Catechol and benzenediol antimicrobial, antibiotic adjuvant and therapeutic compounds

Abstract

The present disclosure provides pharmaceutical compositions and methods for treating microbial infections by administering synergistic combinations of catechol and benzenediol derivatives and antibiotics. These compositions enhance microbial susceptibility to antibiotics, overcoming drug resistance and improving treatment outcomes. The present disclosure also provides pharmaceutical compositions and methods for treating microbial infections by administering catechol and benzenediol derivatives acting as standalone antimicrobials. The invention encompasses formulations comprising catechol and benzenediol derivatives in various forms, including oral, injectable, and topical administration. Additionally, the disclosure includes controlled-release mechanisms and dosage regimens optimized for therapeutic efficacy, including co-administration protocols with bicarbonate buffers or other agents. The compositions are designed for use in human and veterinary applications, providing effective solutions for managing resistant microbial strains and managing disorders related to polyamine detoxification enzymes in human and animals.

Description

CATECHOL AND BENZENEDIOL ANTIMICROBIAL, ANTIBIOTIC ADJUVANT AND THERAPEUTIC COMPOUNDSFIELD

[0001] The present application relates to the methods of treating microbial infections or human or animal diseases or disorders using, for example, catechol-based and benzenediol-based compounds. In particular the application relates to the use of catecholbased and benzenediol-based compounds to treat bacterial and fungal infections and polyamine detoxification (SATI )-related diseases and disorders.BACKGROUND

[0002] Bacteria have developed resistance to all currently known clinical antibiotics posing a large risk to public health. The spread of antimicrobial resistance (AMR) has exacerbated the threat of bacterial infections, with 1.27 million deaths being attributed to bacterial AMR in 2019, alone (Murray, Ikuta et al. 2022). As AMR continues to spread, the discovery and development of antimicrobials with novel chemical structures or cellular targets has drastically slowed (Beyer and Paulin 2020, Miethke, Pieroni et al. 2021 ).

[0003] Bacteria encounter a myriad of chemicals at the infection site that may alter their antibiotic susceptibility or virulence (El-Halfawy and Valvano 2013, Perry, Meirelles et al. 2022). The potential effects of such chemicals are not typically captured in standard antibiotic susceptibility assays, whose conditions follow the guidelines issued by standardizing agencies such as the Clinical & Laboratory Standards institute (CLSI). One important class of chemicals present at the infection sites are polyamines, especially since they are overproduced by the host during infection (Zhang, Wang et al. 2000). Natural polyamines, such as spermine, spermidine, and putrescine, are small polycationic molecules found in almost all living organisms and are critical for their viability (Agostinelli, Arancia et al. 2004). During infections, the host's polyamine biosynthesis is upregulated in regenerating tissues at the inflammatory site; polyamines accumulate in the lungs during pneumonia infections, suggesting a role in suppressing infection (Zhang, Wang et al. 2000, Liao, Lasbury et al. 2006, Bjelakovic, Stojanovic et al. 2010). Additionally, polyamines play an immunomodulatory role, where spermine exhibits macrophage suppression alongside anti-inflammatory activity (Zhang, Wang et al. 2000).

[0004] Certain polyamines can alter antibiotic susceptibility in a wide range of Grampositive and Gram-negative bacteria (Kwon and Lu 2006, Kwon and Lu 2007, El-Halfawy and Valvano 2012, El-Halfawy and Valvano 2013, El-Halfawy and Valvano 2014, Hasan, Pottenger et al. 2022). For example, certain polyamines increased resistance of Pseudomonas aeruginosa and Burkholderia cenocepacia to antibiotics including polymyxins and fluoroquinolones (Kwon and Lu 2006, El-Halfawy and Valvano 2013, El- Halfawy and Valvano 2014), conversely, others increased the susceptibility of Staphylococcus aureus and enteric bacteria, such as Escherichia coli, to 0-lactam antibiotics (Kwon and Lu 2007).

[0005] For Gram-negative bacteria, the amount of therapeutic options is limited, making it one of the largest public health problems; consequentially Gram-negative bacteria make up the majority of World Health Organizations (WHO) priority pathogens (Nikaido 2003, Ak, Batirel et al. 2011 , WHO 2017). Gram-negative bacteria have lipopolysaccharides, an integral component of the outer membrane which prevents antibiotic permeation. Klebsiella pneumoniae is a Gram-negative bacteria listed as one of the WHO priority pathogens gaining international concern due to the high prevalence of hypervirulent and drug resistant strains (WHO 2017) With the constant rise in AMR and the inadequate antibiotic drug discovery pipeline ((WHO) 2019), there is a dire need for new strategies to combat Gram-negative resistant strains (Antimicrobial Resistance 2022).

[0006] Newer classes of macrolides such as azithromycin are clinically advantageous due to their extensive and rapid distribution into tissues and intracellular compartments yet minimal accumulation in fat and muscle (Kohno 2003). The pharmacokinetics of macrolides, in addition to being orally bioavailable make them a highly prescribed antibiotic class in America (Lalak and Morris 1993, Kohno 2003, Magill, O'Leary et al. 2021 ). Typically, macrolides are used to treat Gram-positive infections due to their poor penetration of the outer membrane (Wilson 2009). However, macrolide testing under certain infection-relevant conditions show increased potency against Gram-negative bacteria. E. coli in the presence of the physiologically relevant buffer bicarbonate exhibit heightened sensitivity to macrolides; bicarbonate interferes with the proton motive force increasing transport of the drug through the inner membrane (Farha, French et al. 2018).Understanding the effects of macrolides against Gram-negative bacteria such as K. pneumoniae may expand its spectrum to this group of bacteria where clinical need is high.

[0007] For Gram-positive bacteria, Staphylococcus aureus has become a leading cause of hospital and community-acquired infections, resulting in considerable morbidity and mortality rates (WHO 2017). S. aureus can cause multiple infections including: skin and soft tissue infections (SSTI), infective endocarditis, osteomyelitis, prosthetic-device infections, community-acquired pneumonia, and other systemic infections (Taylor and Unakal 2023). Some strains of S. aureus have developed resistance to [3-lactam antibiotics, coined methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains have spread rapidly, and in 2019, it was estimated that more than 100 000 deaths were directly attributable to MRSA infections (Murray, Ikuta et al. 2022). Notably, the MRSA strain USA300 has become the most common circulating strain of S. aureus; it is a major cause of SSTIs and has been recovered from cases of endocarditis, community-acquired pneumonia, and other systemic infections (Tenover and Goering 2009, Planet, LaRussa et al. 2013).

[0008] Most bacteria can produce polyamines endogenously, with S. aureus being one of few exceptions that cannot produce endogenous spermine or spermidine (Joshi, Spontak et al. 2011 , Li, Maezato et al. 2019, Seravalli, Portugal et al. 2023). Although polyamines are required for cell functions, at high concentrations these compounds become toxic and are bactericidal towards most S. aureus strains; however, USA300 is resistant to exogenous spermine and spermidine (Joshi, Spontak et al. 2011 ). Polyamines are overproduced at the site of infection (Yao and Lu 2014), and can be found at millimolar concentrations in eukaryotes (Sagar, Tarafdar et al. 2021 ), suggesting that they play a role in aiding the immune system in clearing infections. USA300’s virulence and prevalence has partially been attributed to the acquisition of the arginine catabolic mobile element (ACME) (Diep, Stone et al. 2008, Planet, LaRussa et al. 2013, Thurlow, Joshi et al. 2013). The ACME locus was horizontally transferred to S. aureus from Staphylococcus epidermidis (Planet, LaRussa et al. 2013), and encodes speG a member of the GCN5-related N- acetyltransferase (GNAT) family with homology to the A / 1-spermine / spermidine acetyltransferase of Escherichia coli (Joshi, Spontak et al. 2011 , Planet, LaRussa et al. 2013, Thurlow, Joshi et al. 2013, Li, Maezato et al. 2019). SpeG acetylates spermine andspermidine, rendering them less toxic to the cell, resulting in the observed resistance of bacteria to exogenous polyamines. The AMCE also encodes the arginine-deaminase (arc) system which converts arginine to ornithine while producing ATP and ammonia allowing USA300 to withstand acidic environments, such as those found on the skin (Thurlow, Joshi et al. 2013). Ornithine produced via the ACME-Arc system can then be converted to polyamines, and the ACME-Arc system was shown to drive excessive host polyamine biosynthesis in a murine SSTI model (Thurlow, Joshi et al. 2013). Therefore, by encoding speG, S. aureus can resist the excessive host polyamine production, increasing its virulence. SpeG also seems to be involved in increasing biofilm formation; exogenous polyamines increase biofilm formation, an effect lost in a AspeG mutant (Planet, LaRussa et al. 2013).

[0009] Accordingly, there is a need to develop new and effective antibacterial treatments, particularly against resistant bacterial strains.SUMMARY

[0010] The application discloses methods of using catechol-based and benzenediol- based compounds to improve the efficacy of antibiotics (as antibiotic adjuvant) or to suppress microbial (such as bacterial or fungal) virulence and eradicate and inhibit their growth (as standalone antimicrobial compounds) for the treatment of microbial infections.

[0011] Therefore, the present application includes a method of treating a microbial infection in a subject in need thereof, comprising administering to the subject an effective amount of an antibiotic and an effective amount of one or more compounds selected from a compound of Formula IV, V, and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V, or VI is as follows:(IV);(V); orwherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

[0012] In some embodiments, the above method of the application includes administering the antibiotic and the one or more compounds of the application to the subject in a pharmaceutical composition comprising said antibiotic, the one or more compounds of the application or a pharmaceutically acceptable salt and / or solvate thereof, and a pharmaceutically acceptable carrier or vehicle.

[0013] The present application also includes a method of treating a microbial infection in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds selected from a compound of Formula IV, V and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V or VI is as follows:(V); orwherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

[0014] In some embodiments, the above method of the application includes administering the one or more compounds of Formula IV, V and VI to the subject in a pharmaceutical composition comprising the one or more compounds of Formula IV, V and VI or a pharmaceutically acceptable salt and / or solvate thereof, and a pharmaceutically acceptable carrier or vehicle.

[0015] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole.DRAWINGS

[0016] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0017] Figure 1 shows the results of a high throughput screen to identify potent spermine synergists and a putative target. A) Workflow undertaken to identify a chemical inhibitor of polyamine resistance. B) Replica plot of chemical screens, performed to identify an inhibitor of polyamine detoxification in S. aureus USA300. C) Chemogenomic screen of sub-inhibitory concentrations of spermine against the Nebraska Transposon Mutant Library(NTML). D) Representative checkerboard assay of exemplary compound IV-1 and spermine against S. aureus USA300. E) Representative checkerboard assay of exemplary compound IV-1 and spermine against AspeG. F) Representative checkerboard assay of V- 1 and spermine against USA300 and G) Representative checkerboard assay of exemplary compound V-1 and spermine against AspeG.

[0018] Figure 2 shows the results of the representative checkerboard assays of spermine and exemplary compound V-1 against S. aureus A) USA300 JE2, B) CMRSA-10 C) AspeG, D) COL, and E) NCTC8325.

[0019] Figure 3 shows A / 1-acetyltransferase activity of SpeG with spermine, spermidine, and putrescine. Colorimetric assay performed using purified SpeG and a heat- inactivated enzyme control with A and B) spermine, C and D) spermidine, and E and F) putrescine as substrates validates sperm ine / spermidine acetyltransferase activity of SpeG. Substrate saturation curves (B, D, and F) were obtained by fitting the Michaelis-Menten equation or allosteric sigmoidal equation in Graphpad Prism 10. Results are grouped from three independent experiments (A, n=3) and two independent experiments (B and C, n=4) and reported as mean + standard error of the mean G) Table of estimated SpeG kinetic parameters towards spermine and spermidine.

