Mucin-inspired glycopolypeptides for managing microbial infections

Abstract

Described herein are synthetic glycopolypeptides that contain sugars identified as active in mucin. These compounds reduce the virulence of high-priority opportunistic pathogens, including, but not limited to, Salmonella enterica subsp. enterica serovar Typhimurium, Candida albicans, and Gardnerella vaginalis, without reducing the viability of beneficial Lactobacillus crispatus. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

MUCIN-INSPIRED GLYCOPOLYPEPTIDES FOR MANAGING MICROBIAL INFECTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Application No. 63 / 702,624, filed on October 2, 2024, the contents of which are incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under W91 INF-19-2-0026 awarded by the U.S. Army Research Office and R35 GM147262 and EB017755 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Mucus, an abundantly secreted hydrogel that coats all wet epithelia in the human body, is the largest interface for host-microbe interactions and one of the first environments encountered by invading microbes. Its major gel -forming components, mucin gly copolymers, are emerging as important regulators of microbial virulence. For instance, media containing natively purified mucins are sufficient to prevent surface attachment and biofilm formation by several pathogens.1-3

[0004] These mucin glycoproteins are grafted with a diversity' of complex glycan structures that potentially encode a wealth of biological information. While these glycans are grafted to the mucin backbone in nature, it has been shown that free mucin glycans chemically isolated from mucin suppress key virulence pathways, including quorum sensing, siderophore biosynthesis, and toxin secretion, in many high priority pathogens without directly killing the bacteria4-8. setting the stage for the design of mucus-inspired therapeutic strategies that treat opportunistic infection without driving resistance. The identification of specific bioactive mucin glycans and their mechanism of action, however, has remained an obstacle for developing glycan-based therapies. Additionally, how to best present and deliver these glycans to the site of infection while retaining activity was unknown. Accordingly, thereremains a need for the identification of mucin sugar structures that serve as anti-virulence molecules and methods of making and using same.SUMMARY

[0005] In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to synthetic glycopolypeptides that contain sugars identified as active in mucin. As detailed herein, these compounds reduce the virulence of high-priority opportunistic pathogens, including, but not limited to, Salmonella enterica subsp. enterica serovar Typhimurium, Candida cdbicans, and Gardnerella vaginalis, without reducing the viability of beneficial Lactobacillus crispatus.

[0006] Thus, disclosed are glycopolypeptides comprising a peptide backbone having from about 5 to 1,000 amino acid residues thereon, wherein from about 5% to about 50% of the amino acid residues are glycosylated amino acid residues, wherein the glycosylated amino acid residues are selected from GalNAc residues, GlcNAc residues, galactose residues, mannose residues, and fucose residues, or a mixture thereof.

[0007] Also disclosed are pharmaceutical compositions comprising an effective amount of one or more disclosed glycopolypeptides and a pharmaceutically acceptable salt thereof.

[0008] Also disclosed are methods of treating a bacterial-mediated disease a subject in need thereof, the method comprising administering to the subject one or more of the disclosed glycopolypeptides.

[0009] Also disclosed are compositions comprising a polypeptide, wherein the polypeptide comprises: (a) sialylated GalNAc moieties from .S' typhimurium covalently linked to a peptide backbone by a2,3 or a2,6 linkages; (b) O-glycans from Candida albicans selected from Core 1, Core 1+fucose. and Core 2+galactose, wherein the O-glycans are covalently- linked to a peptide backbone; and (c) galactose and core 1 (galactose-GalNAc) moities from G. vaginalis, wherein the galactose and core 1 are covalently attached to a peptide backbone. In various aspects, the sialylated GalNAc-moiety is selected from:

[0010] Also disclosed are methods of producing a synthetic mucin mimic, the method comprising: (a) identifying a natural mucin from a mucus that attenuates virulence of a microbe; (b) solating active molecule (e.g., a mucin -O-glycan) that attenuates microbial virulence; (c) reformatting the mucin into a scalable synthetic mimic; and (d) testing the ability of the mucin to replicate the anti-virulence of the natural mucin. In various aspects, the native mucin comprises native porcine gastric mucin (MUC5AC) from pig stomachs and / or porcine intestinal mucin (MUC2) from pig small intestines.

[0011] Also disclosed are methods of treating bacterial vaginosis (BV) or vulvovaginal candidiasis (VVC) by administering the disclosed composition to a subject in need thereof, optionally in combination (synergy) with a conventional therapy (e.g., an antibiotic).

[0012] Also disclosed are methods of treating traveler’s diarrhea (e.g., caused by Escherichia coll or Salmonella enterica) by administering the disclosed composition. In various aspects, the treatment does not reduce the viability of beneficial microbes (e.g., Lactobacillus crispatus).

[0013] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0015] FIG. 1A-H show representative data illustrating that gastrointestinal mucins are an anti-infective signal. Specifically, FIG. 1A shows a schematic of SPI-1 -mediated invasion (SCV = Salmonella containing vacuole; Graphic created with BioRender). FIG. IB shows a scanning electron microscopy image of S. Typhimurium (false-colored in purple) grown with purified MUC2 (0.4% w / v). FIG. 1C shows host-cell invasion, as determined by a gentamicin protection assay of S. Typhimurium grown with purified MUC2 (0.2% w / v), of HT-29 cells infected at a multiplicity of infection (MOI) of 20. A one-sample / -test was performed to evaluate whether the change in invasion was significantly different than 0. Exact p values reported. FIG. ID shows the effect of MUC2 on SPI-1 gene expression, as measured by qPCR. A two-way ANOVA was conducted to evaluate the effect of MUC2 concentration and Salmonella strain on SPI-1 gene expression. As show, there was a significant effect of MUC2 concentration on SPI-1 expression. Exact p values reported. FIG. IE shows representative gene expression changes, as determined by RNA sequencing, of Salmonella LT2 cultured with MUC2 (0.1% w / v) relative to medium alone (n=3 biologically independent replicates). FIG. IF shows Salmonella LT2 grow th in the presence of MUC2 (0. 1 % w / v). Data points represent the median CFU / mL (n=3 biologically independent replicates) and error bars represent the 95% confidence interval. FIG. 1G shows host-cell invasion, as determined by a gentamicin protection assay of S. Typhimurium grown with purified MUC5AC (0.2% w / v), of HT-29 cells infected at a multiplicity of infection (MOI) of 20. A one-sample / -test was performed to evaluate whether the change in invasion w as significantly different than 0. Exact p values are reported. FIG. 1H shows the effect of MUC5AC (0.1% w / v) on SPI-1 gene expression, as measured by qPCR.

[0016] FIG. 2A-E show representative data illustrating that MUC2-mediated inhibition of S. Typhimurium virulence is HilD-dependent. Specifically, FIG. 2A shows a schematic illustrating how MUC2 inhibits gene expression of SPI-1 -encoded regulators and downstream genes but not regulators encoded outside of SPI-1. Gene expression changes were determined by RNA sequencing with MUC2 (0. 1% w / v) relative to medium alone, overlaying the SPI-1 regulatory network. FIG. 2B show's the effect of MUC2 (0.1% w / v) on hilA gene expression in different mutant backgrounds or the S. Typhimurium parent strain14028s (WT), as measured by qPCR. A one-sample / -test was performed to evaluate whether the change in expression was significantly different than 0. Exact p values reported. FIG. 2C shows Autodock vina predicted binding affinity of monosaccharides to HilD. FIG. 2D shows that GalNAc monomers are predicted to bind directly at residue N44 within the HilD ligand binding pocket. FIG. 2E shows the effect of HilD binding site mutations on SP1-1 gene expression, measured by qPCR, in response to GalNAc or GlcNAc (0.2% w / v). A two-way ANOVA with Tukey’s multiple comparison’s test was conducted to evaluate the effect HilD mutations on HexNAc-mediated suppression of SPI-1 gene expression. Exact p values reported.

