans upregulated the transcription of amino-acid biosynthetic and metabolic processes and downregulated pathways associated with filamentation, biofilm formation, and interspecies interactions (
The 233 upregulated genes include, among others, ACO1, ADE12, ADE13, ADE4, ADE5,7, ADE6, AFL1, AGP2, AIP2, AQY1, ARO3, ARO4, ASN1, ASR1, ATF1, BAT22, BIO2, BMT4, BNA31, BNA32, BUL1, CAN1, CAN2, CAR1, CAR2, CAT8, CDC19, CDR4, CHT3, COX15, CTA4, CWH8, DAG7, DAP1, DED81, DQD1, DUR1,2, DUR3, EBP1, EHT1, ENG1, FAD3, FBA1, FDH3, FGR41, FMA1, FRE10, GCN4, GCV1, GCV2, GCV3, GDH2, GDH3, GIS2, GLN1, GLT1, GNP1, GPD2, GPM2, GRS1, HAK1, HEM1, HEM13, HGT7, HIS4, HIS5, HIS7, HOM3, HOM6, HOS3, HRQ2, HSP12, HSP21, HSP60, ICL1, IDP1, IFG3, ILV1, ILV2, ILV3, ILV5, ILV6, IMH3, LEU1, LEU2, LEU4, LEU42, LYS4, MEP1, MIA40, MIS11, MNN1, MNN22, MNN4, NAR1, NRG1, OAC1, OPT1, OPT2, OPT3, OPT4, PDX3, PGA14, PGA38, PGA45, PGA6, PHO87, PIR1, PMA1, PRO3, PTR22, PUT1, PUT2, PYC2, RBE1, RHD1, RHD3, RHR2, RME1, RNH1, RNR1, RNR22, ROA1, RPL10, RPL10A, RPL11, RPL12, RPL15A, RPL17B, RPL2, RPL21A, RPL23A, RPL24A, RPL25, RPL3, RPL30, RPL35, RPL38, RPL42, RPL4B, RPL5, RPL82, RPL8B, RPL9B, RPP2A, RPP2B, RPS12, RPS19A, RPS20, RPS21, RPS24, RPS3, RPS42, RPS4A, RPS6A, RPS7A, RPS9B, RTA3, SAP9, SCW11, SDH12, SDH2, SER1, SHM2, SIM1, SNZ1, SOD1, SOU1, SSA2, STF2, STP1, THR4, TNA1, TOP1, TPO3, TPO4, TRP5, TRX1, TYE7, UBA4, UBI3, URA1, VAS1, WH11, YWP1, and ZPR1.
The 308 downregulated genes include, among others, ADH2, AGO1, AHR1, ALG5, ALS3, AMS1, ARG3, ARO9, ARP1, ASM3, ASR3, ATO1, ATP9, AUT7, AYR2, BEM1, BEM2, BMT1, BMT3, BMT6, BRG1, BUD14, BUD2, CAS4, CCC2, CDC11, CDC12, CDC3, CDC4, CFL2, CFL5, CHS4, CHT2, CIP1, CLA4, CLG1, CPA2, CSA1, CSH1, CSR1, CTR1, CUP1, DCK1, DDR48, DEF1, DFI1, DPP3, ECE1, ECM4, EFG1, FAV1, FDH1, FET31, FET34, FRE30, FRE7, FRP1, FRP2, FTR1, GAC1, GAD1, GAP4, GDB1, GDE1, GDI1, GIN4, GLG2, GLX3, GPH1, GPX2, GRE2, HAC1, HAP3, HAP43, HET1, HGC1, HGT1, HGT18, HGT6, HMX1, HWP1, HYR1, IDH2, IFD6, IHD1, IHD2, IQG1, IRA2, IRO1, IST2, KEX2, KIP4, KRE1, LAP3, LMO1, LSP1, MAC1, MAL31, MCD4, MED15, MET10, MET14, MET15, MET3, MKC1, MKK2, MNN12, MNN15, MNN24, MRF1, MSB2, MYO2, NCE102, PCL1, PEP1, PGA13, PGA4, PGA54, PGA59, PGA63, PGA7, PGI1, PHM7, PHR1, PIN3, PLB3, PLD1, POL93, PRA1, PRN1, PRN2, PRN3, PRN4, PRX1, PST1, PST2, PTP3, RAS1, RAX1, RCT1, RFG1, RFX2, RGA2, RIB3, RIM101, RIM9, ROB1, RTA4, SAL6, SAP5, SEC24, SEP7, SFL2, SHE3, SIP5, SIR2, SKN1, SLK19, SLM2, SMF12, SOD3, SOD5, SRD1, SSD1, SSU81, STB3, STE23, SUN41, SUR7, TEC1, TFS1, TKL1, TSA1, TSA1B, TUB1, TUP1, UME6, VAC8, VPS1, WOR3, WSC1, WSC2, YCK2, YHB5, YKE2, YOX1, YVC1, ZCF20, ZCF27, ZDS1, ZFU2, ZRC1, ZRT1, ZRT2, and ZWF1.
