Patent Description:
The present application relates to antimicrobial and antiviral effects of C<NUM>-C<NUM> alkyl boronic acids.

Bacterial and viral infections are associated with acute and chronic diseases worldwide. Infections with E. coli, Salmonella, and Shigella cause widespread morbidity and mortality, and infections with Klebsiella and Clostridium ssp are increasingly recognized. Changes in the resident intestinal bacterial flora "dysbiosis" are linked to chronic intestinal inflammation (IBD) and cancer. Acute infections with Coronavirus (CoV) cause respiratory infections, which a can be lethal in forms such as COVID-<NUM>, SARS (severe acute respiratory syndrome)-CoV, and MERS (Middle East Respiratory Syndrome)-CoV in humans, and cause diseases such as FIPV (Feline Infectious Peritonitis Virus) in animals.

There is a need for effective methods for treatment, prevention, and management of both viral and bacterial diseases. Numerous enteropathic bacteria cause disorders and conditions associated with inflammation and damage of the gastrointestinal tract including infectious disease, diarrhea, gut dysbiosis, and cancer. The situation is further exacerbated by the rise of multi-drug resistant strains. New approaches, especially treatments with improved selectivity against growth and virulence of pathogens while sparing beneficial microbes are needed.

To better elucidate ligand interactions with PvdQ (relevant enzyme for P. aeruginosa), Clevenger KD et al. determined the Ki values for a series of boronic acids bearing n-alkyl substituents <NUM>-<NUM> carbons in length (<NPL>).

Trippier PC and McGuigan C summarized recent developments in relation to the use of boronic acids in medicinal chemistry (<NPL>).

The present application is directed to overcoming these and other deficiencies in the art.

Any subject-matter falling outside the scope of the claims is provided for information only. Methods of treatment of the human body by therapy, referred to in this description, are not part of the present invention as such, but are described here in relation to compounds and pharmaceutical compositions for use in said methods for treatment of the human body by therapy, according to the present invention.

One aspect of the present application is directed to a method of suppressing bacterial growth, wherein the bacteria are present on an ex vivo solid surface, said method comprising: providing a C<NUM>-C<NUM> alkyl boronic acid and administering the C<NUM>-C<NUM> alkyl boronic acid to bacteria, wherein said administering comprises applying the C<NUM>-C<NUM> alkyl boronic acid to the ex vivo surface, to suppress growth of the bacteria on the ex vivo solid surface.

Another aspect of the present application is directed to a method of altering bacterial virulence, wherein the bacteria are present on an ex vivo solid surface, said method comprising: providing a C<NUM>-C<NUM> alkyl boronic acid and administering the C<NUM>-C<NUM> alkyl boronic acid to bacteria, wherein said administering comprises applying the C<NUM>-C<NUM> alkyl boronic acid to the ex vivo surface, to alter virulence of the bacteria on the ex vivo surface.

A further aspect of the present application is directed to a C<NUM>-C<NUM> alkyl boronic acid for use in a method of treating a diarrheal disease, an intestinal inflammatory condition, or an intestinal cancer in a subject. This method involves selecting a subject with a diarrheal disease, an intestinal inflammatory condition, or an intestinal cancer, and administering a C<NUM>-C<NUM> alkyl boronic acid to the subject to treat the diarrheal disease, the intestinal inflammatory condition, or the intestinal cancer.

Another aspect of the present application is directed to a C<NUM>-C<NUM> alkyl boronic acid for use in a method of treating a viral infection, said method comprising: selecting a subject with a viral infection, wherein the viral infection is an infection by SARS-Cov-<NUM> or Feline Infectious Peritonitis virus, and administering the C<NUM>-C<NUM> alkyl boronic acid to the subject to treat the viral infection.

Described herein is the discovery that C<NUM>-C<NUM> alkyl boronic acids have antibacterial, anti-inflammatory, anti-cancer, and anti-viral properties. C<NUM>-C<NUM> alkyl boronic acids have been surprisingly shown to inhibit the growth of Gram Negative enteropathogens (E. coli, Salmonella, and Klebsiella) Fusobacterium and Gram Positive Listeria while sparing probiotic species (Lactobacillus, and Bifidobacterium). These data herein indicates that C2-C7 alkyl boronic acids represent a tunable spectrum of molecules targeting a wide group of enteropathogens, while being able to spare healthy resident bacteria and non-target species.

C<NUM>-C<NUM> alkyl boronic acids, such as the ethyl boronic acid ("EBA") and propionyl ("PBA"), are shown to have a previously- unrecognized activity against enteropathogenic bacteria associated with inflammation, chronic diarrhea, intestinal inflammation, and cancer. They are active even in the face of resistance to multiple conventional antimicrobial compounds (e.g. multiple drug resistant E. EBA and PBA are superior to boronic acid ("BA") and long chain alkyl boronic acids that were evaluated, and differed in their mechanism of action, potency, and selectivity compared to previously described aromatic boronic acid <NUM>-fluro-<NUM>-methlyphenylboronic acid ("FMPBA"). In vivo studies indicate that EBA abrogated microbial driven inflammation, lacked toxicity, and did not induce dysbiosis in healthy mice. C<NUM>-C<NUM> alkyl boronic acids show promise for therapeutic intervention against enteropathogenic bacteria associated with diarrhea, microbial driven intestinal inflammation, and cancer.

Furthermore, C<NUM>-<NUM> and C<NUM> alkyl boronic acids (EBA, PBA, butyl boronic acid ("BBA"), and hexyl boronic acid ("HBA")) are able to inhibit replication and syncitium formation by the Coronavirus, Feline Infectious Peritonitis Virus (FIPV-Black), in infected cell lines without causing cytotoxicity. This discovery indicates that C<NUM>-<NUM> alkyl boronic acids have anti FIPV activity, and that this antiviral activity could extend to other Coronaviruses and additional viruses. Due to the anti-inflammatory effect of C<NUM>-<NUM> alkyl boronic acids, these compounds can also abrogate the hyperinflammatory response induced by Coronavirus independent of antiviral effects (e.g. via Nuclear Factor kappa-light-chain-enhancer of activated B cells ("nF-κB")).

Alkyl boronic acids ("ABA") are stable aldehyde mimics and electrophiles capable of covalently linking to enzymes. Related molecules such as aromatic boronic acid proteasome inhibitor Bortezomib® inhibit enzymes in other biological systems and have a favorable safety profile of related compounds as pharmaceutical agents. Boric acid ("BA") has the structure shown in <FIG>. In alkyl boronic acids, "alkyl" refers to straight chain alkyl groups having, for example, <NUM>-<NUM> carbon atoms. Examples of C<NUM>-C<NUM> alkyl boronic acids, as described herein, include C<NUM> methyl boronic acid ("MBA"), C<NUM> ethyl boronic acid ("EBA"), C<NUM> propionyl boronic acid ("PBA"), C<NUM> butyl boronic acid ("BBA"), C<NUM> pentyl boronic acid ("PeBA"), C<NUM> hexyl boronic acid ("HBA"), C<NUM> heptyl boronic acid ("SBA"), C<NUM> octyl boronic acid ("OBA"), C<NUM> nonyl boronic acid ("NBA"), C<NUM> decyl boronic acid ("DBA"), C<NUM> undecyl boronic acid ("UBA"), and C<NUM> dodecyl boronic acid ("DoDBA"). A review of boronic acids can be found in <NPL>). In some embodiments, the ABA is C<NUM> ethyl boronic acid ("EBA"), C<NUM> propionyl boronic acid ("PBA"), C<NUM> butyl boronic acid ("BBA"), C<NUM> pentyl boronic acid ("PeBA"), C<NUM> hexyl boronic acid ("HBA"), C<NUM> heptyl boronic acid ("SBA") or any combination thereof. In some embodiments, the ABA is C<NUM> ethyl boronic acid ("EBA"), C<NUM> propionyl boronic acid ("PBA"), or any combination thereof.