[0020] Figure 4 shows V-1 activity as a SpeG inhibitor. A-H) Colorimetric enzymatic assay of SpeG activity with increasing acetyl-CoA concentrations in the presence of V-1 at A) 0 pM, B) 500 pM, C) 750 pM, D) 1000 pM, E) 1500 pM, F) 2000 pM, G) 2500 pM, and H) 3000 pM. I) Substrate saturation curve of SpeG with a fixed concentration of spermine and variable acetyl-CoA concentrations in the presence of V-1 . Data was fit with the competitive inhibitor equation in GraphPad Prism 10. J) Colorimetric enzymatic assays conducted with 0.75 mM Acetyl Coenzyme A and 1500 pM spermine in the reaction mixture to evaluate the inhibition of V-1 (n=3). Data is represented as mean + SEM for three independent experiments. K) Lineweaver-Burk transformation of data in I) exhibiting an increase in KM and constant Vmax with increasing V-1 , suggesting competitive inhibition. Data is represented as mean + SEM for three independent experiments n=3.

[0021] Figure 5 shows A) DiSC3(5) membrane permeabilization assay of V-1 against S. aureus USA300. Results represented as mean + SEM for three independent experiments (n=8).

[0022] Figure 6 shows A) the acetyltransferase activity of SAT 1 was measured under steady-state condition with an excess of AcCoA (0.25 mM) and variable concentrations of spermine, via a colorimetric enzymatic assay. B) Estimated kinetic parameters of SAT1 acetylation of spermine. Data is represented as mean + SEM from two independent experiments (n=4). C) Substrate saturation curves of SAT1 with a fixed concentration of spermine and variable Acetyl-Coenzyme A concentrations in the presence of V-1 . Data is represented as mean + SEM from two independent experiments (n=2) and was fit with the competitive inhibitor equation in Graphpad Prism 10.

[0023] Figure 7 shows effects of A) V-1 (n=16) on the lysis of sheep red blood cells. V-1 shows no observable lysis at the tested concentration range. Data is represented as mean + SEM for three independent experiments.

[0024] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.DESCRIPTION OF VARIOUS EMBODIMENTSI. Definitions

[0025] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0026] All features disclosed in the specification, including the claims, abstract, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise.

[0027] As used in this application and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0028] The term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0029] The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and / or steps.

[0030] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0031] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds.

[0032] In embodiments comprising an “additional” or “second” component or effect, such as an additional or second compound, the second compound as used herein is different from the other compounds or first compound. A “third” compound is different from the other, first, and second compounds, and further enumerated or “additional” compounds are similarly different.

[0033] When “one or more” molecules or materials are referenced (such as one or more compound), it is understood that this is in reference to the “type” or “identity of the molecule or material. Therefore, a second molecule or material is different from the one, or first, molecule or material. Similarly, a “third” molecule or material is different from the one, first, and second molecules or materials, and further enumerated or “additional” molecules or materials are similarly different.

[0034] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present. The term “and / or” with respect to enantiomers, prodrugs, salts and / or solvates thereof means that the compounds of the application exist as individual enantiomers, prodrugs, salts and hydrates, as well as a combination of, for example, a salt of a solvate of a compound of the application.

[0035] The term “compound of the application” or “compound of the present application” and the like as used herein refers to a compound of Formula IV, V or VI, or a salt and / or solvate thereof.

[0036] The term “composition of the application” or “composition of the present application” and the like as used herein refers to a composition comprising one or more compounds of the application and a carrier, and optionally an antibiotic.

[0037] The term “method of the application” as used herein refers to the method of treating a bacterial infection as described herein.

[0038] The term “suitable” as used herein means that the selection of the particular compound or conditions would depend on the specific synthetic manipulation to be performed, the identity of the molecule(s) to be transformed and / or the specific use for the compound, but the selection would be well within the skill of a person trained in the art.

[0039] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0040] The term “protecting group” or “PG” and the like as used herein refers to a chemical moiety which protects or masks a reactive portion of a molecule to prevent side reactions in those reactive portions of the molecule, while manipulating or reacting a different portion of the molecule. After the manipulation or reaction is complete, the protecting group is removed under conditions that do not degrade or decompose the remaining portions of the molecule. The selection of a suitable protecting group can be made by a person skilled in the art. Many conventional protecting groups are known in the art, for example as described in “Protective Groups in Organic Chemistry” McOmie, J.F.W. Ed., Plenum Press, 1973, in Greene, T.W. and Wuts, P.G.M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3rdEdition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).

[0041] The term “inert organic solvent” as used herein refers to a solvent that is generally considered as non-reactive with the functional groups that are present in the compounds to be combined together in any given reaction so that it does not interfere with or inhibit the desired synthetic transformation. Organic solvents are typically non-polar and dissolve compounds that are non soluble in aqueous solutions.

[0042] The term “alkyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cni-n2”. For example, the term Ci-ioalkyl means an alkyl group having 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. All alkyl groups are optionally fluoro-substitued unless otherwise indicated.

[0043] The term “alkenyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkyl groups containing at least one double bond. The number of carbon atoms that are possible in the referenced alkylene group are indicated by the prefix “Cni-n2”. For example, the term C2-ealkenyl means an alkenyl group having 2, 3, 4, 5 or 6 carbon atoms and at least one double bond. All alkenyl groups are optionally fluoro-substitued unless otherwise indicated.

[0044] The term “alkynyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkynyl groups containing at least one triple bond. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cni-n2”. For example, the term C2-ealkynyl means an alkynyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0045] The suffix “ene” at the end of a group (for example “alkylene”, “heterocycloalkylene” and “cycloalkylene”) means that the group is bivalent, that is that it is bonded to two variables each on a different end of or location on the group.

[0046] The term “cycloalkyl,” as used herein, whether it is used alone or as part of another group, means a saturated carbocyclic group containing one or more rings. The number of carbon atoms that are possible in the referenced cycloalkyl group are indicated by the numerical prefix “Cni-n2”. For example, the term Cs-wcycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0047] The term “heterocyclic ring” as used herewith, whether it is used alone or as part of another group, refers to cyclic groups containing at least one non-aromatic ring in which one or more of the atoms are a heteroatom selected from O, S and N. Heterocyclic rings are either saturated or unsaturated (i.e. contain one or more double bonds).

[0048] The term “available”, as in “available hydrogen atoms” or “available atoms” refers to atoms that would be known to a person skilled in the art to be capable of replacement by a substituent, such as a fluorine atom.

[0049] It is to be clear that all available hydrogen atoms in the compounds of the application, and all embodiments thereof, are optionally substituted with a fluorine atom unless otherwise indicated.

[0050] The term “cell” as used herein refers to a single cell or a plurality of cells and includes a cell either in a cell culture or in a subject.

[0051] The term “subject” as used herein includes all members of the animal kingdom including mammals such as a mouse, a rat, a dog, a human and birds / poultry. Thus, the methods and uses of the present application are applicable to both human therapy and veterinary applications.

[0052] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[0053] The term “pharmaceutically acceptable carrier” means a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e. , a dosage form capable of administration to a subject.

[0054] The term “pharmaceutically acceptable salt” means either an acid addition salt or a base addition salt which is suitable for, or compatible with the treatment of subjects.

[0055] The term “solvate” as used herein means a compound, or a salt and / or prodrug of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered.

[0056] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treating” and “treatment” as used herein also include prophylactic treatment. For example, a subject with early cancer can be treated to prevent progression, or alternatively a subject in remission can be treated with a compound or composition of the application to prevent recurrence. Treatment methods comprise administering to a subject a therapeutically effective amount of one or more of the compounds of the application and optionally consist of a single administration, or alternatively comprise a series of administrations.

[0057] “Palliating” a disease or disorder means that the extent and / or undesirable clinical manifestations of a disorder or a disease state are lessened and / or time course of the progression is slowed or lengthened, as compared to not treating the disorder.

[0058] The term “prevention” or “prophylaxis”, or synonym thereto, as used herein refers to a reduction in the risk or probability of a patient becoming afflicted with a disease, disorder or condition.

[0059] The term “to inhibit the growth of bacterium” and variations thereof as used herein means any detectable inhibition of the growth of or killing of the bacterium in the presence of one or more compounds and optionally an antibiotic or compositions of the application compared to otherwise the same conditions except in the absence of the one or more compounds of the application.

[0060] As used herein, the term “effective amount” or “therapeutically effective amount” means an amount of one or more compounds, or one or more compositions, of the application that is effective, at dosages and for periods of time necessary to achieve the desired result.

[0061] The term “to suppress bacterial virulence” as used herein, refers to any detectable reduction in the virulence of the bacterium in the presence of one or more compounds and optionally an antibiotic or compositions of the application compared to otherwise the same conditions except in the absence of the one or more compounds of the application.

[0062] The term “administered” as used herein means administration of a therapeutically effective amount of one or more compounds, or a composition of the application to a cell, a tissue or an organ in vivo or in vitro, or a subject.

[0063] The term “adjuvant” as used herein refers to an agent which enhances the pharmaceutical effect of an antibiotic.II. Methods of the Application

[0064] The present application includes a method of treating a microbial (such as bacterial and fungal) infection in a subject in need thereof, comprising administering to the subject an effective amount of an antibiotic and an effective amount of one or more compounds selected from a compound of Formula IV, V and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V or VI is as follows:wherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

[0065] In some embodiments, the one or more compounds are compounds of Formula IV:(IV) or a pharmaceutically acceptable salt and / or solvate thereof.

[0066] In some embodiments, the one or more compounds are selected from the compound of Formula V:(V) or a pharmaceutically acceptable salt and / or solvate thereof, wherein R17is as defined for Formula V.

[0067] In some embodiments, the one or more compounds of Formula V are selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

[0068] In some embodiments, the one or more compounds are selected from the compound of Formula VI:or a pharmaceutically acceptable salt and / or solvate thereof, wherein R18 and R19 are as defined for Formula VI;

[0069] In some embodiments, the one or more compounds of Formula VI are selected from:VI-1 or a pharmaceutically acceptable salt and / or solvate thereof.

[0070] The present application also includes a method of treating a microbial infection in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds selected from a compound of Formula IV, V and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V or VI is as follows:wherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

[0071] In some embodiments, the one or more compounds are compounds of Formula IV:(IV) or a pharmaceutically acceptable salt and / or solvate thereof.

[0072] In some embodiments, the one or more compounds are selected from the compound of Formula V:(V) or a pharmaceutically acceptable salt and / or solvate thereof, wherein R17is as defined for Formula V.

[0073] In some embodiments, the one or more compounds of Formula V are selected from:V-1 ; andV-2, or a pharmaceutically acceptable salt and / or solvate thereof.

[0074] In some embodiments, the one or more compounds are selected from the compound of Formula VI:or a pharmaceutically acceptable salt and / or solvate thereof, wherein R18 and R19 are as defined for Formula VI;

[0075] In some embodiments, the one or more compounds of Formula VI are selected from:VI-1 or a pharmaceutically acceptable salt and / or solvate thereof.

[0076] It will be understood that one or more compounds selected from a compound of Formula IV, V and VI can be selected from the same or different Formula of compounds. For example, the one or more compounds can be selected from Formula IV or Formula V.

[0077] It will be understood that any component defined herein as being included may be explicitly excluded by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

[0078] In some embodiments, the salt and / or solvate of the compound of the application is a pharmaceutically acceptable salt and / or solvate. In some embodiments the pharmaceutically acceptable salt is an acid addition salt or a base addition salt. Theselection of a suitable salt may be made by a person skilled in the art (see, for example, S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci. 1977, 66, 1-19).