[0017] FIG. 3A-D show' representative data illustrating that presenting GalNAc on a peptide backbone promotes its activity. Specifically, FIG. 3A shows a heat map of SPI-1 genes with genomic orientation and function indicated. Gene expression changes w ere determined by RNA sequencing of Salmonella cultured with an equal-parts mixture of Galactose, GalNAc, GlcNAc, Neu5Ac, and fucose (0.1% w / v, n = 2 biologically independent replicates), GalNAc (0.2% w / v, n = 3), or GlcNAc (0.2% w / v, n = 3) relative to medium alone on SPI-1 gene expression. FIG. 3B shows the design of glycopolypeptides using the established N- carboxy anhydride (NCA) polymerization strategy. FIG. 3C shows the effect of MUC2, GalNAc. or GalNAc-peptides (25% glycan density. 0.2% w / v) on hilA (left) and prgH (right) gene expression in S. Typhimurium 14028s, as measured by qPCR. A one-sample / -test was performed to evaluate whether the change in expression w as significantly different than 0. Exact p values reported. FIG. 3D shows the design of sialylated glycopeptides. FIG. 3E shows the effect of sialylated or unsialylated GalNAc-peptides (25% glycan density, 0.1% w / v) on hilA (left) and prgH (right) gene expression in S. Typhimurium LT2 or 14028s, as measured by qPCR. A two-way ANOVA with Dunnett’s multiple comparison’s test was conducted to evaluate the effect sialylation on GalNAc-peptide-mediated suppression of SPI- 1 gene expression. Exact p values reported.

[0018] FIG. 4A-D show representative data illustrating that core-1 glycosylated polypeptides inhibit filamentation of Candida albicans. Specifically, brightfield images of Candida albicans cultured in RPMI medium alone (FIG. 4A) or with purified MUC5AC (image from Takagi et al. 2022) (FIG. 4B), soluble core 1 glycans (FIG. 4C), or a core-1 polypeptide (25% glycan- threonine density, 0. 1% w / v) (FIG. 4D) are shown.

[0019] FIG. 5A and FIG. 5B show representative data illustrating inhibition of Gardnerella biofilm by glycopolypeptide prototy pe. Specifically, as shown, preliminary studies indicatethat Core 1 glycopolypeptides can decrease biofdm formation relative to untreated media- only conditions to a similar degree as D-galactose without impacting the viability of either G. vaginalis (FIG. 5A) or Lactobacillus crispatus (FIG. 5B).

[0020] FIG. 6 shows a representative schematic illustrating the glycopolymer design process.

[0021] FIG. 7 shows representative Brightfield images of Candida albicans cultured in RPMI filamentation medium alone or soluble core 1 glycans (right side) as compared to core- 1 polypeptides with varied density of Core 1 -threonine density, 0.5% w / v. The images show that Core-1 glycosylated polypeptides inhibit filamentation of Candida albicans.

[0022] FIG. 8 shows representative Brightfield images of Candida albicans cultured in RPMI medium with 25% glycosylated polypeptides displaying GalNAc or Gal attached to Ser or Thr, 1.0-0.5% w / v. The images show that various glycosylated polypeptides inhibit filamentation of Candida albicans.

[0023] FIG. 9 shows representative Brightfield images of Candida albicans cultured in RPMI medium with 25% glycosylated polypeptides displaying alpha2,3-Sia-Corel, alpha2,6- Sia-Corel, or Lac glycans attached to Ser or Thr, 1.0-0.5% w / v. The images show that various glycosylated polypeptides inhibit filamentation of Candida albicans.

[0024] FIG. 10 shows representative Brightfield images of Candida albicans cultured in RPMI medium with 25% glycosylated polypeptides displaying alphal,2-Fuc-Corel, alphal,3-Fuc-Corel, or alphal,4-Fuc-Corel glycans attached to Thr, 1.0-0.5% w / v. The images show that various glycosylated polypeptides inhibit filamentation of Candida albicans.

[0025] FIG. 11 shows representative Brightfield images of Candida albicans cultured in RPMI medium with Glu / Ala / Pro polypeptides, 1.0 w / v. The images show that the polypeptide backbones plays a role in inhibition of filamentation of Candida albicans.

[0026] FIG. 12 shows representative Brightfield images of Candida albicans cultured in RPMI medium with GalNAc-Ser / Glu / Ala / Pro polypeptides with D vs L stereochemistry, 0.5 w / v. The images show that the polypeptide backbone plays a role in inhibition of filamentation of Candida albicans.

[0027] FIG. 13 shows representative data showing disruption of S', aureus biofilms by Core- 1 glycosylated polypeptides. S. aureus biofilms, induced in TBSg media, are disrupted by 0.1% w / v polypeptide displaying 25% core 1 glycans but not GalNAc glycans.

[0028] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0029] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0030] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0031] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0032] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for thematerial contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.A. DEFINITIONS

[0033] As used in the specification and the appended claims, the singular forms “a,” '‘an’’ and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

[0034] As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0035] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12. 13, and 14 are also disclosed.

[0036] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or otherquantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0037] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by w eight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0038] A weight percent (wt. %) of a component, unless specifically stated to the contrary. is based on the total weight of the formulation or composition in which the component is included.

[0039] As used herein, “ICso” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, an ICso can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein. In a further aspect. ICso refers to the half-maximal (50%) inhibitory concentration (IC) of a substance.

[0040] As used herein, “ECso” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% agonism of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, an ECso can refer to the concentration of a substance that is required for 50% agonism in vivo, as further defined elsewhere herein. In a further aspect, ECso refers to the concentration of agonist that provokes a response halfway between the baseline and maximum response.

[0041] As used herein, the term “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0042] As used herein, the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow. cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, thesubject is a mammal. A patient refers to a subject afflicted with a disease, disorder, or condition. The term “patient” includes human and veterinary subjects.

[0043] As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g, a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it: (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, z.e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term “subject” also includes domesticated animals (e.g, cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

[0044] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0045] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.

[0046] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration,intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0047] As used herein, the terms "effective amount” and "amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the condition being treated and the severity of the condition; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0048] As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. A dosage forms can comprise inventive a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharmaceutically acceptableexcipient, such as a preservative, buffer, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonyl phenol, sodium desoxy cholate), solution and / or cryo / lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g.. salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2- phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). A dosage form formulated for injectable use can have a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, suspended in sterile saline solution for injection together with a preservative.

[0049] As used herein, "kit" means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0050] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents and are meant to include future updates.

[0051] As used herein, the term '‘therapeutic agent” includes any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and / or physiologic effect by local and / or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14thedition), the Physicians' Desk Reference (64thedition), and The Pharmacological Basis of Therapeutics (12thedition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term "therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; anti-cancer and anti-neoplastic agents such as kinase inhibitors, poly ADP ribose polymerase (PARP) inhibitors and other DNA damage response modifiers, epigenetic agents such as bromodomain and extra-terminal (BET) inhibitors, histone deacetylase (HDAc) inhibitors, iron chelotors and other ribonucleotides reductase inhibitors, proteasome inhibitors and Nedd8-activating enzyme (NAE) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, traditional cytotoxic agents such as paclitaxel, dox, irinotecan, and platinum compounds, immune checkpoint blockade agents such as cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibody (mAB), programmed cell death protein 1 (PD-l) / programmed cell death-ligand 1 (PD-L1) mAB, cluster of differentiation 47 (CD47) mAB, toll-like receptor (TLR) agonists and other immune modifiers, cell therapeutics such as chimeric antigen receptor T-cell (CAR-T) / chimeric antigen receptor natural killer (CAR-NK) cells, and proteins such as interferons (IFNs), interleukins (ILs), and mAbs; anti-ALS agents such as entry inhibitors, fusion inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors. NCP7 inhibitors, protease inhibitors, and integrase inhibitors; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents,anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as w ell as other areas. The term “therapeutic agent” also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

[0052] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, z.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0053] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g. a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0054] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as w ell as sterilepowders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-poly glycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0055] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acres Organics (Morris Plains, N.J.), Strem Chemicals (Newbury port, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry' of Carbon Compounds, Volumes 1-5 andsupplemental volumes (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0056] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0057] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D. B-E, B-F. C-D. C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C- E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0058] It is understood that the compounds and compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.B. GLYCOPOLYPEPTIDES

[0059] In one aspect, disclosed are glycopolypeptides comprising a peptide backbone having from about 5 to 1,000 amino acid residues thereon, wherein from about 5% to about 50% of the amino acid residues are glycosylated amino acid residues, wherein the glycosylated amino acid residues are selected from GalNAc residues, GlcNAc residues, galactose residues, mannose residues, and fucose residues, or a mixture thereof.