As described herein, C<NUM>-C<NUM> alkyl boronic acids are superior to BA and ABA having larger alkyl groups (especially C<NUM> and C<NUM>) in terms of their potency against most enteropathogens. The effects of C<NUM>-C<NUM> alkyl boronic acids are distinct from and more specific than BA and aromatic FMPBA, because C<NUM>-C<NUM> alkyl boronic acids have greater effects on the growth and virulence of enteropathogens (Ecoli, Salmonella, Klebsiela) and smaller effects on probiotic and non-enteropathogenic species (Lactobacilus, Bifidobacterium). This modulation of the virulence and pathogenicity of enteropathogens without killing them is a phenomenon called "bacterial taming". The selective effect of C<NUM>-C<NUM> alkyl boronic acids on growth and virulence of bacteria are also distinct from the bactericidal activity conferred by aromatic boronic acids FMPBA and <NUM>-benzyloxyphenylboronic acid ("BOPBA").

Also described herein, short chain alkyl boronic acids are non-disruptive of the microbiome, have low toxicity, are active against cancer associated E. coli and Fusobacterium and diarrheagenic enteropathogens. Alkyl boronic acid have the ability to reduce intestinal inflammation in murine models of inflammatory bowel disease ("IBD"), whereas BA do not decrease inflammation. A group of E. coli associated with intestinal inflammation and dysbiosis across species (Adherent and invasive E. coli, ("AIEC")) are adapted to use inflammation associated chemicals, particularly those related to the carboxysome associated metabolism of ethanolamine utilizing ("eut") and propanediol utilizing ("pdu") carboxysomes. More pathogenic bacteria (e.g. E. coli, Shigella, Salmonella, Klebsiella, Clostridium and Fusobacterium) are enriched in these carboxysomal pathways compared to non -pathogenic residents. This led to the hypothesis that chemical inhibitors targeting the eut and pdu pathways, such as alkyl boronic acids, could selectively antagonize AIEC and enteropathogenic bacteria through metabolic inhibition as one potential mode of action, without being bound to any particular theory.

C<NUM>-C<NUM> alkyl boronic acids also have antiviral properties. BA and the C<NUM>-C<NUM> alkyl boronic acids were able to inhibit replication and syncytium formation by the Coronavirus, Feline Infectious Peritonitis Virus (FIPV-Black) in infected cell lines. The action of C<NUM>-C<NUM> alkyl boronic acids on viruses indicated additional modes of action for the C<NUM>-C<NUM> alkyl boronic acid compounds.

Accordingly, the present application provides methods for the treatment of bacterial growth and bacterial and viral virulence through the use of C<NUM>-C<NUM> alkyl boronic acids, as defined by the claims.

In at least one embodiment of the present application, an ex vivo method of suppressing bacterial growth is disclosed. Suppressing bacterial growth refers to the reduction or prevention of the ability of the bacteria to grow. Said method comprising: providing a C<NUM>-C<NUM> alkyl boronic acid and administering the C<NUM>-C<NUM> alkyl boronic acid to bacteria, wherein said administering comprises applying the C<NUM>-C<NUM> alkyl boronic acid to the ex vivo surface, to suppress growth of the bacteria on the ex vivo solid surface.

The term "bacteria" refers to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. Bacteria included within this definition include cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms that are Gram Negative or Gram Positive. "Gram Negative" and "Gram Positive" refer to staining patterns with the Gram-staining process, which is well known in the art. Examples of bacteria include, but are not limited to, bacterial cells of a genus of bacteria selected from the group comprising Salmonella, Shigella, Escherichia, Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus, Micrococcus, Staphylococcus, Vibrio, Aeromonas, Plessiomonas, Haemophilus, Actinobacillus, Pasteurella, Mycoplasma, Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus, Enterococcus, Aerococcus, Gemella, Lactococcus, Leuconostoc, Pedicoccus, Bacillus, Corynebacterium, Arcanobacterium, Actinomyces, Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Neisseria, Campylobacter, Arcobacter, Wolinella, Helicobacter, Achromobacter, Acinetobacter, Agrobacterium, Alcaligenes, Chryseomonas, Comamonas, Erwinea, Eikenella, Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas, Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella, Brucella, Legionella, Afipia, Bartonella, Calymmatobacterium, Cardiobacterium, Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia, Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus, Bifidobacterium, Eubacterium, Lactobacillus, Rothia, Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Bilophila, Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella, Norcardia, Actinomadura, Norcardiopsis, Streptomyces, Micropolysporas, Thermoactinomycetes, Mycobacterium, Treponema, Borrelia, Leptospira, and Chlamydiae.

In some embodiments, the bacteria are enteric bacteria. Enteric bacteria are bacteria that reside in the enteric tract of animals. In some embodiments, the enteric bacteria are selected from the group consisting of E. coli, Shigella, Listeria, Salmonella, Klebseilla, Clostridium, and Fusobacterium.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid achieves bacterial taming by having high activity against enteric pathogens and low activity against probiotic and nonenteropathic Gram Positive bacteria.

In some embodiments the bacteria are present on an ex-vivo solid surface. Ex vivo surfaces include organic surfaces (e.g., food products, surfaces of animals, surfaces of plants etc.) and inorganic surfaces (e.g., medical devices, countertops, clothing, liquids, etc.). Methods of applying a C<NUM>-C<NUM> alkyl boronic acid to a surface include, but are not limited to, spraying, misting, submerging, and coating. In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered to the ex vivo solid surface to suppress growth of the bacteria on the solid surface.

In some embodiments, the bacteria are present in vivo within a subject. In this and other embodiments, the term "subject" may be taken to mean any living organism that may be treated with C<NUM>-C<NUM> alkyl boronic acid. As such, the term "subject" may include, but is not limited to, any non-human animal or human. In some embodiments, the "subject" is an animal, such as mice, rats, other rodents, fish, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, or humans. In some embodiments, the subject is an adult, child, or infant. In some embodiments, the subject is a human. In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered to the subject to suppress growth of the bacteria with the subject.

