Polyamine analog based antimicrobial, antibiotic adjuvant and therapeutic compounds

Abstract

: The present application discloses methods and compositions for enhancing antibiotic efficacy and treating microbial infections through the use of polyamine analog compounds. These compounds function as antimicrobial agents and antibiotic adjuvants, sensitizing bacteria to antibiotics by disrupting bacterial membrane integrity, inhibiting polyamine detoxification enzyme activity, and mitigating resistance mechanisms. The disclosed methods include administering to a subject an effective amount of an antibiotic in combination with one or more polyamine analog compounds of Formula I, II, or III, or their pharmaceutically acceptable salts or solvates. The compositions are effective against a wide range of Gram-positive and Gram-negative bacteria, including drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae, and fungi such as Candida albicans. The application also describes pharmaceutical formulations, dosing strategies, and synergistic effects achieved with these compounds, addressing the urgent need for innovative solutions to combat antimicrobial resistance and improve the treatment of severe bacterial infections. 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

POLYAMINE ANALOG BASED 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, various polyamine and polyamine analog compounds. In particular the application relates to the use of polyamine and polyamine analogs to treat bacterial and fungal infections and polyamine detoxification (SATl)-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, Stoj anovic 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 Gram-positive 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 P-lactam antibiotics, coined methicillin-resistant Staphylococcus aureus (MRSA). MRS A 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^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 and spermidine, 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 US A300 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 ispeG 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 present application discloses methods of using polyamines and polyamine analog compounds for the treatment of microbial (such as bacterial or fungal) infections. The polyamine analogs of the application sensitize bacteria and fungi and resistant bacterial and fungal strains to the effect of the antibiotics, reduce bacterial and fungal resistance to the antibiotics and improve the efficacy of the antibiotics.

[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 I, II, and III, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula I, II, or III is as follows:R2R1-bZ 'R3(i); wherein:R1is selected from Ce-iealkyl; andR2and R3are independently selected from H and Ci-iealkyl, orR2and R3are linked together with the nitrogen atom to which they are attached to form a 3- to 6- membered heterocyclic ring, or one of R2and R3is H or Ci-4alkyl and the other is selected from C(NR4)NR5Ci-4alkyl, C(NR4)NR5C2- 4alkenyl and C(NR4)NR5C2-4alkynyl; andR4and R5are independently selected from H and Ci-ealkyl;(II); wherein:R6, R7, R8and R9are independently selected from H and Ci-iealkyl, orR6and R7and / or R8and R9are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R6and R7and / or one of R8and R9is H or Ci-4alkyl and the other is selected from C(NR10)NR11Ci-4alkyl, C(NR10)NR11C2-4alkenyl and C(NR10)NR11C2-4alkynyl;R10and R11are independently selected from H and Ci-ealkyl;X1is selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R6, R7, R8and R9are all H, then X1is not n-butylene;wherein:R12, R13, R14, R15and R16are independently selected from H and Ci-isalkyl, orR12and R13and / or R15and R16are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R12and R13and / or one of R15and R16is H or Ci-4alkyl and the other is selected from C(NR17)NR18Ci-4alkyl, C(NR17)NR18C2.4alkenyl and C(NR17)NR18C2.4alkynyl;R17and R18are independently selected from H and Ci-ealkyl;X2and X3are independently selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R12, R13, R14, R15and R16are all H, then X2and X3are not both n-butylene;

[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] 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 illustrationonly 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 THE DRAWINGS:

