Antibiotic alkaloid and methods of generating and use thereof

Abstract

The present invention provides a compound useful for the treatment of microbial pathogens and related conditions, compositions and formulations comprising the compound thereof, and methods of treating using the compound thereof.

Description

[0001] TITLE OF THE INVENTION

[0002] ANTIBIOTIC ALKALOID AND METHODS OF GENERATING AND USE THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority to U.S. Provisional Application No. 63 / 670,455, filed July 12, 2024, which is incorporated by reference herein in its entirety.

[0005] BACKGROUND OF THE INVENTION

[0006] It is estimated that there were 13.7 million bacterial infection-related deaths in 2019. One in eight deaths globally are linked to common bacterial infections, making these infections the second-leading cause of death in 2019. Further, antibiotic resistance remains a hurdle for treatment of common infections, thereby spurring the interest in the development of new therapeutic strategies.

[0007] There is a need in the art for new compositions and methods for treating infections. The present invention satisfies this need.

[0008] SUMMARY OF THE INVENTION

[0009] In one aspect, the present invention provides a compound represented by Formula (I), or a tautomer, ion, salt, and / or solvate thereof,

[0010] Formula (I).

[0011] In some embodiments, the compound is an inner salt. In some embodiments, the compound is zwitterionic. In some embodiments, the compound is protonated. In some embodiments, the compound is deprotonated. In some embodiments, the compound is selected from the group consisting of: tautomers, salts, and solvates thereof.

[0012] In some embodiments, the present invention provides a pharmaceutical formulation comprising a compound of the present invention.

[0013] In some embodiments, the present invention provides pharmaceutical formulation, comprising: a therapeutically effective amount of a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,

[0014] Formula (I); and a pharmaceutically acceptable excipient.

[0015] In some embodiments, the pharmaceutical formulation is in the form of a particle, bead, tablet, capsule, or pill. In some embodiments, the pharmaceutical formulation is in the form of a lotion, liquid, aerosol, or gel. In some embodiments, the pharmaceutical formulation further comprises one or more antimicrobial agents. In some embodiments, the pharmaceutical formulation further comprises one or more antibiotic agents. In some embodiments, the pharmaceutical formulation further comprises one or more antifungal agents.

[0016] In some embodiments, the present invention provides a method of treating or preventing a microbial infection in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a compound of Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,

[0017] Formula (I).

[0018] In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the subject is exposed to or infected with a bacterium. In some embodiments, the subject is exposed to or infected with a gram-negative bacterium. In some embodiments, the subject is exposed to or infected with a bacterium from an Acmetobacter species, a Vibrio species, or an Escherichia species. In some embodiments, the subject is exposed to or infected with a bacterium selected from the group consisting of an Acmetobacter species, Enterococcus species, Staphylococcus species, Klebsiella species, Pseudomonas species, Enterobacter species, Burkholderia species, and any combination thereof. In some embodiments, the subject is exposed to or infected with a bacterium or pathogen selected from the list consisting of Acmetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, Mycobacterium tuberculosis, Salmonella species, Shigella species, Enterococcus faecium, Pseudomonas aeruginosa, Enterobacter species, Burkholderia species, Neisseria gonorrhoeae, Citrobacter species, Proteus species, Serratia species, Group A Streptococci, Group B Streptococci, Streptococcus pneumoniae , Haemophilus influenzae, Morganella species, Vibrio cholerae, and any combination thereof. In some embodiments, the subject is exposed to or infected with a bacterium selected from the group consisting of Acmetobacter baumannii, Escherichia coli, and / or Vibrio cholerae. In some embodiments, the subject is exposed to or infected with a grampositive bacterium.

[0019] In some embodiments, the subject is exposed to or infected with a bacterium that is resistant to one or more antibiotic agents. In some embodiments, the one or more antibiotic agents are selected from the group consisting of a bacteriostatic antibiotic and a bactericidal antibiotic. In some embodiments, the one or more antibiotic agents are selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, and tebipenem.

[0020] In some embodiments, the method further comprises the step of administering one or more additional antibiotic agents. In some embodiments, the one or more additional antibiotic agents is selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, tebipenem, and combinations thereof.

[0021] In some embodiments, the microbial infection is a fungal infection. In some embodiments, the subject is exposed to or infected with a fungal pathogen selected from the group consisting of Cryptococcus neoformans. Candida auris, Aspergillus fumigaius. Candida albicans, Nakaseomyces / Candida glabrata, Histoplasma species, a Eumycetoma causative agent, Mucorales species, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Lomentospora prolificans, Coccidioides species, Pichia kudriavzeveii / Candida krusei, Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, Paracoccidioides species, and any combination thereof. In some embodiments, the subject is exposed to or infected with a fungal pathogen that is resistant to one or more antifungal agents selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof. In some embodiments, the method further comprises the step of administering one or more antifungal agents. In some embodiments, the one or more antifungal agents is selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

[0022] In some embodiments, the subject is exposed to or infected with a human pathogen. In some embodiments, the subject is exposed to or infected with an agricultural pathogen.

[0023] In some embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with sepsis, pneumonia, abscesses, furuncles, cellulitis, folliculitis, bloodstream infections, medical device infections, pneumonia, osteomyelitis, skin and soft tissue infections, endocarditis, impetigo, septic arthritis, brain abscess, bum wounds, venous ulcers, diabetic foot ulcers, surgical wounds, post-operation infections, carbuncles, meningitis, bacteremia, necrotizing pneumonia, endocarditis, osteomyelitis, or necrotizing fasciitis.

[0024] In some embodiments, the step of administering comprises topical, ophthalmic, and / or optic administration of a therapeutically effective amount of a compound of Formula (I), or pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, to the subject. In some embodiments, the step of administering comprises internally administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, is internally administered by a means selected from the group consisting of oral, intravenous, intramuscular, intraperitoneal, subcutaneous, mucosal, percutaneous, sublingual, intrathecal, intraocular, inhalation, rectal, and nasal administration.

[0025] In some embodiments, the present invention provides a method of treating or preventing a microbial infection in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an extract or one or more compounds in an extract, wherein the extract comprises at least one compound derived from a Streptomyces bacterium; and at least one of the one or more compounds is a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,

[0026] Formula (I).

[0027] In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the subject is exposed to or infected with a bacterium. In some embodiments, the subject is exposed to or infected with a gram-negative bacterium. In some embodiments, the subject is exposed to or infected with a bacterium from an Acinetobacter species, a Vibrio species, or an Escherichia species. In some embodiments, the subject is exposed to or infected with a bacterium selected from the group consisting of an Enterococcus species, Staphylococcus species, Klebsiella species, Acinetobacter species, Pseudomonas species, Enterobacter species, Burkholderia species, and any combination thereof. In some embodiments, the subject is exposed to or infected with a bacterium or pathogen selected from the list consisting of Klebsiella pneumoniae Escherichia coli, Acinetobacter baumannii, Mycobacterium tuberculosis, Salmonella species, Shigella species, Enterococcus faecium, Pseudomonas aeruginosa, Enterobacter species, Neisseria gonorrhoeae, Staphylococcus aureus, Citrobacter species, Proteus species, Serratia species, Group A Streptococci, Group B Streptococci, Streptococcus pneumoniae, Haemophilus influenzae, Morganella species, Vibrio cholerae, any generation thereof, and any combination thereof. In some embodiments, the subject is exposed to or infected with a bacterium selected from the group consisting of Acinetobacter baumannii, Escherichia coli, and / or Vibrio cholerae.

[0028] In some embodiments, the subject is exposed to or infected with a gram-positive bacterium.

[0029] In some embodiments, the subject is exposed to or infected with a bacterium that is resistant to one or more antibiotic agents. In some embodiments, the one or more antibiotic agents are selected from the group consisting of a bacteriostatic antibiotic and a bactericidal antibiotic. In some embodiments, the one or more antibiotic agents are selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, and tebipenem.

[0030] In some embodiments, the method further comprises the step of administering one or more additional antibiotic agents. In some embodiments, the one or more additional antibiotic agents is selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, tebipenem, and any combination thereof.

[0031] In some embodiments, the microbial infection is a fungal infection. In some embodiments, the subject is exposed to or infected with a fungal pathogen selected from the group consisting of Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces / Candida glabrata, Histoplasma species, a Eumycetoma causative agent, Mucorales species, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Lomentospora prolificans, Coccidioides species, Pichia kudriavzeveii / Candida krusei, Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii , Paracoccidioides species, and any combination thereof. In some embodiments, the subject is exposed to or infected with a fungal pathogen that is resistant to one or more antifungal agents selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

[0032] In some embodiments, the method further comprises the step of administering one or more antifungal agents. In some embodiments, the one or more antifungal agents is selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

[0033] In some embodiments, the subject is exposed to or infected with a human pathogen. In some embodiments, the subject is exposed to or infected with an agricultural pathogen.

[0034] In some embodiments, the subject is at risk of, exhibiting symptoms of, or diagnosed with sepsis, pneumonia, abscesses, furuncles, cellulitis, folliculitis, bloodstream infections, medical device infections, pneumonia, osteomyelitis, skin and soft tissue infections, endocarditis, impetigo, septic arthritis, brain abscess, bum wounds, venous ulcers, diabetic foot ulcers, surgical wounds, post-operation infections, carbuncles, meningitis, bacteremia, necrotizing pneumonia, endocarditis, osteomyelitis, or necrotizing fasciitis.

[0035] In some embodiments, the step of administering comprises topical, ophthalmic, and / or optic administration of a therapeutically effective amount of a compound of Formula (I), a tautomer or ion thereof, or pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, to the subject. In some embodiments, the step of administering comprises internally administering to the subject a therapeutically effective amount of a compound of Formula (I), a tautomer or ion thereof, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof. In some embodiments, the compound of Formula (I), a tautomer or ion thereof, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, is internally administered by a means selected from the group consisting of oral, intravenous, intramuscular, intraperitoneal, subcutaneous, mucosal, percutaneous, sublingual, intrathecal, intraocular, inhalation, rectal, and nasal administration.

[0036] In some embodiments, the present invention provides a method of producing an extract comprising the steps of: providing a Streptomyces bacterium; culturing the Streptomyces bacterium in a culture medium under conditions conducive to provide overexpression of the extract; and optionally isolating the extract from the culture medium. In some embodiments, the extract comprises a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,

[0037] Formula (I).

[0038] In some embodiments, the step of isolating the extract from the culture medium comprises lyophilizing the culture medium and performing activity guided fractionation. In some embodiments, the activity guided fractionation comprises: loading the lyophilized culture medium onto a solid phase sorbent; performing a first fractionation step comprising at least two sequential solid phase extractions of the loaded solid phase sorbent with aqueous methanol, with each sequential extraction comprising a higher concentration of methanol to a final sequential extraction of 100% methanol; examining the activity of the fractions and selecting the fraction with the greatest activity for further fractionation; performing a second fractionation step comprising reverse-phase liquid chromatography of the fraction with the greatest activity; and examining the activity of the fractions and selecting the fraction with the greatest activity.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following detailed description of various embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings illustrative embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0041] Figure 1, comprising Figure 1A and Figure IB, depicts representative results of MIC testing with supernatant from isolate 9005BA. Figure 1A depicts representative antibacterial activity of raw supernatant when bacterial cells are cultured with a percentage of 9005BA demonstrating activity against 5. aureus, E. coli, and A. baumannii. Values are given as volume / volume percentage. Figure IB depicts representative antibacterial activity of lyophilized supernatant against A. baumannii, which showed the greatest susceptibility to raw supernatant in Figure 1A.