In some embodiments, an effective amount of the C<NUM>-C<NUM> alkyl boronic acid is administered. As used herein, the term "effective amount" refers to the amount of a C<NUM>-C<NUM> alkyl boronic acid composition sufficient to effect a beneficial or desired result (e.g., reducing growth, bacterial taming, reducing virulence of bacteria and/or viruses). An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. Procedures for determining the effective amount are well known to those skilled in the art. Each dosage should contain a quantity of the compositions comprising the C<NUM>-C<NUM> alkyl boronic acids calculated to produce the desired effect (e.g., suppressing growth bacterial taming, altering virulence of bacteria or viruses). The potency of ABA increases from C<NUM>-C<NUM>, therefore a reduction in dose for longer chain alkyl boronic acids can be used. ABA are effective at doses that are not associated with cytotoxicity in vitro or with in vivo toxicity in mice. EBA has been safely administered to mice without adverse effects at <NUM>. Exemplary dosages of C<NUM>-C<NUM> alkyl boronic acids include, without limitation, EBA up to <NUM>, PBA up to <NUM>, BBA up to <NUM>, PeBA up to <NUM>-<NUM>, HBA up to <NUM>-<NUM>, and HeBA up to <NUM>.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered in a composition which further comprises probiotic cells. Exemplary probiotic cells include, without limitation, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus brevis, Lactobacillus breve, Streptococcus thermophilus, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium coagulans, Bifidobacterium breve, Bifidobacterium animalis, Bifidobacterium subtilis, Saccharomyces boulardi, and combinations thereof. In some embodiments, the one or more probiotic cells comprises from about <NUM> million colony forming units (CFUs) to about <NUM> billion CFUs.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered in a composition which further comprises prebiotics. Prebiotics are capable of changing microbiota composition and promoting intestinal barrier integrity. Prebiotics are poorly absorbed carbohydrates that can reach into the colon after ingestion and are used by intestinal bacteria as a source of energy for growth. Prebiotics include, without limitation, resistant starch, psyllium, inulin, pectin, natural oligofructoses, fructo-oligosaccharides (FOS), lactulose, galactomannan, indigestible polydextrose, acemannan, various gums, indigestible dextrin and partial hydrolysates thereof, trans-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS), beta-glucan and partial hydrolysates thereof, together if desired with phytosterol/phytostanol components and their suitable esters. See also <NPL>).

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered in a composition which further comprises bacterial culture supernatants and/or secreted products. In some embodiments, the bacterial culture supernatants and/or secreted products are from probiotic cell cultures. In some embodiments, the secreted products are from purified secreted products from F. prauznitzii, Lactobacillus and Bifidobacterium spp.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered in a composition which further comprises one or more other antibacterial agents. Other antibacterial agents can include, without limitation, lysozyme, protamine, antibiotics such as erythromycin, oxytetracycline, tetracycline, chloramphenicol, fusidic acid, micamycin, kanamycin, gentamicin, fradiomycin, gramicidin, streptomycin, polymyxin, colistin, bacitracin, biguanide compounds such as chlorhexidine, benzethonium, benzalkco, compounds having surface activity such as nium, lauryl sulfate, alkylpolyaminoethylglycine, fatty acids, phenol derivatives such as phenol, hexachlorophene, resorcin, iodine compounds such as iodine, iodoform, and povidone iodine, metals such as gold, silver, copper, mercury, metal compounds such as thimerosal and methylobromine, acrinol, methyl rosary, antimicrobial dye compounds such as down, mafenide acetate, sulfadiazine, Surufisomijin, like sulfa drugs such as sulfamethoxazole. These antibacterial substances may be a salt compound such as sodium salt, potassium salt, magnesium salt, calcium salt, hydrochloride, sulfate, gluconate and the like, and two or more kinds of antibacterial agents may be used in combination with the C<NUM>-C<NUM> alkyl boronic acid.

The C<NUM>-C<NUM> alkyl boronic acids used according to the methods of the present application can be administered alone or as a pharmaceutical composition, which includes the compound(s) and a pharmaceutically-acceptable carrier. The C<NUM>-C<NUM> alkyl boronic acids of the present can be provided as a pharmaceutical composition. The pharmaceutical composition can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions. Typically, the composition will contain from about <NUM> to <NUM> percent, preferably from about <NUM> to <NUM> percent of C<NUM>-C<NUM> alkyl boronic acids, together with the carrier(s).

The C<NUM>-C<NUM> alkyl boronic acids of the present application, when optionally combined with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers, whether in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions, can be administered orally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by implantation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, transdermally, or by application to mucous membranes, for example, that of the nose, throat, and bronchial tubes (i.e., by inhalation). In some embodiments, administration is orally, parenterally, intranasally, by nebulization, or topically.

In some embodiments the C<NUM>-C<NUM> alkyl boronic acid is coated with an enteric coating. The formulation can be coated in any suitable enteric coating that permits transit through the stomach to the small intestine before the medication is released. Thus, the enteric formulation can comprise any suitable substance that aids in permitting transit through the stomach to the small intestine. For example, without limitation, the enteric formulation can comprise a chitosan, a fiber, a cellulose derivative (e.g., cellulose acetate phthalate), a polyvinyl acetate (e.g., polyvinyl acetate phthalate), a hydroxypropyl-methyl cellulose derivative (e.g., hydroxypropyl-methyl cellulose phthalate or hydroxypropyl-methyl cellulose acetate succinate), an acrylic acid copolymer (e.g., ethylacrylate methacrylic acid copolymer or methylmethacrylate methacrylic acid copolymer), shellac, dextran sulfate, galacturonic acid, alginates, mannuronic acid, guluronic acid, sodium hyaluronate, chondroitin sulfates, heparin, chitin, glycosaminoglycans, proteoglycans or any combination thereof.

In some embodiments the C<NUM>-C<NUM> alkyl boronic acid is administered as a solid or as a solution or suspension in liquid form. The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the compounds of the present application and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, the C<NUM>-C<NUM> alkyl boronic acids are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered as an aerosol. For use as aerosols, the C<NUM>-C<NUM> alkyl boronic acids in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The C<NUM>-C<NUM> alkyl boronic acids of the present application also may be administered in a non-pressurized form such as in a nebulizer or atomizer. A surfactant can be used to improve release and dissolution rates of the C<NUM>-C<NUM> alkyl boronic acids from the coatings. In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is encapsulated in a surfactant.

Solutions or suspensions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol, hyaluronan and its derivatives, carboxymethyl cellulose and other soluble polysaccharide derivatives, or polyethylene glycol, are preferred liquid carriers, particularly for injectable solution.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered transdermally. For transdermal routes, the compound is present in a carrier which forms a composition in the form of a cream, lotion, solution, and/or emulsion. The composition can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose. In some embodiments the C<NUM>-C<NUM> alkyl boronic acid is encapsulated in a liposome.

Another aspect of the present application is an ex vivo method of altering bacterial virulence. The method involves providing C<NUM>-C<NUM> alkyl boronic acid and administering the C<NUM>-C<NUM> alkyl boronic acid to bacteria, wherein said administering comprises applying the C<NUM>-C<NUM> alkyl boronic acid to the ex vivo surface, to alter virulence of the bacteria on the ex vivo surface.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acids alter the virulence of the bacteria. As used herein, "alter" or "altering" the virulence of a bacteria means reducing the severity or harmfulness of the bacterial. In some embodiments, the virulence is reduced expression of virulence genes. In some embodiments the bacterial virulence is selected from the group consisting of bacterial growth, bacterial invasion, bacterial motility, bacterial persistence and bacterial adhesion.