[0060] In various aspects, the glycosylated amino acid residues are linked to the peptide backbone via an a2,3 linkage or an a2,6 linkage. Thus, in various further aspects, the glycosylated amino acid residues are linked to the peptide backbone via an a2,3 linkage. In various further aspects, the glycosylated amino acid residues are linked to the peptide backbone via an a2,6 linkage.

[0061] In various aspects, wherein the glycosylated amino acid residues are linked to the peptide backbone via a residue selected from Ser, Thr, Cys, homoSer, homoThr, Lys, Om, Gly, and Asp. In various further aspects, the glycosylated amino acid residues are linked to the peptide backbone via a residue selected from Ser and Thr.

[0062] In various aspects, from about 5% to about 50% of the amino acid residues are glycosylated amino acid residues. Thus, in various aspect, from about 5% to about 45%, from about 5% to about 40%, from about 5% to about 35%. from about 5% to about 30%, from about 5% to about 25%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%, from about 10% to about 50%, from about 15% to about 50%, from about 20% to about 50%, from about 25% to about 50%, from about 30% to about 50%, from about 35% to about 50%, from about 40% to about 50%, from about 45% to about 50%, from about 10% to about 45%, from about 15% to about 40%, from about 20% to about 35%, or from about 25% to about 30% of the amino acid residues are glycosylated amino acid residues. In a further aspect, from about 10% to about 40% of the amino acid residues are glycosylated amino acid residues. In a still further aspect, from about 15% to about 35% of the amino acid residues are glycosylated amino acid residues.

[0063] In various aspects, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% or about 50% of the amino acid residues are glycosylated amino acid residues. In a further aspect, about 25% of the amino acid residues are glycosylated amino acid residues.

[0064] In various aspects, at least a portion of the glycosylated amino acid residues are sialylated glycosylated amino acid residues. In various further aspects, each of the glycosylated amino acid residues are sialylated glycosylated amino acid residues. In various further aspects, the glycosylated amino acid residues comprise sialylated glycosylated amino acid residues.

[0065] In various aspects, one or more of the glycosylated amino acid residues are sialylated glycosylated amino acid residues.

[0066] In various aspects, at least a portion of the glycosylated amino acid residues are O- glycan residues. In various further aspects, each of the glycosylated amino acid residues are O-glycan residues, various further aspects, the glycosylated amino acid residues comprise O- glycan residues.

[0067] In various aspects, one or more of the glycosylated amino acid residues are O-glycan residues.

[0068] In various aspects, the glycosylated amino acid residues are selected from Core 1 (Gal-GalNAc) glycan residues. Gal residues, and GalNAc residues, or a mixture thereof. In a further aspect, the glycosylated amino acid residues are a mixture of Core 1 (Gal-GalNAc) glycan residues and Gal residues.

[0069] In various aspects, one or more of the glycosylated amino acid residues are GalNAc residues. In various aspects, each of the glycosylated amino acid residues are GalNAc residues. In a further aspect, one or more of the glycosylated amino acid residues are sialylated GalNAc residues. In a still further aspect, one or more of the sialylated GalNAc residues have a structure selected from:or a mixture thereof, wherein each occurrence of m is 0.25. and wherein n is a 100-mer, or a pharmaceutically acceptable salt thereof.

[0070] In various aspects, the glycosylated amino acid residues are sialylated glycosylated amino acid residues linked to the peptide backbone via an a2,3 linkage or an a2,6 linkage. In a further aspect, the sialylated glycosylated amino acid residues are sialylated GalNAc residues. In a still further aspect, the sialylated GalNAc residues have a structure selected from:or a mixture thereof, wherein each occurrence of m is 0.25, and wherein n is a 100-mer, or a pharmaceutically acceptable salt thereof.

[0071] In various aspects, one or more of the glycosylated amino acid residues are Core 1 glycan residues. In a further aspect, the Core 1 glycan residues are selected from Core 1 glycan residues. Core 1 glycan + fucose residues, Core 1 glycan + galactose residues. Core 1 glycan + N-acetylgalactosamine residues, Core 1 glycan + lactose residues, Core 1 glycan +fucosylated residues, and Core 1 glycan + sialylated residues, or a mixture thereof. In a still further aspect, the Core 1 glycan residues are selected from Core 1 glycan residues, Core 1 glycan + fucose residues, and Core 1 glycan + galactose residues, or a mixture thereof. In yet a further aspect, the Core 1 glycan residue comprises a structure selected from:andor a pharmaceutically acceptable salt thereof.

[0072] In various aspects, the glycopolypeptide comprises a structure selected from:andor a pharmaceutically acceptable salt thereof.C. PHARMACEUTICAL COMPOSITIONS

[0073] In one aspect, disclosed are pharmaceutical compositions comprising an effective amount of one or more of the disclosed glycopolypeptides and a pharmaceutically acceptable salt thereof. In a further aspect, the glycopolypeptide comprises a peptide backbone having from about 5 to 1,000 amino acid residues thereon, wherein from about 5% to about 50% of the amino acid residues are glycosylated amino acid residues, wherein the glycosylated amino acid residues are selected from GalNAc residues, GlcNAc residues, galactose residues, mannose residues, and fucose residues, or a mixture thereof.

[0074] In various aspects, the glycopolypeptides and compositions of the invention can be administered in pharmaceutical compositions, which are formulated according to the intended method of administration. The glycopolypeptides and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, a pharmaceutical composition can be formulated for local or systemic administration, e.g., administration by drops or injection into the ear, insufflation (such as into the ear), intravenous, topical, or oral administration.

[0075] The nature of the pharmaceutical compositions for administration is dependent on the mode of administration and can readily be determined by one of ordinary skill in the art. In various aspects, the pharmaceutical composition is sterile or sterilizable. The therapeutic compositions featured in the invention can contain carriers or excipients, many of which are known to skilled artisans. Excipients that can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols,ascorbic acid, phospholipids, polypeptides (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. The nucleic acids, polypeptides, small molecules, and other modulatory compounds featured in the invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. A modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, for injection, or for ingestion; gels or powders can be made for ingestion or topical application. Methods for making such formulations are well known and can be found in. for example, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed.. Mack Publishing Co., Easton, PA 1990.

[0076] In various aspects, the disclosed pharmaceutical compositions comprise a disclosed glycopolypeptide (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0077] In various aspects, the pharmaceutical compositions of this invention can include a pharmaceutically acceptable carrier and a glycopolypeptide or a pharmaceutically acceptable salt of the glycopolypeptides of the invention. The glycopolypeptides of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.

[0078] The pharmaceutical carrier employ ed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and w ater. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0079] In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystallinecellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0080] A tablet containing the composition of this invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free- flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0081] The pharmaceutical compositions of the present invention comprise a glycopolypeptide of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0082] Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

[0083] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0084] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0085] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0086] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including antioxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound of the invention, and / or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

[0087] In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount.

[0088] In a further aspect, the pharmaceutical composition is administered to a mammal. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient.

[0089] In various aspects, the pharmaceutical composition further comprises an antibacterial agent. Examples of antibacterial agents include, but are not limited to, amoxicillin, ampicillin, azithromycin, aztreonam, azlocillin, bacitracin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, chloramphenicol, cilastin, ciprofloxacin, clarithromycin, clavulanic acid, clinafloxacin, clindamycin, clofazimine, cloxacillin, colistin, dalbavancin, dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doxycycline, ery thromycin, enrofloxacin, enoxacin, enviomycin, ertepenem, ethambutol, flucloxacillin, fosfomycin, furazolidone, gatifloxacin, gentamicin, imipenem. isoniazid, kanamycin, linezolid, lomefloxacin, loracarbef, mafenide, moxifloxacin, meropenem, metronidazole, mezlocillin, minocycline, mupirocin, nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oritavancin, oxytetracycline, penicillin, piperacillin, platensimycin, polymixin B, quinupristin, retapamulin, rifabutin, rifampin, rifapentine. roxithromycin, sparfloxacin. spectinomycin, sulbactam, sulfacetamide, sulfamethizole, sulfamethoxazole, teicoplanin, telithromycin, telavancin, temafloxacin, tetracycline, thioacetazone, thioridazine, ticarcillin, tinidazole, tobramycin, torezolid, tosufloxacin, trimethoprim, troleandomycin, trovafloxacin, and vancomycin.