[0014] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which: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 11-1 and spermine against S. aureus USA300. E) Representative checkerboard assay of exemplary compound 11-1 and spermine against AspeG.Figure 2 shows the results of the representative checkerboard assays of spermine and exemplary compound II- 1 against S. aureus A) US A300 JE2, B) CMRSA-10, C) sspeG, D) COL, and E) NCTC8325.Figure 3 shows A1-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 spermine / 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.Figure 4 shows the inhibitory effects of exemplary compounds 1-1, 1-2, 11-4, II-3, 1-3, 11-2, I- 4, and ILL Inhibition of purified SpeG by A and B) 1-1, C and D) 1-2, E and F) 11-4, G and H)II-3, I and J) 1-3, K and L) II-2, M and N) 1-4, and O and P) II-l. Colorimetric spermine / spermidine acetyltransferase (SSAT) assays were conducted with 1 mM Acetyl Coenzyme A and 1500 pM spermine in the reaction mixture. Data are represented as the mean ± SEM from four independent experiments n=4 with the exception of O and P where n=7.Figure 5 shows the inhibitory effects of exemplary compounds 1-1, 1-2, II-4, II-3, 1-3, II-2, I- 4, and II-l . Inhibition of purified SAT1 by A and B) 1-1, C and D) 1-2, E and F) II-4, G and H) II-3, 1 and J) 1-3, K and L) II-2, M and N) 1-4, and O and P) II-l. Data are represented as the mean ± SEM from three independent experiments n=3.Figure 6 shows A) DiSCs(5) membrane permeabilization assay of II-l against S. aureus USA300. Results represented as mean relative fluorescence units (RFU) ± SEM for three independent experiments (n=l 1). B) A representative DiSCs(5) membrane permeabilization assay performed as a mini-checkerboard evaluating the combined effects of II-l and spermine.Figure 7 shows representative checkerboard assays of exemplary compound II-l and spermine against A) B. subtilis 168 (n=4), B) / / . coli BW25113 (n=4), C)K. pneumoniae MKP103 (n=4), D) S. Typhimurium 14028s (n=4), E) E. faecalis TX1322 (n=3), F) E. faecium ERV165 (n=3), G) S. epidermidis NRS6 (n=3), H) S. epidermidis NIH04008 (n=3), I) B. cenocepacia K56-2 (n=3), J) M. smegmatis (n=3), and K) C. albicans SC5314 (n=3). L) Putrescine and II-l checkerboard against baumannii ATCC 19606 (n=3). Representative checkerboard assays of spermine and II-l against spermine / spermidine acetyltransferase mutants M) B. subtilis bltD (n=4), N) E. coli l.speG (n=3), O) K. pneumoniae speG'. :Tn30 (n=3), P) S. Typhimurium speG. '.Cva (n=3). FICI values are represented as mean ± standard deviation for the indicated number of replicates.Figure 8 shows PaiAsa as a spermine / spermidine acetyltransferase in S. aureus. A-C) Steadystate enzymatic assays of PaiAsa evaluate the acetylation activity in the presence of substrates A) spermine (n=6), B) spermidine (n=6), and C) putrescine (n=6). D) Enzymatic inhibition assay of PaiAsa with spermine as a substrate by II-l (n=3). Results are represented as a mean ± SEM of three independent experiments. Representative checkerboard assays of spermineand II- 1 against S. aureus E) USA300 (n=l 1), F) paiA .Gn (n=3). FICI values are represented as mean ± standard deviation.Figure 9 shows representative checkerboard assays of spermine and vancomycin (A-C), kanamycin (D-F), and rifampicin (G-I) against S. aureus A, D, G) US A300, B, E, H) speG, and C, F, I) USA300 supplemented with a sub-inhibitory concentration of II-l where II-l abolishes the protective effect of polyamines from the antibiotics. Representative checkerboard assay of II-l and cefuroxime (J) and azithromycin (K) against S. aureus US A300, where II-l potentiates the antibiotic activity. FICI values are represented as mean ± standard deviation for the indicated number of replicates: A) n=10, B) n=7, C) n=4, D) n=5, E) n=5, F) n=3, G) n=8, H) n=5, 1) n=3, J) n=3, and K) n=4.Figure 10 shows representative checkerboard assays of spermine and vancomycin (A-C), 1-4 and vancomycin (D), spermine and kanamycin (E-H), OES2-0052 and kanamycin (I), spermine and rifampicin (J-L), and 1-4 and rifampicin against S. aureus A, D, E, H, I, and L) US A300, B, F, J) speG, and C, G, K) US A300 supplemented with a sub-inhibitory concentration of 1-4 where 1-4 abolishes the protective effect of polyamines from the antibiotics. Representative checkerboard assay of 1-4 and cefuroxime (M) and azithromycin (N) against S. aureus USA300, where OES2-0052 potentiates the antibiotic activity. FICI values are represented as mean ± standard deviation n=10 (A), n=7 (B), n=2 (C, G, H, K, and L), n=8 (I), n=5 (E, F, and J), and n=3 (D, M and N).Figure 11 shows the acetyltransferase activity of SAT1 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) Enzymatic inhibition assay of II-l performed against human SAT1 with excess AcCoA (0.25 mM) and 75 pM spermine (« KM; n=3). Results are represented as a mean ± SEM of three independent experiments.Figure 12 shows hemolytic effects of A) 1-1, B) 1-2, C) II-4, D) II-3, E) 1-3, F) II-2, G) 1-4, and H) II- 1. Cells begin to lyse at concentrations of II- 1 greater than those required to inhibit bacterial growth. Data are represented as the mean ± SEM from three independent experiments A-D) n=16 and E-F) n=20 H) n=24.Figure 13 shows A-C) A