This aspect of the present application can be carried out with any of the methods and embodiments described above.

A further aspect of the present application is a method of treating a diarrheal disease, an intestinal inflammatory condition, or an intestinal cancer in a subject. This method involves selecting a subject with a diarrheal disease, an intestinal inflammatory condition, or an intestinal cancer, and administering a C<NUM>-C<NUM> alkyl boronic acid to the subject to treat the diarrheal disease, the intestinal inflammatory condition, or the intestinal cancer.

Enteropathogenic bacteria are associated with acute and chronic diarrheal diseases worldwide. Acute infections with E. coli, Salmonella, and Shigella cause widespread morbidity and mortality, and infections with Klebsiella and Clostridium spp are increasingly recognized. Changes in the resident intestinal bacterial flora "dysbiosis" are increasingly linked to chronic intestinal inflammation (IBD) and cancer. Inflammatory disorders in humans or other mammals, include Inflammatory Bowel Disease (IBD). IBD is also termed Crohn's Disease, ileitis, colitis, ulcerative colitis (UC) or enteritis. Symptoms of IBD include abdominal pain, diarrhea or constipation or alternating diarrhea and constipation, gas, bloating, nausea, weight loss, rectal bleeding, fatigue, and decreased appetite. Children suffering from IBD also experience delayed growth and development. Subjects suffering from IBD have symptoms similar to subjects suffering from Irritable Bowel Disease (also known as Irritable Bowel Syndrome) or ulcerative colitis. Certain types of E. coli and Fusibacterium are also associated with intestinal cancer. In some embodiments, a subject is treated for an intestinal inflammatory condition selected from the group consisting of intestinal dysbiosis, irritable bowel syndrome, and inflammatory bowel disease. In some embodiments, the subject is treated for an inflammatory bowel disease selected from the group consisting of ulcerative colitis and Crohn's Disease. In some embodiments, the subject is treated for an intestinal cancer. In some embodiments, the subject is treated for a diarrheal disease. In some embodiments, the subject is human.

Another aspect of the present application is a method of of treating a viral infection, said method comprising: selecting a subject with a viral infection, wherein the viral infection is an infection by SARS-Cov-<NUM> or Feline Infectious Peritonitis virus, and administering the C<NUM>-C<NUM> alkyl boronic acid to the subject to treat the viral infection.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered orally, parenterally, intranasally, or by nebulization.

In some embodiments, the C<NUM>-C<NUM> alkyl boronic acid is administered in a composition which further comprises additional antiviral or antibiotic compounds. Exemplary antiviral agents include without limitation, 3TC (lamivudine), AZT (zidovudine), FTC (<NUM>-fluoro-<NUM>-[<NUM>-(hydroxymethyl)-<NUM>,<NUM>-oxathiolan-<NUM>-yl]cytosine), d4T (<NUM>',<NUM>'-dideoxy-<NUM>',<NUM>'-didehydrothymidine, stavudine and Zerit), nevirapine, DMP-<NUM>, nelfinavir, delavirdine, <NUM>-[(<NUM>-hydroxymethyl)-<NUM>,<NUM>-dioxolan-<NUM>-yl]guanine, <NUM>-amino-<NUM>-[(<NUM>-hydroxymethyl)-<NUM>,<NUM>-dioxolan-<NUM>-yl]adenine, MKC-<NUM>, 1592U89 (abacavir), 141W94, MK-<NUM>, BMS-<NUM>, PNU-<NUM>, ABT-<NUM>, DMP-<NUM>, Indinavir , saquinavir, ritonavir, efavirenz (sustiva), TIBO, HEPT, BHAP, α-APA, TSAO, calanolides, L-<NUM>,<NUM>, <NUM>',<NUM>'-dideoxycytidine (ddC or zalcitabine), <NUM>',<NUM>'-dideoxyadenosine, <NUM>',<NUM>'-dideoxyinosine (ddI or didanosine), <NUM>'-deoxythymidine and <NUM>,<NUM>'-dideoxy-<NUM>',<NUM>'-didehydrocytidine and ribavirin; acyclic nucleosides such as acyclovir, ganciclovir, interferons such as alpha-, beta-and gamma-interferon; glucuronation inhibitors such as probenecid; nucleoside transport inhibitors such as dipyridamole; immunomodulators such as interleukin II (IL2) and granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin, ampligen, thymomodulin, thymopentin, foscarnet, glycosylation inhibitors such as <NUM>-deoxy-D-glucose, castanospermine, <NUM>-deoxynojirimycin; and inhibitors of HIV binding to CD4 receptors such as soluble CD4, CD4 fragments, CD4-hybrid molecules, inhibitors of the HIV aspartyl protease such as L-<NUM>,<NUM>, or combinations thereof.

In some embodiments, the treatment encompasses reducing viral virulence, said viral virulence is selected from the group consisting of viral replication, viral infection, viral persistence, and syncytium formation.

The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.

Bacteria were grown in proper liquid culture medium such as Luria-Bertani broth ("LB"), Brain Heart Infusion broth ("BHI"), or Man Rogosa Sharpe broth ("MRS") overnight at <NUM> with shaking. Overnight cultures were diluted <NUM>:<NUM> into fresh medium containing NaCl (control), BA or C<NUM>-C<NUM> alkyl boronic acids at specified concentration in a <NUM> well-plate (Growth Curve, USA). On top of the growth medium in each well, <NUM>µl of mineral oil was gently added to achieve a microaerophilic growth environment. Bacterial growth was monitored with OD600 for <NUM> to <NUM> hours at <NUM> in a BioScreen C system (Growth Curve, USA). Growth curves were generated with OD600 as the function of time. Where needed, the area under each growth curve (AUC) was calculated with Graphpad Prism7. Anaerobic bacteria (i. e Bifidobacterium, Fusobacterium, and Clostridium) were grown in an anaerobic chamber at <NUM> in a proper medium (i. BHI, MRS, etc.) ± NaCl, boric or boronic acid as indicated in the Figures. <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, <FIG>, and <FIG> show quantitative growth data. Fusobacteria nucleatum was cultured in BHI medium, and grown in anaerobic chamber at <NUM> for <NUM> hours in the presence of boronic acid at concentration indicated in the figure. The control sample contained <NUM> NaCl. The bacterial growth was monitored at every <NUM> by measuring OD600. OD600 readings were taken at different time points. Area under the curves were calculated with Graphpad Prism7.

Differences in growth, gene expression, motility, adhesion, invasion, and cytokine production between control and boric or boronic acid treated samples were analyzed by <NUM>-way ANOVA with Turkey's test for multiple comparisons. All statistical analyses were performed with GraphPad Prism <NUM> software and p < <NUM> was considered significant.