[0042] Figure 2 depicts representative results of MIC testing with 100% methanol extract of lyophilized 9005BA supernatant against ESKAPE pathogens, demonstrating a 10-fold increase in activity against A. baumannii compared to raw lyophilized supernatant.

[0043] Figure 3, comprising Figure 3A and Figure 3B, depicts a representative isolation scheme of compound 1. Figure 3 A depicts a small-scale fractionation into 96-well microplate for compound identification. Figure 3B depicts large-scale semi-prep fractionation to produce compound 1 for biological assays.

[0044] Figure 4, comprising Figure 4A and Figure 4B, depicts representative results of MIC testing with isolated compound 1. Figure 4A depicts representative results of MIC testing of isolated compound 1 against ESKAPE pathogens. The MIC for all ESKAPE pathogens were greater than 20 pg / mL, however, S. aureus and E.coli both displayed altered bacteria morphology (lack of pellet, only cloudy media), indicating the MIC is greater than 20 pg / mL but close to it. Figure 4B depicts representative results re-testing the MIC against A. baumannii, confirming the MICD is 0.625ug / mL, a 200-fold improvement as compared to lyophilized supernatant.

[0045] Figure 5, comprising Figure 5A through Figure 5E, depicts representative nuclear magnetic resonance (NMR) spectra for isolated compound 1 in methanol-r / y. Figure 5A depicts a representative 'H NMR spectrum (top) and zoomed-in view of the aromatic region (bottom). Figure 5B depicts a representative13C NMR spectrum. Figure 5C depicts a representative correlated spectroscopy (COSY) spectrum. Figure 5D depicts a representative heteronuclear single quantum coherence (HSQC) spectrum. Figure 5E depicts a representative heteronuclear multiple bond correlation (HMBC) spectrum.

[0046] Figure 6, comprising Figure 6A through Figure 6E, depicts representative nuclear magnetic resonance (NMR) spectra for isolated compound 1 in acetone-rL. Figure 6A depicts a representative!H NMR spectrum (top) and zoomed-in view of the aromatic region (bottom). Figure 6B depicts a representative13C NMR spectrum. Figure 6C depicts a representative 'H-'H correlated spectroscopy (COSY) spectrum. Figure 6D depicts a representative heteronuclear single quantum coherence (HSQC) spectrum. Figure 6E depicts a representative heteronuclear multiple bond correlation (HMBC) spectrum.

[0047] Figure 7 depicts a representative HPLC-MS analysis of compound 1, including the predicted molecular formula (top), an MS2spectrum, and the UV-Vis absorption profde.

[0048] Figure 8 depicts a representative structure of compound 1 obtained by small-angle X-ray diffraction (SXRD).

[0049] Figure 9 depicts the structure of compound 1 and tautomer forms la and lb and Key correlated spectroscopy (COSY) and key heteronuclear multiple bond correlation (HMBC) correlations of la, lb and lb'.

[0050] Figure 10 depicts a representative image demonstrating the low solubility of compound 1 in acetone-tfc.

[0051] Figure 11 depicts representative images of 9005BA being cultured in liquid (left) or on a plate (right). Note that white colonies on the plate are immature and have yet to produce compound 1.

[0052] DETAILED DESCRIPTION

[0053] The present invention is based, in part, on the unexpected discovery of a novel antibiotic compound from a Streptomyces bacterium. In some embodiments, the present invention provides compositions comprising an of a Streptomyces bacterium with antimicrobial activity. In some embodiments, the present invention provides a compound isolated from an extract of a Streptomyces bacterium with antimicrobial activity. In some embodiments, compositions of the invention comprise a compound derived from . Streptomyces bacterium and a pharmaceutically acceptable excipient. In some embodiments, the compound or composition has antibiotic activity. In some embodiments, the antibiotic compound or composition of the invention can be used in the treatment of bacterial infections. In some embodiments, the compound can be produced upon culture of a bacterium under conditions conducive to provide overexpression of the compound. Thus, in one aspect, the invention further provides methods for producing and isolating the compounds.

[0054] Definitions

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

[0056] As used herein, each of the following terms has the meaning associated with it in this section.

[0057] The articles A and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0058] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0059] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0060] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0061] A disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0062] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0063] “Parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i . v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0064] The term, “biologically active” or “bioactive” can mean, but is in no way limited to, the ability of an agent or compound to effectuate a physiological change or response. The response may be detected, for example, at the cellular level, for example, as a change in growth and / or viability, gene expression, protein quantity, protein modification, protein activity, or combination thereof; at the tissue level; at the systemic level; or at the organism level. For example, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, antibiotics, fungicides, anti-viral agents, antiinflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, and the like.

[0065] An “effective amount” or “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

[0066] “Isolated” means altered or removed from the natural state. For example, a compound naturally present in a living animal is not “isolated,” but the same compound partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated compound can exist in substantially purified form.

[0067] The term “pharmacological composition,” “therapeutic composition,” “therapeutic formulation” or “pharmaceutically acceptable formulation” can mean, but is in no way limited to, a composition or formulation that allows for the effective distribution of an agent provided by the invention, which is in a form suitable for administration to the physical location most suitable for their desired activity, e.g., systemic administration.

[0068] Non-limiting examples of agents suitable for formulation with the, e.g., compounds provided by the instant invention include: cinnamoyl, PEG, phospholipids or lipophilic moieties, phosphorothioates, P-glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Em erich, D F et al, 1999, Cell Transplant, 8, 47-58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).

[0069] The term “pharmaceutically acceptable” or “pharmacologically acceptable” can mean, but is in no way limited to, entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.

[0070] As used herein, a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, or composition, such as a liquid or solid fdler, diluent, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, and not injurious to the patient. Carriers or excipients may have additional properties that allow it to perform additional activities including, but not limited to, binding, controlling release, dilution, disintegration, effervescence, emulsification, film formation, flavoring, lubrication, penetration enhancement, permeation enhancement, pH modification, plasticizing, preserving, solubilizing, solvating, surfactant activity, taste masking, thickening, viscosity modification, blending, filling, compaction, compression, granulation, extrusion, rapid release, increasing bioavailability, dispersion, and stabilization. Some examples of materials that can serve as pharmaceutically acceptable carriers / excipients include, but are not limited to: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; cocoa butter; suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” also includes any and all coatings, absorption delaying agents, and the like that are compatible with the activity of the compound and are physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the compositions.

[0071] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids, organic acids, solvates, hydrates, or clathrates thereof.

[0072] As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease. Disease and disorder are used interchangeably herein.

[0073] As used herein, the term “medical intervention” means a set of one or more medical procedures or treatments that are required for ameliorating the effects of, delaying, halting or reversing a disease or disorder of a subject. A medical intervention may involve surgical procedures or not, depending on the disease or disorder in question. A medical intervention may be wholly or partially performed by a medical specialist, or may be wholly or partially performed by the subject himself or herself, if capable, under the supervision of a medical specialist or according to literature or protocols provided by the medical specialist.

[0074] As used herein, the term “subject” refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like) that can have a disease, disorder, or condition; or be at risk for developing a disease, disorder, or condition; but may or may not have a disease, disorder, or condition or be at risk for developing a disease, disorder, or condition. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an “individual” or a “patient.” The terms “individual” and “patient” do not denote a particular age.

[0075] As used here, “biocompatible” refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal. A biocompatible material, when introduced into an individual, is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the mammal.

[0076] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0077] As used herein, “treating a disease or disorder” means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.

[0078] The phrase “therapeutically effective amount,” as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases.

[0079] The term “compound,” as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein. In some embodiments, the term also refers to stereoisomers and / or optical isomers (including racemic mixtures) or enantiomerically enriched mixtures of disclosed compounds.

[0080] As used herein, “derivatives” are compositions formed from the native compounds either directly, by modification, or by partial substitution. As used herein, “analogs” are compositions that have a structure similar to, but not identical to, the native compound.

[0081] As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e. Ci-6 means one to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl”, “haloalkyl” and “homoalkyl”. As used herein, the term “substituted alkyl” means alkyl, as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, -N(CH3)2, -C(=O)OH, trifluoromethyl, -ON, -C(=O)O(Ci-C4)alkyl, -C(=O)NH2, -SO2NH2, - C(=NH)NH2, and -NO2, preferably containing one or two substituents selected from halogen, - OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and -C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxy cyclopentyl and 3 -chloropropyl.

[0082] As used herein, the term “alkylene” by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified by (-CH2-)n. By way of example only, such groups include, but are not limited to, groups having 24 or fewer carbon atoms such as the structures -CH2CH2- and -CH2CH2CH2CH2-. The term “alkylene,” unless otherwise noted, is also meant to include those groups described below as “heteroalkylene.”

[0083] As used herein, the terms “alkoxy,” “alkylamino” and “alkylthio” are used in their conventional sense, and refer to alkyl groups linked to molecules via an oxygen atom, an amino group, a sulfur atom, respectively.

[0084] As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (Ci-C3) alkoxy, particularly ethoxy and methoxy.

[0085] As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

[0086] As used herein, the term “cycloalkyl” refers to a mono cyclic or polycyclic non-aromatic radical, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl group is saturated or partially unsaturated. In another embodiment, the cycloalkyl group is fused with an aromatic ring. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but are not limited to, the following moieties:

[0087] Monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicyclic cycloalkyls include, but are not limited to, tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycyclic cycloalkyls include adamantine and norbornane. The term cycloalkyl includes “unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups, both of which refer to a nonaromatic carbocycle as defined herein, which contains at least one carbon-carbon double bond or one carbon-carbon triple bond.

[0088] As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, Si, P, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.

[0089] As used herein, the term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that consists of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or nonaromatic in nature. An example of a 3 -membered heterocycloalkyl group includes, and is not limited to, aziridine. Examples of 4-membered heterocycloalkyl groups include, and are not limited to, azetidine and a beta lactam. Examples of 5-membered heterocycloalkyl groups include, and are not limited to, pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6- membered heterocycloalkyl groups include, and are not limited to, piperidine, morpholine and piperazine. Other non-limiting examples of heterocycloalkyl groups are:

[0090] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2, 3 -dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran,

[0091] 2.3 -dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine,

[0092] 1.3-dioxepane, 4,7-dihydro-l ,3-dioxepin and hexamethyleneoxide.

[0093] As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized p (pi) electrons, where n is an integer.

[0094] As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl.