Over 20% of the downregulated genes were associated with filamentous growth, which was suppressed by intact mucins. It was found that isolated MUC5AC glycans suppressed filamentation across the three medium conditions without altering growth, while medium alone supported the formation of extensive hyphae (
Hyphal morphogenesis in C. albicans is induced by environmental signals acting via multiple signaling cascades, including a cAMP-dependent pathway and a MAPK pathway23. The RNA-sequencing data revealed that the transcription of many filamentation activators and key outputs of these pathways (including hyphal-specific proteins Ume6, Eed1, and Hgc124) were significantly downregulated in the presence of mucin glycans (
To assess whether mucin glycans suppress hyphal formation by preventing activation of the major transcriptional activators of filamentation, mutants that constitutively activate major positive filamentation regulators (Ras1, Cph1, and Efg1) were screened for the filamentation-suppression response to mucin glycans. Ras1 cycles between inactive and active states. The RAS1G13V strain is locked in an active state, leading to hyperfilamentation25. If mucin glycans act upstream or directly via Ras1 activation, the dominant active RAS1G13V strain should remain filamentous in the presence of mucin glycans, being unable to respond to mucin glycans. However, in the presence of mucin glycans, cells from both the RAS1G13V strain and wild-type strain retained the yeast morphology (
To determine whether mucin glycans act via the cAMP-PKA pathway, whether mucin glycans suppress filamentation was tested in a strain with a phosphomimetic mutation controlled by the glucose-repressible PCK1 promoter26,27 (PCKpr-efg1-T206E), which simulates constitutive signaling of Efg1, a downstream transcription factor in the cAMP-PKA pathway23. In the presence of mucin glycans, cells from a constitutively expressed Efgl transcription factor transitioned to a yeast morphology, as observed for the wild-type strain in Spider medium (
To explore alternate regulation pathways, it was examined whether mucin glycans act via transcriptional repressors of filamentation, which inhibit the yeast-to-hyphal transition23. The focus was on NRG1 and TUP1, as their loss leads to constitutive filamentation and upregulation of hyphal genes, even in non-inducing conditions28. Specifically, 30 min after exposure to mucin glycans (
To further explore this function of mucin glycans, RNA sequencing of the wild-type strain and Δ/Δnrg1 mutant strain was performed after 2 h in the presence or absence of mucin glycans to detect early transcriptional changes during hyphal morphogenesis. In the wild-type strain, mucin glycans downregulated 45 and upregulated 64 genes (P<0.05) after 2 h (
The 45 downregulated genes include, among others, AHR1, ALS3, AMS1, ASM3, CFL2, CSH1, CTR1, DDR48, DUR1,2, EFG1, FDH1, FET31, FET34, FRE10, FRE30, FRE7, FTR1, HGC1, HMX1, HWP1, IHD1, LAP3, LYS1, LYS2, LYS22, MAC1, MED16, MNN24, OPT4, PGA54, PLB1, POL93, PRA1, RFG1, RIB3, SFL2, SHM1, WOR3, YVC1, ZRT1, and ZRT2.