[0079] An acid addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic acid addition salt of any basic compound. Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids, as well as acidic metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono-, di- and tricarboxylic acids. Illustrative of such organic acids are, for example, acetic, trifluoroacetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, mandelic, salicylic, 2- phenoxybenzoic, p-toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. In an embodiment, the mono- or di-acid salts are formed, and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection criteria for the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts such as but not limited to oxalates may be used, for example in the isolation of compounds of the application for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0080] A base addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic compounds that form a basic addition salt include, for example, compounds comprising a carboxylic acid group. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide as well as ammonia. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. The selection of the appropriate salt may be useful, for example, so that an ester functionality, if any, elsewhere in a compound is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art.

[0081] Solvates of a compound of the application include, for example, those made with solvents that are pharmaceutically acceptable. Examples of such solvents include water (resulting solvate is called a hydrate) and ethanol and the like. Suitable solvents are physiologically tolerable at the dosage administered.

[0082] In some embodiments of the present application, the compounds described herein have at least one asymmetric center. In some embodiments, where compounds possess more than one asymmetric center, they exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (for example, less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the present application having an alternate stereochemistry. It is intended that any optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof are included within the scope of the present application.

[0083] In some embodiments, the compounds of the present application exist in different tautomeric forms and it is intended that any tautomeric forms which the compounds form, as well as mixtures thereof, are included within the scope of the present application.

[0084] In some embodiments, the compounds of the present application exist in varying amorphous and polymorphic forms and it is contemplated that any amorphous forms, polymorphs, or mixtures thereof, which form are included within the scope of the present application.

[0085] In some embodiments, the compounds of the present application comprise one or more alternate isotopes to the naturally occurring isotope for a particular atom. For example, in some embodiments the compounds of the invention comprise one or more deuterium atoms.

[0086] The present application also includes a use of an antibiotic and one or more compounds of formula IV, V or VI for treating a bacterial infection in a subject in need thereof.

[0087] The present application also includes a use of an antibiotic and one or more compounds of formula IV, V or VI for preparation of a medicament for treating a bacterial infection in a subject in need thereof.

[0088] It has been shown that exemplary compounds of the application increased the efficacy of antibiotics against bacterium, including drug-resistant bacterium. As such, in some embodiments, the one or more compounds of the application improve the efficacy of the antibiotic for the treatment of the bacterial infection in the subject in need thereof. Thus, in some embodiments, in the method of the application, the efficacy of the antibiotic administered with one or more compounds of the application is greater than the efficacy of the antibiotic administered in the absence of the one or more compounds of the application.

[0089] The exemplary compounds of the application showed synergistic activity with antibiotics, which resulted in increased efficacy of the antibiotics against bacteria, such as drug-resistant bacteria S. aureus USA300. As such, in some embodiments, the improved efficacy of the antibiotic is achieved by synergistic activity of the one or more compounds of the application with the antibiotic.

[0090] It has been further shown that exemplary compounds of the application sensitize Gram-negative and Gram-positive bacteria to the effects of the antibiotic, thus functioning as an antibiotic adjuvant. Specifically, exemplary compounds of the application inhibited bacterial detoxification of natural polyamines present at the site of infection and / or disrupted membrane integrity of the bacterium. Therefore, the improved efficacy of the antibiotic is also achieved by the sensitization of the bacteria to the effects of the antibiotic.

[0091] As such, in some embodiments, the one or more compounds of the application inhibit bacterial detoxification of natural polyamines present at the site ofinfection. In some embodiments, the one or more compounds of the application disrupt membrane integrity of the bacterium, in particular a Gram-negative bacterium.

[0092] In some embodiments, the one or more compounds of the application inhibit bacterial detoxification of natural polyamines present at the site of infection by inhibiting polyamine detoxification enzyme activity in bacterium.

[0093] In some embodiments, the one or more compounds of the application inhibit bacterial ribosome.

[0094] One or more compounds of the application inhibit human sperm ine / spermidine acetyltransferase SAT1

[0095] Compounds inhibiting human sperm ine / spermidine acetyltransferase may be used to treat SAT1 -related dysfunction or disorders related to SAT1 dysfunction in humans and animals

[0096] Compounds inhibiting human sperm ine / spermidine acetyltransferase may be used to treat SAT1 -related dysfunction or disorders related to SAT1 dysfunction, including, but not limited to, cancer, osteoporosis, and ischemia / reperfusion leading to acute kidney injury.

[0097] In addition, it has been shown that exemplary compounds abolished the resistance of S. aureus USA300 to the antibiotics. As such, in some embodiments, the one or more compounds of the application reduce or eliminate polyamine-mediated antibiotic resistance of the bacterium to the antibiotic. In some embodiments, the one or more compounds of the application reduce or eliminate polyamine-mediated antibiotic resistance of the bacterium to the antibiotic by inhibiting polyamine detoxification enzyme activity in bacterium.

[0098] In some embodiments, the polyamine detoxification enzyme is acetyltransferase enzyme. In some embodiments, the acetyltransferase enzyme is SpeG.

[0099] In some embodiments, the natural polyamines present at the site of infection are spermine, spermidine and / or putrescine.

[0100] In some embodiments, the antibiotic is selected from a macrolide, a tetracycline, a lipopeptide, a glycopeptide, a beta-lactam, a penicillin, a cephalosporin, amonobactam, a carbapenem, an aminoglycoside, a rifamycin, a ketolide, an oxazolidinone, a glycylcycline, an aminocoumarin, sulfonamide, a trimethoprim, a lincomycin, a streptogramin, a chloramphenicol, a quinolone, a fluoroquinolone, an antimicrobial peptide and any other antibiotic, or a combination thereof.

[0101] In some embodiments, the antibiotic is selected from amikacin, neomycin, tobramycin, paromomycin, streptomycin, spectinomycin, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cefalothin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, lipopeptide, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, linezolid, posizolid, radezolid, torezolid, amoxicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, penicillin g, temocillin, ticarcillin, bacitracin, colistin, polymyxin b, besifloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, sulfonamidochrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin / dalfopristin, thiamphenicol, tigecycline, tinidazole, trimethoprim, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, nitrofurantoin, methenamine, and streptomycin, or a pharmaceutically acceptable salt thereof, or a combination thereof.

[0102] In some embodiments, the antibiotic is selected from azithromycin, cefuroxime, ciprofloxacin, daptomycin, rifampicin, vancomycin and colistin, or a combination thereof.

[0103] The present application also includes a use of one or more compounds of formula IV, V or VI for treating a microbial infection in a subject in need thereof.

[0104] The present application also includes a use of one or more compounds of formula IV, V or VI for preparation of a medicament for treating a microbial infection in a subject in need thereof.

[0105] It has been shown that exemplary compounds IV, V and VI exhibited growth inhibitory effects against bacterium and fungus, including drug-resistant bacterium and fungus. As such, in the method and the use of the one or more compounds of formula IV, V or VI for treating a bacterial or fungal infection in a subject in need thereof, the one or more compounds of formula IV, V or VI inhibit growth of a bacterium or fungus.

[0106] It has been also shown that exemplary compounds IV, V and VI suppress bacterial and fungal virulence. As such, in the method and the use of the one or more compounds of formula IV, V or VI for treating a bacterial or fungal infection in a subject in need thereof, the one or more compounds of formula IV, V or VI suppress virulence of bacterium or fungus.

[0107] In some embodiments, the virulence of the bacterium and fungus is suppressed by inhibiting bacterial / fungal detoxification of natural polyamines present at the site of infection and / or by disrupting membrane integrity of the bacterium / fungus.

[0108] In some embodiments, the one or more compounds IV, V and VI inhibit bacterial or fungal detoxification of natural polyamines present at the site of infection. In some embodiments, the one or more compounds IV, V and VI disrupt membrane integrity of the bacterium or fungus.

[0109] In some embodiments, the one or more compounds IV, V and VI inhibit bacterial or fungal detoxification of natural polyamines present at the site of infection by inhibiting polyamine detoxification enzyme activity in bacterium or fungus.

[0110] In some embodiments, the polyamine detoxification enzyme is acetyltransferase enzyme. In some embodiments, the acetyltransferase enzyme is SpeG.

[0111] In some embodiments, the natural polyamines present at the site of infection are spermine, spermidine and / or putrescine.

[0112] In some embodiments, the infection is caused by Gram-positive or Gramnegative bacterium or pathogenic fungus.

[0113] In some embodiments, the bacterium is a Gram-negative bacterium. In some embodiments, the Gram-negative bacterium is a species of Acetic acid bacteria, Acidaminococcus, Anaerobiospirillum, Arcobacter, Bacteroides, Bacteroidetes, Bdellovibrio, Brachyspira, Burkholderia, Campylobacter, Christensenella, Cyanobacteria, Cytophaga, Dialister, Enterobacter, Enterobacteriaceae, Enterobacteriales, Escherichia, Flavobacterium, Haemophilus, Helicobacter, Legionella, Megamonas, Megasphaera, Meiothermus, Moraxella, Pectinatus, Pelosinus, Propionispora, Proteobacteria, Pseudomonas, Salmonella, Samsonia, Selenomonadales, Shigella, Shimwellia, Spirochaeta, Spirochaetaceae, Sporomusa, Stenotrophomonas, Thorselliaceae, Vampirococcus, Verminephrobacter, Vitreoscilla, Wolbachia, Yersiniaceae, or Zymophilus or a combination thereof.

[0114] In some embodiments, the Gram-negative bacterium is Acinetobacter baumannii, Agrobacterium tumefaciens, Akkermansia muciniphila, Anaerobiospirillum, Anaerolinea thermolimosa, Anaerolinea thermophile, Arcobacter skirrowii, Armatimonas rosea, Azotobacter salinestris, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides ureolyticus, Bartonella japonica, Bartonella koehlerae, Bartonella taylorii, Bradyrhizobium japonicum, Burkholderia cenocepacia, Caldilinea aerophila, Cardiobacterium hominis, Chaperone-Usher fimbriae, Chthonomonas calidirosea, Coxiella burnetii, Dehalogenimonas lykanthroporepellens, Desulfurobacterium atlanticum, Devosia pacifica, Devosia psychrophila, Devosia soli, Devosia subaequoris, Devosia submarina, Devosia yakushimensis, Dictyoglomus thermophilum, Dinoroseobacter shibae, Enterobacter cloacae, Enterobacter cowanii, Enterobacteriales, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Fimbriimonas ginsengisoli, Flavobacterium akiainvivens, Francisella novicida, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium polymorphum, Gluconacetobacter diazotrophicus, Haemophilus felis, Haemophilus haemolyticus, Haemophilus influenza, Haemophilus pittmaniae, Helicobacter typhlonius, Helicobacter pylori, Kingella kingae, Klebsiella pneumoniae, Kluyvera ascorbata, Kluyvera cryocrescens, Kozakia baliensis, Legionella clemsonensis, Legionella pneumophila, Leptonema mini, Leptotrichia buccalis, Levilinea saccharolytica, Luteimonas aestuarii, Luteimonas aquatic, Luteimonas composti, Luteimonas lutimaris, Luteimonas marina, Luteimonas mephitis, Luteimonas vadosa, Meiothermus timidus, Methylobacteriumfujisawaense, Morax-Axenfeld diplobacilli, Moraxella bovis, Moraxella osloensis, Morganella morganii, Mycoplasma spumans, Neisseria cinerea, Neisseria gonorrhoeae, Neisseria meningitides, Neisseria polysaccharea, Neisseria sicca, Nitrosomonas eutropha, Nitrosomonas halophile, Nitrosomonas stercoris, Pedobacter heparinus, Proteus mirabilis, Proteus penneri, Pseudomonas aeruginosa, Pseudomonas luteola, Pseudomonas teessidea, Pseudoxanthomonas broegbernensis, Pseudoxanthomonas japonensis, Rickettsia rickettsia, Riemerella anatipestifer, Salinibacter ruber, Salmonella bongori, Salmonella enterica, Salmonella Typhimurium, Selenomonas noxia, Serratia marcescens, Solobacterium moorei, Sorangium cellulosum, Sphaerotilus natans, Sphingomonas gei, Stenotrophomonas nitritireducens, Thermotoga neapolitana, Vibrio adaptatus, Vibrio azasii, Vibrio campbellii, Vibrio cholera, Victivallis vadensis, or Yersinia pestis, or a combination thereof.