[0090] In a further aspect, the pharmaceutical composition is used to treat a bacterial- mediated disease such as, for example, a vaginal disease (e.g., bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC)) or traveler’s diarrhea.

[0091] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.D. METHODS OF MAKING A GLYCOPOLYPEPTIDE

[0092] In one aspect, disclosed are methods of producing a synthetic mucin mimic (z.e., a glycopolypeptide as disclosed herein), the method comprising: (a) identifying a natural mucin from a mucus that attenuates virulence of a microbe; (b) isolating the natural mucin that attenuates microbial virulence: (c) reformatting the mucin into a scalable synthetic mimic; and (d) testing the ability of the mucin to replicate the anti-virulence of the natural mucin.

[0093] Additional methods of preparing the disclosed glycopolypeptide are detailed herein in the Examples section.E. METHODS OF TREATING A BACTERIAL-MEDIATED DISEASE

[0094] In one aspect, disclosed are methods of treating a bacterial-mediated disease a subject in need thereof, the method comprising administering to the subject one or more of the disclosed glycopolypeptides. In a further aspect, the glycopolypeptide comprises a peptidebackbone having from about 5 to 1,000 amino acid residues thereon, wherein from about 5% to about 50% of the amino acid residues are glycosylated amino acid residues, wherein the glycosylated amino acid residues are selected from GalNAc residues, GlcNAc residues, galactose residues, mannose residues, and fucose residues, or a mixture thereof.

[0095] In one aspect, disclosed are methods of treating bacterial vaginosis (BV) or vulvovaginal candidiasis (VVC) in a subject in need thereof, the method comprising administering to the subject a disclosed composition, optionally in combination (synergy ) with a conventional therapy (e.g., an antibiotic or antibacterial agent).

[0096] In one aspect, disclosed are methods of treating traveler's diarrhea (e.g., caused by Escherichia coli or Salmonella ent erica) in a subject in need thereof, the method comprising administering to the subject a disclosed composition. In a further aspect, the treatment does not reduce the viability of beneficial microbes (e.g., Lactobacillus crispatus).

[0097] In various aspects, the bacterial-mediated disease is due to a bacterium selected from S. Typhimurium, C. albicans, G. vaginalis, and S. aureus.

[0098] In various aspects, the bacterial-mediated disease is a vaginal disease. Examples of vaginal diseases include, but are not limited to, bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC).

[0099] In various aspects, the bacterial-mediated disease is traveler s diarrhea.

[0100] In various aspects, the method further comprises administering to the subject an antibacterial agent.

[0101] In various aspects, the glycopolypeptide and the antibacterial agent are administered sequentially. In various further aspects, the glycopolypeptide and the antibacterial agent are administered simultaneously. In various further aspects, the glycopolypeptide and the antibacterial agent are co-formulated.

[0102] In various aspects, the subject is a mammal. In various further aspects, the subject is a human.

[0103] In various aspects, the subject has been diagnosed with a need for treatment of the bacterial-mediated disease prior to the administering step.

[0104] In various aspects, the method further comprises the step of identifying a subject in need of treatment of the bacterial-mediated disease.

[0105] In various aspects, the effective amount is a therapeutically effective amount. In various further aspects, the effective amount is a prophylactically effective amount.F. EXAMPLES

[0106] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0107] The Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way.

[0108] Herein, the identification of a subset of mucin sugar structures that serve as anti-virulence molecules is described. When displayed on a polypeptide backbone, these structures retain their activity or have enhanced potency against key virulence features of Candida albicans, Gardnerella vaginalis, and Salmonella enterica subsp. enterica serovar Typhimurium. Moreover, the specific mechanism of action against Salmonella enterica has been identified. Without wishing to be bound by theory, these results indicate that the technology to design synthetic mucins that mimic the potent virulence-attenuating functions of mucin for targeted high-priority' pathogens has been developed. The modular, high- precision, and scalable nature of this invention makes it an ideal system for tackling a broad range of infectious threats.1. RESULTS AND DISCUSSION a. DESIGN OF GLYCOPOLYPEPTIDES AGAINST SALMONELLA ENTERICA TYPHIMURIUM

[0109] A detailed study of how enteric pathogens interact w ith mucin matrices was performed in order to rationally design bio-inspired materials for stabilizing microbiota that are disrupted by pathogens. The initial focus was on S. Typhimurium, which is a highly gut- adapted pathogen that can disrupt gut microbiota and compete with beneficial probiotic species. S. Typhimurium uses its ty pe III secretion system (T3SS) to inject effector proteins into host cells, aiding in invasion (FIG. 1A). The protective role of mucin MUC2 was explored by reconstituting it in SPI-1 inducing medium and measuring its impact on S.Typhimurium. Scanning electron microscopy showed close interaction between the mucin matrix and S. Typhimurium cells (FIG. IB). When HT-29 monolayers were cultured with exogenous MUC2 and infected with S. Typhimurium, fewer bacteria invaded the epithelial cells (FIG. 1C). qPCR revealed a dose-dependent decrease in the expression of SPI-1 genes hilA and prgH (FIG. ID). RNA sequencing showed that MUC2 downregulated 190 genes, including the entire SPI-1, while 96 genes were upregulated (FIG. IE). Mucin addition did not alter bacterial grow th (FIG. IF). This virulence suppression was also observed with gastric mucin MUC5AC, which reduced cell invasion and downregulated SPI-1 genes (FIG. 1G and FIG. 1H). Without wishing to be bound by theory, these results suggest that a conserved feature of gastrointestinal mucins can inhibit S. Typhimurium invasion genes.

[0110] Salmonella regulates SPI-1 gene expression through various factors to optimize virulence. It was hypothesized that regulators like Mlc, which control sugar uptake, or the FimYZ system might sense mucins. Transcriptomic analysis revealed that MUC2 downregulated the central SPI-1 regulator HilD and its downstream genes (FIG. 2A). Testing hilA expression in 14028s mutants showed that MUC2 suppressed hilA in all except the \hil!) mutant (FIG. 2B), indicating HilD's role in mucin response. HilD is a transcriptional regulator with a putative carbohydrate-binding domain. Virtual screening identified N- acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) as potential HilD ligands (FIG. 2C) Structural modeling showed GalNAc binding residues that are important for sensing intestinal signals (FIG. 2D). A HilD mutant impaired in ligand binding was unresponsive to GalNAc and GlcNAc. showing no SPI-1 gene downregulation (FIG. 2E). Without wishing to be bound by theory, this suggests that mucin-derived sugars inhibit SPI-1 expression through HilD.

[0111] RNA sequencing was performed on LT2 with 0.2% GalNAc or GlcNAc. These sugars partially inhibited SPI-1, but less effectively than complex mucins (FIG. 3 A). Next, to leverage these identified sugars with a known target in S. Typhimurium, the development of a bio-enabled mucin mimic that could reduce the virulence of microbiotadisrupting threats was explored. Using the information on the active components of mucin, the reformatting of active sugars w as directed onto polypeptide backbones. The first material examined was a GalNAc-Serine polypeptide mimicking natural sugar presentation on mucins (FIG. 3B). Despite lower GalNAc content, these glycopolypeptides showed similar inhibitory activity to intact MUC2 (FIG. 3C), highlighting the importance of native sugar presentation. The role of sialic acids capping mucin glycans w as also explored. SialylatedGalNAc-polypeptides with a2,3 or a2,6 linkages abolished the inhibitory effect on SPI-1 genes in both LT2 and 14028s strains (FIG. 3D and FIG. 3E). Without wishing to be bound by theory, this demonstrates the importance of glycan identity and presentation in mucin function. b. DESIGN OF GLYCOPOLYPEPTIDES AGAINST CANDIDA ALBICANS

[0112] Candida albicans is a significant human pathogen that causes a range of infections, including oral thrush, vulvovaginal yeast infections, and sometimes lifethreatening systemic disease. Filamentation plays a critical role in Candida albicans pathogenesis by enabling the transition from a benign yeast form to an invasive hyphal form, which enhances tissue invasion, immune evasion, and biofilm formation, key factors in infection severity.