The 64 upregulated genes include, among others, KCH1, PGA6, RME1, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL15A, RPL16A, RPL17B, RPL18, RPL2, RPL20B, RPL21A, RPL23A, RPL24A, RPL25, RPL27A, RPL28, RPL29, RPL3, RPL30, RPL32, RPL35, RPL37B, RPL43A, RPL4B, RPL6, RPL8B, RPL9B, RPP2A, RPS1, RPS10, RPS12, RPS14B, RPS15, RPS16A, RPS17B, RPS19A, RPS20, RPS21, RPS22A, RPS24, RPS25B, RPS26A, RPS27, RPS5, RPS6A, RPS7A, RPS9B, SOD1, TMA19, UBI3, YST1, and YWP1.
The transcription of several hyphal-specific genes (ALS3, HWP1, EFG1, and HGC1) was downregulated; further, YWP1, a marker for yeast cells, was upregulated (
Several genes involved in ion homeostasis were differentially downregulated in the Δ/Δnrg1 mutant strain and wild-type strain (
A virulence attribute of C. albicans is its ability to form robust biofilms6. Biofilm cells are highly resistant to conventional antifungal therapeutics and the host immune system, making them highly pathogenic6. In C. albicans, biofilm development requires six master transcriptional regulators (Efg1, Tec1, Bcr1, Ndt80, Brg1, and Rob1)29. The first step in biofilm formation involves cell adherence to a surface, mediated by the master regulator Bcr1 and its downstream target genes30.
It was observed that MUC5AC glycans significantly downregulated the transcription of several genes involved in adherence and biofilm initiation, while equivalent amounts of monosaccharides had no effect (
After initial adherence and biofilm initiation, the next step in biofilm formation is biofilm maturation, where hyphal cells grow and all cells become encased in extracellular matrix29. The RNA-sequencing data revealed significant downregulation of several transcriptional regulators of biofilm maturation, including genes encoding Efg1, Tec1, Brg1, and Rob1 (
In a host, C. albicans is generally part of a larger multispecies microbial community6,31. It was found that genes involved in microbial interspecies interactions were differentially regulated by mucin glycans, suggesting that mucins influence microbial community dynamics. C. albicans was often found in the presence of the bacterial pathogen Pseudomonas aeruginosa, both as part of the normal microbiota and during infection31. In vitro work has shown that these two microbes have an antagonistic relationship when grown together in C. albicans filamentation-inducing conditions32, where P. aeruginosa forms biofilms on C. albicans hyphal cells and secretes small molecules that result in fungal cell death (
Because mucins suppressed filamentation10, it was hypothesized that in filamentation-inducing conditions, mucin glycans would increase the viability of C. albicans in coculture with P. aeruginosa. As previously reported32, cocultures grown under filamentation-inducing conditions in the absence of mucin glycans showed reduced C. albicans CFUs and eventual eradication of C. albicans cells (
To determine whether filamentation suppression is the dominant factor promoting microbial coexistence, the C. albicans Δ/Δnrg1 mutant strain, which remains filamentous in the presence of mucin glycans, was cocultured with P. aeruginosa in the presence or absence of mucin glycans. The increased viability of C. albicans in the presence of mucin glycans was eliminated in the Δ/Δnrg1 mutant strain (
Research on mucus has traditionally focused on the role of mucins as scaffolding polymers. Here, the findings that mucin O-glycans potently inhibit a range of virulence behaviors, suggest that they can be leveraged for therapeutic applications. Specifically, mucin glycans across three major niches are found to be potent regulators of C. albicans filamentation (