representative checkerboard assay of K. pneumoniae (MKP103) treated with polyamine-azithromycin combination (putrescine, spermine, spermidine respectively). D) A scatter plot representing the distribution of FICI values across the K. pneumoniae diversity panel (Martin, M. J., 2023). E) Representative checkerboard assays of a select few clinical isolates from the diversity panel. F) A checkerboard assay of K. pneumoniae (MKP103) in 25 mM bicarbonate buffer with putrescine and azithromycin in combination. Dark blue regions represent high cell density determined by ODeoo. Red dotted lines refer to the clinical break point as set by CLSI. Mean of the FICI and SEM are displayed on each representative checkerboard, n=3.Figure 14 shows A) The screening process to identify genetic determinants of azithromycinputrescine synergy. B) An Index plot of the primary screen shown as % growth of mutants from K. pneumoniae (MKP103) grown in 32 pg / mL of azithromycin compared to a control. Data points below the red line mark significant hits, where a cut-off of 1.5x the standard deviation of all datapoints below the interquartile mean was used to set the cutoff. C-F) Representative putrescine-azithromycin checkerboards of the K. pneumoniae (MKP103) mutants identified from the chemogenomic screen. Dark blue regions represent high cell density determined by ODeoo. Mean of the FICI and SEM are displayed on each representative checkerboard, n=3.Figure 15 shows: A) Results of a kinetic assay of three combined independent assays conducted as triplicates displaying N-phenyl-naphthylamine (NPN) uptake in K. pneumoniae (MKP103) induced by perturbation of the outer membrane by putrescine (error bars shown as standard error). B) Results of a kinetic assay of three combined independent assays conducted as triplicates displaying the difference in NPN uptake between K. pneumoniae wild-type and K. pneumoniae waaD::Tn. C) Results of a kinetic absorbance assay of three combinedindependent assays conducted as triplicates displaying the difference in nitrocefin hydrolysis by periplasmic P-lactamase in K. pneumoniae wild-type and K. pneumoniae waaD::Tn. D)-E) Representative checkerboard assays of triplicate experiments showing the combination of putrescine and azithromycin against K. pneumoniae (MKP103) in the absence and presence of 1 ug / mL polymyxin B nonapeptide; Dark blue regions represent high cell density determined by ODeoo, error shown as standard error. F) Results of a kinetic absorbance assay of ONPG hydrolysis in E. coli ML-35 representing rate of inner membrane perturbation. Mean of the FICI and SEM are displayed on each representative checkerboard, n=3. All error bars denote SEM.Figure 16 shows a heat map with average FICI values of duplicate checkerboard assays in K. pneumoniae (MKP103) with various drug combinations. B-H) representatives of triplicate checkerboard assays run independently. B-D) in K. pneumoniae (MKP103) wild-type comparing the phenotypes of B) putrescine-azithromycin, C) tetracycline-azithromycin and D) putrescine-tetracy cline. E-F) comparing the phenotypes of K. pneumoniae rpsO::Tn in combination of E) putrescine-azithromycin and F) tetracycline-azithromycin. G-H) comparing the phenotypes of K. pneumoniae rpsR::Tn in combination of G) tetracycline-azithromycin and H) putrescine-azithromycin. All FICI values are shown as the average with the associated standard error. Dark blue regions represent high cell density determined by ODeoo. Mean of the FICI and SEM are displayed on each representative checkerboard, n=3.Figure 17 shows the results of the fluorometrical monitoring (433 / 475 and 610 / 650 nm respectively) in black 384 well plates using the NEBExpress cell-free protein expression system in 200 mM HEPES buffer with an engineered plasmid encoding deGFP with a malachite green mRNA aptamer. Results shown are after two hours incubation and were normalized by dividing each well’s RFU deGFP value by the malachite green RFU value.Figure 18 shows A) A replica plot displaying the OD readings of K. pneumoniae MKP103 in the presence of the polyamine analog alone (screen 1 on the y-axis) and in the presence of both polyamine analogs and 32 pg / mL of azithromycin (screen 2 on the x-axis). B)-E) Representative checkerboard assays against K. pneumoniae (MKP103) of exemplarycompounds II-3, 1-2, 1-5 and II- 1. All FICI values are shown as the average with the associated standard error. Dark blue regions represent high cell density determined by ODeoo.Figure 19 shows the results of a kinetic assay displaying NPN uptake in K. pneumoniae (MKP103) induced by perturbation of the outer membrane by the exemplary compounds A) II- 1, B) 1-2, C) II-3, and D) 1-5 respectively (error bars shown as standard error).Figure 20 shows the results of a kinetic assay displaying absorbance of ONPG hydrolysis in E. coli ML-35 pBR322 induced by perturbation of the inner membrane by the exemplary compounds A) II-l, B) 1-5, C) II-3, D) 1-2, and E) putrescine (error bars shown as standard error).Figure 21 shows the checkerboard assay of K. pneumoniae treated with 1-1 and 1-5 in combination with colistin. Dark blue regions represent high cell density determined by ODeoo.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