[0113] Mucins, large gel-forming polymers within the mucus barrier, inhibit the yeast-to-hypha transition, a key virulence factor of this fungal pathogen. In Takagi et al. (2022), mucins were found to contain an abundance of virulence-attenuating molecules in the form of mucin O-glycans. Over 100 mucin O-glycans were isolated from three major mucosal surfaces, and they were shown to inhibit filamentation and other infection-relevant traits, including surface adhesion, biofilm formation, and competition between C. albicans and Pseudomonas aeruginosa. Three specific O-glycan structures (Core 1, Core 1+fucose, and Core 2+galactose) were identified as potent inhibitors of filamentation. comparable to the effect of the full O-glycan pool.

[0114] Based on these insights, mucins were reformatted into a scalable synthetic form using the same chemical approaches previously applied to Salmonella. As shown in Figure 4A-D, the Core 1 (Gal-GalNAc) glycan remained effective in inhibiting C. albicans filamentation when displayed on a polypeptide backbone, despite the lower overall concentration of Core 1 glycans compared to the pool of soluble glycans. Without wishing to be bound by theory, this suggests that glycan display on a peptide backbone enhances efficacy, potentially improving the scalability and delivery' of gly can-based therapies. It was determined that a range of glycan densities are tolerated while maintaining filamentation inhibition. Polypeptides can be composed of 5-50% residues that bear a Core 1 glycan as shown in FIG. 7.

[0115] Aside from polypeptides that display Core 1 glycans, it was found that a range of alternate glycan structures inhibit filamentation and pathogenic hyphae formation asshown in FIG. 8-10. Similar antifungal efficacy was found for structures with display of galactose (Gal), N-acetylgalactosamine (GalNAc), lactose (Lac), and fucosylated (Fuc), and sialylated (Sia) glycans on Corel, Gal, or GalNAc. It was determined that the residue to which the glycan is attached has structural tolerance while maintaining activity; both Ser and Thr inhibit fllamentation.

[0116] As shown in FIG. 11, the polypeptide backbone itself plays a role in inhibition since a reduction in hyphae content is observed for a polypeptide composed of Glu, Ala, and Pro. FIG. 12 also indicates that the peptide backbone plays a role in inhibition since reversal of stereochemistry to the non-native D-amino acid configuration results in reduction in activity. Overall, these findings underscore their potential as therapeutic agents for managing fungal infections. c. DESIGN OF GLYCOPOLYPEPTIDES AGAINST GARDNERELLAVAGINALIS

[0117] Mucins, soluble mucin glycans isolated from native mucus, and specific sugar monomers that can suppress toxin expression and protect human endocervical cells from the key bacterial vaginosis (BV) pathogen Gardnerella vaginalis have been identified previously. Additionally, these findings indicate that these protective glycan structures not only suppress biofilm formation but also enhance antibiotic efficacy against G. vaginalis.

[0118] The highly desirable properties of mucins and certain mucin glycans provide motivation to create mimetics that simplify the structural complexity of mucin while preserving its functional characteristics.

[0119] Herein, mucin-mimetics designed for the targeted delivery and sustained presence of these therapeutic glycans at the mucosal surface have been successfully developed and described.

[0120] As backbones for these mucin mimetics, testing of glycan attachments to polypeptides was initiated. These polypeptides are designed to retain the biocompatibility and mechanical properties necessary for therapeutic applications. Based on the recent insights into which mucin-derived glycans exhibit the highest bioactivity against G. vaginalis, developed galactose (Gal) and core 1 (Gal- GalNAc) functionalized polypeptide prototypes were developed. Initial studies with the core 1 -polypeptide demonstrated that this mucin mimic can inhibit G. vaginalis biofilm while maintaining the viability of the vaginal commensal Lactobacillus crispatus (FIG. 5A and FIG. 5B).d. DESIGN OF GLYCOPEPTIDES AGAINST STAPHYLOCOCCUS A UREUS

[0121] Staphylococcus aureus is a microbe that can cause deadly infections in skin and mucosal epithelia, including the respiratory and gastrointestinal (GI) systems and wounds. There is a crucial need for novel, non-antibiotic, host-directed therapies that both enhance host defense and suppress pathogens such as S. aureus. The findings herein show that mucin glycans inhibit S. aureus biofilm formation and clumping, two phenotypes known to be critical for pathogenesis. Biofilms are particularly problematic because they enable microbes to evade the host immune response by concealing their presence and reducing the expression of acute virulence determinants. Further, microbes in biofilms develop tolerance to antimicrobial concentrations that are otherwise effective against planktonic cells, hindering the eradication of biofilm-associated infections. Biofilm formation can also promote the emergence of antibiotic-resistant pathogens. e. SUMMARY OF COMPOSITIONS THAT CAN BE USED TO MANAGE MICROBIAL INFECTIONS

[0122] A panel of glycopolypeptide structures that can be used to inhibit microbial infections have been prepared. These structures are prepared by established NCA polymerization methodologies that result in statistical distributions of amino acids within the peptide chains (random mixture of sequences). Glycans are typically attached to Ser or Thr. However, glycans could be attached to analog amino acids including but not limited to Cys, homoSer, homoThr, Lys, Om, Glu, Asp or a fully synthetic amino acid with a click chemistry group or other reactive group. The polypeptide chain can contain 5-1000 amino acids and maintain anti-infective or prebiotic activity. The portion of the amino acids that must be glycosylated ranges from 5-50%. The glycans can be Gal. GalNAc. Corel. Lac or sialylated or fucosylated Gal, GalNAc, Corel, Lac or analog glycan structures. The remaining 50-95% of the residues is ty pically 1 :1 :1 Glu:Ala:Pro. However, this 50-95% can vary to include other naturally occurring amino acids. Inhibition of Candida Albicans can be achieved using polypeptides composed of Glu / Ala / Pro where the Glu or Pro content must be at least 20% and no glycan is present. In all cases the polypeptide chain end can be a free amine or can be modified with a targeting or tracking group such as a fluorophore, biotin, a peptide sequence, a glycan, or other carbon containing group.2. METHODS a. DESIGN STRATEGY

[0123] As shown in FIG. 6, the design process follows a strategic, step-by-step approach aimed at developing an effective, scalable therapeutic. First, how a microbe’s virulence is attenuated by the presence of natural mucin was observed, leading to identification of a promising interaction to investigate. Next, the mucus was deconstructed to isolate and identify the specific active molecules — typically, mucin O-glycans — that are responsible for suppressing microbial virulence. Once the active component is identified, it was reformatted into a scalable synthetic mimic, ensuring that it can be produced efficiently and in a form suitable for therapeutic use. Finally, the efficacy of the synthetic mucin mimic was validated by testing its ability to replicate the original anti-virulence effects, ensuring that it effectively neutralizes the targeted pathogen without harmful side effects. Without wishing to be bound by theory, this process allows for the systematic development mucin-inspired therapeutics that are both biologically effective and commercially viable. b. MUCIN PURIFICATION