[0095] As used herein, the term “aryl-(Ci-C4)alkyl” means a functional group wherein a one to three carbon alkylene chain is attached to an aryl group, e.g., -CPECEE-phenyl . Preferred is aryl- CH2- and aryl-CH(CH3)-. The term “substituted aryl-(Ci-C4)alkyl” means an aryl-(Ci-C4)alkyl functional group in which the aryl group is substituted. Preferred is substituted aryl(CH2)-. Similarly, the term “heteroaryl-(Ci-C4)alkyl” means a functional group wherein a one to three carbon alkylene chain is attached to a heteroaryl group, e.g., -CtbCEh-pyridyl. Preferred is heteroaryl -(CH2)-. The term “substituted heteroaryl-(Ci-C4)alkyl” means a heteroaryl-(Ci-C4)alkyl functional group in which the heteroaryl group is substituted. Preferred is substituted heteroaryl-(CH2)-.

[0096] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0097] Examples of polycyclic heterocycles include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0098] The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.

[0099] As used herein, the term “amino aryl” refers to an aryl moiety which contains an amino moiety. Such amino moieties may include, but are not limited to primary amines, secondary amines, tertiary amines, masked amines, or protected amines. Such tertiary amines, masked amines, or protected amines may be converted to primary amine or secondary amine moieties. Additionally, the amine moiety may include an amine-like moiety which has similar chemical characteristics as amine moieties, including but not limited to chemical reactivity.

[0100] As used herein, the term “substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. For aryl, aryl-(Ci-C4)alkyl and heterocyclyl groups, the term “substituted” as applied to the rings of these groups refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In some embodiments, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two. In yet another embodiment, the substituents are independently selected from the group consisting of Ci-6 alkyl, - OH, Ci-6 alkoxy, halo, amino, acetamido and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of Ci-6 alkyl, Ci-6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being preferred.

[0101] As used herein, the term “optionally substituted” means that the referenced group may be substituted or unsubstituted. In some embodiments, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.

[0102] In some embodiments, the substituents are independently selected from the group consisting of oxo, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, alkyl (including straight chain, branched and / or unsaturated alkyl), substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, fluoro alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy, -S-alkyl, S(=O)2alkyl, -C(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -C(=O)N[H or alkyl]2, - OC(=O)N[substituted or unsubstituted alkyl]2, -NHC(=O)NH[substituted or unsubstituted alkyl, or substituted or unsubstituted phenyl], -NHC(=O)alkyl, -Nfsubstituted or unsubstituted alkyl]C(=O)[substituted or unsubstituted alkyl], -NHC(=O)[substituted or unsubstituted alkyl], - C(OH) [substituted or unsubstituted alkyl]2, and -C(NH2)[substituted or unsubstituted alkyl]2. In another embodiment, by way of example, an optional substituent is selected from oxo, fluorine, chlorine, bromine, iodine, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -CH3, -CH2CH3, -CH(CH3)2, - CF3, -CH2CF3, -OCH3, -OCH2CH3, -OCH(CH3)2, -OCF3, - OCH2CF3, -S(=O)2-CH3, - C(=O)NH2, -C(=O)-NHCH3, -NHC(=O)NHCH3, -C(=O)CH3, -ON(O)2, and -C(=O)OH. In yet one embodiment, the substituents are independently selected from the group consisting of Ci-6 alkyl, -OH, Ci-6 alkoxy, halo, amino, acetamido, oxo and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of Ci-6 alkyl, Ci-6 alkoxy, halo, acetamido, and nitro. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic.

[0103] As used herein, the term “analog,” “analogue,” or “derivative” is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog can be a structure having a structure similar to that of the small molecule therapeutic agents described herein or can be based on a scaffold of a small molecule therapeutic agents described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative can also be a small molecule that differs in structure from the reference molecule, but retains the essential properties of the reference molecule. An analog or derivative may change its interaction with certain other molecules relative to the reference molecule. An analog or derivative molecule may also include a salt, an adduct, tautomer, isomer, or other variant of the reference molecule.

[0104] The term “tautomer,” as used herein, refers to one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. It will be apparent to those skilled in the art that certain compounds of the present invention may exist in tautomeric forms, and that all such tautomeric forms of the compounds are within the scope of the present invention.

[0105] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0106] Description

[0107] The present invention is based, in part, on the unexpected discovery of a novel antibiotic compound from a Streptomyces bacterium. In some embodiments, the present invention provides a compound with antimicrobial activity. In some embodiments, the present invention provides compositions with antimicrobial activity. In some embodiments, the compound or composition has antibiotic activity. In some embodiments, the antibiotic compound or composition of the invention can be used in the treatment of bacterial infections. In certain embodiments, the use of the antibiotic compound or composition of the invention in the treatment of bacterial infections optionally includes a pharmaceutically acceptable excipient.

[0108] In some embodiments, the compound can be produced upon culture of a bacterium under conditions conducive to provide overexpression of the compound. Thus, in one aspect, the invention further provides methods for producing and isolating the compounds.

[0109] Compounds and Extracts

[0110] In one aspect, the present invention provides a compound represented by Formula (I), or a tautomer, ion, salt, and / or solvate thereof,

[0111] Formula (I).

[0112] In some embodiments, the compound is an inner salt. In some embodiments, the compound is zwitterionic. In some embodiments, the compound is protonated. In some embodiments, the compound is cationic. In some embodiments, the compound is deprotonated. In some embodiments, the compound is anionic.

[0113] In some embodiments, the compound is selected from the group consisting of:

[0114] In some embodiments, the compound is substituted with a substituent selected from the group consisting of deuterium, fluorine, chlorine, bromine, iodine, and any combination thereof.

[0115] In certain embodiments, the present invention provides an extract which comprises the compound represented by Formula (I). In some embodiments, the extract comprises at least one compound derived from a bacterial isolate. Examples of bacteria from which compounds may be derived include, but are not limited to, Streptomyces species.

[0116] In some embodiments, the extract is from a culture of a bacteria. Methods of culturing bacteria include, but are not limited to, solid culture (e.g., agar plate), liquid culture, hybrid culture (e.g., solid-liquid interface culture), and biphasic liquid culture (e.g., liquid-liquid interface culture). In some embodiments, the method of culturing the bacteria comprises one or more external stimuli which induce increased production of a compound of interest. Examples of external stimuli include, but are not limited to, alterations in temperature, alterations the ambient atmosphere (e.g., aerobic culture, anaerobic culture, etc.), electromagnetic radiation, viral infection, bacterial co-culture, fungal co-culture, pathogen associated molecular patterns (PAMPs), altered media composition (e.g., increased or decreased concentration of a nutrient / salt / buffer / etc., substitution of a nutrient source / salt / buffer / etc., change in pH, etc.), exogenous biomolecules (e.g., proteins, nucleic acids, small molecules, etc.), pharmaceuticals, and combinations thereof.

[0117] In some embodiments, the extraction process comprises extraction of the bacteria grown in the culture. In some embodiments, the extraction process comprises extraction of the culture medium. In some embodiments, the bacteria and / or culture medium is processed prior to extraction. Examples of processing include, but are not limited to, sterilization (e.g., filtration, heat, chemical, radiation, etc.), sonication, freezing, grinding, and lyophilization. In certain embodiments, the extracting process comprises obtaining a liquid culture medium and performing direct liquid phase extraction of the medium at least one time with at least one solvent. The at least one solvent used for extraction each time may be the same at least one solvent or may be a distinct solvent. When more than one extraction is performed, each resulting extract may be kept separate or combined with at least one additional extract. Examples of suitable solvents include, but are not limited to, pentanes, hexanes, heptanes, octanes, nonanes, decanes, pentanols, hexanols, heptanols, octanols, nonanols, decanols, methyl tert-butyl ether, diethyl ether, diisopropyl ether, tetrahydrofuran, tetrahydropyran, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethanes, trichloroethylene, benzene, toluene, xylenes, and combinations thereof. In some embodiments, extraction solvents are pH modified by the addition of an acid or base. Examples of suitable acids and bases include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, pentanoic acid, ammonia, methylamines (e.g., methylamine, dimethylamine, triethylamine, ethyl methylamine, propyl methylamine, isopropyl methylamine, dimethyl ethylamine, etc.) ethylamines (e.g., ethylamine, di ethylamine, tri ethylamine, ethyl methylamine, ethyl propylamine, ethyl isopropylamine, diethyl methylamine, diethyl propylamine, diethyl isopropylamine, diisopropyl ethylamine, etc.) , propylamines (e.g., propylamine, isopropylamine, dipropylamine, diisopropylamine, dipropyl-isopropylamine, tripropylamine, triisopropylamine, dipropyl methylamine, diisopropyl methylamine, dipropyl ethylamine, diisopropyl ethylamine, etc.), and butylamines (e.g., butylamine, isobutylamine, sec-butylamine, tert-butylamine, dibutylamine, diisobutyl amine, di-(sec-butyl)amine, di-(tert-butyl)amine, butyl methylamine, butyl ethylamine, butyl propylamine, butyl isopropylamine, dibutyl methylamine, dibutyl ethylamine, etc.), and combinations thereof.

[0118] In some embodiments, the extracting process comprises obtaining a solid sample (e.g., bacterial pellet, lyophilized culture media, etc.) and performing direct solid phase extraction of the sample at least one time with at least one solvent. The at least one solvent used for extraction each time may be the same at least one solvent or may be a distinct solvent. When more than one extraction is performed, each resulting extract may be kept separate or combined with at least one additional extract. Examples of suitable solvents include, but are not limited to, water, pentanes, hexanes, heptanes, octanes, nonanes, decanes, methanol, ethanol, propanols, butanols, pentanols, hexanols, octanols, nonanols, decanols, dimethyl sulfoxide, dimethyl formamide, acetone, methyl ethyl ketone, ethyl acetate, propyl acetate, butyl acetate, acetonitrile, ethyl methyl ether, methyl butyl ether, methyl tert-butyl ether, diethyl ether, tetrahydrofuran, tetrahydropyran, dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethanes, trichloroethylene, benzene, toluene, xylenes, and combinations thereof. In some embodiments, extraction solvents are pH modified by the addition of an acid or base. Examples of suitable acids and bases include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, pentanoic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, ammonia, methylamines (e.g., methylamine, dimethylamine, triethylamine, ethyl methylamine, propyl methylamine, isopropyl methylamine, dimethyl ethylamine, etc.) ethylamines (e.g., ethylamine, diethylamine, triethylamine, ethyl methylamine, ethyl propylamine, ethyl isopropylamine, diethyl methylamine, diethyl propylamine, diethyl isopropylamine, diisopropyl ethylamine, etc.) , propylamines (e.g., propylamine, isopropylamine, dipropylamine, diisopropylamine, dipropylisopropyl amine, tripropylamine, triisopropylamine, dipropyl methylamine, diisopropyl methylamine, dipropyl ethylamine, diisopropyl ethylamine, etc.), and butylamines (e.g., butylamine, isobutylamine, sec-butylamine, tert-butylamine, dibutylamine, diisobutyl amine, di- (sec-butyl)amine, di-(tert-butyl)amine, butyl methylamine, butyl ethylamine, butyl propylamine, butyl isopropylamine, dibutyl methylamine, dibutyl ethylamine, etc.), sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, magnesium hydroxide, N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid, sodium bicarbonate, sodium carbonate, and combinations thereof.