[0015] 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.

[0016] 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 featuredisclosed 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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 ormaterial. 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.

[0024] 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.

[0025] The term “compound of the application” or “compound of the present application” and the like as used herein refers to a compound of Formula I, II, or III, or a salt and / or solvate thereof.

[0026] 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.

[0027] The term “method of the application” as used herein refers to the method of treating a bacterial infection as described herein.

[0028] 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.

[0029] 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.

[0030] 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 OrganicSynthesis”, John Wiley & Sons, 3rdEdition, 1999 and in Kocienski, P. Protecting Groups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas).

[0031] 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.

[0032] 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.

[0033] 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 C2-6alkenyl 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.

[0034] 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 C2-6alkynyl means an alkynyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0035] 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.

[0036] 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-iocycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 (whetherpartial 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.

[0047] “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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The term “adjuvant” as used herein refers to an agent which enhances the pharmaceutical effect of an antibiotic.II. Methods of the Application

[0054] 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 I, II, and III or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula I, II, or III is as follows:(i); wherein:R1is selected from Ce-iealkyl; andR2and R3are independently selected from H and Ci-iealkyl, orR2and R3are linked together with the nitrogen atom to which they are attached to form a 3- to 6- membered heterocyclic ring, or one of R2and R3is H or Ci-4alkyl and the other is selected from C(NR4)NR5Ci-4alkyl, C(NR4)NR5C2- 4alkenyl and C(NR4)NR5C2-4alkynyl; andR4and R5are independently selected from H and Ci-ealkyl;R6R8'N-X1-N'R7k9(II); wherein:R6, R7, R8and R9are independently selected from H and Ci-iealkyl, orR6and R7and / or R8and R9are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R6and R7and / or one of R8and R9is H or Ci-4alkyl and the other is selected from C(NR10)NR11Ci-4alkyl, C(NR10)NR11C2-4alkenyl and C(NR10)NR11C2-4alkynyl;R10and R11are independently selected from H and Ci-ealkyl;X1is selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R6, R7, R8and R9are all H, then X1is not n-butylene;wherein:R12, R13, R14, R15and R16are independently selected from H and Ci-isalkyl, orR12and R13and / or R15and R16are linked together with the nitrogen atom to which they are attached to form a 3 - to 6-membered heterocyclic ring, or one of R12and R13and / or one of R15and R16is H or Ci-4alkyl and the other is selected from C(NR17)NR18Ci-4alkyl, C(NR17)NR18C2.4alkenyl and C(NR17)NR18C2-4alkynyl;R17and R18are independently selected from H and Ci-ealkyl;X2and X3are independently selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R12, R13, R14, R15and R16are all H, then X2and X3are not both n-butylene;

[0055] In some embodiments, the one or more compounds are selected from the compound of Formula I:or a pharmaceutically acceptable salt and / or solvate thereof, wherein R1, R2and R3are as defined for Formula I.

[0056] In some embodiments, R2and R3are independently selected from H and Ci-iealkyl. In some embodiments, R2and R3are both H. In some embodiments, one of R2and R3is H and the other one is selected from Ci-ioalkyl.

[0057] In some embodiments, R2and R3are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring. In some embodiments, R2and R3are linked together with the nitrogen atom to which they are attached to form a 4, 5 or 6-membered heterocyclic ring.