[0115] In some embodiments, the Gram-negative bacterium is Klebsiella pneumoniae, Salmonella Typhimurium, Burkholderia cenocepacia or Escherichia coli, or a combination thereof.

[0116] In some embodiments, the bacterium is a Gram-positive bacterium. In some embodiments, the Gram-positive bacterium is a species of Actinobacteria, Actinomyces, Arcanobacterium, Bad Hales, Bacillus, Bavariicoccus, Brachybacterium, Carnobacteriaceae, Clostridium, Cnuibacter, Coriobacteriia, Corynebacterium, Enterococcus, Janibacter, Lactobacillales, Listeriaceae, Nocardia, Pasteuria, Pilibacter, Roseburia, Sarcina, Solibacillus, Sporosarcina, Staphylococcus, Streptococcus, or Tepidibacter, or a combination thereof.

[0117] In some embodiments, the Gram-positive bacterium is Actinomyces bovis, Actinomyces georgiae, Actinomyces gerencseriae, Actinomyces israelii, Actinomyces neuii, Actinomyces radicidentis, Actinomyces viscosus, Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, Alicyclobacillus aeris, Alicyclobacillus contaminans, Alicyclobacillus cycloheptanicus, Alicyclobacillus dauci, Alicyclobacillus disulfidooxidans, Alicyclobacillus fastidiosus, Alicyclobacillus ferrooxydans, Alicyclobacillus kakegawensis, Alicyclobacillus macrosporangiidus, Alicyclobacillus sacchari, Alicyclobacillus shizuokensis, Alicyclobacillus tolerans, Bacillus mojavensis, Bacillus subtilis, Bacillusweihenstephanensis, Brachybacterium alimentarium, Brachybacterium aquaticum, Brachy bacterium conglomeratum, Brachybacterium faecium, Brachybacterium fresconis, Brachybacterium ginsengisoli, Brachybacterium horti, Brachybacterium huguangmaarense, Brachybacterium massiliense, Brachybacterium muris, Brachybacterium nesterenkovii, Brachybacterium paraconglomeratum, Brachybacterium phenoliresistens, Brachybacterium rhamnosum, Brachybacterium tyrofermentans, Clostridium acetobutylicum, Clostridium aerotolerans, Clostridium argentinense, Clostridium autoethanogenum, Clostridium baratii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium botulinum, Clostridium butyricum, Clostridium cadaveris, Clostridium cellobioparum, Clostridium cellulolyticum, Clostridium cellulovorans, Clostridium chauvoei, Clostridium clostridioforme, Clostridium colicanis, Clostridium difficile, Clostridium estertheticum, Clostridium fallax, Clostridium formicaceticum, Clostridium histolyticum, Clostridium innocuum, Clostridium kluyveri, Clostridium ljungdahlii, Clostridium novyi, Clostridium paradoxum, Clostridium paraputrificum, Clostridium pasteurianum, Clostridium perfringens, Clostridium phytofermentans, Clostridium piliforme, Clostridium ragsdalei, Clostridium ramosum, Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium scatologenes, Clostridium septicum, Clostridium sordellii, Clostridium sporogenes, Clostridium stercorarium, Clostridium sticklandii, Clostridium straminisolvens, Clostridium tertium, Clostridium tetani, Clostridium thermosaccharolyticum, Clostridium tyrobutyricum, Clostridium uliginosum, Cory nebacteri urn amycolatum, Cory nebacteri urn bovis, Cory nebacteri urn diphtheria, Corynebacterium efficiens, Cory nebacteri urn glutamicum, Cory nebacteri urn granulosum, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium ulcerans, Cutibacterium acnes, Deinococcus marmoris, Desulfitobacterium dehalogenans, Enterococcus faecium, Enterococcus faecalis, Fervidobacterium changbaicum, Fervidobacterium gondwanense, Fervidobacterium islandicum, Georgenia ruanii, Microbispora coralline, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, and Nontuberculosis Mycobacteria (NTM) also referred to as atypical mycobacteria, mycobacteria other than tuberculosis (MOTT), or environmental mycobacteria, including M. abscessus and M. abscessus complex, M. avium and M. avium complex, M. kansasii, M. fortuitum, M. xenopi, M.malmoense, M. szulgai, and M. simiae, Nocardia asteroids, Nocardia brasiliensis, Nocardia farcinica, Nocardia ignorata, Rathayibacter toxicus, Rhodococcus equi, Rothia dentocariosa, Sporosarcina aquimarina, Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus pettenkoferi, Staphylococcus pseudintermedius, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus succinus, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus canis, Streptococcus downei, Streptococcus equi, Streptococcus bovis, Streptococcus gordonii, Streptococcus iniae, Streptococcus lactarius, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophiles, Streptococcus tigurinus, Streptococcus uberis, Streptococcus vestibularis, Syntrophomonas curvata, Syntrophomonas palmitatica, Syntrophomonas sapovorans, Syntrophomonas wolfei, Syntrophomonas zehnderi, or Viridans streptococci, or a combination thereof.

[0118] In some embodiments, the Gram-positive bacterium is Enterococcus faecium, Enterococcus faecalis, Mycobacterium smegmatis, Staphylococcus epidermidis or Bacillus subtilis, or a combination thereof.

[0119] In some embodiments, the Gram-positive bacterium is Staphylococcus aureus. In some embodiments, the bacterium is S. aureus USA300.

[0120] In some embodiments, the Gram-positive bacterium is a drug-resistant or a multidrug-resistant bacterium. In some embodiments, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).

[0121] In some embodiments, the Gram-positive bacterium is a species of Mycobacterium, for example, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, and Nontuberculosis Mycobacteria(NTM) also referred to as atypical mycobacteria, mycobacteria other than tuberculosis (MOTT), or environmental mycobacteria, including M. abscessus and M. abscessus complex, M. avium and M. avium complex, M. kansasii, M. fortuitum, M. xenopi, M. malmoense, M. szulgai, and M. simiae,

[0122] In some embodiments, the infection is caused by pathogenic fungi

[0123] In some embodiments the fungus is Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. auris, and Saccharomyces cerevisiae

[0124] In some embodiments, the one or more compounds are compounds of Formulae:VI-1 , or a pharmaceutically acceptable salt and / or solvate thereof, wherein the infection is caused by Gram-positive or Gram-negative bacterium or fungus.

[0125] In some embodiments, the antibiotic is antimicrobial peptide. In some embodiments, the antimicrobial peptide is colistin.

[0126] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a bird, fish or reptile.

[0127] The one or more compounds of the present application and optionally the antibiotic may be suitably formulated in a conventional manner into compositions using one or more carriers. Accordingly, in the method of the present application, the one or more compounds and optionally the antibiotic are administered to the subject in a composition comprising the one or more compounds or a pharmaceutically acceptable salt and / or solvate thereof, optionally the antibiotic and a carrier. The compounds of the application may be suitably formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in the method of the present application, the one or more compounds and optionally the antibiotic are administered to the subject in a pharmaceutical composition comprising said one or more compounds or a pharmaceutically acceptable salt and / or solvate thereof, optionally the antibiotic, and a pharmaceutically acceptable carrier or vehicle. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 20thedition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0128] In some embodiments, when the one or more compounds of the application are administered with the antibiotic, the compounds and the antibiotic are administered simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. In some embodiments, the one or more compounds of the application are administered to subjects prior or subsequent to a course of treatment with antibiotics, so as to sensitize the bacteria to the antibiotics. In some embodiments, the subject can be treated with antibiotics for a period of time prior to the administration of the one or more compounds of the application, which then can be administered to sensitize the bacteria or resistant bacteria to the antibiotics. Thus, the one or more compounds of the application can be administered to a subject concurrently with the antibiotics, prior to the antibiotics, and / or subsequent to the antibiotics.

[0129] In some embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, inert solid fillers or diluents and sterile aqueous or organic solutions. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, aqueous and non-aqueous solutions. Pharmaceutically acceptable carriers can be aqueous or non- aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents suitable for use in the present application include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers suitable for use in the present application include, but are not limited to, water, ethanol, alcoholic / aqueous solutions, glycerol, emulsions or suspensions, including saline and buffered media.

[0130] In some embodiments, the compounds of the application and optionally the antibiotic are administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. In some embodiments, the compounds of the application and antibiotic are administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal administration and the pharmaceutical compositions formulated accordingly. Administration can be by means of a pump for periodic or continuous delivery. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington’s Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. In some embodiments, when the compounds of the application are administered with the antibiotic, the compounds of the application and antibiotic are administered in the same pharmaceutical composition. In some embodiments, the compounds of the application and antibiotic are administered in the separate pharmaceutical compositions. When separate pharmaceutical compositions are used, the form of administration may be the same or different.

[0131] Parenteral administration includes intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary (for example, by use of an aerosol), intrathecal, rectal and topical (including the use of a patch or other transdermal delivery device) modes of administration. In some embodiments, parenteral administration is by continuous infusion over a selected period of time.

[0132] In some embodiments, topical administration includes liquid, ointment, cream, gel, hydrogel, cataplasm, pomade, liniment, milk, lotion, emulsion, spray, aerosol, collyrium, drops and powder forms of administration. In some embodiments, for topical administration, suitable excipients include, but are not limited to animal and plant oils, mineral oils, synthetic oils, ester oils, waxes, linear higher alcohols, fatty acids, surfactants, phospholipids, gelling and / or thickening agents, alcohol, polyols (including glycerine and propylene glycol), fillers such as clay minerals, soft-focus powders, preservatives, fragrances, pigments, purified water, polysaccharides, such as for example mannans, gluco mannans, galactomannans, fucomannans, proteoglycans, glucosaminoglycans, chitins and chitomannans. In some embodiments, topical administration includes a medical device comprising one or more compounds or compositions of the application. In some embodiments, the medical device is in the form of a dressing, bandage, transdermic medical device, controlled drug release medical device, or a drug-eluting stent. Suitable dressings include, without any limitation, hydrocolloid dressings, hydrocellular dressings, alginate dressings, hydrogel dressings, chitosan-based dressings, cellulose derivatives dressings and any other type of dressing. By "transdermic medical device" it is meant a device for slow liberation via transdermic process of a substance, such as for example adhesive patch. By "drug-eluting stent", also called "coated" or "medicated" stent, it is meant a stent that has been coated with the active substance, such as for example "protein 156A".

[0133] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability exists.

[0134] In some embodiments, the one or more compounds of the application and / or antibiotic are orally administered, for example, with an inert diluent or with an assimilable edible carrier, or are enclosed in hard or soft shell gelatin capsules, or are compressed into tablets, or are incorporated directly with the food of the diet. In some embodiments, for oral therapeutic administration, the compounds and / or the antibiotics are incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers,aqueous solutions and suspensions, and the like. In the case of tablets, carriers that are used include lactose, corn starch, sodium citrate and salts of phosphoric acid.Pharmaceutically acceptable excipients include binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). In some embodiments, the tablets are coated by methods well known in the art. In the case of tablets, capsules, caplets, pellets or granules for oral administration, pH sensitive enteric coatings, such as Eudragits™ designed to control the release of active ingredients are optionally used. Oral dosage forms also include modified release, for example immediate release and timed-release, formulations. Examples of modified-release formulations include, for example, sustained-release (SR), extended- release (ER, XR, or XL), time-release or timed-release, controlled-release (CR), or continuous-release (CR or Contin), employed, for example, in the form of a coated tablet, an osmotic delivery device, a coated capsule, a microencapsulated microsphere, an agglomerated particle, e.g., as of molecular sieving type particles, or, a fine hollow permeable fiber bundle, or chopped hollow permeable fibers, agglomerated or held in a fibrous packet. Timed-release compositions can be formulated, e.g. liposomes or those wherein the compound and the antibiotic is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. Liposome delivery systems include, for example, small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. For oral administration in a capsule form, useful carriers or diluents include lactose and dried corn starch.