[0119] In some embodiments, the method comprises suspending or dissolving a sample in a first solvent, applying the suspension or solution to a solid phase extraction substrate to “load” the substrate, and performing solid phase extraction on the loaded substrate one or more times with one or more solvents. The at least one solvent used for extraction each time may be the same at least one solvent or may be a distinct solvent. When more than one extraction is performed, each resulting extract may be kept separate or combined with at least one additional extract. Examples of suitable solvents include, but are not limited to, water, pentanes, hexanes, heptanes, octanes, nonanes, decanes, methanol, ethanol, propanols, butanols, pentanols, hexanols, octanols, nonanols, decanols, dimethyl sulfoxide, dimethyl formamide, acetone, methyl ethyl ketone, ethyl acetate, propyl acetate, butyl acetate, acetonitrile, ethyl methyl ether, methyl butyl ether, methyl tert-butyl ether, diethyl ether, tetrahydrofuran, tetrahydropyran, dioxane, ethyl acetate, propyl acetate, butyl acetate, dichloromethane, chloroform, carbon tetrachloride, dichloroethanes, trichloroethylene, benzene, toluene, xylenes, and combinations thereof. In some embodiments, extraction solvents are pH modified by the addition of an acid or base. Examples of suitable acids and bases include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, pentanoic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, ammonia, methylamines (e.g., methylamine, dimethylamine, triethylamine, ethyl methylamine, propyl methylamine, isopropyl methylamine, dimethyl ethylamine, etc.) ethylamines (e.g., ethylamine, di ethylamine, tri ethylamine, ethyl methylamine, ethyl propylamine, ethyl isopropylamine, diethyl methylamine, diethyl propylamine, diethyl isopropyl amine, diisopropyl ethylamine, etc.) , propylamines (e.g., propylamine, isopropylamine, dipropylamine, diisopropylamine, dipropyl-isopropylamine, tripropylamine, triisopropylamine, dipropyl methylamine, diisopropyl methylamine, dipropyl ethylamine, diisopropyl ethylamine, etc.), and butylamines (e.g., butylamine, isobutylamine, sec-butylamine, tert-butylamine, dibutylamine, diisobutyl amine, di-(sec-butyl)amine, di-(tert-butyl)amine, butyl methylamine, butyl ethylamine, butyl propylamine, butyl isopropylamine, dibutyl methylamine, dibutyl ethylamine, etc.), sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, magnesium hydroxide, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, sodium bicarbonate, sodium carbonate, and combinations thereof.

[0120] In some embodiments, the at least one extract is examined for activity. The extract may be examined for any activity including, but not limited to, antibacterial activity, antifungal activity, anthelminthic activity, quorum sensing activity, herbicidal activity, pesticidal activity, anti-inflammatory activity, anti-cancer activity, antioxidant activity, protein activation or agonism activity, protein inhibition or antagonism activity, and any combination thereof.

[0121] In some embodiments, an extract with an activity is further purified. Examples of purification methods include, but are not limited to, liquid phase extraction, solid phase extraction, normal phase liquid chromatography, reverse phase liquid chromatography, normal phase thin layer chromatography, reverse phase liquid chromatography, size exclusion chromatography, ion exchange chromatography, trituration, dialysis, distillation, and crystallization. In some embodiments, chromatography is used to separate an active extract into a plurality of fractions. In some embodiments, the chromatography is liquid chromatography. Stationary phases suitable for liquid chromatography include, but are not limited to, silica, alumina, hydrocarbon-bonded silica, fluorocarbon-bonded silica, cyano-bonded silica, aminobonded silica, and combinations thereof. Liquid phases suitable for liquid chromatography include, but are not limited to, water, acetonitrile, methanol, ethanol, propanol, isopropanol, tetrahydrofuran, ethyl acetate, pentanes, hexanes, dichloromethane, diethyl ether, acetone, and combinations thereof. One or more additional compounds may be added to the liquid phase to improve chromatography. Examples of compounds added to liquid phases to adjust the pH include, but are not limited to, formic acid, acetic acid, trifluoroacetic acid, heptafluorobutyric acid, sodium formate, sodium acetate, and ammonia.

[0122] In some embodiments, the fractions resulting from the liquid chromatography are examined for activity, typically the same activity that was demonstrated by the original extract. In some embodiments, a fraction identified to have the desired activity is selected for use. In some embodiments, the fraction is put through at least one additional round of purification and activity analysis.

[0123] Pharmaceutical Compositions and Formulations

[0124] The invention also encompasses pharmaceutical compositions and / or formulations comprising a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,

[0125] Formula (I); and a pharmaceutically acceptable excipient.

[0126] In some embodiments, the pharmaceutical composition comprises an extract, wherein the extract comprises at least one compound derived from a Streptomyces bacterium. In some embodiments, the pharmaceutical composition comprises an extract, wherein the extract comprises at least one compound of Formula (I) derived from a Streptomyces bacterium. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0127] Pharmaceutical compositions of the present invention may comprise at least one compound of the invention, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof in a form suitable for administration to a subject and at least one additional ingredient, or the pharmaceutical composition may comprise at least one compound of the invention, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, and at least one pharmaceutically acceptable excipient, optionally with at least one additional ingredient, or some combination of these. The compound of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0128] Pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0.

[0129] The compounds (and extracts) of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent’s site of action in the body of the organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0130] In general, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium Ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, a standard reference text in this field.

[0131] In some embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In some embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound or conjugate of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that are useful, include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0132] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. In some embodiments, the pharmaceutically acceptable carrier is not DM SO alone.

[0133] The present invention also provides pharmaceutical compositions comprising one or more of the compositions described herein. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for administration to subject. The pharmaceutical compositions may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0134] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fdlers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0135] In some embodiments, the composition includes an antioxidant that inhibits the degradation of one or more components of the composition. Exemplary antioxidants for some compounds are BHT, BHA, alpha-tocopherol and ascorbic acid in the range of about 0.01% to 0.3%, Other suitable and equivalent antioxidants may be substituted as would be known to those skilled in the art.

[0136] Liquid suspensions may be prepared using conventional methods to achieve suspension of the HMW-HA or other composition of the invention in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.

[0137] Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

[0138] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0139] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

[0140] The active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U.S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful, include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund’s adjuvant, and IL-12. Other components may include a polyoxypropylenepolyoxyethylene block polymer (Pluronic®), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Patent No. 4,606,918).

[0141] Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

[0142] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof.

[0143] Accordingly, the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in the body of a subject to achieve a particular effect (see, e g., Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

[0144] The active ingredients of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term “unit dosage form” as used herein refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host.

[0145] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0146] When the therapeutic agents of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.

[0147] Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

[0148] The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0149] Thus, the therapeutic agent may be formulated for parenteral administration (e g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0150] It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

[0151] In some embodiments, the pharmaceutical formulation further comprises one or more antimicrobial agents. Examples of antimicrobial agents include antiviral agents, antibacterial and antibiotic agents, anti-archaeal agents, antifungal agents, and antiprotozoal s.

[0152] In some embodiments, the pharmaceutical formulation comprises one or more antibiotic agents. Exemplary antibiotic agents include, but are not limited to, albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, and penicillin. Other examples include sulfadiazine, sulfones, such as dapsone (DDS) and para-aminosalicylic (PAS), sulfanilamide, sulfamethizole, sulfamethoxazole, sulfapyridine, trimethoprim, pyrimethamine, nalidixic acids, norfloxacin, cinoxacin, enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin, trovafloxacin, penicillins (amoxicillin, ampicillin, azlocillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, hetacillin, oxacillin, mezlocillin, penicillin G, penicillin V, piperacillin), cephalosporins (cefacetrile, cefadroxil, cefalexin, cefalonium, cefalotin, cefapirin, cefatrizine, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, ceforanide, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefotaxime, cefotiam, cefpiramide, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefepime), carbapenems (imipenem, ertapenem, meropenem) monobactams (aztreonam) oxytetracycline, chlortetracycline, clomocycline, demeclocycline, tetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, chloramphenicol, amikacin, gentamicin, framycetin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, colistin, bacitracin, tyrothricin, furazolidone, metronidazole, tinidazole, isoniazid, pyrazinamide, ethionamide, nystatin, amphotericin-B, hamycin, miconazole, clotrimazole, ketoconazole, fluconazole, lincomycin, clindamycin, spectinomycin, fosfomycin, loracarbef, polymyxin B, polymyxin B Sulfate, ramoplanin, teicoplanin, vancomycin, and / or nitrofurantoin.

[0153] In some embodiments, the pharmaceutical formulation comprises one or more antifungal agents. Exemplary antifungal agents include, but are not limited to, anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, clotrimazole, econazole, miconazole, amphotericin, itraconazole, ketoconazole, naftifine, terbinafme, 5 -fluorocytosine, nystatin, amphotericin B, pimaricin, griseofulvin, amorolfine, and combinations thereof.

[0154] Administration of the therapeutic agent in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the subject, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art.

[0155] The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0156] In some embodiments, the pharmaceutical compositions useful for practicing the methods of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 500 mg / kg / day.

[0157] Typically, dosages which may be administered in a method of the invention to a mammal, preferably a human, range in amount from 0.5 pg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In some embodiments, the dosage of the compound will vary from about 1 pg to about 10 mg per kilogram of body weight of the mammal. In some embodiments, the dosage will vary from about 3 pg to about 5 mg per kilogram of body weight of the mammal.

[0158] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0159] Administration of the compositions of the present invention to a subject, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to prevent or treat disease. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0160] The compound may be administered to a subject as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of compound dosed per day may be administered, in nonlimiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0161] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

[0162] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0163] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease in a subject.

[0164] In some embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound or conjugate of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound or conjugate to treat, prevent, or reduce one or more symptoms of a disease in a subject.

[0165] Routes of administration of any of the compositions of the invention include oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0166] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

[0167] The pharmaceutical composition can be formulated for oral administration as, for example but not limited to, drug powders, crystals, granules, small particles (which include particles sized on the order of micrometers, such as microspheres and microcapsules), particles (which include particles sized on the order of millimeters), beads, microbeads, pellets, pills, microtablets, compressed tablets or tablet triturates, molded tablets or tablet triturates, and in capsules, which are either hard or soft and contain the composition as a powder, particle, bead, solution or suspension. The pharmaceutical composition can also be formulated for oral administration as a solution or suspension in an aqueous liquid, as a liquid incorporated into a gel capsule or as any other convenient formulation for administration, or for rectal administration, as a suppository, enema or other convenient form.

[0168] These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

[0169] Methods of Treatment

[0170] In one aspect, the invention provides methods of treating or preventing a microbial infection in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a compound, extract, or pharmaceutical composition of the present invention. In some embodiments, the microbial infection is a bacterial infection. In some embodiments, the subject is exposed to or infected with a bacterium.