[0058] In some embodiments, one of R2and R3is H or Ci-4alkyl and the other is selected from C(NR4)NR5Ci-4alkyl, C(NR4)NR5C2-4alkenyl and C(NR4)NR5C2-4alkynyl. In some embodiments, one of R2and R3is H or CH3 and the other is selected from C(NH)NHCi-4alkyl, C(NH)NHC2-4alkenyl and C(NH)NHC2-4alkynyl. In some embodiments, one of R2and R3is H and the other isC(NH)NHC2alkynyl.

[0059] In some embodiments, the compound of Formula I is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

[0060] In some embodiments, the one or more compounds are selected from the compound ofFormula II:or a pharmaceutically acceptable salt and / or solvate thereof, wherein R6, R7, R8, R9and X1are as defined for Formula I.

[0061] In some embodiments, R6, R7, R8and R9are independently selected from H and Ci- lealkyl. In some embodiments, one of R6, R7, R8and R9is H and the other three are selected from Cn lealkyl. In some embodiments, two of R6, R7, R8and R9are H and the other two are selected from Cn lealkyl. In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Ci-iealkyl. In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Ce-iealkyl. In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Cs-ualkyl. In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Cio-ualkyl. In some embodiments, all four of R6, R7, R8and R9are H.

[0062] In some embodiments, R6and R7and / or R8and R9are linked together with the nitrogen atom to which they are attached to form a 3 - to 6-membered heterocyclic ring. In some embodiments, R6and R7or R8and R9are linked together with the nitrogen atom to which they are attached to form a 4, 5 or 6-membered heterocyclic ring. In some embodiments, R6and R7or R8and R9are linked together with the nitrogen atom to which they are attached to form a 5 membered heterocyclic ring.

[0063] In some embodiments, one of R6and R7and / or one of R8and R9is H or Ci-4alkyl and the other is selected from C(NR10)NRnCi-4alkyl, C(NR10)NRnC2-4alkenyl and C(NR10)NRnC2- 4alkynyl. In some embodiments, one of R6and R7and one of R8and R9is H or CH3 and the other is selected from C(NRH)NHCi-4alkyl, C(NH)NHC2-4alkenyl and C(NH)NHC2-4alkynyl. In some embodiments, one of R6and R7and one of R8and R9is H and the other is C(NH)NHC2alkynyl.

[0064] In some embodiments, X1is Ci-ioalkylene. In some embodiments, X1is C2-salkylene.In some embodiments, X1is C2-3alkylene. In some embodiments, X1is Csalkylene. In some embodiments, X1is Ci-ioalkylene optionally interrupted by cyclohexane. In some embodiments, X1is cyclohexane.

[0065] In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Ce-iealkyl and X1is C2-salkylene. In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Cs-ualkyl and X1is C2-3alkylene. In some embodiments, three of R6, R7, R8and R9are H and the remaining one is selected from Cio-walkyl and X1is Csalkylene.

[0066] In some embodiments, the compound of Formula II is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

[0067] In some embodiments, the one or more compounds are selected from the compound of Formula III:or a pharmaceutically acceptable salt and / or solvate thereof, wherein R12, R13, R14, R15, R16, X2and X3are as defined for Formula I.

[0068] In some embodiments, R12, R13, R14, R15and R16are independently selected from H and Ci-ioalkyl. In some embodiments, R12, R13, R14, R15and R16are independently selected from H and Ci-salkyl. In some embodiments, R12, R13, R14, R15and R16are independently selected from H and CH3. In some embodiments, one of R12, R13, R14, R15and R16is H and the remaining three are CH3. In some embodiments, two of R12, R13, R14, R15and R16are H and the remaining two are CH3. In some embodiments, three of R12, R13, R14, R15and R16are H and the remaining one is CH3. In some embodiments, all four R12, R13, R14, R15and R16are H. In some embodiments, all four R12, R13, R14, R15and R16are CH3.

[0069] In some embodiments, R12and R13and / or R15and R16are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring. In some embodiments, R12and R13or R15and R16are linked together with the nitrogen atom to which they are attached to form a 4, 5 or 6 membered heterocyclic ring.