[0135] In some embodiments, the compounds of the application and optionally the antibiotic can be formulated in any nano-scale formulations known in the art, such as nanoparticles, nanofibers and the like. In some embodiments, the compounds of the application are encapsulated, loaded or embedded within these nano-scale formulations. In some embodiments, the compounds of the application are also chemically-conjugated to any of these dosage forms and drug delivery systems, via direct conjugation or via a linker group as would be known to those skilled in the art.

[0136] In some embodiments, liquid preparations for oral administration take the form of, for example, solutions, syrups or suspensions, or they are suitably presented as a dry product for constitution with water or other suitable vehicle before use. When aqueous suspensions and / or emulsions are administered orally, the compound of the application and the antibiotic are suitably suspended or dissolved in an oily phase that is combined with emulsifying and / or suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents may be added. Such liquid preparations for oral administration may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycols.

[0137] It is also possible to freeze-dry the one or more compounds of the application and / or the antibiotic and use the lyophilizates obtained, for example, for the preparation of products for injection.

[0138] In some embodiments, the one or more compounds of the application and / or the antibiotic are administered parenterally. Solutions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. For parenteral administration, sterile solutions are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. In some embodiments, for ocular administration, ointments or droppable liquids are delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzyl chromium chloride, and the usualquantities of diluents or carriers. For pulmonary administration, diluents or carriers will be selected to be appropriate to allow the formation of an aerosol.

[0139] In some embodiments, the one or more compounds of the application and / or antibiotic are formulated for parenteral administration by injection, including using conventional catheterization techniques or infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In some embodiments, the compositions take such forms as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability exists. Alternatively, the one or more compounds of the application and / or antibiotic are suitably in a sterile powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0140] In some embodiments, compositions for nasal administration are conveniently formulated as aerosols, drops, gels and powders.

[0141] For intranasal administration or administration by inhalation, the one or more compounds of the application and / or antibiotic are conveniently delivered in the form of a solution, dry powder formulation or suspension from a pump spray container that is squeezed or pumped by the patient or as an aerosol spray presentation from a pressurized container or a nebulizer. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or nonaqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, in some embodiments, the sealed container is a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. Suitable propellants include but are not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide or another suitable gas. Inthe case of a pressurized aerosol, the dosage unit is suitably determined by providing a valve to deliver a metered amount. In some embodiments, the pressurized container or nebulizer contains a solution or suspension of the antibiotic and the compound of the application. In some embodiments, capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator are formulated containing a powder mix of the antibiotic and a compound of the application and a suitable powder base such as lactose or starch. The aerosol dosage forms can also take the form of a pump-atomizer.

[0142] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0143] Suppository forms of the one or more compounds of the application and / or antibiotic are useful for vaginal, urethral and rectal administrations. Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature. The substances commonly used to create such vehicles include but are not limited to theobroma oil (also known as cocoa butter), glycerinated gelatin, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. See, for example: Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp. 1530-1533 for further discussion of suppository dosage forms.

[0144] In some embodiments, the one or more compounds of the application and / or antibiotic are also coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, in some embodiments, the antibiotic and the compounds of the application are coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

[0145] In some embodiments, depending on the mode of administration, the pharmaceutical composition will comprise from about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt%, of the one or more compounds of the application and / or the antibiotics, and from about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of one or more pharmaceutically acceptable carriers, all percentages by weight being based on the total composition.

[0146] In some embodiments, the one or more compounds of the application and / or antibiotic are administered with a bicarbonate buffer simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present application provides a single unit dosage form comprising an antibiotic, one or more compounds of the application, a bicarbonate buffer, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions of the application further comprise a bicarbonate buffer.

[0147] In some embodiments, the one or more compounds of the application and optionally the antibiotic are administered with an additional therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present application provides a single unit dosage form comprising the one or more compounds of the application, optionally the antibiotic, an additional therapeutic agent, and a pharmaceutically acceptable carrier. As such, in some embodiments, the pharmaceutical compositions of the application further comprise an additional therapeutic agent. Treatment methods comprise administering to a subject a therapeutically effective amount of the one or compounds of the application (either in separate or the same composition), and optionally the antibiotic, and optionally consist of a single administration, or alternatively comprise a series of administrations, and optionally comprise concurrent administration or use of one or more other therapeutic agents. For example, in some embodiments, the one or more compounds of the application and optionally the antibiotic are administered at least once a week. In some embodiments, the one or more and optionally the antibiotic compounds are administered to the subject fromabout one time per two or three weeks, or about one time per week to about once daily for a given treatment. In another embodiment, the one or more compounds and optionally the antibiotic are administered 2, 3, 4, 5 or 6 times daily. The length of the treatment period depends on a variety of factors, such as the seventy of the disease, disorder or condition, the age of the subject, the concentration and / or the activity of the conjugates of the application, and / or a combination thereof. It will also be appreciated that in some embodiments, the effective dosage of the one or more compounds used for the treatment is increased or decreased over the course of a particular treatment regime. In some embodiments, changes in dosage result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration is required. For example, the one or more compounds and optionally the antibiotic are administered to the subject in an amount and for duration sufficient to treat the subject. In some embodiments treatment comprise prophylactic treatment.

[0148] In some embodiments, effective amounts vary according to factors such as the disease state, age, sex and / or weight of the subject. The amount of the compounds and optionally the antibiotic that will correspond to such an amount will vary depending upon various factors, such as the given compound and antibiotic, the pharmaceutical formulation, the route of administration, the type of condition, disease or disorder, the identity of the subject being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[0149] In some embodiments, references above to an antibiotic can also refer to one or more antibiotics.

[0150] In some embodiments, the compounds of the application are used as probes or research tools in in vitro assays, for example, to test for the presence of a bacterial or fungal infection, to test the efficacy of another antimicrobial compound, or to inhibit an enzyme or a cellular component. In these embodiments, the compounds of the application may be labelled, for example with a radiolabel.III. Preparation Methods of the Compounds of the Application

[0151] The compounds of the present application can be prepared by various synthetic processes or commercially available products can be used. Some startingmaterials for preparing the compounds of the present application are available from commercial chemical sources. Other starting materials, are readily prepared from available precursors using straightforward transformations that are well known in the art. The selection of a particular process to prepare a given compound of the application is within the purview of the person of skill in the art.

[0152] The antibiotics used on the present application are commercially available antibiotics.

[0153] Salts of the compounds of the application can be generally formed by dissolving the neutral compound in an inert organic solvent and adding either the desired acid or base and isolating the resulting salt by either filtration or other known means.

[0154] The formation of solvates of the compounds of the application will vary depending on the compound and the solvate. In general, solvates can be formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions to form a particular solvate can be made by a person skilled in the art. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”.

[0155] It will be understood that any component defined herein as being included may be explicitly excluded by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.Examples

[0156] The following non-limiting examples are illustrative of the present application:Example 1: Catechol and benzenediol derivative compounds with broad spectrum activityMaterials and methodsBacterial strains and reagents

[0157] Table 1 and 2 list strains, plasmids and primers used in this work. Bacteria were grown in Mueller Hinton medium at 37°C. Except for Enterococcus faecalis andEnterococcus faecium, which were grown in Tryptic Soy Broth and Mycobacterium smegmatis, which was grown in Luria Broth.

[0158] Table 1 . Strains and plasmids used in this study:

[0159] Table 2. Primers used in this studyAntimicrobial susceptibility testing

[0160] Minimum inhibitory concentration (MIC) and checkerboard assays were performed using the Clinical & Laboratory Standards Institute (CLSI) broth microdilution technique (CLSI 2012). The fractional inhibitory concentration indices (FICI) were calculated as FICI=(MICdrug A in combination / MICdrug Aalone)+(MICdrug B in combination) / MICDrug B alone). FICI values were interpreted as synergy when FICI < 0.5, no interaction when 1 < FICI < 4, and antagonism when FICI > 4.0.Chemical screen and polyamine analog library assembly

[0161] We assembled a library of polyamine analogs by selecting commercially available (Aldrich Market select) analogs with at least 60% structural similarity to substrates and products of polyamine biosynthesis and detoxification enzymes in bacteria. This librarywas screened at 20 pM in the presence and absence of spermine at 1 / 4th the wild-type MIC. Library compounds were added to 96-well plates and filled with inoculated MHB. We also screened a library of previously approved drugs and natural product derivatives (Spectrum collection, MicroSource Inc.) at 20 pM against the wild-type USA300 strain in the presence of spermine at 1 / 4th the wild-type MIC. Compounds were administered to 384- well plates containing inoculated MHB with spermine using the Biomatrix BM5-BC (S&P Robotics inc.). Plates were incubated at 37°C, and bacterial growth was determined turbidim etrically after 24 hours.S. aureus Nebraska Transposon Mutant Library (NTML) screen

[0162] Overnight cultures of the NTML (Fey, Endres et al. 2013) were prepared in 384-well plates using Biomatrix BM5-BC (S&P Robotics inc.) in MHB containing 5 pg / mL erythromycin. The following day, MHB containing 1 / 8thand 1 Z16ththe wild-type MIC of spermine were inoculated using the Biomatrix BM5-BC. Plates were grown at 37°C, and ODeoo was read after 24 h.General molecular techniques

[0163] Unmarked in-frame deletion of speG was performed using the temperaturesensitive allelic exchange plasmid pJB38 (Bose, Fey et al. 2013). Roughly 1-kb upstream and downstream of speG were amplified through polymerase chain reaction (PCR) and the upstream fragment digested with EcoRI, and BamHI (New England Biolabs), and the downstream fragment digested with EcoRI and Sall (New England Biolabs). pJB38 was digested with BamHI and Sall and fragments were ligated with T4 DNA ligase. The resultant deletion plasmid pJB38-ZIspeG was passaged through E. coli DH5a and E. coli DC10B, before electroporation into S. aureus USA300 JE2 (Grosser and Richardson 2016). Deletion of speG was confirmed through PCR.Membrane permeabilization assay

[0164] Fluorometric Disc3(5) membrane permeabilization assays were performed as described in (Farha, Verschoor et al. 2013).Hemolysis Assay

[0165] The hemolysis assay was performed using defibrinated sheep blood (Thermo Fisher Scientific, and Cedarlane) as previously described (Barker, Chandler et al. 2019).Protein overexpression and purification

[0166] speG, SAUSA300_2083, SAUSA300_0441 , and paiA were cloned into pET28a(+) and overexpressed under conditions previously described (Li, Maezato et al. 2019). cDNA of SAT1 was codon-optimized, synthesized from, and cloned into pET28a(+) (Bio Basic Canada). Lysis was achieved using a Branson Sonifier 450 and the supernatant was isolated from the insoluble fraction by centrifugation at 10 000 xg for 30 minutes at 4°C. His-tag batch purification was performed using His-select nickel-affinity gel (Sigma- Aldrich). Purified proteins were detected on a 10% acrylamide gel (Bio-Rad) by PageBlue staining solution (Fisher Scientific). Proteins were quantified using the Bradford method with protein assay dye reagent concentrate (Bio-Rad), following dialysis using SnakeSkin™ dialysis tubing (Fisher Scientific).In vitro SSA T enzymatic assaysSteady state kinetics.