The ability of C<NUM>-C<NUM> C<NUM>, and C<NUM> alkyl boronic acids, Boric Acid (BA) and Aromatic BA (FMPBA) to impact bacterial growth was evaluated using a temperature controlled <NUM>-well multiplate incubator at <NUM> to culture bacteria over <NUM>-48hrs. Growth of bacteria with or without addition of C<NUM>-C<NUM> C<NUM>, and C<NUM> alkyl boronic acids, BA, or FMPBA was monitored by readings at OD<NUM>. The concentrations used for each boronic acid was <NUM> (BA, EBA, PBA), <NUM> (BBA), <NUM> (PeBA), <NUM> (HBA, SBA), <NUM> (OBA), <NUM> (DBA), <NUM> (DoDBA), or <NUM> (FMPBA). The area under the curve ("AUC") of the growth curve was calculated and the results were expressed as AUC + test compound : AUC- test compound. <FIG> shows a heat map graphical representation of the data showing this ratio, with blue representing the lowest growth of bacteria i.e. the highest antibacterial activity, whereas red indicates highest growth i.e. lowest antibacterial activity.

BA selectively inhibited the growth (i. e bacteriostatic) of enteropathogenic bacteria, E. coli, (diarrheagenic, AIEC, GC-associated, cancer inducing, APEC, EXPEC), Salmonella (e. g Typhimurium) , Listeria monocytogenes, and Klebsiella (oxytoca, pneumoniae) while sparing healthy resident bacteria including probiotic Lactobacillus and Bifidobacteria. The antibacterial potency of ABA against enteric pathogens increased from C<NUM>-<NUM> alkyl boronic acids EBA, PBA, BBA, PeBA, HBA, and SBA. However, for ABA greater than C<NUM>, there was an increase in the inhibition of probiotic Lactobacillus and Gram Positive species such as Streptococccus, and Staphylococcus. The selectivity of ABA appeared highest for Gram Negative enteropathogens with ABA C<NUM>,C<NUM>, C<NUM>, and C<NUM>. C<NUM>-C<NUM> alkyl boronic acid and longer alkyl boronic acids had activity against the Gram Positive enteropathogen Listeria monocytogenes, with selectivity apparent for ABA C<NUM>,C<NUM>, C<NUM>. Thus C<NUM>-C<NUM> alkyl boronic acids represented a "tunable spectrum" of molecules targeting a wide spectrum of enteropathogens while sparing healthy resident bacteria and non-target species. ABA molecules with more than six carbons (C<NUM>, C<NUM>, and C<NUM>) were much less soluble and precipitated out of solution after solubilization in ethanol. This resulted in a marked decrease in antibacterial activity of ABA > C<NUM> vs ABA <C<NUM> (e.g. OBA and DBA) as shown in <FIG>.

In a further comparative analysis of the experiment in Example <NUM>, it was found that C<NUM>-C<NUM> alkyl boronic acids were more potent than BA against enteric pathogens. EBA was more selective than BA versus Gram Positive aerobes. EBA and ABA C<NUM> and C<NUM> were more selective than BA against Probiotic spp (<FIG>).

C<NUM>-C<NUM> alkyl boronic acids were found to be more potent against enteric pathogens than DoDBA. Also, EBA was found to be more selective than DoDBA with less impact on gram positive aerobes and probiotic Lactobacillus (<FIG>).

EBA was found to be more selective than C<NUM>-C<NUM> alkyl boronic acids with less impact on Gram Positive aerobes and probiotic species (<FIG>).

Aromatic FMPBA had more generalized and potent antibacterial activity than C<NUM>-C<NUM> alkyl boronic acid compounds. However, FMPBA was not selective and killed more Gram Positive aerobes and probiotic species than C<NUM>-C<NUM> alkyl boronic acid compounds (<FIG>).

A cluster analysis further demonstrates the differences in the antimicrobial potency and selectivity of BA, aromatic BA and ABA molecules (<FIG>). Cluster analysis indicates that FMPBA had more potent and generalized antibacterial activity than BA, and ABA C<NUM>, C<NUM> and C<NUM>. FMPBA was bacteriocidal and minimally selective. EBA (ABA C<NUM>) and PBA (ABA C<NUM>) clustered together, and were distinct from BA and DoDBA having higher potency against enteropathogens and less impact on probiotic Lactobacillus, Bacillus, and Gram Positive aerobes (Streptococcus zooepidemicus and Stapholococcus aureus) (<FIG>).

Inhibition of bacterial growth was measured using different concentrations of BA, EBA, and PBA on bacterial in M9 media using ethanolamine ("EA") or NH<NUM>Cl as nitrogen sources and glucose or glycerol as carbon sources (<FIG>). BA and EBA were tested at <NUM>, <NUM>, <NUM>, and <NUM> concentrations, whereas PBA was tested at <NUM> and MBA at <NUM>.

EBA was much more effective against adherent invasive E. coli ("AIEC". "<NUM>-<NUM>") than BA and MBA. EBA was also effective against cancer associated E. coli ("HM288") and Klebsiella pneumonia ("13Dk") (<FIG>).

This in vitro analysis of the impact of BA and C<NUM>-C<NUM> alkyl boronic acids on the growth of AIEC E. coli revealed that C<NUM>-C<NUM> alkyl boronic acids were able to reduce growth of AIEC E. coli in defined media containing ethanolamine. This result supported the hypothesis that ABA have the ability to antagonize the ethanolamine utilizing ("eut") carboxysome. However, BA and C<NUM>-C<NUM> alkyl boronic acids were also effective in suppressing AIEC growth in complex media lacking ethanolamine or fucose, suggesting antibacterial activity of ABA beyond simply antagonizing carboxysomal metabolism.

<FIG> shows the effect of <NUM> BA, EBA and PBA on the growth of diarrheagenic and APEC E. coli in LB media. Diarrheagenic E. coli pathogroups differ by their preferential host colonization sites, virulence mechanisms, and clinical symptoms. This experiment demonstrated that BA, EBA, and PBA have activity versus the diarrheagenic E. coli and APEC that varies by pathogroup (APEC-<NUM> avian pathogenic, EPEC-<NUM> or <NUM> enteropathogenic, EIEC-<NUM> enteroinvasive, or <NUM>, and EAEC-<NUM> or <NUM> enteroaggregative) (<FIG>). EBA and PBA were more effective in inhibiting bacterial growth than BA overall.

<FIG> shows the effect of BA, EBA, and PBA on the growth of five Salmonella strains. Overall EBA and PBA inhibit the growth of multiple serovars of Salmonella more effectively than BA.

<FIG> shows the effect of EBA (<NUM>), PBA (<NUM>), BBA (<NUM>), PeBA (<NUM>), HBA (<NUM>), HeBA (<NUM>), OBA (<NUM>), DBA (<NUM>) and DoBA (<NUM>) on the growth of Fusobacterium nucleatum.

<FIG> shows the effect of BA, MBA, EBA, and PBA on the growth of three Lactobacillus strains. In contract to their effect on pathogenic bacteria, ABA C1-<NUM> MBA, EBA, and PBA) do not suppress growth of a variety of probiotic Lactobacillus spp.