[0070] In some embodiments, one of R12and R13and / or one of R15and R16is H or Ci-4alkyl and the other is selected from C(NR17)NR18Ci-4alkyl, C(NR17)NR18C2-4alkenyl and C(NR17)NR18C2- 4alkynyl. In some embodiments, one of R12and R13and one of R15and R16is H or CH and the other is selected from C(NR.H)NHCi-4alkyl, C(NH)NHC2-4alkenyl and C(NH)NHC2-4alkynyl. In some embodiments, one of R12and R13and one of R15and R16is H and the other is C(NH)NHC2alkynyl.

[0071] In some embodiments, X2and X3are independently selected from Ci-ioalkylene. In some embodiments, X2and X3are independently selected from Ci-ealkylene. In some embodiments, X2and X3are different. In some embodiments, X2and X3are the same. In some embodiments, both X2and X3are Csalkylene.

[0072] In some embodiments, the compound of Formula III is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

[0073] It will be understood that one or more compounds selected from a compound of Formula I, II, and III can be selected from the same or different Formula of compounds. For example, the one or more compounds can be selected from Formula I or the one or more compounds can be selected from Formula I and Formula II.

[0074] 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.

[0075] 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. The selection 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).

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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 compoundlisted 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] The present application also includes a use of an antibiotic and one or more compounds of formula I, II, or III for treating a bacterial infection in a subject in need thereof.

[0084] The present application also includes a use of an antibiotic and one or more compounds of formula I, II, or III for preparation of a medicament for treating a bacterial infection in a subject in need thereof.

[0085] 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.

[0086] The exemplary compounds of the application showed synergistic activity with antibiotics, which resulted in increased efficacy of the antibiotics against bacteria, such as drugresistant 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.

[0087] It has been further shown that exemplary compounds of the application sensitize Gramnegative 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.

[0088] As such, in some embodiments, the one or more compounds of the application inhibit bacterial detoxification of natural polyamines present at the site of infection. In some embodiments, the one or more compounds of the application disrupt membrane integrity of the bacterium, in particular a Gram-negative bacterium.

[0089] 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.

[0090] In some embodiments, the one or more compounds of the application inhibit bacterial ribosome.

[0091] One or more compounds of the application inhibit human spermine / spermidine acetyltransferase SAT1

[0092] Compounds inhibiting human spermine / spermidine acetyltransferase may be used to treat SATl-related dysfunction or disorders related to SAT1 dysfunction in humans and animals.

[0093] Compounds inhibiting human spermine / spermidine acetyltransferase may be used to treat SATl-related dysfunction or disorders related to SAT1 dysfunction, including, but not limited to, cancer, osteoporosis, and ischemia / reperfusion leading to acute kidney injury.

[0094] 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.

[0095] In some embodiments, the polyamine detoxification enzyme is acetyltransferase enzyme. In some embodiments, the acetyltransferase enzyme is SpeG.

[0096] In some embodiments, the natural polyamines present at the site of infection are spermine, spermidine and / or putrescine.

[0097] In some embodiments, 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, an antimicrobial peptide and any other antibiotic, or a combination thereof.

[0098] 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, cioxacillin, dicloxacillin, flucioxacillin, 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, thi amphenicol, 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.

[0099] In some embodiments, the antibiotic is selected from azithromycin, cefuroxime, ciprofloxacin, daptomycin, rifampicin, vancomycin and colistin, or a combination thereof.

[0100] In some embodiments, the antibiotic resistance and 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.

[0101] In some embodiments, the polyamine detoxification enzyme is acetyltransferase enzyme. In some embodiments, the acetyltransferase enzyme is SpeG.

[0102] In some embodiments, the natural polyamines present at the site of infection are spermine, spermidine and / or putrescine.

[0103] In some embodiments, the infection is caused by Gram-positive or Gram-negative bacterium or pathogenic fungus.

[0104] 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.

[0105] 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 illini, Leptotrichia buccalis, Levilinea saccharolytica, Luteimonas aestuarii, Luteimonas aquatic, Luteimonas composti, Luteimonas lutimaris, Luteimonas marina, Luteimonas mephitis, Luteimonas vadosa, Meiothermus timidus, Methylobacterium fujisawaense, 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.

[0106] In some embodiments, the Gram-negative bacterium is Klebsiella pneumoniae, Salmonella Typhimurium, Burkholderia cenocepacia or Escherichia coir or a combination thereof.

[0107] In some embodiments, the bacterium is a Gram-positive bacterium. In some embodiments, the Gram-positive bacterium is a species of Actinobacteria, Actinomyces, Arcanobacterium, Bacillales, 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.