[0167] Purified enzymes were used in sperm ine / spermidine / V-acetyltransferase (SSAT) colorimetric enzymatic assays as described previously (Lin, Lien et al. 2010, Li, Maezato et al. 2019), with modifications. Briefly, 25 pL of enzyme solution (200 ng enzyme, 100 mM Tris-HCI, pH 7.5, 1 mM EDTA) were added to a 96 well plate then 50 pL of freshly prepared Ellman’s reagent [2 mM 5, 5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) (Sigma Aldrich) 100 mM Tris-HCI, pH 7.5, 1 mM EDTA)] was added to each well. Finally, 25 pL of substrate solution was added to begin the reaction. The substrate solution consisted of a final concentration of 1 mM acetyl-CoA (Sigma Aldrich) with SpeG, PaiAsa, SAUSA300_2083, and SAUSA300_0441 , and 0.25 mM acetyl-CoA with SAT1 in 100 mM Tris-HCI, pH 7.5, 1 mM EDTA with variable concentrations of polyamines. Each reaction plate contained serial dilutions of Coenzyme A (5-150 nmol) as a standard. Reactions were allowed to proceed at 37°C and A4i2nm readings were taken each minute for 45 min. Kinetic parameters were estimated by fitting with the Michaelis-Menten equation or allosteric sigmoidal equation in Graphpad Prism 10.Enzymatic inhibition assays

[0168] The colorimetric SSAT assay was performed where 25 pL of enzyme solution containing 200 ng enzyme, 100 mM Tris-HCI, pH 7.5, 1 mM EDTA and a dilution series of inhibitor was added to a 96-well plate. The enzyme-inhibitor solution was incubated at room temperature for 30 min before adding 50 pL Ellman’s reagent and 25 pL substrate solution to initiate the reaction. V-1 inhibition was assayed with a substrate solution including 1500 pM spermine and an AcCoA concentration at ~Km. The reaction was monitored each minute at A4i2nm for 45 minutes. We evaluated the decrease in absorbance when the reaction was supplemented with the inhibitors compared to a positive control containing only spermine and acetyl-CoA. Data for ICso plots were fit to a nonlinear regression inhibition model in Graphpad Prism 10 and Ki was estimated from equation 1 (Cheng and Prusoff 1973). The colorimetric SSAT assay was also performed in a 96-well plate where enzyme activity was monitored in the presence of the respective inhibitor across 8 concentrations of substrate and data were fit to a competitive inhibition model in Graphpad Prism 10.

[0169] Equation ! :Results and DiscussionA high-throughput chemical screen identifies potential polyamine detoxification inhibitors

[0170] To identify inhibitors of polyamine detoxification mechanisms, we undertook two screens against S. aureus USA300. In an unbiased approach, we screened a library of 2560 small molecules (Spectrum collection, MicroSource Discovery Systems, Inc.), comprised of previously approved drugs and natural product derivatives, at 20 pM in the presence of !ththe minimum inhibitory concentration (MIC) of spermine against USA300. In parallel, we took a rationale-based approach by assembling a collection of 83 polyamine analogs with at least 60% structural similarity to substrates and products of polyamine biosynthesis and detoxification enzymes in bacteria, reasoning that such analogs have a higher probability of interacting with, and potentially inhibiting, the polyamine-related enzymes of interest. Notably, several polyamine biosynthesis inhibitors are analogous to natural polyamines or substrates of their biosynthetic enzymes (Kallio and McCann 1981 ,Mattila, Honkanen-Buzalski et al. 1984, El-Halfawy and Valvano 2013), which supports this rationale-based approach. We screened the polyamine analog library at 20 pM (similar to the Spectrum collection screen) in the presence of spermine at1 / 4ththe MIC. The screen was performed with the hypothesis that a compound inhibiting polyamine detoxification would exhibit synergy with spermine. Combined, the screens resulted in 187 primary hits showing at least 80% growth inhibition. We then excluded known antimicrobials in the Spectrum collection yielding 129 hits (Figure 1 A and B). We tested these hits in follow-up dose-response assays in the presence and absence of a single spermine concentration; only 8 compounds showed a reduction in MIC in the presence of spermine (Figure 1 A). We then tested these eight compounds in checkerboard assays with spermine; six compounds, V-1 , IV-1 , OES1-1639, OES1-1238, and OES2-0017, showed synergy with spermine (Figure 1 D and F). Synergy was determined by calculating the fractional inhibitory concentration index (FIC I), with an FICI value < 0.5 defined as a synergistic interaction. V-1 exhibited the strongest synergistic interaction with spermine with an FICI value of 0.15 + 0.03 followed by IV-1 (0.28 + 0.07), OES2-0017 (0.33 + 0.06), OES1-1639 (0.42 + 0.06), OES1 -1238 (0.46 + 0.12), and finally OES2-0052 (0.50 + 0) (Figure 1 D and F). IV, V-1 , and VI-1 are benzenediols, OES1-1238 is an acridine, and OES2-0052 and OES2-0017 are amines. In addition to synergizing with polyamines, the six hits exhibited growth inhibitory effects against USA300 with OES2-0017 being the most potent at an MIC of 20 pM (Figure 1 D and F). Of note, we prioritized OES2-0017 and V-1 (isoproterenol) for further characterization based on their relative potencies (spermine synergy at FICI<0.35) and as representatives of different chemical classes (polyamine and catechols, respectively). We confirmed the structures of the two prioritized compounds by NMR.A chemogenomic screen to uncover the putative mechanism of spermine synergy

[0171] We undertook a genome-wide approach to identify the putative mechanism of spermine synergy by the screen hits, reasoning that a mutant with disruption in their putative target would exhibit both increased spermine susceptibility and loss or reduction in spermine-hit compound synergy. First, we sought to uncover the determinants of polyamine detoxification whose mutants would exhibit increased susceptibility to spermine byscreening the Nebraska Transposon Mutant Library (NTML), a sequence-defined transposon mutant library covering the non-essential genome of S. aureus USA300 (Fey, Endres et al. 2013), against spermine at 1 / 8thand 1 / 16ththe wild-type MIC. speG::Tn was sensitized to spermine, in agreement with previous reports (Joshi, Spontak et al. 2011 ), thus serving as an internal control for the screen. Mutants in another four determinants not previously linked to the response to polyamines, fcaA:Tn, c / s::Tn, prmC n, and pyrP n, were also sensitized to spermine (Figure 1 C). Dose-response assays showed an 8-fold reduction in spermine MIC in speG::Tn, and a decrease of the other four mutants at MIC relative to the wild-type strain. We constructed an in-frame unmarked deletion of speG given that its transposon mutant had the most pronounced spermine MIC shift; AspeG confirmed the observed spermine susceptibility shift. tcaA encodes a predicted transmembrane protein and is implicated in teicoplainin resistance (Maki, McCallum et al. 2004). cis encodes a cardiolipin synthase involved in modulating the phospholipid composition of the membrane (Tsai, Ohniwa et al. 2011 ). prmC encodes a release factor methyltransferase, and pyrP an uracil-xanthine permease. The molecular basis linking these determinants to polyamine response warrants further investigation, which falls beyond the scope of this study. Notably, sbnB: :Tn, disrupted in a gene encoding an enzyme involved in the siderophore staphyloferrin B biosynthesis (Beasley, Cheung et al. 2011 ), was initially identified in the screen and showed an 8-fold spermine MIC reduction; however, sbnB mutant was not sensitized to spermine (Supplementary Fig. 3). Wholegenome sequencing and subsequent PCR analysis of the sbnB n mutant revealed an additional truncation of 46.8 kb comprising the SCCmec cassette and the ACME locus, including speG and mecA hence, sbnB was excluded from the putative determinants list.

[0172] Next, we resupplied the hit compounds and conducted mini-checkerboard assays with spermine against the five identified polyamine resistance determinants to detect loss or reduction in synergy. The spermine synergistic interaction with OES2-0017 and V-1 , and I V-1 was lost or reduced in the speG mutant (Figure 1 E and G), suggesting that SpeG is the putative target of these compounds.

[0173] We performed checkerboard assays with spermine and OES2-0017 or V-1 against other speG+and speG- S. aureus strains. The tested strains included another USA300 strain CMRSA-10 that encodes speG as well as two strains that do not encodespeG including the MRSA strain COL and the methicillin-sensitive Staphylococcus aureus (MSSA) strain NCTC8325. The speG- strains, whether MRSA or MSSA, displayed a loss or reduction in synergy between spermine and OES2-0017 or V-1 , phenocopying the USA300 AspeG mutant (Figure 2 C-E). For both OES2-0017 and V-1 , CMRSA-10 displayed a similar synergistic phenotype to that observed in the other wild-type USA300 strain, JE2 (Figure 2 A, B). These results further suggest that OES2-0017 and V-1 are inhibiting SpeG activity, and that strains must encode speG to observe the spermine synergistic phenotype.SpeG as the target of V-1 and GES2-0017

[0174] We sought to confirm the SpeG inhibitory activity of the hit compounds in in vitro assays using purified His-tagged recombinant SpeG. We used a colorimetric SSAT assay based on the quantification of CoA-SH formed during the transfer of the acetyl group from acetyl-CoA, which is proportional to the amount of the acetylated substrate (AcCoA) (Lin, Lien et al. 2010). Under these experimental conditions, we observed acetyltransferase activity for the native and not the heat-inactivated SpeG when both spermine and spermidine were used as the test substrates. Based on our results and previous analysis (Tsimbalyuk, Shornikov et al. 2023), SpeG displays Michaelis-Menten kinetics with spermine as a substrate, allosteric kinetics with spermidine, and no activity with putrescine, thus we estimated catalytic efficiency of each as Km and So.s, respectively. The SpeG Km with spermine was 1312 + 348.1 pM and So.s with spermidine was 8669 + 3031 pM (Figure3). Catalytic efficiency of SpeG with spermine and spermidine as substrates were 5.58E+03 and 2.51 E+02 M’1s’1, respectively (Figure 3G). These results agree with previous reports showing that spermine is the preferred substrate of SpeG, followed by spermidine and that SpeG does not acetylate putrescine (Li, Maezato et al. 2019, Tsimbalyuk, Shornikov et al. 2023).

[0175] Next, we confirmed that OES2-0017 and V-1 inhibited acetyltransferase activity of SpeG in vitro by performing enzymatic inhibition assays testing a dilution series of each of the hits in the presence of either a single concentration or multiple concentrations of substrate in a checkerboard-style format estimating ICso and Ki, respectively. The ICso of OES2-0017 against SpeG was 34.82 + 6.810 pM at ~ 1 x Km ofspermine and the Ki was 20.20 + 3.446 pM when varying spermine concentrations both at a fixed saturating AcCoA concentration. The ICso of V-1 was 5508 + 1890 pM (at ~1 x Km of AcCoA and a spermine ~ 1 x Km concentration; (Figure 4 A-J). V-1 did not exhibit inhibitory activity under a fixed AcCoA concentration and variable spermine concentrations; however, at a fixed spermine concentration and variable AcCoA, V-1 inhibited SpeG with an estimated Ki of 5153 + 2126 pM (Figure I and J). Ki calculations were based on fitting the checkerboard-style data to the competitive inhibition model for both inhibitors, which was further supported by Lineweaver-Burk transformations (Figure 4 K). These values are slightly higher than expected when compared to the concentrations exhibiting spermine synergy interactions in whole-cell assays. However, this could be attributed to the in vitro cell-free experimental conditions (i.e., buffer concentration, pH, temperature, etc.) that do not perfectly match the conditions in vivo, which may be more optimal for catalysis. Of note, we use a high buffer concentration (100 mM Tris-HCI) to prevent polyamine high pH- mediated cleavage of Ellman’s reagent, which is sensitive to high pH (Zhu, Dhimitruka et al. 2004). These data confirm that both OES2-0017 and V-1 inhibit SpeG with OES2-0017 being more potent as a SpeG inhibitor, matching with the whole-cell assay results (Figure 4).Uncovering the mechanism of growth inhibitory effects of OES2-0017.