<FIG> shows a cluster analysis of alkyl boronic acid, aromatic boronic acids and boric acid on bacterial growth. FMPBA was tested at <NUM> and <NUM>; DoDBA was tested at <NUM>:<NUM>,<NUM>, <NUM>:<NUM>,<NUM> and <NUM>:<NUM>,<NUM>, and BA, EBA and PBA were tested at <NUM>. Inhibitors clustered according to the type of compound independent of concentrations evaluated.

The ability of C<NUM>-C<NUM> alkyl boronic acids and BA to impact the growth of MDR E. coli isolated from people and dogs was evaluated using a temperature controlled <NUM>-well multiplate incubator at <NUM> over 48hrs. Growth in media (LB broth) with and without ABA C2-<NUM> (<NUM>) was monitored by readings at OD<NUM> with the AUC of the growth curve calculated.

<FIG> shows the effect of C<NUM>-C<NUM> alkyl boronic acids and BA for multi-drug resistant ("MDR") strains associated with Crohn's disease (MDR strains are: T75, LF82, <NUM>-<NUM>, <NUM>-<NUM>, 41CB-<NUM>, <NUM>-<NUM>, and 35MN-<NUM>) isolated from people. Non-MDR strains <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM> were also evaluated. EBA and PBA were more effective than BA over a wider range of AIEC, including MDR and non-MDR strains.

<FIG> shows the effect of ABA C1-<NUM> and BA for multi-drug resistant ("MDR") strains associated with Granulomatous colitis in dogs (MDR strains are: BB1, DCH1, DFW1, DK1, DLU3, and DMG1). Non-MDR strains KD1, KD2, and KD3 were also evaluated. EBA and PBA were more effective than BA over a wider range of AIEC, including MDR and non-MDR strains.

Overall, ABA C2 and <NUM> (EBA and PBA) were more effective than ABA C1 and BA against a wider range of MDR and sensitive AIEC.

The ability of ABA Cl-<NUM> and BA to impact the motility of E. coli and Salmonella was evaluated using sloppy agar treated with and without BA and C<NUM>-C<NUM> alkyl boronic acids. Motility is strongly linked to the virulence phenotype of these bacterial strains. coli was grown overnight at <NUM> in LB broth. Soft agar plates (<NUM>% tryptone, <NUM>% NaCl, <NUM>% agar) were prepared the day before the assay was carried out. Sterile NaCl (control), boric or boronic acid stock solution was added into the agar right before pouring the plates. The overnight cultures of E. coli were transferred (<NUM>µl) on to the center of each plate, followed by incubation of the plates at <NUM> for <NUM>. coli motility was quantified by measuring the diameter of the circular swarming area formed by the growing motile bacteria. In <FIG>, BA is compound A, MBA is compound B, EBA is compound C, PBA is compound D.

As shown in <FIG>, EBA and PBA (compounds C and D) were highly effective at blocking motility of AIEC and cancer associated E. coli and were found to be more potent than BA (A) and MBA (B). EBA and PBA (C, D) were highly effective at blocking motility of AIEC NC101 and <NUM>-<NUM>. BA and MBA (C, D) had less impact.

As shown in <FIG>, the motility of multiple strains of E. coli and Salmonella was expressed as the % motility of the control. It was found that EBA and PBA were highly effective at blocking motility of Salmonella and AIEC E. They were more potent than BA.

Overall, EBA and PBA were found to modulate the virulence of pathogenic E. coli and Salmonella without killing them and were more effective than BA and MBA.

HEK-Blue KD-TLR5 cells were used to detect the promotor activation of nF-κB by E. coli infection with or without boric or boronic acid treatment. nF-κB is the prototypical transcription factor for pro-inflammatory cytokines in epithelium. Briefly, cells were seeded in <NUM>-well plates at a density of <NUM> × <NUM><NUM> cells per well. coli was diluted into fresh cell medium containing either NaCl (Control), boric or boronic acid at an m. i of <NUM> as 10X inocula, followed by addition of this inoculum (<NUM>µl) into each well containing <NUM>µl of medium for a final m. i of <NUM>. At <NUM> post infection, the cell medium was carefully removed from each well, and replaced with <NUM>µl of fresh medium containing gentamycin (<NUM>µg mL-<NUM>). At <NUM> post infection, the spent medium was collected, and centrifuged at <NUM>,<NUM> r. for <NUM> to remove any particulate matter. QUANTI-Blue Kit (InvivoGen, San Diego, CA, USA) was used to detect the reporter protein SEAP (secreted alkaline phosphatase) following the manufacturer's instructions. The SEAP activity was detected as optical density at <NUM>. BA, EBA and PBA reduced the induction of nF-κB by E.

As shown in <FIG>, BA, EBA, and PBA reduced the induction of nF-κB by E.

HEK-Blue KD-TLR5 cells were also used to detect the induction of NF-κB by LPS (lipopolysaccharides). Briefly, cells were seeded in <NUM>-well plates at a density of <NUM> × <NUM><NUM> cells per well. LPS was added into each well with a multichannel pipettor to a final concentration of <NUM>µg/ml, followed by addition of NaCl (Control), or boronic acid for a final concentration of <NUM> (NaCl, EBA, PBA), <NUM> (BBA, HeBA), or <NUM> (HBA, HeBA). At <NUM> post treatment, the spent medium was collected, and centrifuged at <NUM>,<NUM> r. for <NUM> to remove any particulate matter. QUANTI-Blue Kit (InvivoGen, San Diego, CA, USA) was used to detect the reporter protein SEAP (secreted alkaline phosphatase) following the manufacturer's instructions. The SEAP activity was detected as optical density at <NUM>.

<FIG> shows a direct anti-inflammatory effect on host cells of the C<NUM>-C<NUM> alkyl boronic acids due to inhibition of LPS mediated nF-κB activation.

coli was cultured overnight in LB at <NUM> with shaking. Bacterial pellets were re-suspended in PBS before dilution in cell culture media ± <NUM> boronic acid or alkyl boronic acid to an m. i (multiplicity of infection) of <NUM>. Caco-<NUM> cells were infected with the diluted E. coli for <NUM> at <NUM> before washing with PBS, then lysed with <NUM>% Triton X-<NUM>. Serial dilutions of the lysates were made in PBS and plated on LB agar. The total number of colonies recovered was used to calculate the number of adherent bacteria. For invasion assays, cells were treated with gentamicin (<NUM>µg mL-<NUM>) for one hour after initial infection and 3X wash with PBS to kill extracellular bacteria. Cells were then washed 3X after gentamicin treatment, lysed and plated as described above.

Supernatants of Caco-<NUM> (at <NUM> post infection) cell cultures were collected and centrifuged to remove any cells or cell debris. The concentrations of IL-<NUM> secreted by Caco-<NUM> cells were analyzed by ELISA methods, using Human IL-<NUM> Antibody Pair Kit (Invitrogen) per manufacturer's instructions.

As shown in <FIG>, BA, EBA, and PBA suppress adhesion and invasion of Caco-<NUM> by AIEC <NUM>-<NUM> and LF82. Other strains tested included <NUM>-<NUM>, T75, <NUM> -<NUM>, <NUM>-<NUM>, 24LW-<NUM>, HM288, HM334, HM497,HM580, and NC101, which showed a similar response.