[0124] Native porcine gastric mucin (MUC5AC) was purified from 10-20 pig stomachs, and porcine intestinal mucin (MUC2) was purified from 6-10 pig small intestines (each of approximately 5 to 10 m in length) as previously described1 G. Organs and mucus scrapings were kept on ice throughout processing. Stomachs were opened by cutting greater curvature; then excess food was discarded before scraping mucus from the tissue. Intestines were cut into about 30 cm length sections, cut open along the long axis, and scraped to collect mucus. Scrapings were then diluted at a ratio of 1 :5 (500 mL scrapings to 2.5 L) using Milli- Q water. NaCl (to 0.2 M), NaN3 (to 0.05%), Benzamidine HO (to 5 mM), dibromoacetophenone (to 1 mM), phenylmethylsulfonylfluoride (PMSF, to 1 mM), and EDTA pH 7 (to 5mM) were added to the diluted scrapings. Scrapings were stirred at 4 °C overnight to solubilize mucus. Coarse tissue and food debris were removed by low-speed centrifugation of solubilized mucus at 8000 x g RCF (7,000 rpm Sorvall GS-3 rotor) for 30 minutes at 4 °C. Supernatant was recovered and then centrifuged on an ultracentrifuge at 32,000 x g RCF for 30 min at 15 °C (20,000 rpm, Beckman 45 Ti rotor). The central fraction was collected, avoiding the accumulated fats and lipids at the top and the pelleted material at the bottom. Using a Buchner funnel, the supernatant was filtered through 2 wetted Whatmanfilters. The filtered sample was then concentrated up to 5x, washed twice with 0.2 M NaCl and 0.05% NaN3, and ultracentrifuged for a second time. The collected supernatant (500-800 mL) was then loaded on a Sepharose CL-2B column at 3-5 mL / min. Columns were run and are stored in 0.2 M NaCl, 0.05% NaN3. After sample loading, the column was eluted, and 45 mL fractions were collected. Elution peaks were monitored by UV absorbance at 215 nm or 280 nm. Typical 215:280 ratios of the mucin fractions are 5-8: 1. Mucin fractions can be confirmed by dot blot. Mucin-containing fractions were collected and pooled, transferred to a Millipore filtration cell containing a pre- wet, prewashed Pall membrane (100,000 MWCO), and concentrated at 4 °C. Samples were then iteratively washed with filtered Milli-Q water (until samples were diluted at least 1: 1000) to remove salts. Twenty -five mL samples were aliquoted into 50 ml tubes, flash-frozen, and lyophilized. Lyophilized purified mucin was stored at < -20 °C until needed. The mucin yield from 10 stomachs or 10 intestines is typically on the order of 100 mg MUC5AC or up to 1 g MUC2. For experiments involving purified mucins, the material was weighed and diluted into the appropriate medium and then solubilized by gentle shaking overnight at 4°C. c. MUCIN GLYCAN ISOLATION

[0125] Mucin glycans were isolated from purified MUC2 from porcine intestinal mucus. Glycans were cleaved from the protein mucin backbone via a non-reductive ammonolysis procedure as previously described5,7. A saturated ammonium hydroxide reagent was prepared by mixing 10 mL NH4OH with 3 g (NH4)2CO3 and mixed for 3 h in a chemical hood. The ammonia hydroxide reagent was added to the lyophilized mucin to a concentration of 30 mg / mL (e.g., 60 mg mucin in 2 mL ammonia solution). An equal volume of the ammonia solution was added to an empty tube to serve as a blank control. The mucin- ammonia mixture was then incubated in a 50 °C sand bath for 60 h. Every 12 hours, 0.1 mg (NH4)2CO3 was added per 1 mL sample and vigorously vortexed. The mucin- ammonium mix was then dried by centrifugal evaporation in a SpeedVac with a vapor trap. Dried samples were iteratively washed by resuspending the sample in H2O and drying by centrifugal evaporation until all (NH4)2CO3 was removed from the blank. These glycosylamines were then converted to free, reducing glycans by adding 1.0 ml of 0.5M boric acid to each sample and incubating at 37 °C for 1 h. Glycans were then dried by centrifugal evaporation and then iteratively washed by resuspending the sample in methanol and dry ing by centrifugal evaporation until the blank control was empty. Glycans were then separatedfrom the peptide backbone by spin dialysis. Specifically, all samples were dissolved in HPLC-grade water and centrifuged at 4°C against a pre- washed Amicon 10k MWCO membrane. The concentration of the glycan solution was then quantified using a Phenol / Sulfuric Assay and a glucose standard curve ranging from 0 to 10 mg / mL glucose. For the Phenol / Sulfuric Assay. 10 pL of standards or sample was added to the wells of a 96- well plate and mixed with 100 pL of concentrated sulfuric acid and then 30 pL of 5 % phenol. The plate was incubated at room temperature for 15 minutes to allow the color to develop, and then the absorbance was measured at 420 nm on a plate reader. The weight percentage of the glycans was calculated based on the linear relationship of the glucose concentration to absorbance. Samples were then aliquoted to the desired mass per tube, dried on the SpeedVac, and stored at -20 °C. d. GLYCOPOLYPEPTIDE PREPARATION, CHEMOENZYMATIC SYNTHESIS

[0126] aGalNAc(OAc)3Ser9, Pro10, tBuGlu , and Ala9A-carboxyanhydrides (NCAs) were prepared according to literature procedures. All NCA polymerizations were performed in an N2 filled glovebox. NCAs were suspended at 50 mg / mL in anhydrous THF and combined at a molar ratio of 1 : 1 : 1 : 1 of each NCA. Polymerizations were initiated via rapid addition of 30 mg / mL in dry THF of (PMe3)4Co catalyst at a monomer to initiator ratio of 30: 1. Polymerizations were allowed to proceed at ambient temperature in the glovebox for 16 hours. Reaction progress was monitored via ATR-FTIR on a Bruker Alpha Spectrophotometer. Upon reaction completion, polymers were analyzed via tandem gel permeation chromatography / light scattering (GPC / LS) with an Agilent 1260 Infinity liquid chromatography pump equipped with Wyatt DAWN HELEOS-II light scattering (LS) and Wyatt Optilab T-rEX refractive index (RI) detectors. SynMUCs were passed through 105, 104, and 103A Phenominex Phenogel 5 pm columns in an eluent of 0.1 M LiBr in DMF at 60 °C at a concentration of 3 mg / mL. SynMUC chemical protecting groups were removed according to literature protocols11. GalNAc groups were sialylated using a one-pot multienzyme procedure adapted from literature protocols12 13. Glycopolypeptides were treated with sialic acid aldolase (GenBank WP_000224714). CMP-sialic acid synthetase (GenBank WP_002215295), and either a2,3 sialyltransferase (GenBank NC_002663) or a2,6 sialyltransferase (GenBank AAK02272). One equiv. of synMUC (0.02-2 mg) was combined with 50 equiv. ManNAc. 7.5 equiv. sodium pyruvate, and 10 equiv. CTP in lOOmM Tris- HC1, pH 7.5 with 20mM MgC12. Sialyltransferase was added at 0.2 mg / mL, as well as 0.1mg / mL CMP-sialic acid synthetase and 0.2 mg / mL aldolase in a final volume of 300 pL for a 20 pg SynMUC reaction. Sialylations were allowed to proceed overnight at room temperature with gentle agitation at 100 RPM. For all reactions, polyhistidine-tagged enzymes were removed from the reaction mixture via incubation with NEB Express Ni-NTA magnetic beads according to manufacturer protocols. Finally. synMUCs were purified via dialysis against ultrapure water in 2kDa tubing for 4 water changes every 4-24 hours, condensed by lyophilization, and analyzed by a commercial sialic acid quantification kit.3. ADVANTAGES AND IMPROVEMENTS OVER EXISTING METHODS

[0127] The global surge in antibiotic resistance poses a serious threat and calls for the development of innovative treatments that do not contribute to resistance. Designing new anti-virulence therapies that target infections without killing the microbes offers a significant advantage over current therapeutic approaches, as these treatments would avoid the selective pressure that drives antibiotic resistance. Moreover, broad-spectrum antibiotics can have unintended effects on the composition and function of the human microbiota. Developing therapeutics based on human biology, such as mucin mimics, may reduce these off-target side effects compared to traditional antibiotics. By creating a polypeptide that presents the specific glycans identified as most bioactive pathogens, the highest therapeutic potency can be achieved, and these sugars can be delivered directly to the infection site in a form similar to their natural state. This platform will further allow for chemical modifications of these glycans, potentially improving drug delivers' and efficacy.4. COMMERCIAL APPLICATIONS

[0128] It is anticipated that synthetic mucins could serve as a treatment and preventive measure for opportunistic infections, either as an alternative to or in combination with traditional antibiotic therapies. Unlike antibiotics, which promote resistance through selective pressure, virulence-neutralizing agents do not kill microbes and are therefore less likely to drive the development of resistance. Furthermore, these treatments would preserve beneficial members of the microbiome, addressing a significant limitation of broad-spectrum antibiotics that often have unintended impacts on microbiota composition and function.