To determine the glycan structures regulating C. albicans physiology and the therapeutic potential of novel glycan-based drugs for C. albicans infection, mucin glycan libraries were screened for filamentation suppression. Glycans isolated from human saliva (MUC5B), porcine gastrointestinal mucus (MUC2), and porcine gastric mucin (MUC5AC) all suppressed filamentation (
To distinguish unique and shared structural features, NSI-MSn was used to analyze released, permethylated glycans from these three mucin pools (Example 1). Glycans at 83 distinct mass/charge (m/z) ratios were identified, approximately ⅓rd of which produced MS fragmentation, indicating the presence of 2-3 isomeric glycan structures (
Beyond the core structures, 23% and 15% of MUC5AC and MUC5B glycans, respectively, were more than seven sugars long, versus <3% of MUC2 glycans (Tables 4-6). The shortened length of MUC2 glycans may result from degradation through microbial feeding or differences in endogenous glycosyltransferase expression levels in the intestinal tract15. Consistent with previous reports18,34, mucin glycans from all three sources were heavily fucosylated, with >35% of structures containing at least one fucose, with minimal sialyation (
Because glycan structure compositions vary across mucin types, glycan structures that were highly abundant across the mucin species examined here were focused on. In total, six glycan structures (
Rather than fractionating glycan pools down to the single-glycan level, which poses technical challenges36, a synthetic approach was developed to obtain these six highly abundant mucin glycans (
To elucidate the role of glycan composition, the effects of four modified Core structures: Core 1+fucose (3), Core 1+sialic acid (4), Core 2+fucose (5), and Core 2+galactose (6) (
The phenotypic filamentation assay confirmed this conclusion: while medium alone supported the formation of extensive hyphal filaments, Core 1, Core 1+fucose, and Core 2+galactose most potently blocked filamentation, as evidenced by the predominance of yeast cells in culture with these structures compared with the monosaccharide pool and medium alone (
By characterizing bioactive glycans across mucin types, prominent Core-1- and Core-2-modified structures commonly found across mucosal surfaces were identified and synthesized. It was demonstrated that Core 1, Core 2+galactose, and Core 1+fucose individually suppress filamentation at potencies comparable to those of native mucins. These findings highlight that O-glycans can control virulence traits (
Given the complexity and diversity of mucin glycans18,34 and dynamic glycosylation changes based on cell type40, developmental stage41, and disease state42, structural changes in host signals may activate or inhibit the function of specific O-glycans. Accordingly, it was determined that Core 1, Core 1+fucose, and Core 2+galactose effectively suppress filamentation, while Core 1+sialic acid significantly dampens this response. This suggests sialic acid, which is ubiquitously expressed on host cells43, has an unappreciated role in modulating virulence. Changes in glycosylation in disease states may mask or eliminate mucins' protective functions: the presentation of complex glycan structures in mucus contributes to a healthy mucosal environment, while degradation or modification of mucin glycans may trigger C. albicans to transition from commensal to pathogenic.
Nrg1 regulation is temporally coordinated by two central signaling pathways mediating cell growth, leading to transient NRG1 downregulation and degradation of Nrg1 protein followed by occlusion of Nrg1 from hyphal-specific promoters that sustain hyphal development24. Mucin glycans may potentially function as ligands to mimic nutrient signaling pathways or may bind directly to C. albicans adhesins, thus modulating morphogenesis44.