[0108] 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, Bacillus weihenstephanensis, Brachybacterium alimentarium, Brachybacterium aquaticum, Brachybacterium 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, Corynebacterium amycolatum, Corynebacterium bovis, Corynebacterium diphtheria, Corynebacterium efficiens, Corynebacterium glutamicum, Corynebacterium 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, Rothiadentocariosa, 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.

[0109] In some embodiments, the Gram-positive bacterium is Enterococcus faecium, Enterococcus faecalis, Mycobacterium smegmatis, Staphylococcus epidermidis o Bacillus subtilis, or a combination thereof.

[0110] In some embodiments, the Gram-positive bacterium is Staphylococcus aureus. In some embodiments, the bacterium is S. aureus USA300.[0011 1 ] In some embodiments, the Gram-positive bacterium is a drug-resistant or a multidrugresistant bacterium. In some embodiments, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA).

[0112] In some embodiments, the Gram-positive bacterium is a species of Mycobacterium, for example, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium lepromatosis, r / 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,

[0113] In some embodiments, the infection is caused by pathogenic fungi

[0114] In some embodiments the fungus is Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. auris, and Saccharomyces cerevisiae

[0115] In some embodiments, the one or more compounds is the compound of formula II- 1 :II-l, wherein said compound reduces or eliminates polyamine-mediated antibiotic resistance of the bacterium or fungus to the antibiotic. In some embodiments, the bacterium is a drug-resistant Grampositive bacterium. In some embodiments, the bacterium is S. aureus USA300. In some embodiments, the antibiotic is cefuroxime or azithromycin.

[0116] In some embodiments, the one or more compounds are compounds of Formulae:1-5, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the bacterial infection is caused by Gram-negative bacterium.

[0117] In some embodiments, the Gram-negative bacterium is K. pneumoniae. In some embodiments, the antibiotic is macrolide antibiotic. In some embodiments, the macrolide is azithromycin.

[0118] In some embodiments, the one or more compounds is the compound of Formula 1-5:1-5, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the bacterial infection is caused by Gram-negative bacterium.

[0119] In some embodiments, the Gram-negative bacterium is K. pneumoniae. It has been shown that exemplary compound 1-5 sensitizes Gram-negative bacterium to Gram-negative and Grampositive antibiotics. Exemplary compound 1-5 showed synergy with macrolides, such as azithromycin,colistin and other antibiotics. As such, in some embodiments, the antibiotic is anti-Gram-negative or anti-Gram-positive antibiotic. In some embodiments, the antibiotic is azithromycin, novobiocin, rifampicin, doxycycline, tetracycline, vancomycin, oxacillin, erythromycin or trimethoprim. In some embodiments, the antibiotic is azithromycin.

[0120] In some embodiments, the antibiotic is antimicrobial peptide. In some embodiments, the antimicrobial peptide is colistin.

[0121] 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.

[0122] 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.

[0123] 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 theapplication can be administered to a subject concurrently with the antibiotics, prior to the antibiotics, and / or subsequent to the antibiotics.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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".

[0128] 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.

[0129] 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, com 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 calciumphosphate); 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), timerelease 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.

[0130] 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.

[0131] 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 hydrogenatededible 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.

[0132] 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.

[0133] 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 usual quantities of diluents or carriers. For pulmonary administration, diluents or carriers will be selected to be appropriate to allow the formation of an aerosol.

[0134] 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.

[0135] In some embodiments, compositions for nasal administration are conveniently formulated as aerosols, drops, gels and powders.

[0136] 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 non-aqueous 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, di chlorotetrafluoroethane, heptafluoroalkanes, carbon dioxide or another suitable gas. In the 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.

[0137] 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.

[0138] 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.

[0139] 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, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, poly cyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

[0140] 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.

[0141] 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.

[0142] 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 inseparate 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 from about 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 severity 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.

[0143] In some embodiments, effective amounts vary according to factors such as the disease state, age, sex and / or weight of the subj ect. 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.

[0144] In some embodiments, references above to an antibiotic can also refer to one or more antibiotics.

[0145] 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

[0146] The compounds of the present application can be prepared by various synthetic processes or commercially available products can be used. Some starting materials 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.

[0147] The antibiotics used on the present application are commercially available antibiotics.

[0148] 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.

[0149] 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”.