[0171] In one aspect, the invention provides compositions and methods for treating and / or preventing a disease or disorder related to the detrimental growth and / or proliferation of a bacterial cell in vivo, ex vivo or in vitro. In certain embodiments, the method comprises administering a composition comprising an effective amount of a compound, extract, or pharmaceutical composition provided by the invention to a subject, wherein the compound, extract, or pharmaceutical composition is effective in inhibiting or preventing the growth and / or proliferation of a bacterial cell.

[0172] In certain embodiments, the bacterial cell is a gram-negative bacterium, e.g., a bacteria of a genera such as Acmetobacter, Citrobacter , Enterobacter , Enterococcus, Escherichia, Helicobacter, Hemophilus, Klebsiella, Legionella, Moraxella, Neisseria, Proteus, Pseudomonas, Salmonella, Staphylococcus, Burkholderia, and Yersinia.

[0173] In certain embodiments, the bacterial cell is a gram-positive bacterium, e.g., a bacteria of a genera such as Staphylococcus, Streptococcus, Enterococcus, Bacillus, Corynebacterium, Nocardia, Clostridium, Actinobacteria, Listeria, Mollicutes, bacteria-like Mycoplasma, and Actinobacteria.

[0174] The compounds, extracts, and pharmaceutical compositions of the invention may thus be for use in the treatment of bacterial infections by gram-negative or gram-positive bacteria.

[0175] In some embodiments, the subject is exposed to or infected with an Acmetobacter species. In some embodiments, the subject is exposed to or infected with a Vibrio species. In some embodiments, the subject is exposed to or infected with an Escherichia species. In some embodiments, the subject is exposed to or infected with an Enterococcus species. In some embodiments, the subject is exposed to or infected with a Staphylococcus species. In some embodiments, the subject is exposed to or infected with a Klebsiella species. In some embodiments, the subject is exposed to or infected with a Pseudomonas species. In some embodiments, the subject is exposed to or infected with an Enterobacter species. In some embodiments, the subject is exposed to or infected with a Burkholderia species. In some embodiments, the bacterium is Acmetobacter baumannii. In some embodiments, the bacterium is Vibrio cholerae. In some embodiments, the bacterium is Escherichia colt. Exemplary bacterium or pathogens include, but are not limited to, Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, Mycobacterium tuberculosis, Salmonella species, Shigella species, Enterococcus faecium, Pseudomonas aeruginosa, Enterobacter species, Neisseria gonorrhoeae, Staphylococcus aureus, Citrobacter species, Proteus species, Serratia species, Group A Streptococci, Group B Streptococci, Streptococcus pneumoniae, Haemophilus influenzae, Morganella species, Vibrio cholerae, and any combination thereof.

[0176] Other exemplary bacterial infections that may be treated by way of the present invention include, but are not limited to, infections caused by bacteria from the taxonomic genus of Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia. In some embodiments, the bacterial infection is an infection of Acinetobacter baumannii, Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia cepacian, Burkholderia mallei, Burkholderia pseudomallei, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella species, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Moraxella species, Moraxella osloensis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae , Neisseria meningitidis, Proteus species, Proteus vulgaris, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus species, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enter ocolitica, or Yersinia pseudotuberculosis. Exemplary diseases caused by bacterial infections which may be treated using compositions of the present invention, include but are not limited to, sepsis, pneumonia, bacterially mediated meningitis, sinus tract infections, endocarditis, pancreatitis, appendicitis, gastroenteritis, biliary tract infections, soft tissue infections, urinary tract infections, cystitis, pyelonephritis, osteomyelitis, bacteremia, actinomycosis, whooping cough, secondary bacterial pneumonia, Lyme disease (B. burgdorferi), relapsing fever, brucellosis, enteritis, bloody diarrhea, Guillain-Barre syndrome, atypical pneumonia, trachoma, neonatal conjunctivitis, neonatal pneumonia, nongonococcal urethritis(NGU), urethritis, pelvic inflammatory disease, epididymitis, prostatitis, lymphogranuloma venereum (LGV), psittacosis, botulism, muscle weakness and paralysis, pseudomembranous colitis, anaerobic cellulitis, gas gangrene, acute food poisoning, tetanus, diphtheria, abscesses, furuncles, cellulitis, folliculitis, bloodstream infections, medical device infections, skin infections, impetigo, septic arthritis, brain abscess, bum wounds, venous ulcers, diabetic foot ulcers, surgical wounds, post-operation infections, carbuncles, necrotizing pneumonia, and necrotizing fasciitis.

[0177] However, the invention should not be limited to only treating bacterial infection. The invention encompasses compounds having an antimicrobial activity including but not limited to antibacterial, antimycobacterial, antifungal, antiviral, anti-archaeal, antiprotozoal, and the like.

[0178] In some embodiments, the subject is exposed to or infected with a bacterium that is resistant to one or more antibiotic agents. In certain embodiments, the one or more antibiotic agents are a bacteriostatic antibiotic, a bactericidal antibiotic, and / or a beta-lactam antibiotic. Exemplary antibiotic agents include, but are not limited to albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, and penicillin. In some embodiments, the antibiotic agent is selected from the group consisting of imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, and tebipenem.

[0179] Other non-limiting examples of the at least one additional antibiotic agents include levofloxacin, doxycycline, neomycin, clindamycin, minocycline, gentamycin, rifampin, chlorhexidine, chloroxylenol, methylisothiazolinone, thymol, a-terpineol, cetylpyridinium chloride, hexachlorophene, triclosan, nitrofurantoin, erythromycin, nafcillin, cefazolin, imipenem, aztreonam, gentamicin, sulfamethoxazole, vancomycin, ciprofloxacin, trimethoprim, rifampin, metronidazole, clindamycin, teicoplanin, mupirocin, azithromycin, clarithromycin, ofloxacin, lomefloxacin, norfloxacin, nalidixic acid, sparfloxacin, pefloxacin, amifloxacin, gatifloxacin, moxifloxacin, gemifloxacin, enoxacin, fleroxacin, minocycline, linezolid, temafloxacin, tosufloxacin, clinafloxacin, sulbactam, clavulanic acid, amphotericin B, fluconazole, itraconazole, ketoconazole, nystatin, penicillins, cephalosporins, carbapenems, betalactams antibiotics, aminoglycosides, macrolides, lincosamides, glycopeptides, tetracylines, chloramphenicol, quinolones, fucidines, sulfonamides, trimethoprims, rifamycins, oxalines, streptogramins, lipopeptides, ketolides, polyenes, azoles, echinocandines, and any combination thereof.

[0180] In certain embodiments, the compounds and compositions / formulations described herein are used to treat fungal infections. Exemplary fungal infections and pathogens include, but are not limited to, Cryptococcus ueoformans. Candida auris, Aspergillus fumigatus. Candida albicans, Nakaseomyces / Candida glabrata, Histoplasma species, a eumycetoma causative agent, Mucorales species, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Lomentospora prolificans, Coccidioides species, Pichia kudriavzeveii / Candida krusei, Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, Paracoccidioides species, and any combination thereof.

[0181] In some embodiments, the subject is exposed to or infected with a fungal pathogen that is resistant to one or more antifungal agents selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

[0182] In certain embodiments, the method further comprises the step of administering one or more additional antibiotic agents as mentioned above. In certain embodiments, the method further comprises the step of administering one or more additional antifungal agents as mentioned above.

[0183] In some embodiments, the subject is exposed to or infected with a human pathogen. In some embodiments, the subject is exposed to or infected with an agricultural pathogen.

[0184] The present invention can be used to treat subjects at risk of, exhibiting symptoms of, or diagnosed with toxic shock syndrome, scalded skin syndrome, abscesses, furuncles, cellulitis, folliculitis, bloodstream infections, medical device infections, pneumonia, osteomyelitis, staphylococcal food poisoning, skin and soft tissue infections, endocarditis, eczema, atopic dermatitis, psoriasis, impetigo, septic arthritis, brain abscess, bum wounds, venous ulcers, diabetic foot ulcers, surgical wounds, post-operation infections, carbuncles, meningitis, bacteremia, necrotizing pneumonia, sepsis, endocarditis, osteomyelitis, or necrotizing fasciitis.

[0185] The composition of the invention may be administered to a patient or subject in need in a wide variety of ways, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient internally, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the composition is administered systemically to the subject. In some embodiments, the compositions of the present invention are administered to a patient by i.v. injection. In some embodiments, the composition is administered locally to the subject. In some embodiments, the compositions of the present invention are administered to a patient topically. In some embodiments, the compositions of the present invention are administered to a patient ophthalmically. In some embodiments, the compositions of the present invention are administered to a patient optically. In some embodiments, the compositions of the present invention are internally administered by a means selected from the group consisting of oral, intravenous, intramuscular, intraperitoneal, subcutaneous, mucosal, percutaneous, sublingual, intrathecal, intraocular, inhalation, rectal, and nasal administration. Any administration may be a single application of a composition of invention or multiple applications. Administrations may be to single site or to more than one site in the individual to be treated. Multiple administrations may occur essentially at the same time or separated in time.

[0186] In one aspect, the compositions of the invention may be in the form of a coating that is applied to the surface of a medical device or the surface of a subject’s body. In some embodiments, the coating prevents or hinders microorganisms and / or biofilm-embedded microorganisms from growing and proliferating on at least one surface of the medical device or at least one surface of the subject’s body. In another embodiment, the coating facilitates access of antimicrobial agents to the microorganisms and / or biofilm-embedded microorganisms, thus helping prevent or hinder the microorganisms and / or biofdm-embedded microorganisms from growing or proliferating on at least one surface of the medical device or at least one surface of the subject’s body. The compositions of the invention may also be in the form of a liquid or solution, used to clean the surface of medical device or the surface of a subject’s body, on which microorganisms and / or biofilm-embedded microorganisms live and proliferate. Such cleaning of the medical device or body surface may occur by flushing, rinsing, soaking, or any additional cleaning method known to those skilled in the art, thus removing at least a portion of or reducing the number of microorganisms and / or biofilm-embedded microorganisms attached to at least one surface of the medical device or at least one surface of the subject’s body.

[0187] In some embodiments, the compositions of the invention find use in removing at least a portion of or reducing the number of microorganisms and / or biofilm-embedded microorganisms attached to the surface of a medical device or the surface of a subject’s body (such as the skin of the subject, or a mucous membrane of the subject, such as the vagina, anus, throat, eyes or ears). In some embodiments, the compositions of the invention find further use in coating the surface of a medical device, thus inhibiting or disrupting microbial growth and / or inhibiting or disrupting the formation of biofilm on the surface of the medical device. The compositions of the invention find further use in preventing or reducing the growth or proliferation of microorganisms and / or biofilm-embedded microorganisms on the surface of a medical device or on the surface of a subject’s body. However, the invention is not limited to applications in the medical field. Rather, the invention includes using a compound or an analog thereof as an antimicrobial and / or antibiofilm agent in any setting.

[0188] Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including but not limited to non-human mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0189] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject’s disease, although appropriate dosages may be determined by clinical trials.

[0190] When “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, disease type, extent of disease, and condition of the patient (subject). EXPERIMENTAL EXAMPLES

[0191] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0192] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.