General methods. All commercial reagents were used as supplied unless otherwise stated, and solvents were dried and distilled using standard techniques. Thin layer chromatography was performed on silica-coated glass plates (TLC Silica Gel 60 F254, Merck) with detection by fluorescence, charring with 5% H2SO4(aq), or staining with a ceric ammonium molybdate solution. Organic solutions were concentrated and/or evaporated to dry under vacuum in a water bath (<50° C.). Molecular sieves were dried at 400° C. under vacuum for 20-30 minutes prior to use. Amberlite IR-120H resin was washed extensively with MeOH and dried under vacuum prior to use. Medium-pressure liquid chromatography (MPLC) was performed using a CombiFlash Companion equipped with RediSep normal-phase flash columns, and solvent gradients refer to sloped gradients with concentrations reported as % v/v. NMR spectra were recorded on a Bruker Avance DMX-500 (500 MHz) spectrometer, and assignments achieved with the assistance of 2D gCOSY, 2D gTOCSY, 2D gHSQC, and 2D gHMBC; chemical shifts are expressed in ppm and referenced to either Si(CH3)4 (for CDCl3), residual CHD2OD (for CD3OD), or a MeOH internal standard (for D2O). Low resolution electron-spray ionization mass spectrometry (ESI-MS) was performed using a Waters micromass ZQ. High resolution mass spectrometry was performed using an Agilent 1100 LC equipped with a photodiode array detector, and a Micromass QTOF I equipped with a 4 GHz digital-time converter. Optical rotation was determined in a 10-cm cell at 20° C. using a Perkin-Elmer Model 341 polarimeter. HPLC analysis was performed using an Agilent 1100 LC equipped with an Atlantis T3 (3 μm, 2.1×100 mm) C18 column and ELSD detection.
Acetyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranoside (8). D-Galactosamine hydrochloride (7; 12.35 g, 57.27 mmol) in anhydrous pyridine (80 mL) was cooled to 0° C., and then Ac2O (40 mL) added dropwise over 15 min and the flask slowly warmed to rt. After 16 hours, the reaction mixture was concentrated to a syrup via co-evaporation with toluene (2×50 mL), and then the crude material was purified via MPLC on silica gel using 0→60% acetone-CH2Cl2 to afford the pure product as a white solid (21.63 g, 55.55 mmol, 97% yield). Rf=0.11 (3:7 acetone:CH2Cl2). [α]D20: +9.8° (c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz): δH 5.70 (d, 1H, J=8.8 Hz, H-1), 5.43 (d, 1H, J=9.5 Hz, NH), 5.38 (dd, 1H, J=3.3, <1 Hz, H-4), 5.09 (dd, 1H, J=11.3, 3.3 Hz, H-3), 4.45 (ddd, 1H, J=11.2, 9.2, 9.2 Hz, H-2), 4.17 (dd, 1H, J=11.3, 6.6 Hz, H-6a), 4.12 (dd, 1H, J=11.3, 6.5 Hz, H-6b), 4.02 (ddd, 1H, J=6.5, 6.5, 1.0 Hz, H-5), 2.17 (s, 3H, OAc), 2.13 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.94 (s, 3H, NHAc). 13C NMR (CDCl3, 125 MHz): δC 170.97 (C═O), 170.62 (C═O), 170.47 (C═O), 170.39 (C═O), 169.79 (C═O), 93.28 (C-1), 72.11 (C-5), 70.55 (C-3), 66.56 (C-4), 61.52 (C-6), 50.06 (C-2), 23.54 (NHAc), 21.11 (OAc), 20.89 (2×OAc), 20.86 (OAc). LRMS m/z calc'd for C16H23NNaO10 (M+Na)+: 412.12; found: 412.10.
Methyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-α-
Methyl 2,3,4,6-tetra-O-benzoyl-β-
Methyl β-
Methyl 2,3,4,6-tetra-O-benzoyl-β-
The crude material was purified via MPLC on silica gel using 0→20% acetone (containing 0.05% v/v NH4OH(aq))—CH2Cl2 to afford the pure product as a white solid (156 mg, 0.127 mmol, 76% yield). Rf=0.58 (0.01:19.99:80 NH4OH:acetone:CH2Cl2). [α]D20: +123° (c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz): δH 8.09-8.06 (m, 2H, Ar), 8.02-7.99 (m, 2H, Ar), 7.93-7.90 (m, 2H, Ar), 7.87-7.71 (m, 6H, Ar), 7.65-7.59 (m, 2H, Ar), 7.52-7.46 (m, 5H, Ar), 7.43-7.40 (m, 1H, Ar), 7.39-7.35 (m, 2H, Ar), 7.24-7.20 (m, 2H, Ar), 5.93 (dd, 1H, J=3.2, <1 Hz, Gal_H4), 5.82 (dd, 1H, J=10.7, 9.1 Hz, GlcN_H3), 5.76 (dd, 1H, J=10.4, 8.0 Hz, Gal_H2), 5.56 (dd, 1H, J=10.4, 3.4 Hz, Gal_H3), 5.33 (d, 1H, J=8.4 Hz, GlcN_H1), 5.18 (dd, 1H, J=9.6, 9.6 Hz, GlcN_H4), 5.06 (d, 1H, J=8.9 Hz, NH), 4.85 (d, 1H, J=8.0 Hz, Gal_H1), 4.53-4.50 (m, 2H, Gal_H6a and Gal_H6b), 4.35 (dd, 1H, J=12.2, 4.2 Hz, GlcN_H6a), 4.35-4.29 (m, 2H, GalN_H2 and Gal_H5), 4.29 (dd, 1H, J=10.8, 8.5 Hz, GlcN_H2), 4.22 (d, 1H, J=3.5 Hz, GalN_H1), 4.19 (dd, 1H, J=12.2, 2.0 Hz, GlcN_H6b), 3.96-3.95 (m, 1H, GalN_H4), 3.87 (ddd, 1H, J=10.1, 4.3, 2.3 Hz, GlcN_H5), 3.76 (dd, 1H, J=10.6, 2.4 Hz, GalN_H6a), 3.69 (dd, 1H, J=10.7, 8.5 Hz, GalN_H6b), 3.65-3.61 (m, 2H, GalN_H3 and GalN_H5), 2.81 (s, 3H, OCH3), 2.76 (d, 1H, J=<1 Hz, GalN_4-OH), 2.10 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.86 (s, 3H, OAc), 1.28 (s, 3H, NHAc). 13C NMR (CDCl3, 125 MHz): δC 170.91 (C═O), 170.28 (C═O), 169.87 (C═O), 169.77 (C═O), 167.71 (C═O), 166.18 (C═O), 165.79 (C═O), 165.72 (C═O), 164.84 (C═O), 134.52 (Ar), 134.01 (Ar), 133.74 (Ar), 133.71 (Ar), 133.60 (Ar), 131.68 (Ar), 130.27 (Ar), 129.96 (Ar), 129.38 (Ar), 129.26 (Ar), 128.94 (Ar), 128.86 (Ar), 128.76 (Ar), 128.72 (Ar), 128.53 (Ar), 102.02 (Gal_C1), 99.13 (GlcN_C1), 98.23 (Fuc_C1), 79.92 (GalN_C3), 72.19 (Gal_C5), 72.05 (GlcN_C5), 71.61 (Gal_C3), 71.09 (GalN_C6), 70.83 (GalN_C3), 69.82 (Gal_C2), 69.19 (GlcN_C4), 68.54 (GalN_C5), 68.25 (GalN_C4), 68.16 (Gal_C4), 62.57 (Gal_C6), 62.24 (GlcN_C6), 54.89 (GlcN_C2), 54.50 (OCH3), 47.88 (GalN_C2), 22.62 (NHAc), 20.99 (OAc), 20.86 (OAc), 20.67 (OAc). LRMS m/z calc'd for C63H62N2NaO24 (M+Na)+: 1253.36; found: 1253.32.