[0129] The applications of these peptides for vaginitis, including bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC), which is a prevalent issue in the U.S., resulting in over 10 million office visits annually, are of particular interest. BV is the most common causeof vaginal symptoms among women aged 15-44, with a prevalence of 21.7 million (29.2%). VVC, the second most common vaginal infection after BV, leads to approximately 1.4 million outpatient visits per year, and 75% of women experience at least one episode in their lifetime. Complications of BV and VVC include heightened risk of sexually transmitted infections and adverse pregnancy outcomes. This technology offers a promising solution for two primary markets: 1) individuals currently affected by BV or VVC, and 2) those at high risk for developing these conditions. Since vaginitis is often recurrent, requiring repeated courses of antibiotics, patients are at increased risk of developing antimicrobial-resistant infections. This virulence-reducing platform (VRP) could be prescribed as a standalone treatment or alongside traditional therapies to help prevent recurrent infections. This product would specifically target pathogens involved in BV or VVC and, to remain competitive, should be prescribed in appropriate doses or in synergy with existing treatments. Future versions may enhance antibiotic or probiotic delivery and retention, offering additional therapeutic benefits.

[0130] Beyond vaginal health, synthetic mucins have broader potential in treating traveler's diarrhea, a condition often triggered by pathogens like Escherichia coli and Salmonella enlerica, for which antibiotics can exacerbate resistance and microbiome disruption. By neutralizing the virulence of pathogens rather than killing them, these glycopolymers could be incorporated into travel products or dietary supplements aimed at preventing gastrointestinal infections during travel.

[0131] Additionally, this glycan-based strategy for neutralizing virulence could be applied across a range of health, wellness, and beauty products. Given the growing concern over antibiotic resistance, which is estimated to cost the U.S. healthcare system $55 billion annually, and potentially $100 trillion globally by 2050, synthetic mucins offer a groundbreaking approach to infection management. By focusing on their anti-virulence properties, these glycopolymers could enter the >$82 billion prebiotics market for maintaining gut health, the >$84 billion anti-fouling coatings market for medical devices, and the >$110 billion skincare market by replicating the hydrating properties of natural mucins.G. REFERENCES1. Caldara, M., Friedlander, R.S., Kavanaugh, N.L., Aizenberg, J., Foster, K.R., and Ribbeck, K. (2012). Mucin biopolymers prevent bacterial aggregation by retaining cells inthe free-swimming state. Curr Biol 22, 2325-2330. https: / / doi.Org / 10.1016 / j.cub.2012.10.028.2. Co, J.Y., Carcamo-Oyarce, G., Billings, N., Wheeler, K.M., Grindy, S.C., Holten- Andersen, N., and Ribbeck, K. (2018). Mucins trigger dispersal of Pseudomonas aeruginosa biofilms, npj Biofilms Microbiomes 4, 1-8. https: / / doi.org / 10.1038 / s41522-018-0067-0.3. Kavanaugh, N.L., Zhang, A.Q., Nobile, C.J., Johnson, A.D., and Ribbeck, K. (2014). Mucins suppress virulence traits of Candida albicans. mBio 5, e01911. https: / / doi.org / 10.1128 / mBio.01911-14.4. Wang, B.X., Takagi, J., McShane, A., Park, J.H., Aoki, K., Griffin, C., Teschler, J., Kitts, G., Minzer, G., Tiemeyer, M., et al. (2023). Host-derived O-glycans inhibit toxigenic conversion by a virulence-encoding phage in Vibrio cholerae. EMBO J 42, el 11562. https: / / doi.org / 10. 15252 / embj.2022111562.5. Wheeler, K.M., Carcamo-Oyarce, G., Turner, B.S., Dellos-Nolan, S., Co. J.Y., Lehoux, S., Cummings, R.D., Wozniak, D.J., and Ribbeck, K. (2019). Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection. Nature Microbiology 4, 2146-2154. https: / / doi.org / 10.1038 / s41564-019-0581-8.6. Takagi, J., Aoki, K., Turner, B.S., Lamont, S., Lehoux, S., Kavanaugh, N., Gulati, M., Valle Arevalo. A., Lawrence, T.J.. Kim. C.Y.. et al. (2022). Mucin O-glycans are natural inhibitors of Candida albicans pathogenicity. Nat Chem Biol 18, 1^'2-113. https : / / doi. org / 10. 1038 / s41589-022- 01035-1.7. Wang, B.X., Wheeler, K.M., Cady, K.C., Lehoux, S., Cummings, R.D., Laub, M.T., and Ribbeck. K. (2021). Mucin Glycans Signal through the Sensor Kinase RetS to Inhibit Virulence- Associated Traits in Pseudomonas aeruginosa. Curr Biol 31, 90-102.e7. https: / / doi.Org / 10.1016 / j.cub.2020.09.088.8. Werlang, C.A., Chen, W.G., Aoki, K., Wheeler, K.M., Tymm, C., Mileti, C.J., Burgos, A.C., Kim, K., Tiemeyer, M., and Ribbeck. K. (2021). Mucin O-glycans suppress quorum-sensing pathways and genetic transformation in Streptococcus mutans. Nat Microbiol 6, 574-583. https: / / doi.org / 10.1038 / s41564-021-00876-l.9. Kramer, J.R., Onoa, B., Bustamante, C., and Bertozzi, C.R. (2015). Chemically tunable mucin chimeras assembled on living cells. Proceedings of the National Academy of Sciences 112, 12574-12579. https: / / doi.org / 10.1073 / pnas.1516127112.10. Detwiler, R.E., McPartlon, T.J., Coffey, C.S., and Kramer, J.R. (2023). Clickable Polyprolines from Azido-proline N-Carboxyanhydride. ACS Polym. Au 3, 383-393. https: / / doi.org / 10.1021 / acspolymersau.3c00011.11. Clauss, Z.S., Wardzala, C.L., Schlirf, A.E., Wright, N.S., Saini, S.S., Onoa. B., Bustamante, C., and Kramer, J.R. (2021). Tunable, biodegradable grafting-from glycopolypeptide bottlebrush polymers. Nat Commun 72, 6472. https: / / doi.org / 10.1038 / s41467-021-26808-5.12. Yu, EL, Huang, S., Chokhawala, H., Sun, M., Zheng, H., and Chen, X. (2006). Highly Efficient Chemoenzymatic Synthesis of Naturally Occurring and Non-Natural a-2.6-Linked Sialosides: A P. damsela a-2,6-Sialyltransferase with Extremely Flexible Donor-Substrate Specificity. Angewandte Chemie International Edition 45, 3938-3944. https: / / doi.org / 10.1002 / anie.200600572.13. Yu. H., Chokhawala. H., Karpel. R., Yu. H.. Wu, B., Zhang, J., Zhang, Y., Jia, Q., and Chen, X. (2005). A multifunctional Pasteurella multocida sialyltransferase: a powerful tool for the synthesis of sialoside libraries. J Am Chem Soc 127, 17618-17619. https: / / doi.org / 10.1021 / ja0561690.

[0132] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMSWhat is claimed is:1 . A glycopolypeptide comprising a peptide backbone having from about 5 to 1,000 amino acid residues thereon, wherein from about 5% to about 50% of the amino acid residues are glycosylated amino acid residues, wherein the glycosylated amino acid residues are selected from GalNAc residues, GlcNAc residues, galactose residues, mannose residues, and fucose residues, or a mixture thereof.

2. The glycopolypeptide of claim 1, wherein the glycosylated amino acid residues are linked to the peptide backbone via an a2,3 linkage or an a2,6 linkage.

3. The glycopolypeptide of claim 1 or claim 2, wherein the glycosylated amino acid residues are linked to the peptide backbone via a residue selected from Ser, Thr, Cys, homoSer, homoThr, Lys, Om, Gly, and Asp.

4. The glycopolypeptide of claim 1 or claim 2, wherein the glycosylated amino acid residues are linked to the peptide backbone via a residue selected from Ser and Thr.

5. The glycopolypeptide of any one of claims 1 to 4. wherein from about 10% to about 40% of the amino acid residues are glycosylated amino acid residues.