[0176] OES2-0017 and V-1 exhibit growth-inhibitory effects at concentrations higher than those required to synergize with spermine; therefore, we hypothesized that this activity occurs via a second mode of action. Given its growth inhibitory activity at low micromolar range, we prioritized OES2-0017 to determine if growth inhibition was mediated through an essential protein target. We confirmed activity against Bacillus subtilis, a Gram-positive organism with high degree of genetic conservation with S. aureus. Then, we screened a knock-down library covering the essential genes of B. subtilis against three sub-inhibitory concentrations of OES2-0017, spanning1 / 4th-1 / 16ththe wild-type MIC. While some mutants showed reduced growth in the presence of one or more of the tested sub-inhibitory concentrations, follow-up dose-response assays revealed that none of those mutants was more susceptible to OES2-0017 relative to the wild-type, suggesting the absence of an essential protein target.Next, we hypothesized that the growth-inhibitory activity of OES2-0017 is due to an effect on membrane integrity in the absence of an essential protein target. Using a DiSC3(5) membrane integrity assay (Farha, Verschoor et al. 2013), we found that OES2- 0017 permeabilized the membrane of USA300 at concentrations starting from its MIC (> 20 pM) in a concentration-dependent manner, suggesting membrane activity at high concentrations as the mechanism of growth inhibitory effects of OES2-0017. Notably, a DiSC3(5) assay with V-1 did not have an observable effect on membrane integrity (Figure 5).Next, we sought to check whether membrane disruption contributed to the observed synergy between OES2-0017 and spermine. We conducted a checkerboard-style DiSC3(5) assay with a combination of spermine and OES2-0017. OES2-0017 concentrations that correspond to the range of synergy (2.5-10 pM) resulted in no observable permeabilization. Like OES2-0017, spermine disrupted membrane integrity of USA300 at concentrations starting from its MIC against USA300. We observed the similar membrane permeabilization with spermidine and putrescine. Large polyamine molecules were previously shown to permeabilize the outer membrane of Gram-negative bacteria (Yasuda, Ohmizo et al. 2004); however, we found no reports on similar effects on Gram-positive membrane integrity. Importantly, there was no significant effect on membrane integrity in the presence of the combination of OES2-0017 and spermine at concentrations where synergy is observed, suggesting that membrane permeabilization does not contribute to the observed synergy. Taken together, we revealed a dual mode of action of OES2-0017 with high concentrations leading to growth-inhibitory effects due to membrane perturbation and low concentrations synergizing with polyamines through inhibiting SpeG and other SSAT activity.Evaluation of OES2-0017 and V-1 potential eukaryotic activity.

[0177] Given the membrane activity of OES2-0017 against S. aureus (Figure 7 A and B), we sought to evaluate its potential effects on mammalian cell membranes. To that end, we assessed the hemolytic activity of OES2-0017 against sheep red blood cells. OES2-0017 exhibited hemolytic effects, which are first observed at 160 pM with an increase at 640 pM reaching an absorbance of roughly that of the positive control (Triton X100). The hemolytic effects do occur at a concentration significantly higher than what isnecessary to inhibit bacterial growth (8-fold higher than the MIC) or to synergize with spermine through SpeG inhibition (512-fold higher than the lowest point of synergy with spermine). Notably, V-1 did not exhibit hemolytic effects at the concentrations tested (Figure 7).

[0178] Next, we assessed the activity of OES2-0017 and V-1 against the human sperm ine / spermidine acetyltransferase, SAT1. SAT1 is a dimer in solution with one monomer seemingly self-acetylating a lysine residue and the other binding polyamines for acetylation (Bewley, Graziano et al. 2006). First, we overexpressed and purified SAT1 enzyme and confirmed its acetyltransferase activity in vitro with spermine. SAT1 demonstrated higher substrate specificity (KM = 73.10 + 8.328 pM) and high catalytic efficiency (Kcat / KM = 1.28E+05 M’1S’1) towards spermine than SpeG (KM = 1312 + 348.1 pM and Kcat / KM = 5.58E+03 M’1s’1; Figure 6 A and B). Our results are in agreement with previous kinetic characterization and the relative activities of both enzymes (Hegde, Chandler et al. 2007, Li, Maezato et al. 2019). We then tested the ability of OES2-0017 and V-1 to inhibit SAT 1 . The ICso of OES2-0017 against SAT 1 was 26.39 + 6.411 pM, whereasV-1 did not inhibit SAT1 under any of the tested conditions (Figure 6 C). Although, OES2- 0017 can inhibit SAT1 activity, this may not prove detrimental to human cells. Multiple studies have employed the SSAT knockout mice and the non-specific SSAT inhibitor Berenil in in vivo mouse models, observing no toxic effects to mice (Zahedi, Lentsch et al. 2009, Jin, Xu et al. 2024, Xu, Ruan et al. 2024). On the other hand, SAT1 inhibition might have other potential therapeutic uses. Altered polyamine metabolism has been implicated in many human diseases and conditions, including cancer, osteoporosis, and ischemia / reperfusion leading to acute kidney injury (Russell 1971 , Zahedi, Lentsch et al. 2009, Maksymiuk, Sitar et al. 2018, Zahedi, Barone et al. 2019, Jin, Xu et al. 2024) with SAT1 being previously suggested as a promising drug target (Wallace and Fraser 2004, Bewley, Graziano et al. 2006).Conclusions

[0179] We undertook a chemical screen to identify inhibitors of polyamine detoxification, and uncovered two lead compounds, OES2-0017 and V-1 , that show growthinhibition and synergy with spermine. A chemogenomic screen and in vitro enzymatic assays revealed that both compounds inhibit the sperm ine / spermidine acetyltransferase SpeG. The most potent of the two, OES2-0017 with activity at the low micromolar concentration range, inhibited bacterial growth via membrane perturbation. We also observed the growth inhibitory and spermine synergistic effects of OES2-0017 in a broadspectrum of Gram-positive and Gram-negative bacteria, including E. faecalis, E. faecium, B. cenocepacia, K. pneumoniae, and A. baumannii, and the pathogenic yeast C. albicans. Synergy between spermidine and OES2-0017 was not completely lost in a USA300 AspeG mutant, suggesting other underlying polyamine detoxification targets, which prompted us to search for SSATs homologous to B. subtilis PaiA and BltD. We found PaiAsa, characterized its SSAT activity in vitro, and showed that OES2-0017 inhibited its activity. Further, OES2- 0017 synergized with cefuroxime and potentiated azithromycin activity against S. aureus. This potentiation effect with azithromycin is similar to previous work in K. pneumoniae (Adams, Moulding et al. 2024). OES2-0017 also abolished the ability of S. aureus to use exogenous polyamines to resist antibiotic treatment. Finally, we performed a small-scale SAR study and identified compounds with differential activity towards the human SAT1 and S. aureus SpeG, including OES2-0086 and OES2-0052. Together, the growth inhibitory effects of the inhibitors identified herein suggest their potential use as stand-alone antimicrobials and their SSAT inhibitory effects, suggest they may serve as antivirulence agents. The synergy with some antibiotics and ability to prevent exogenous polyam inemediated resistance to other antibiotics in S. aureus suggest OES2-0017 may also be used as an antibiotic adjuvant.

[0180] Polyamine analogs have previously been demonstrated to have antiparasitic, anticancer (Wallace and Fraser 2004, Boncher, Bi et al. 2007, Egorov, Yu et al. 2021 ), and antibacterial properties (Blanchet, Borselli et al. 2016, Douglas, Alkhzem et al. 2022, Chen, Cadelis et al. 2023); however, the previously tested analogs differ in structure from OES2-0017 and the other polyamine analogs tested in this study. Antibacterial polyamines described to date include mono- and bis-acyl polyamines, amine steroidal derivatives (Blanchet, Borselli et al. 2016), and diacylpolyamines with aromatic head groups (Chen, Cadelis et al. 2023). One study tested linear polyamine analogs, similar to OES2-0017, and revealed bactericidal and 0-lactam potentiating activity;however, the mechanism of growth inhibition and antibiotic potentiation were not discussed (Douglas, Alkhzem et al. 2022). Notably, several polyamine analogs were shown to inhibit polyamine biosynthesis, transport, and catabolism (Boncher, Bi et al. 2007, Egorov, Yu et al. 2021 ). As such, in addition to screening a diverse small molecule library, we also undertook a targeted approach of screening a library of polyamine analogs for activity against polyamine detoxification enzymes. Indeed, this rationale-based approach showed a higher hit rate and higher potencies compared to the unbiased approach although both screens identified SSAT bioactives.

[0181] Specific bacterial SSAT inhibitors have not been identified previously.Notably, berenil (diminazene aceturate) was shown to inhibit SSAT and has been used in vivo in murine models for this purpose (Jin, Xu et al. 2024). In vitro assays against human SSAT suggested berenil is a potent competitive inhibitor of spermidine with a Ki of 2.0 pM (Libby and Porter 1992); however, berenil also inhibits rat S-adenosyl-L-methionine decarboxylase and mouse polyamine oxidase (Karvonen, Kauppinen et al. 1985, Libby and Porter 1992). Activity against multiple enzymes may explain the toxic effects observed during intramuscular injection of camels with 10 mg / kg berenil (Homeida, El Amin et al. 1981 ); however no toxic effects were observed in mice dosed at 16 mg / kg weekly for six weeks (Jin, Xu et al. 2024) or with a single intraperitoneal or intramuscular dose of 3.5, 10, or 20 mg / kg (Homeida, El Amin et al. 1981 , Elamin, Homeida et al. 1982). Aside from the SSAT activity, berenil was shown to have other activities, including immunomodulatory(Passaglia, Kanashiro et al. 2024) and cardioprotective(Coutinho, Santos-Miranda et al. 2022) activities, and is used in veterinary medicine mostly for parasitic infections albeit the exact underlying mode of such activity is not fully elucidated but has been associated with the connection of its aromatic diamidine to the kinetoplast DNA (kDNA) of the trypanosomes(da Silva Oliveira and de Freitas 2015). Our study provides insights that may lead to the development of specific bacterial SSAT inhibitors. Although OES2-0017 caused hemolytic activity at 4X MIC against S. aureus USA300, SpeG-inhibitory activity of the compound was observed as low as 1 / 32ndthe MIC, suggesting a concentration range within which OES2-0017 may remain efficacious while avoiding potential adverse effects. Future studies will further assess the potential toxicity ofOES2-0017 and its analogs and their interaction with other polyamine-related enzymes using natural polyamines as substrate.

[0182] We also identified V-1 (isoproterenol) as a spermine synergist.Isoproterenol is a 01- 02-adrenergic agonist used in the treatment of heart block, heart failure, and cardiac arrest (Isoproterenol 2024). Its identification in our chemical screen serves as an example of potential drug repurposing for the treatment of bacterial infections. Time and money spent on drug development are drastically reduced by repurposing as pharmacodynamic, pharmacokinetic, and toxicity profiles of the drugs are already known (Farha and Brown 2019). Our results indicate that isoproterenol also inhibits SpeG, albeit with much lower potency than OES2-0017, and exhibits growth inhibitory activity. Importantly, it did not show detectable activity against the human SAT1 and no hemolytic activity.