[0193] Example 1: \ Streptomyces bacterium elicits antibacterial activity

[0194] The majority of antibiotics in clinical use today are natural products derived from microorganisms in the environment. The present work is drawn from a screen of over a Strepiomyces isolate to discover potent antibiotics candidates from an uncharacterized microbial source.

[0195] Antimicrobial Screen

[0196] Recognizing that bacterial natural products may be temporally expressed in response to changes in growth-phase and / or exogeneous stressors, a Streptomyces isolate (9005BA) was cultured in four different conditions: i. 24 °C, ii. 30 °C, iii. 30 °C in the presence of heat killed A. baumannii, and iv. 37 °C for a total of 7 days. Aliquots were removed at days 1, 3, and 7 of incubation for each culture condition, the supernatant was collected, filter sterilized, and spot plated onto lawns of A. baumannii, S. aureus, P. aeruginosa, and E. coli to learn which organisms the sample displayed antimicrobial properties. 9005BA displayed antimicrobial activity against A. baumannii, S. aureus, and E. coli.

[0197] The supernatant from 9005BA was then re-tested for growth inhibition toward antibiotic resistant strains, including strains that are resistant to antibiotics commonly produced by natural product isolates. If a supernatant displays a loss of antimicrobial performance toward a given antibiotic resistant strain, then the supernatant’s antimicrobial properties are likely to be modulated by that particular antibiotic.

[0198] Accordingly, 9005BA was retested for activity against S. aureus strains resistant to the antibiotics streptomycin, erythromycin, rifampicin, tetracycline, fosfomycin, albocycline or methicillin and A. baumannii strains with tetracycline or rifampicin resistance. After testing, it was revealed that the supernatant of 9005BA retained activity against all resistant strains, suggesting that its antimicrobial performance is not mediated by those antibiotics.

[0199] Whole Genome Sequencing

[0200] Whole genome sequencing (WGS) was used as a more comprehensive means to learn whether the antimicrobial performance of the 9005BA is likely to be mediated by a novel antimicrobial. More directly, the genomic sequence of each genome was analyzed using antiSMASH software version 7.0 to identify synthetic gene-clusters of known natural product antimicrobials.

[0201] The materials and methods employed are described herein.

[0202] Culturing Conditions of Isolates: 9005BA was collected by swabbing microbes colonizing a rock surface directly onto nutrient limited G-agar (1% Dextrose (D-glucose), 0.5% N-Z amine A, 0.1% calcium carbonate, 2% agar). Plates were incubated at 37 °C for 2-3 weeks, during which rapidly appearing colonies were heat-killed with a heated inoculation loop to enrich for outgrowth of slow growing microbial species. Resulting slow-growing microbes were then streak plated on fresh G-agar plates and were transferred to 40% glycerol media for archiving (-80 °C). For culturing in liquid media, a single colony was used to inoculate 5 mL of G-broth (1% Dextrose (D-glucose), 0.5% N-Z amine A, 0.1% calcium carbonate). The culture was incubated for 7 days in a rotating drum at 37 °C and processed.

[0203] Preparation of Strains: 5. aureus, V. cholerae, and P. aeruginosa were propagated in Mueller- Hinton broth (MHB). Albocycline-, Fosfomycin-, and rifampicin-resistant derivatives of S. aureus strain UAMS-1 were created either by directly plating on MHB agar plates supplemented with 8 pg / mL rifampicin to obtain the rifampicin resistant strain UAMS-1 R (rifampicin MIC of X) or stepwise cultured in 0.5*, 1 x, 2*, and 4* MIC of albocycline or fosfomycin to yield strains UAMS-1 AB (albocycline MIC of Y) and UAMS-1 F (Fosfomycin MIC ofZ), respectively. USA300 is a well characterized methicillin resistant S. aureus (MRSA) lineage, whereas VRSA1 is a vancomycin resistant S. aureus (VRSA) clinical isolate (Tenover). E. coh and A. baumannii were grown in Luria-Bertani (LB) broth.

[0204] Screen for Antimicrobial Activity: Independently, a single colony of 9005BA was used to inoculate 5 mL of G-broth, incubated for 14 days at 37 °C and used to inoculate (1 : 100 dilution) four fresh 3 mL G-broth tubes. Tubes were incubated at either (A) 26 °C, (B) 30 °C, (C) 30 °C in the presence of 105heat-killed A. baumannii strain 98-37-09, or (D) 37 °C. Aliquots (0.5 mL) were removed on days 1, 3, and 7 from each of the incubation conditions (A-D), centrifuged, sterilized by passage through 0.22 pm filters (Pall Corporation Life Sciences, Port Washington, NY), and stored at 4 °C.

[0205] For antimicrobial testing, 20 pL of each sterilized aliquot was spot-plated onto Mueller- Hinton plates that were seeded with 104to 105of P. aeruginosa strain PA01, S. aureus strain UAMS-1, E. coli strain Top 10, A. baumannii strain 98-37-09, M. abscessus strain PM3044, M. smegmatis strain MC2155, or V. cholerae strain AM-19226. Plates were incubated for 16 hours at 37 °C and zones of bacterial growth inhibition were determined by the un-aided eye. Aliquots that displayed antimicrobial activity were retested for antimicrobial activity against antibiotic resistant strains by spot plating on MH plates seeded with antimicrobial resistant strains. The antimicrobial activity was also measured in microtiter liquid format, in which individual wells of a 96-well microtiter plate containing 88 pL MH media was inoculated with 104colony forming units (10 pL) of the indicated bacterial species and 2 pL of various concentrations of sterilized supernatant (0, 1, 2, 4, 8, 16, 32, 64, or 100%). Plates were incubated for 16 hours at 37 °C; the lowest concentration of supernatant that inhibited bacterial growth was considered the relative minimal inhibitory concentration.

[0206] Whole Genome Sequencing: Genomic DNA was isolated and sequenced for indicated collection members that displayed antimicrobial activity. Assembled genomes were queried for known antibiotic producing loci / gene clusters using antiSmash version 7.0. Minimum Inhibitory Concentration: Filter-sterilized supernatants were serially diluted 1 :2 with autoclaved water, leaving 90 uL of diluted supernatant in each well (96 well plates). Tested bacteria were grown overnight in Mueller Hinton Broth (MHB) and sub-cultured to an OD of 108CFUs / mL and diluted 1 :100 with sterile MHB. 10 pL of 106CFUs / mL (104cells seeded) of tested pathogen was inoculated into the 90 pL supernatant and grown for 16 hours at 37 °C under static conditions. The MIC was characterized by percent of the original supernatant.

[0207] Antimicrobial Overexpressor Strains: An antimicrobial molecule overexpressor of 9005BA was generated by serial passage of cultures displaying high antimicrobial activity. Wild-type cultures were inoculated into G-media and grown for one week. Aliquots of supernatants were tested using the identified MIC and the lowest MIC cultures were selected to be reinoculated into G- media. Passaging was repeated until a stable mutant that consistently generated high antimicrobial activity was found.

[0208] Resistant Mutants: 9005BA was tested against P. aeruginosa strain PA01, S. aureus strain UAMS-1, E. coli strain Top 10, A. baumannii strain 98-37-09, M. abscessus strain PM3044, M. smegmatis strain MC2155, and V. cholerae strain AM-19226 in an MIC test. Resistant mutants for susceptible pathogens were generated by serial passaging at subinhibitory concentrations. WT pathogens were grown overnight and sub-cultured to 108CFUs / mL. The subculture was diluted 1 : 100 to 106CFUs / mL, 0.5 MIC of sterilized, active supernatant was added to 5 mL subcultures, and grown 16 hours at 37 °C under shaking conditions. The passaged mutant had the MIC retested, with the WT strain serving as a control. Serial passage of the mutant was repeated four times (1 *,2*,4x) and MIC retested.

[0209] Example 2; Identification of a Potent Antibiotic Alkaloid from Streptomyces Bacteria

[0210] As the lyophilized 9005BA culture supernatant was found to exhibit a relatively strong minimum inhibitory concentration (MIC) of 125 pg / mL against Acinetobacter baumannii, 9005BA was selected for further isolation and structural elucidation studies on the active compound (Figure 1). To define the antimicrobial agent produced by 9005BA, an overproducer strain was identified by individually screening over 500 individual colonies for those that produced increased antimicrobial activity toward A. baumannii. The process was repeated three additional times to yield a 9005BA derivative, 9005B A+ that appeared to produce increased antimicrobial agent in comparison to 9005BA.

[0211] The culture supernatant resulting from 9005BA+ was subjected to an initial round of fractionation through differential extraction. The lyophilized supernatant was loaded on to a solid phase extraction (SPE) cartridge and subsequently extracted with 30% aqueous methanol and 100% ethanol. The two extracts were then tested for activity against ESKAPE pathogens (E. coll, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae), with the 100% methanol extract demonstrating an improved efficacy against A. baumannii (12.5 gg / mL, Figure 2). The active extract was then subjected to activity guided fractionation through microfractionation (Figure 3) using high-performance liquid chromatography with diode-array detection (HPLC-DAD) to target the active compound. ESKAPE pathogens were exposed to the re-fractionated extract, with the isolated compound demonstrating an MIC of 0.625 gg / mL, a 200-fold improvement in activity compared to the original lyophilized supernatant (Figure 4 and Table 1).

[0212] Table 1. 9005BA Extract and isolated compound activity.

[0213] SN, supernatant; 100M, 100% Methanol Solid Phase Extract

[0214] In parallel to HPLC-based fractionation, HPLC coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF MS) in positive ion mode was used for the early detection and prediction of the active compound presented in the extract and fraction. Substantive compound isolation was achieved by solid phase extraction and further semi-preparative HPLC and its structure was established by carrying out ID and 2D nuclear magnetic resonance (NMR) experiments (Figure 5 and Figure 6) along with high -resolution mass spectrometry (HRMS; Figure 7), and small-angle X-ray diffraction (SXRD; Figure 8).

[0215] The compound (Compound 1; Figure 9) was isolated as a dark blue pigment. The molecular formula identified by HRESIMS was suggested as C13H10N2O2 (m / z 227.0826 [M + H]+, calculated for C13H11N2O2, 227.0810), with 10 indices of hydrogen deficiency. Its UV spectrum showed maximum absorption at 271, 318, 371, 458 and 785 nm, suggesting the existence of a long-conjugated system. The 'H,13C, ^^H correlated spectroscopy (COSY), edited-heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) spectra acquired in methanol-d4 solvent revealed that the molecule consists of two separated spin systems, and these two-spin system coexist as a keto-enol tautomer (Figure 9). The1H NMR data (Table 2) showed the presence of the first spin system containing two symmetrical 1,2,3-trisubstituted aromatic rings [8H 7.87 (t, J = 8.4 Hz), 6.89 (d, J = 8.0 Hz), 6.75 (d, J = 8.4 Hz)] and a N-methyl proton [8H 4.14 (s)]. The second spin system separated from the first spin system was composed of two symmetrical 1,2,3-trisubstituted aromatic rings [8H 7.78 (t, J = 8.1 Hz), 7.61 (d, J = 8.7 Hz), 7.13 (d, J = 7.5 Hz)] and a N-methyl proton [8H 1.59 (s)].