6. The glycopolypeptide of any one of claims 1 to 4, wherein from about 15% to about 35% of the amino acid residues are glycosylated amino acid residues.

7. The glycopolypeptide of any one of claims 1 to 4, wherein about 25% of the amino acid residues are glycosylated amino acid residues.

8. The glycopolypeptide of any one of claims 1 to 7, wherein one or more of the glycosylated amino acid residues are sialylated glycosylated amino acid residues.

9. The glycopolypeptide of any one of claims 1 to 7. wherein one or more of the glycosylated amino acid residues are O-glycan residues.

10. The glycopolypeptide of any one of claims 1 to 7, wherein the glycosylated amino acid residues are selected from Core 1 (Gal-GalNAc) gly can residues, Gal residues, and GalNAc residues, or a mixture thereof.1 1. The glycopolypeptide of any one of claims 1 to 7, wherein the glycosylated amino acid residues are a mixture of Core 1 (Gal-GalNAc) glycan residues and Gal residues.

12. The glycopolypeptide of any one of claims 1 to 7, wherein one or more of the glycosylated amino acid residues are GalNAc residues.

13. The glycopolypeptide of claim 12, wherein one or more of the glycosylated amino acid residues are sialylated GalNAc residues.

14. The glycopolypeptide of claim 13, wherein the sialylated GalNAc residues have a structure selected from:or a mixture thereof. wherein each occurrence of m is 0.25, and wherein n is a 100-mer, or a pharmaceutically acceptable salt thereof.

15. The glycopolypeptide of any one of claims 1 to 14, wherein the glycosylated amino acid residues are sialylated glycosylated amino acid residues linked to the peptide backbone via an a2,3 linkage or an a2,6 linkage.

16. The glycopolypeptide of claim 15, wherein the sialylated glycosylated amino acid residues are sialylated GalNAc residues.

17. The glycopolypeptide of claim 16, wherein the sialylated GalNAc residues have a structure selected from:or a mixture thereof, wherein each occurrence of m is 0.

25. and wherein n is a 100-mer, or a pharmaceutically acceptable salt thereof.

18. The glycopolypeptide of any one of claims 1 to 7, wherein or more of the glycosylated amino acid residues are Core 1 glycan residues.

19. The glycopolypeptide of claim 18, wherein the Core 1 glycan residues are selected from Core 1 glycan residues, Core 1 glycan + fucose residues, Core 1 glycan + galactose residues, Core 1 glycan + N-acetylgalactosamine residues, Core 1 glycan + lactose residues, Core 1 glycan + fucosylated residues, and Core 1 glycan + sialylated residues, or a mixture thereof.

20. The glycopolypeptide of claim 18, wherein the Core 1 glycan residues are selected from Core 1 glycan residues, Core 1 glycan + fucose residues, and Core 1 glycan + galactose residues, or a mixture thereof.

21. The glycopolypeptide of claim 18, wherein the Core 1 glycan residue comprises a structure selected from:or a pharmaceutically acceptable salt thereof.

22. The glycopolypeptide of any one of claims 1 to 21, wherein the glycopolypeptide comprises a structure selected from:or a pharmaceutically acceptable salt thereof.

23. A pharmaceutical composition comprising an effective amount of one or more of the glycopolypeptides of any one of claims 1 to 22 and a pharmaceutically acceptable salt thereof.

24. The pharmaceutical composition of claim 23, further comprising an antibacterial agent.

25. The pharmaceutical composition of claim 24, wherein the antibacterial agent is selected from amoxicillin, ampicillin, azithromycin, aztreonam, azlocillin, bacitracin, carbenicillin. cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastin, ciprofloxacin, clarithromycin, clavulanic acid, clinafloxacin, clindamycin, clofazimine,cloxacillin, colistin, dalbavancin, dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doxycycline, erythromycin, enrofloxacin, enoxacin, enviomycin, ertepenem, ethambutol, flucioxacillin, fosfomycin, furazolidone, gatifloxacin, gentamicin, imipenem, isoniazid, kanamycin, linezolid, lomefloxacin, loracarbef, mafenide, moxifloxacin, meropenem, metronidazole, mezlocillin, minocycline, mupirocin, nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oritavancin, oxytetracycline, penicillin, piperacillin, platensimycin, polymixin B, quinupristin, retapamulin, rifabutin, rifampin, rifapentine, roxithromycin, sparfloxacin, spectinomycin, sulbactam, sulfacetamide, sulfamethizole, sulfamethoxazole, teicoplanin, telithromycin. telavancin, temafloxacin, tetracycline, thioacetazone, thioridazine, ticarcillin, tinidazole, tobramycin, torezolid, tosufloxacin, trimethoprim, troleandomycin, trovafloxacin, and vancomycin, or a combination thereof.

26. A method of treating a bacterial-mediated disease a subject in need thereof, the method comprising administering to the subject one or more of the glycopolypeptides of any one of claims 1 to 22.

27. The method of claim 26, wherein the bacterial-mediated disease is due to a bacterium selected from S. Typhimurium, C. albicans, G. vaginalis, and S. aureus.

28. The method of claim 26 or claim 27. wherein the bacterial-mediated disease is a vaginal disease.

29. The method of claim 28, wherein the vaginal disease selected from bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC).

30. The method of claim 26 or claim 27, wherein the bacterial-mediated disease is traveler’s diarrhea.

31. The method of any one of claims 26 to 30, further comprising administering to the subj ect an antibacterial agent.

32. The method of claim 31, wherein the glycopolypeptide and the antibacterial agent are administered sequentially.

33. The method of claim 31, wherein the glycopolypeptide and the antibacterial agent are administered simultaneously.

34. The method of claim 33, wherein the glycopolypeptide and the antibacterial agent are co-formulated.

35. The method of any one of claims 26 to 34. wherein the subject is a mammal.

36. The method of any one of claims 26 to 34, wherein the subject is a human.

37. The method of any one of claims 26 to 36, wherein the subject has been diagnosed with a need for treatment of the bacterial-mediated disease prior to the administering step.

38. The method of any one of claims 26 to 37, further comprising the step of identifying a subject in need of treatment of the bacterial-mediated disease.

39. The method of any one of claims 26 to 38. wherein the effective amount is a therapeutically effective amount.

40. The method of any one of claims 26 to 38. wherein the effective amount is a prophylactically effective amount.

41. A composition comprising a plurality of polypeptides, wherein each polypeptide comprises one or more selected from:(a) one or more sialylated GalNAc moieties from S. typhimurium covalently linked to a peptide backbone by a2,3 or a2,6 linkages;(b) one or more O-glycans from Candida albicans selected from Core 1, Core 1+fucose, and Core 2+galactose, wherein the O-glycans are covalently linked to a peptide backbone; and(c) one or more galactose and / or core 1 (galactose-GalNAc) moieties from G. vaginalis, wherein each galactose and / or core 1 moiety is covalently attached to a peptide backbone.

42. The composition of claim 41, wherein the sialylated GalNAc-moiety is selected from:

43. A method of producing synthetic mucin mimic, the method comprising:(a) identifying a natural mucin from a mucus that attenuates virulence of a microbe;(b) isolating the natural mucin that attenuates microbial virulence;(c) reformatting the mucin into a scalable synthetic mimic; and(d) testing the ability of the mucin to replicate the anti-virulence of the natural mucin.

44. The method of claim 43, wherein the native mucin comprises native porcine gastric mucin (MUC5AC) from pig stomachs and / or porcine intestinal mucin (MUC2) from pig small intestines.

45. A method of treating bacterial vaginosis (BV) or vulvovaginal candidiasis (VVC) in a subject in need thereof, the method comprising administering to the subject the composition of claim 41, optionally in combination (synergy) with a conventional therapy (e.g., an antibiotic or antibacterial agent).

46. A method of treating traveler’s diarrhea (<?.g., caused by Escherichia coli or Salmonella enterica) in a subject in need thereof, the method comprising administering to the subject the composition of claim 41.

47. The method of claim 45 or claim 46, wherein the treatment does not reduce the viability of beneficial microbes (e.g., Lactobacillus crispatus).

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