[0183] The cellular concentrations of polyamines in mammalian cells vary across tissues and physiological conditions; however, the total polyamine content is generally assessed to be in the mM range (Igarashi and Kashiwagi 2010). Polyamines are known to bind to macromolecules, such as DNA, RNA, phospholipids, and ATP, so levels of free intracellular polyamines are not definitive (Igarashi and Kashiwagi 2010). In polyamine-producing bacteria, the contents of intracellular polyamines have been reported as high as 30 mM (Igarashi and Kashiwagi 2010). Importantly, polyamine concentrations in the host further increase ~2-fold in response to infection (Thurlow, Joshi et al. 2013). As such, bacteria are likely to encounter polyamines during infection or in their surrounding environment at concentrations similar or higher than those tested in this study. Further, the detrimental effects of speG or alternative SSAT mutations on bacterial virulence (Thurlow, Joshi et al. 2013, Martini, Michaux et al. 2015, Fang, Huang et al. 2017) suggest that the levels of polyamines within the host are comparable to those used in vitro at which polyamine susceptibility and synergy with the inhibitors were observed in this study.Therefore, given that OES2-0017 inhibits speG, it will likely reduce the ability of bacteria to withstand host polyamines produced at the site of infection, suggesting it may allow these host compounds to help clear infection serving as antivirulence agent.

[0184] In vitro enzymatic assays suggest that OES2-0017 is relatively equipotent against both SpeG and SAT1 suggesting potential activity against the human enzyme in vivo. SAT1 inhibitors have other potential therapeutic uses including disrupting tumor growth, protection from kidney injury following ischemia-reperfusion injury, and preventing osteoporosis (Wallace and Fraser 2004, Bewley, Graziano et al. 2006, Zahedi, Lentsch et al. 2009, Jin, Xu et al. 2024). Notably, there were two analogs of OES2-0017, OES2-0085 and OES2-0086, that showed inhibitory activity against SpeG but not SAT1 and another two, OES2-0077 and OES2-0052, which were more potent inhibitors of SpeG than SAT1. Although these analogs have lower inhibitory activity than OES2-0017, they and other analogs from our analysis can guide future SAR studies to optimize the inhibition of SpeG while limiting activity against SAT1.

[0185] S. aureus was not previously known to have an SSAT except for strains that acquired SpeG, such as USA300. Notably, the USA300 AspeG and other S. aureus strains that do not harbor speG (e.g., COL and NCTC8325) are only 4-fold and 8- fold more susceptible to spermidine and spermine, respectively, compared to wild-type USA300, suggesting a potential for another detoxification mechanism. Here, we identified PaiAsa, a homolog of B. subtilis spermine / spermidine acetyltransferase PaiA, in S. aureus, providing its first experimental characterization as an additional SSAT in S. aureus. SpeG appears to be more dominant with a greater shift in polyamine susceptibility observed in a AspeG mutant than pa / Asa::Tn; the contribution of PaiAsa to polyamine resistance in whole cells requires assessment in a speG paiAsa double mutant in USA300 or a single paiAsa mutant in a speG- S. aureus strain. We further showed that OES2-0017 can inhibit PaiAsa in vitro.

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Claims

CLAIMS:1 . A method of treating a microbial (such as bacterial or fungal) infection in a subject in need thereof, comprising administering to the subject an effective amount of an antibiotic and an effective amount of one or more compounds selected from a compound of Formula IV, V and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V or VI is as follows:wherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

2. The method of claim 1 , wherein the one or more compounds are compounds of Formula IV:(IV) or a pharmaceutically acceptable salt and / or solvate thereof.

3. The method of claim 1 , wherein the one or more compounds are selected from the compound of Formula V:or a pharmaceutically acceptable salt and / or solvate thereof.

4. The method of claim 3, wherein the one or more compounds of Formula V are selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

5. The method of claim 1 , wherein the one or more compounds are compounds of Formula VI:or a pharmaceutically acceptable salt and / or solvate thereof.

6. The method of claim 5, wherein the one or more compounds of Formula VI are selected from:VI-1 or a pharmaceutically acceptable salt and / or solvate thereof.

7. A method of treating a microbial (such as bacterial or fungal) infection in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds selected from a compound of Formula IV, V and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V or VI is as follows:wherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

8. The method of claim 7, wherein the one or more compounds are compounds of Formula IV:or a pharmaceutically acceptable salt and / or solvate thereof.

9. The method of claim 7, wherein the one or more compounds of Formula V are selected from:V-2, or a pharmaceutically acceptable salt and / or solvate thereof.

10. The method of claim 7, wherein the one or more compounds of Formula VI are selected from:VI-1 or a pharmaceutically acceptable salt and / or solvate thereof.11 . The method of claim 1 , wherein the efficacy of the antibiotic administered with one or more compounds of claim 1 is greater than the efficacy of the antibiotic administered in the absence of the one or more compounds of the application.

12. The method of claims 1 and 7, wherein the one or more compounds of claims 1 and 7 inhibit bacterial detoxification of natural polyamines present at the site of infection.

13. The method of claims 1 and 7, wherein the one or more compounds of claims 1 and 7 inhibit fungal detoxification of natural polyamines present at the site of infection.

14. The method of claim 1 , wherein the one or more compounds of claim 1 reduce or eliminate polyamine-mediated antibiotic resistance of the bacterium or fungus to the antibiotic.

15. The method of any one of claims 1 to 6 and 11 to 14, wherein the antibiotic is selected from a macrolide, a tetracycline, a lipopeptide, a glycopeptide, a beta-lactam, a penicillin, a cephalosporin, a monobactam, a carbapenem, an aminoglycoside, a rifamycin, a ketolide, an oxazolidinone, a glycylcycline, an aminocoumarin, sulfonamide, a trimethoprim, a lincomycin, a streptogramin, a chloramphenicol, a quinolone, a fluoroquinolone and an antimicrobial peptide, or a combination thereof.

16. The method of claim 15, wherein the antibiotic is selected from amikacin, neomycin, tobramycin, paromomycin, streptomycin, spectinomycin, ertapenem, doripenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cefalothin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, lipopeptide, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, linezolid, posizolid, radezolid, torezolid, amoxicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, penicillin g, temocillin, ticarcillin, bacitracin, colistin, polymyxin b, besifloxacin,enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, sulfonamidochrysoidine, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin / dalfopristin, thiamphenicol, tigecycline, tinidazole, trimethoprim, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, nitrofurantoin, methenamine, and streptomycin, or a pharmaceutically acceptable salt thereof, or a combination thereof.

17. The method of claim 7, wherein the one or more compounds of claim or 7 inhibit growth of a bacterium.

18. The method of claim 7, wherein the one or more compounds of claim 1 or 7 suppress virulence of bacterium.

19. The method of claim 7, wherein the one or more compounds of claim 1 or 7 inhibit growth of a fungus.

20. The method of claim 7, wherein the one or more compounds of claim 1 or 7 suppress virulence of fungus.21 . The method of claim 1 or 7, wherein the infection is caused by Gram-positive or Gram-negative bacterium or fungus.

22. The method of claim 21 , wherein the Gram-negative bacterium is a species of Klebsiella, Salmonella, Burkholderia, Pseudomonas or Escherichia, or a combination thereof.

23. The method of claim 21 , wherein the Gram-positive bacterium is a species of Enterococcus, Mycobacterium, Bacillus or Staphylococcus, or a combination thereof.

24. The method of claim 23, wherein the bacterium is S. aureus USA300.

25. The method of claim 21 , wherein the fungus is a species of Candida or Saccharomyces, or a combination thereof.

26. The method of any one of claims 1 or 7, wherein the one or more compounds are compounds of Formulae:V-1 ; andVI-1 , or a pharmaceutically acceptable salt and / or solvate thereof, wherein the infection is caused by Gram-positive or Gram-negative bacterium or fungus.

27. The method of claim 26, wherein the bacterium is a drug-resistant Gram-positive bacterium.

28. The method of claim 27, wherein the bacterium is S. aureus USA300.

29. The method of claim 26, the Gram-negative bacterium is K. pneumoniae.

30. The method of claim 29, wherein the antibiotic is macrolide antibiotic.31 . The method of claim 30, wherein the macrolide is azithromycin.

32. The method of claim 26, wherein the antibiotic is anti-Gram-negative or anti-Gram- positive antibiotic.

33. The method of claim 32, wherein the antibiotic is azithromycin, novobiocin, rifampicin, doxycycline, tetracycline, vancomycin, oxacillin, erythromycin, tobramycin, gentamicin, ciprofloxacin, daptomycin or trimethoprim.

34. The method of claim 32, wherein the antibiotic is colistin.

35. A method of treating a disease, disorder or dysfunction related to polyamine detoxification or Spermine / Spermidine N-acetyltransferase or SAT1 , including but not limited to, cancer, osteoporosis, and ischemia / reperfusion leading to acute kidney injury in a subject in need thereof, comprising administering to the subject an effective amount of one or more compounds selected from a compound of Formula IV, V and VI, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula IV, V or VI is as follows:wherein:R17is selected from H and OCH3;R18 is selected from H, CH3, and any other hydrocarbon;R19 is selected from H, CH3, and any other hydrocarbon; or a pharmaceutically acceptable salt and / or solvate of any of the above.

36. The method of claim 35, wherein the one or more compounds are compounds of Formula IV:or a pharmaceutically acceptable salt and / or solvate thereof.

37. The method of claim 35, wherein the one or more compounds are selected from the compound of Formula V:(V) or a pharmaceutically acceptable salt and / or solvate thereof.

38. The method of claim 37, wherein the one or more compounds of Formula V are selected from:V-2, or a pharmaceutically acceptable salt and / or solvate thereof.

39. The method of claim 35, wherein the one or more compounds are compounds of Formula VI:or a pharmaceutically acceptable salt and / or solvate thereof.

40. The method of claim 39, wherein the one or more compounds of Formula VI are selected from:VI-1or a pharmaceutically acceptable salt and / or solvate thereof.41 . The method of any one of claims 1 to 6, 11 to 16 and 21 to 34, wherein the antibiotic and the one or more compounds are administered to the subject in a pharmaceutical composition comprising said antibiotic, the one or more compounds or a pharmaceutically acceptable salt and / or solvate thereof, and a pharmaceutically acceptable carrier or vehicle.

42. The method of claim 7 to 10, 12 to 13, 17 to 29 and 35 to 40, wherein the one or more compounds are administered to the subject in a pharmaceutical composition comprising the one or more compounds or a pharmaceutically acceptable salt and / or solvate thereof, and a pharmaceutically acceptable carrier or vehicle.

43. The method of claims 41 or 42, wherein the pharmaceutical composition further comprises a bicarbonate buffer.

44. A composition comprising: a) one or more compounds of Formula IV, V, or VI, or a pharmaceutically acceptable salt and / or solvate thereof; and b) a pharmaceutically acceptable carrier, wherein the composition is configured for administration in combination with an antibiotic for treating a bacterial or fungal infection.

45. A pharmaceutical composition comprising: a) an effective amount of one or more compounds selected from a compound of Formula IV, V, or VI, or a pharmaceutically acceptable salt and / or solvate thereof; and b) an effective amount of an antibiotic, wherein the composition is formulated to enhance the efficacy of the antibiotic against Gram-positive or Gram -negative bacteria or fungi.

46. The composition of claim 45, wherein the bacterial infection is caused by a Gramnegative bacterium selected from Klebsiella pneumoniae, Escherichia coli, or Salmonella Typhimurium.

47. The composition of claim 45, wherein the bacterial infection is caused by a Gram-positive bacterium selected from Staphylococcus aureus or Enterococcus faecalis.

48. The composition of claim 45, wherein the one or more compounds are configured to inhibit bacterial polyamine detoxification enzyme activity.

49. A compound of Formula IV, V, or VI, or a pharmaceutically acceptable salt and / or solvate thereof, for use in the treatment of a bacterial and fungal infection and polyamine detoxification (SATl)-related diseases and disorders.

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