Similarly, as shown in <FIG>, EBA suppressed the adhesion and invasion of Salmonella by <NUM>-<NUM>%.

Murine macrophage J774A. <NUM> cells were seeded in <NUM>-well plates (<NUM> × <NUM><NUM> cells/well) and grown for two days at <NUM> with <NUM>% CO<NUM>. coli was cultured in LB broth overnight at <NUM>. The overnight culture was diluted into cell culture media containing either NaCl, <NUM> boric acid or alkyl boronic acid at an m. i of <NUM>. <NUM> cells were infected with E. coli for <NUM> at <NUM>, washed 3X with PBS, and treated with gentamicin (<NUM>µg ml-<NUM>) for <NUM> to kill extracellular bacteria. coli uptake assays, J774A. <NUM> cells were washed 3X with PBS, lysed in <NUM>% Triton X-<NUM> and enumerated by quantitative plating as described above. For intracellular survival assays, after the initial gentamycin treatment (<NUM>µg ml-<NUM>), the cells were kept in medium containing <NUM>µg ml-<NUM> gentamycin overnight at <NUM>. At <NUM> post infection, the cells were washed 3X with PBS, and lysed with <NUM>% Triton X-<NUM>. The number of E. coli that survived was determined by quantitative plating. The bacterial survival rate was calculated as R = (# survived/# uptaken) × <NUM>.

As shown in <FIG>, EBA, PBA, and BA inhibited the uptake of AIEC <NUM>-, LF82, 25LW-<NUM>, <NUM>-<NUM> and CUMT8. Also shown in FIG. 12C, ABA C<NUM> and C<NUM> and BA inhibited the survival of AIEC <NUM>-, LF82, 25LW-<NUM>, <NUM>-<NUM>, and CUMT8 (this was despite not being maintained in media during gentamicin protection. Other strains tested included <NUM>-<NUM>, T75, <NUM>-<NUM>, <NUM>-<NUM>, 24LW-<NUM>, HM288, and NC101, which followed a similar pattern.

coli was grown in media with either NaCl, <NUM> boric acid or alkyl boronic acid to mid log phase. Total RNA was extracted using the Qiagen RNAProtect-RNeasy Kit per manufacturer's protocol. Total RNA was treated with TURBO DNA-Free Kit (Ambion), followed by a two-step qRT-PCR analysis, using Qiagen's QuantiTect Reverse Transcription Kit and QuantiNova SYBR Green PCR Kit. Each qPCR reaction contained <NUM>µl of cDNA, <NUM>µl of each forward and reverse primers (<NUM>), <NUM>µL of 2X SYBR Green Master Mix, <NUM>µl of QN ROX Reference Dye and <NUM>µl of nuclease-free water to make the total volume of <NUM>µl. The reaction was run with ABI7000 (Applied Biosystems). The comparative quantification (ΔCt) method was used to determine the up- or down-regulated genes. The relative change of a targeted gene expression was calculated by using the equation RQ = <NUM>-ΔΔCT.

As shown in <FIG>, EBA inhibited virulence gene expression, an aspect of "Bacterial Taming" more than BA. Down regulated genes included FliC, a gene corresponding with motility in LF82. Upregulation of pduC by BA and EBA in <NUM>-<NUM>, and BA in LF-<NUM> may reflect an effect on the pdu carboxysome with lack of product mediated enzyme suppression leading to upregulation of the gene.

BA, EBA, and PBA were similar in cytotoxity to control when evaluated using trypan blue exclusion and cultured Caco-<NUM> cells. However, the degree of cytopathic effect observed at 24hrs was EBA<BA<PBA. Based on these results, cytotoxicity was tested in mice.

The toxicity of boric and ethylboronic acids were examined with C57B <NUM> mice. At concentrations of <NUM>, <NUM>, <NUM>, and <NUM>, the two chemicals were given to each group (<NUM> mice) in drinking water. The body weight of each mouse was recorded weekly for <NUM> weeks, and fecal samples were also collected weekly for <NUM> sequencing. All animals were sacrificed at the end of <NUM>th week. The length of whole intestine and ileum of each mouse was measured, and sections of the intestine (i. small intestine, ileum, cecum, and large intestine) were taken for histopathological and FISH analysis. Fecal samples were used for <NUM> and lipocalin analysis. <FIG> shows the result of body weight over <NUM> weeks with either BA or EBA. No significant differences were observed.

For lipocalin analysis, mouse fecal pellets were collected and frozen at -80C during toxicity study. To determine lipocalin activity fecal pellets were diluted in microfuge tubes <NUM>/<NUM> with <NUM>%Tween20/PBS and vortexed for <NUM> at maximum speed. Undissolved particles were removed by centrifuging at <NUM>,<NUM> rpm for <NUM> at 4C. Supernatant was removed and placed into a clean microfuge tube. Lipocalin activity was determined using Mouse Lipocalin-<NUM>/NGAL DuoSet ELISA Kit (R&D Systems, DY1857) per manufacturer's instructions. <FIG> shows the intestine length and lipocalin amounts (ng/g feces) in control versus BA and EBA treated mice. There was no evidence of intestinal or systemic toxicity, and fecal <NUM> profiles were not significantly different from control mice in BA and EBA at all doses tested.

On the basis of favorable in vivo toxicity tests, the ability of BA and EBA to reduce the severity of inflammation in murine models of microbially driven IBD was tested. The effect of BA and EBA (<NUM>) on intestinal inflammation was evaluated in mice with dextran sodium sulfate ("DSS") induced colitis. The DSS model has a complex etiopathogenesis that involves host- immune and inflammatory responses and the enteric microbiome BA To test their protecting ability to the gastrointestinal tract, boric and ethylboronic acid were given to C57BL mice at <NUM> concentration respectively in drinking water after DSS (<NUM>%) treatment. Body weight and fecal samples were taken before and after <NUM>-day treatment. At the <NUM>th day, all mice were euthanized for gross necropsy and histopathology. Intestinal sections were collected and embedded for pathological and FISH analysis. Fecal samples were used for <NUM> and lipocalin analysis.

<FIG> shows that the body weight and intestinal lengths were similar in EBA and control mice, whereas body weight was reduced in mice receiving DSS alone and DSS +BA. Intestinal bleeding was observed in in mice receiving DSS alone and DSS +BA, but was absent in mice receiving EBA. EBA had a protective effect against DSS associated weight loss and GI bleeding.

<FIG> shows that the colon length and fecal lipocalin levels were not significantly different in control mice and mice receiving DSS+EBA. In contrast, colon length and fecal lipocalin levels were significantly different in control mice from those receiving DSS alone and DSS +BA. EBA also protected against DSS induced intestinal shortening and intestinal inflammation.

This novel observation of an anti-inflammatory effect of EBA was corroborated in subsequent collaborative studies. EBA markedly reduced intestinal inflammation, assessed by fecal lipocalin and histopathology, in the SIHUMI colonized <NUM>-<NUM>-/- mouse (these mice are colonized with a consortium of human bacteria including AIEC LF82). There were no adverse or pro-inflammatory effects of EBA on SIHUMI colonized wild type mice. These experiments confirm that EBA has an anti-inflammatory effect in two distinct murine models of microbially driven IBD.