[0216] Table 2.13C, and HMBC NMR Spectroscopic Data of la and lb (800 / 200 MHz, in methanol-d4).

[0217] The COSY spectrum supported these cross-peak correlations rings in the first and second individual ring system. Comparing the integral value of protons in the aromatic ring system in the first ring and the second ring system, the ratio of existence was calculated as 73.24% for la having the first ring system, and 26.76% for lb having the second ring system.

[0218] The structure of la and lb were established based on the HMBC experiment. The observed ^^H COSY and HMBC correlations are marked by bold lines and arrows, respectively in Figure 9 for structures la and lb. In structure la, strong HMBC correlation between H-7 (5H 7.87) and C-9 (8c 170.30) together with weak correlation between H-8 (8H 6.75) and C-9 (5c 170.30) suggested the location of carbonyl carbon at C-9. HMBC correlations from H-8 (5H 6.75) and H-6 (5H 6.89) to C-9a (5c 136.95) revealed that the quaternary carbon at C-9a positioned next to carbonyl carbon C-9 (5c 170.30). HMBC correlations from H-7 (5H 7.86) and methyl proton at 5-CHs (5H 4.14) to C-5a (5c 136.27) revealed the location of the quaternary carbon at C-5a. In structure lb, strong HMBC correlation between H-7 (5H 7.78) and C-9 (5c 156.22) together with weak correlation between H-8 (5H 7.13) and C-9 (5c 156.22) suggested the location of C-9 bearing a hydroxy group. HMBC correlations from H-8 (5H 7.13) and H-6 (5H 7.61) to C-9a (5c 135.41) revealed that the quaternary carbon at C-9a positioned next to hydroxyl carbon C-9 (5c 156.22). HMBC correlations from H-7 (6n 7.78) and methyl proton at 5-CH3 (5H 1.59) to C-5a (5C145.15) revealed the location of the quaternary carbon at C-5a.

[0219] The chemical shift of la showed high accordance with those for the pyocyanin, blue pigment isolated from Podophyllum peltatum (Jancheva, M., 2021, J. Am. Chem. Soc., 143, 8344). The NMR data of pyocyanin was also acquired using methanol-d4 NMR solvent. Comparing with pyocyanin, la possesses symmetrical structure by having carbonyl group at position C-l and C-9 side both, and in the same methanol solvent system, exchange the format of tautomer as enol forms as lb. Also, one more NMR solvent system, in acetone-de, was applied to confirm the format of tautomer as shown in lb' in Figure 9. Compound 1 was not dissolved well in acetone solvent as shown in Figure 10, however, in the acetone solvent, tautomerism was hindered and revealed the small amount of dissolved material was enol form. Thus, the structure of compound 1, 9-hydroxy-5-methylphenazin-l(5H)-one, was established as shown in Figure 9 with its tautomer forms.

[0220] The materials and methods used are described herein.

[0221] General Experimental Procedures :Optical rotation of a compound in water was measured on an AUTOPOL® III (AUTOPOL® IV) automatic polarimeter (Rudolph Research Analytical, Hackettstown, NI, USA) using a 10 cm cell at 25 °C using a sodium (tungsten-halogen) lamp (589 nm). UV spectrum was recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd., Surrey, UK). ID and 2D NMR spectra were recorded on a Bruker Avance III HD 800 MHz equipped with a 3 mm TCI CryoProbe (Bruker, Billerica, MA, USA). HRESIMS was performed with an Agilent 6545XT AdvanceBio Q-TOF MS (Agilent Technologies, Inc., Santa Clara, CA, USA), which was equipped with an electrospray ionization (ESI) interface. Solid-phase extraction (SPE) was performed with Oasis HLB 6 cc Vac Cartridge (500 mg, 60 pm) and Oasis HLB 35 cc Vac Cartridge (6 g, 60 pm) (Waters Corporation, Milford, MA, USA). Semipreparative HPLC separations were performed using an Agilent 1260 system equipped with a photodiode array detector (Agilent Technologies, Santa Clara, CA, USA), by a YMC Hydrosphere Cis column (10 mm x 250 mm, 5 pm, YMC CO., Ltd., Kyoto, Japan) with a compatible guard column at a column temperature of 30 °C. HPLC data was evaluated by Agilent OpenLab CDS Chemstation edition software. Solvents for SPE fractionation and HPLC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deuterated solvents for NMR analysis were purchased from Cambridge Isotope Laboratories (Cambridge, MA, USA).

[0222] Microfractionation and Isolation of a Compound: The isolation scheme and fractionation tree of compound 1 is detailed in Figure 3. Representative images of 9005BA being cultured are shown in Fig. 11. Culture supernatant was concentrated in vacuo and then lyophilized. After evaporation of solvent, the residual (977.4 mg) was dissolved in MeOH-TLO (3:7, v / v) and loaded onto 4 Oasis HLB SPE cartridges (500 mg, 60 pm), eluted with 5mL of MeOH-HzO (3:7, v / v) for each cartridge, to yield fraction 9005BA_30M. Subsequently, fraction 9005BA_100M was washed out with 5mL of 100% MeOH for each cartridge. 9005BA_100M was further microfractionated using semi-preparative HPLC (YMC Hydrosphere C18 column (10 mm x 250 mm, 5 pm) with a compatible guard column at a column, MeCN-H2O containing 0.1% formic acid, 5:95 > 100:0, v / v, 4mL / min, monitored at 203 nm, 254 nm, 320 nm, and 365 nm) to collect fractions into 96-well microplate.

[0223] And then, for large-scale separation, the residual (24.0 g) was dissolved in MeOH- UO (3:7, v / v) and loaded onto 4 Oasis HLB SPE cartridges (6 g, 60 pm), eluted with 70mL of MeOHMEO (3:7, v / v) for each cartridge, to yield fraction 9005BA F1. Subsequently, fraction 9005BA F2 was acquired by eluting MeOH-TLO (7:3, v / v) into the cartridge before dark blue band washed out. Lastly, fraction 9005BA_F3 was washed out with lOOmL of 100% MeOH for each cartridge. 9005BA F3 was further separated using semi-preparative HPLC (YMC Hydrosphere Cis column (10 mm x 250 mm, 5 pm) with a compatible guard column at a column, MeCISMLO containing 0.1% formic acid, 5:95— >100:0, v / v, 4mL / min, monitored at 203 nm, 254 nm, 320 nm, and 365 nm) to afford 1 (12.1 mg, tR 18.7 min). Compound 1: blue pigment; [CI] D5= +6 (c 0.8, MeOH); UV (MeOH)max (log e) 271 (4.27), 318 (3.54), 371 (3.15), 458 (2.99), 785 (2.95) nm; ’H and ”C NMR, see Table 2; HRES1MS m / z 227.0826 [M + H]+(calcd. for C13H11N2O2, 227.0810).

[0224] LC-MS Analysis

[0225] The compound (1) was dissolved with MeOH for LC-MS analysis. The LC-MS analysis was performed on an Agilent 1290 Infinity II UHPLC system coupled to an Agilent 6545XT QTOFMS (Agilent Technologies, Santa Clara, CA, USA), which was equipped with a Dual AJS ESI Ion Source (Agilent Technologies). Chromatographic separations were performed on a Zorbax Eclipse XDB-C18 (100 mm x 2.1 mm, 1.8 pm) column coupled with Zorbax Eclipse XDB-C18 (5 mm x 2.1 mm, 1.8 pm) guard column. The mobile phase was comprised of H2O (A) and acetonitrile (B), both of which were acidified with 0.1% formic acid. The column temperature and sample organizer were maintained at 40 °C and 15 °C, respectively. A stepwise gradient method at a constant flow rate of 0.4mL / min was used to elute the column with the following conditions: 5-5% B (0.0-0.5 min); 5-25% B (0.5-6.0 min); 25-100% B (6.0-9.0 min); and 100-100% B (9.0-10.5 min), followed by a return to the starting conditions at 10.6 min and 1.4 min of reconditioning the column (total runtime of 12.0 min). Analyses of the samples (2.0 pL, injection volume) were performed in the positive ion mode in both profile and centroid mode. The ESI conditions were set as follows: the capillary voltage was 3.5 kV, the nozzle voltage was 200 V for negative mode, the fragmentation voltage was 125 V, the drying gas temperature and flow were set to 150 °C and 10 L / min, respectively, and sheath gas temperature and flow were 375 °C and 11 L / min, respectively, and the nebulizer was operating at 35 psi. Nitrogen served both as the nebulizer gas and the dry gas. The Auto-MS / MS mode was used with an MS range of m / z 100-1700 and an MS2 range of m / z 50-1700, at 7 spectra / s and 5 spectra / s, respectively. The narrow isolation (~ 1.3 m / z) width was used. The collision energy was set by the formula based on the m / z and charge of the precursor (condition 1 : slope of 4.0 and an offset of 20, condition 2: slope of 4.5 and an offset of 30, , condition 3: slope of 5.0 and an offset of 40). The maximum precursors per cycle are set to 5, with the absolute precursor threshold set to 500 (relative threshold 0.015%) and active exclusion after 3 scans and released after 0.1 min were performed. MassHunter Workstation Acquisition B.10.00 software and MassHunter Qualitative Analysis 10.0 software (Agilent Technologies) were used for acquiring and processing MS data.

[0226] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A compound represented by Formula (I), or a tautomer, ion, salt, and / or solvate thereof,Formula (I).

2. The compound of claim 1, wherein the compound is an inner salt.

3. The compound of claim 1, wherein the compound is zwitterionic.

4. The compound of any one of claims 1-3, wherein the compound is protonated.

5. The compound of any one of claims 1-3, wherein the compound is deprotonated.

6. The compound of any one of claims 1-5, wherein the compound is selected from the group consisting oftautomers, salts, and solvates thereof.

7. A pharmaceutical formulation comprising a compound of any one of claims 1-6.

8. A pharmaceutical formulation, comprising:a therapeutically effective amount of a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,Formula (I); and a pharmaceutically acceptable excipient.

9. The pharmaceutical formulation of claim 8, wherein the pharmaceutical formulation is in the form of a particle, bead, tablet, capsule, or pill.

10. The pharmaceutical formulation of claim 8, wherein the pharmaceutical formulation is in the form of a lotion, liquid, aerosol, or gel.

11. The pharmaceutical formulation of any one of claims 8-10, wherein the pharmaceutical formulation further comprises one or more antimicrobial agents.

12. The pharmaceutical formulation of any one of claims 8-11, wherein the pharmaceutical formulation further comprises one or more antibiotic agents.

13. The pharmaceutical formulation of any one of claims 8-12, wherein the pharmaceutical formulation further comprises one or more antifungal agents.

14. A method of treating or preventing a microbial infection in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a compound of Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,Formula (I).