Methyl β-
Ethyl 3-O-benzyl-4,6-O-benzylidene-1-thio-β-
Ethyl 2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene-1-thio-β-
Methyl 3-O-benzyl-4,6-O-benzylidene-β-
Methyl 2,3,4-tri-O-benzyl-α-
Methyl α-
Ethyl 2,3-di-O-acetyl-4,6-O-benzylidene-1-thio-β-
3.294 mmol, 96% yield). Rf=0.64 (1:4 acetone:toluene). [α]D20: +28° (c 1.0, CHCl3). 1H NMR (CDCl3, 500 MHz): δH 7.51-7.46 (m, 2H, Ar), 7.40-7.34 (m, 3H, Ar), 5.48 (s, 1H, PhCH), 5.46 (dd, 1H, J=9.9, 9.9 Hz, H-2), 4.98 (dd, 1H, J=10.0, 3.5 Hz, H-3), 4.45 (d, 1H, J=9.8 Hz, H-1), 4.39 (dd, 1H, J=3.5, <1 Hz, H-4), 4.31 (dd, 1H, J=12.5, 1.5 Hz, H-6a), 3.99 (dd, 1H, J=12.5, 1.6 Hz, H-6b), 3.53-3.52 (m, 1H, H-5), 2.87 (dq, 1H, J=12.3, 7.5 Hz, SCHaHbCH3), 2.72 (dq, 1H, J=12.3, 7.5 Hz, SCHaHbCH3), 2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac), 1.28 (dd, 3H, J=7.5, 7.5 Hz, SCH2CH3). 13C NMR (CDCl3, 125 MHz): δC 170.69 (C═O), 169.53 (C═O), 137.72 (Ar), 129.21 (Ar), 128.30 (Ar), 126.46 (Ar), 101.20 (PhCH), 82.86 (C-1), 73.71 (C-4), 73.10 (C-3), 69.80 (C-5), 69.18 (C-6), 66.70 (C-2), 22.90 (SCH42CH3), 20.96 (Ac), 20.95 (Ac), 14.88 (SCH2CH3). LRMS m/z calc'd for C19H24NaO7S (M+Na)+: 419.11; found: 419.11.
Methyl 2,3-di-O-acetyl-4,6-O-benzylidene-β-
Methyl 4,6-O-benzylidene-β-
Methyl O-[methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
Methyl 2,4,6-tri-O-acetyl-3-O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
Methyl 3-O-(sodium 5-acetamido-3,5-dideoxy-
Methyl 2,3,4-tri-O-benzyl-α-
Methyl α-
Ethyl 2-deoxy-2-phthalimido-1-thio-β-
Ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-β-
Ethyl 3-O-benzyl-4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-β-
Ethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-β-
Ethyl 2,3,4,6-tetra-O-benzoyl-β-
Methyl 2,3,4,6-tetra-O-benzoyl-β-
Methyl β-
Methyl β-
Table 2: Glycans released from each mucin were permethylated and analyzed by NSI-MS. Table 3 presents details (glycan reference number, structural representation, composition, GlyTouCan accession, theoretical m/z, detected m/z, amount, and relative abundance) for the glycans released from MUC5AC only.
Table 3: Glycans released from each mucin were permethylated and analyzed by NSI-MS. Table 3 presents details (glycan reference number, structural representation, composition, GlyTouCan accession, theoretical m/z, detected m/z, amount, and relative abundance) for the top 5 sulfated glycans detected from each mucin preparation by negative mode NSI-MS.
In Tables 2-3:
Tables 4-6: Glycans released from each mucin were permethylated and analyzed by NSI-MS. Tables 4-6 present details (glycan reference number, structural representation, composition, GlyTouCan accession, theoretical m/z, detected m/z, amount, and relative abundance) for the glycans released from all three mucins for cross-comparison.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that they are incorporated by reference in their entirety for all purposes as well as for the proposition that is recited. Where any conflict exists between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GeneIDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures), as well as chemical references (e.g., PubChem compound, PubChem substance, or PubChem Bioassay entries, including the annotations therein, such as structures and assays, etc.), are hereby incorporated by reference in their entirety.
This application claims the benefit of U.S. Provisional Application No. 63/269,825, filed on Mar. 23, 2022. The entire teachings of the above application are incorporated herein by reference.
This invention was made with government support under DMR1419807 and PHY1454673 awarded by the National Science Foundation, and under EB017755, GM124594 and GM103694 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63269825 | Mar 2022 | US |