Human intestinal cell line Henle-<NUM> was seeded in a <NUM>-well plate at cell density of <NUM> × <NUM><NUM> cells/well. The cells were incubated at <NUM> with <NUM>% CO<NUM>. Salmonella typhi (expressing CdtB3-Flag) were cultured in LB broth overnight at <NUM> with gentle shaking at <NUM> rpm. The overnight culture was diluted into fresh LB plus <NUM> NaCl at a ratio of <NUM>:<NUM>, then incubated at <NUM> until reaching OD600 = <NUM> (cfu = <NUM>×<NUM><NUM>). The bacteria suspension was diluted to cell culture medium (DMEM with high glucose + <NUM>% FBS) to achieve a moi of <NUM>, with either <NUM> NaCl or C<NUM>-C<NUM> alkyl boronic acid as indicated in <FIG>. After <NUM> infection at <NUM>, the cells were washed 3X with PBS, and treated with <NUM>µg/ml gentamycin for <NUM>. For infection assays, the cells were wash 3X with PBS, lysed with <NUM>% Triton X-<NUM>, and the cell lysate was plated on LB-agar for determination of intracellular bacteria. For toxin assays, the cells were given fresh culture medium containing <NUM>µg/ml gentamycin. At <NUM> post infection, the cells were harvested in cold PBS. The cell pellets were store at -<NUM> for Western Blot assay analysis. <FIG> shows the inhibition of Salmonella typhi adhesion and invasion by C<NUM>-C<NUM> alkyl boronic acids. EBA, PBA, BBA, PeBA, and HeBA inhibited adhesion and invasion of S. typhi in Henle cells. PeBA and HBA were cytotoxic at higher concentrations (as indicated with an asterisk *).

For Western blot analysis, the infected Henle cells were solubilized in <NUM>µl of RIPA Lysis Buffer (<NUM> Tris-HCl, pH <NUM>, <NUM> EDTA, <NUM> EGTA , <NUM>% Triton X-<NUM>, <NUM>% Sodium Deoxycholate, <NUM>% SDS, and <NUM> NaCl). Thirty microliters of the sample were used for electrophoresis with <NUM>% precast polyacrylamide gel. Proteins were transferred onto nitrocellulose membrane and blocked with <NUM>% skim milk. The mouse anti-Flag antibody was used for CdtB3-Flag protein recognition, and mouse anti-β-actin was used for actin binding. The fluorescein conjugated anti-mouse m2 was used as secondary antibody. The fluorescent bands were quantified by Odyssey CLx Imaging System.

<FIG> shows the results of the Western analysis of the inhibition of toxin production (CdtB) after treatment with EBA and PBA (<FIG>) compared to b-actin production (<FIG>). Quantification of the results is shown in <FIG>.

The experiments described in Examples <NUM>-<NUM> revealed C<NUM>-C<NUM> alkyl boronic acids, exemplified by ethylboronic acid (EBA), as promising candidate molecules for therapeutic intervention against a wide variety of enteropathogenic bacteria (including those resistant to current antimicrobials) associated with diarrhea, food poisoning, sepsis, multi-drug resistance and microbially driven intestinal inflammation and cancer. There was a different spectrum of antibacterial activity for C<NUM>-C<NUM> alkyl boronic acids, with BBA and HBA having high potency vs enteropathogens while somewhat sparing probiotic species. BA was found to be more potent than DoDBA, which contradicted previous plate ethanol studies. ABA C<NUM> and C<NUM> had much less potency than C<NUM>-C<NUM> alkyl boronic acids due to insolubility and precipitation. C<NUM>-C<NUM> alkyl boronic acids differ in their potency and spectrum of activity compared to BA, DODBA, and FMBPA. From these experiments, it was determined that BA was less potent and less specific than EBA in inhibiting pathogenic bacteria.

The potent anti-inflammatory activity of EBA in polymicrobial murine models of IBD was a highly significant finding that supports further development of EBA as a novel therapeutic agent.

Monolayers of <NUM> × <NUM><NUM> Feline Lung cells (obtained from the Cornell Animal Health Diagnostic Center) were cultured on multiwell plates at <NUM> for <NUM>. Cell Monolayers were infected with Feline Coronavirus type I feline infectious peritonitis virus (FIPV) Black (TN-<NUM>), at an MO1 of <NUM> and rocked for <NUM> hours, followed by removal of the virus by washing. Boronic acids were diluted in maintenance media (Eagle's Minimum Essential Medium (EMEM), <NUM>% Fetal Bovine Serum (FBS), <NUM>% Nu-serum™ (Corning, Corning, NY), <NUM>% penicillin-streptomycin (PS), <NUM>% <NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazineethanesulfonic acid (HEPES)) and cells were incubated for <NUM> post-infection at <NUM>. At <NUM> hours post-infection, the infected cells were fixed, permeabilized and incubated with blocking solution containing <NUM>% normal goat serum and <NUM>% Triton X-<NUM> in phosphate buffered saline (PBS) at <NUM> overnight. The FIP virus was stained with FIP <NUM>-<NUM> Antibody labeled with Alexa <NUM> (green). Slides were mounted, and examined with an fluorescence microscope. FIPV is shown with green staining and nuclei are stained blue using DAPI.

As shown in <FIG>, C<NUM>-C<NUM> alkyl boronic acids reduced the amount of virus replication and syncytium formation by the Coronavirus, Feline Infectious Peritonitis Virus ("FIPV") in infected cell lines in a dose dependent manner. In <FIG>, treatment with EBA at <NUM> (B), <NUM> (C), <NUM> (E), and <NUM> (F) inhibited replication and syncytium formation of FIPV in infected cells compared to the positive control (D). In <FIG>, treatment with PBA at <NUM> (B), <NUM> (C), <NUM> (E), and <NUM> (F) inhibited replication and syncytium formation of FIPV in infected cells compared to the positive control (D). In <FIG>, treatment with BBA at <NUM> (B), <NUM> (C), <NUM> (E), and <NUM> (F) inhibited replication and syncytium formation of FIPV in infected cells compared to the positive control (D). In <FIG>, treatment with HBA at <NUM> (B), <NUM> (C), <NUM> (E), and <NUM> (F) inhibited replication and syncytium formation of FIPV in infected cells compared to the positive control (D).

Cytotoxicity was not observed at antiviral doses with EBA and PBA. Cytotoxicity was observed with BBA and HBA at higher doses, but antiviral activity was observed at lower doses without cytotoxicity.

Claim 1:
A method of suppressing bacterial growth, wherein the bacteria are present on an ex vivo solid surface, said method comprising:
providing a C<NUM>-C<NUM> alkyl boronic acid and
administering the C<NUM>-C<NUM> alkyl boronic acid to bacteria, wherein said administering comprises applying the C<NUM>-C<NUM> alkyl boronic acid to the ex vivo surface, to suppress growth of the bacteria on the ex vivo solid surface.