15. The method of claim 14, wherein the microbial infection is a bacterial infection.

16. The method of claim 14, wherein the subject is exposed to or infected with a bacterium.

17. The method of any one of claims 14-16, wherein the subject is exposed to or infected with a gram-negative bacterium.

18. The method of any one of claims 14-17, wherein the subject is exposed to or infected with a bacterium from an Acinetobacter species, a Vibrio species, or an Escherichia species.

19. The method of any one of claim 15-18, wherein the bacterium is selected from the group consisting of Acinetobacter baumannii, Vibrio cholerae, and Escherichia coli.

20. The method of any one of claims 14-16, wherein the subject is exposed to or infected with a bacterium selected from the group consisting of an Acinetobacter species, Enterococcus species, Staphylococcus species, Klebsiella species, Pseudomonas species, Enterobacter species, Burkholderia species, and any combination thereof.

21. The method of any one of claims 14-16, wherein the subject is exposed to or infected with a bacterium or pathogen selected from the list consisting of Acinetobacter baumannii, Klebsiella pneumoniae , Escherichia coli, Mycobacterium tuberculosis, Salmonella species, Shigella species, Enterococcus faecium, Pseudomonas aeruginosa, Enterobacter species, Burkholderia species, Neisseria gonorrhoeae, Citrobacter species, Proteus species, Serratia species, Group A Streptococci, Group B Streptococci, Streptococcus pneumoniae, Haemophilus influenzae, Morganella species, Vibrio cholerae, and any combination thereof.

22. The method of any one of claims 14-19 and 21, wherein the subject is exposed to or infected with a bacterium selected from the group consisting of Acinetobacter baumannii, Escherichia coli, and / or Vibrio cholerae.

23. The method of any one of claims 14-16, wherein the subject is exposed to or infected with a gram-positive bacterium.

24. The method of any one of claims 14-23, wherein the subject is exposed to or infected with a bacterium that is resistant to one or more antibiotic agents.

25. The method of claim 24, wherein the one or more antibiotic agents are selected from the group consisting of a bacteriostatic antibiotic and a bactericidal antibiotic.

26. The method of claim 24 or 25, wherein the one or more antibiotic agents are selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, and tebipenem.

27. The method of any one of claims 14-26, wherein the method further comprises the step of administering one or more additional antibiotic agents.

28. The method of claim 27, wherein the one or more additional antibiotic agents is selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin,metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, tebipenem, and combinations thereof.

29. The method of claim 14, wherein the microbial infection is a fungal infection.

30. The method of claim 29, wherein the subject is exposed to or infected with a fungal pathogen selected from the group consisting of Cryptococcus neoformans, Candida auris. Aspergillus fumigatus, Candida albicans, Nakaseomyces Candida glabrala, Histoplasma species, a Eumycetoma causative agent, Mucorales species, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Lomentospora prolificans, Coccidioides species, Pichia kudriavzeveii Candida krusei, Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, Paracoccidioides species, and any combination thereof.

31. The method of claim 29 or 30, wherein the subject is exposed to or infected with a fungal pathogen that is resistant to one or more antifungal agents selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

32. The method of any one of claims 14 and 29-31, wherein the method further comprises the step of administering one or more antifungal agents.

33. The method of claim 32, wherein the one or more antifungal agents is selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

34. The method of any one of claims 14-33, wherein the subject is exposed to or infected with a human pathogen.

35. The method of any one of claims 14-33, wherein the subject is exposed to or infected with an agricultural pathogen.

36. The method of any one of claims 14-35, wherein the subject is at risk of, exhibiting symptoms of, or diagnosed with sepsis, pneumonia, abscesses, furuncles, cellulitis, folliculitis, bloodstream infections, medical device infections, pneumonia, osteomyelitis, skin and soft tissue infections, endocarditis, impetigo, septic arthritis, brain abscess, bum wounds, venous ulcers, diabetic foot ulcers, surgical wounds, post-operation infections, carbuncles, meningitis, bacteremia, necrotizing pneumonia, endocarditis, osteomyelitis, or necrotizing fasciitis.

37. The method of any one of claims 14-36, wherein the step of administering comprises topical, ophthalmic, and / or optic administration of a therapeutically effective amount of a compound of Formula (I), or pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, to the subject.

38. The method of any one of claims 14-36, wherein the step of administering comprises internally administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof.

39. The method of any one of claims 14-36, wherein the compound of Formula (I), or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, is internally administered by a means selected from the group consisting of oral, intravenous, intramuscular, intraperitoneal, subcutaneous, mucosal, percutaneous, sublingual, intrathecal, intraocular, inhalation, rectal, and nasal administration.

40. A method of treating or preventing a microbial infection in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of an extract or one or more compounds in an extract, wherein the extract comprises at least one compound derived from a Streptomyces bacterium; andat least one of the one or more compounds is a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,Formula (I).

41. The method of claim 40, wherein the microbial infection is a bacterial infection.

42. The method of claim 40, wherein the subject is exposed to or infected with a bacterium.

43. The method of any one of claims 40-42, wherein the subject is exposed to or infected with a gram-negative bacterium.

44. The method of claim 43, wherein the subject is exposed to or infected with a bacterium from an Acinetobacter species, a Vibrio species, or an Escherichia species.

45. The method of claim 44, wherein the bacterium is selected from the group consisting of Acinetobacter baumannii, Vibrio cholerae, and Escherichia coli.

46. The method of any one of claims 40-42, wherein the subject is exposed to or infected with a bacterium selected from the group consisting of an Enterococcus species, Staphylococcus species, Klebsiella species, Acinetobacter species, Pseudomonas species, Enterobacter species, Burkholderia species, and any combination thereof.

47. The method of any one of claims 40-42, wherein the subject is exposed to or infected with a bacterium or pathogen selected from the list consisting of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, Mycobacterium tuberculosis, Salmonella species, Shigella species, Enterococcus faecium, Pseudomonas aeruginosa, Enterobacter species,Neisseria gonorrhoeas, Staphylococcus aureus, Citrohacter species, Proteus species, Serratia species, Group A Streptococci, Group B Streptococci, Streptococcus pneumoniae, Haemophilus influenzae, Morganella species, Vibrio cholerae, any generation thereof, and any combination thereof.

48. The method of any one of claims 40-45 and 47, wherein the subject is exposed to or infected with a bacterium selected from the group consisting of Acinetobacter baumannii, Escherichia coli, and / or Vibrio cholerae.

49. The method of any one of claims 40-42, wherein the subject is exposed to or infected with a gram-positive bacterium.

50. The method of any one of claims 40-49, wherein the subject is exposed to or infected with a bacterium that is resistant to one or more antibiotic agents.

51. The method of claim 50, wherein the one or more antibiotic agents are selected from the group consisting of a bacteriostatic antibiotic and a bactericidal antibiotic.

52. The method of claim 50 or 51, wherein the one or more antibiotic agents are selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, and tebipenem.

53. The method of any one of claims 40-52, wherein the method further comprises the step of administering one or more additional antibiotic agents.

54. The method of claim 53, wherein the one or more additional antibiotic agents is selected from the group consisting of albocycline, fosfomycin, oxacillin, rifampicin, streptomycin, tetracycline, tigecycline, doxycycline, minocycline, clindamycin, azithromycin, clarithromycin, erythromycin, linezolid, sulfamethoxazole, tobramycin, gentamicin, amikacin, amoxicillin, cefazolin, meropenem, ciprofloxacin, levofloxacin, moxifloxacin, vancomycin, daptomycin, metronidazole, carbapenem, cephalosporin, fluoroquinolone, vancomycin, methicillin, macrolide, ampicillin, penicillin, imipenem, meropenem, ertapenem, doripenem, panipenem, biapenem, tebipenem, and any combination thereof.

55. The method of claim 40, wherein the microbial infection is a fungal infection.

56. The method of claim 55, wherein the subject is exposed to or infected with a fungal pathogen selected from the group consisting of Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces- Candida glabrata, Histoplasma species, a Eumycetoma causative agent, Mucorales species, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Lomentospora prolificans, Coccidioides species, Pichia kudriavzeveii / Candida krusei. Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, Paracoccidioides species, and any combination thereof.

57. The method of claim 55 or 56, wherein the subject is exposed to or infected with a fungal pathogen that is resistant to one or more antifungal agents selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

58. The method of any one of claims 40 and 55-57, wherein the method further comprises the step of administering one or more antifungal agents.

59. The method of claim 58, wherein the one or more antifungal agents is selected from the group consisting of anidulafungin, caspofungin, fluconazole, micafungin, voriconazole, and any combination thereof.

60. The method of any one of claims 40-59, wherein the subject is exposed to or infected with a human pathogen.

61. The method of any one of claims 40-59, wherein the subject is exposed to or infected with an agricultural pathogen.

62. The method of any one of claims 40-59, wherein the subject is at risk of, exhibiting symptoms of, or diagnosed with sepsis, pneumonia, abscesses, furuncles, cellulitis, folliculitis, bloodstream infections, medical device infections, pneumonia, osteomyelitis, skin and soft tissue infections, endocarditis, impetigo, septic arthritis, brain abscess, bum wounds, venous ulcers, diabetic foot ulcers, surgical wounds, post-operation infections, carbuncles, meningitis, bacteremia, necrotizing pneumonia, endocarditis, osteomyelitis, or necrotizing fasciitis.

63. The method of any one of claims 40-62, wherein the step of administering comprises topical, ophthalmic, and / or optic administration of a therapeutically effective amount of a compound of Formula (I), a tautomer or ion thereof, or pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, to the subject.

64. The method of any one of claims 40-62, wherein the step of administering comprises internally administering to the subject a therapeutically effective amount of a compound of Formula (I), a tautomer or ion thereof, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof.

65. The method of any one of claims 40-62, wherein the compound of Formula (I), a tautomer or ion thereof, or a pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, is internally administered by a means selected from the group consisting of oral, intravenous, intramuscular, intraperitoneal, subcutaneous, mucosal, percutaneous, sublingual, intrathecal, intraocular, inhalation, rectal, and nasal administration.

66. A method of producing an extract comprising the steps of: providing a Streptomyces bacterium;culturing the Streptomyces bacterium in a culture medium under conditions conducive to provide overexpression of the extract; and optionally isolating the extract from the culture medium.

67. The method of claim 66, wherein the extract comprises a compound represented by Formula (I), or a tautomer, ion, pharmaceutically acceptable salt, and / or pharmaceutically acceptable solvate thereof,Formula (I).

68. The method of claim 66 or 67, where the step of isolating the extract from the culture medium comprises lyophilizing the culture medium and performing activity guided fractionation.

69. The method of claim 68, wherein the activity guided fractionation comprises: loading the lyophilized culture medium onto a solid phase sorbent; performing a first fractionation step comprising at least two sequential solid phase extractions of the loaded solid phase sorbent with aqueous methanol, with each sequential extraction comprising a higher concentration of methanol to a final sequential extraction of 100% methanol; examining the activity of the fractions and selecting the fraction with the greatest activity for further fractionation; performing a second fractionation step comprising reverse-phase liquid chromatography of the fraction with the greatest activity; and examining the activity of the fractions and selecting the fraction with the greatest activity.

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