[0150] 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

[0151] The following non-limiting examples are illustrative of the present application: Example 1: Polyamine analog compounds with broad spectrum activity Materials and methodsBacterial strains and reagents

[0152] 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 and Enterococcus faecium. which were grown in Tryptic Soy Broth and Mycobacterium smegmatis, which was grown in Luria Broth.

[0153] Table 1. Strains and plasmids used in this study:

[0154] Table 2. Primers used in this studyAntimicrobial susceptibility testing

[0155] 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 A alone)+(MICdrug B in combination) / MICDrugB 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

[0156] 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 ofpolyamine biosynthesis and detoxification enzymes in bacteria. This library was screened at 20 pM in the presence and absence of spermine at l / 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 l / 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 turbidimetrically after 24 hours.S. aureus Nebraska Transposon Mutant Library (NTML) screen

[0157] 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 l / 8thand 1 / 16ththe 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.B. subtilis CRISPRi essential gene knockdown library screen

[0158] Overnight cultures of the library (Peters, Colavin et al. 2016), were prepared in 384- well plates using Biomatrix BM5-BC (S&P Robotics inc.) in MHB containing 6 pg / mL chloramphenicol. The following day, MHB containing II- 1 at the wild-type MIC and at 2-fold dilutions to 1 / 16ththe MIC, and in the absence of 11-1 were inoculated using the Biomatrix BM5-BC. The experiment was performed with 0.05% xylose (allowing low level of dcas9 expression) and without xylose (allowing basal dcas9 expression). Plates were incubated at 37°C, and ODeoo was read after 24 h.General molecular techniques

[0159] Unmarked in-frame deletion of speG was performed using the temperature-sensitive 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- / ?eG was passaged through E. coli DH5a and A. coli DC 10B, before electroporation into S. aureus ESAJOO JE2 (Grosser and Richardson 2016). Deletion of speG was confirmed through PCR.Membrane permeabilization assay

[0160] Fluorometric Disc3(5) membrane permeabilization assays were performed as described in (Farha, Verschoor et al. 2013).Hemolysis Assay

[0161] 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

[0162] 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 Hisselect 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 SSAT enzymatic assaysSteady state kinetics

[0163] Purified enzymes were used in spermine / spermidine Macety I transferase (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-HCl, 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-HCl, 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 -Co A with SAT1 in 100 mM Tris-HCl, 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 A412 nm 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

[0164] The colorimetric SSAT assay was performed where 25 pL of enzyme solution containing 200 ng enzyme, 100 mM Tris-HCl, 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 L Ellman’s reagent and 25 pL substrate solution to initiate the reaction. II- 1 and analogs in the small-scale structure-activity relationship study were assayed with a substrate solution including a fixed concentration of spermine « Kmand 1 or 0.25 mM acetyl-CoA with SpeG and SAT1, respectively. Substrate solution to evaluate II-l inhibition of PaiAsa contained a final concentration of 1 mM acetyl-CoA and spermine at ~Km. The reaction was monitored each minute at A4i2nmfor 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 IC50 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.

[0165] Equation 1:Results and DiscussionA high-throughput chemical screen identifies potential polyamine detoxification inhibitors

[0166] 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 ’ / 4ththe 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 rationalebased approach. We screened the polyamine analog library at 20 pM (similar to the Spectrum collection screen) in the presence of spermine at ’ / 4ththe MIC. The screen was performed with thehypothesis 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, OES1- 1087, OES1-0507, OES1-1639, OES1-1238, 1-4, and II-l, showed synergy with spermine (Figure ID). Synergy was determined by calculating the fractional inhibitory concentration index (FICI), with an FICI value < 0.5 defined as a synergistic interaction. OES1-1087 exhibited the strongest synergistic interaction with spermine with an FICI value of 0.15 ± 0.03 followed by OES 1-0507 (0.28 ± 0.07), II-l (0.33 ± 0.06), OES1-1639 (0.42 ± 0.06), OES1-1238 (0.46 ± 0.12), and finally 1-4 (0.50 ± 0) (Figure 1 D). OES1-0507, OES1-1087, and OES1-1639 are benzenediols, OES1-1238 is an acridine, and 1-4 and II-l are amines. In addition to synergizing with polyamines, the six hits exhibited growth inhibitory effects against USA300 with II-l being the most potent at an MIC of 20 M (Figure ID). Of note, we prioritized II-l and OES1-1087 (isoproterenol) for further characterization based on their relative potencies (spermine synergy atFICI<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

[0167] 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 by screening the Nebraska Transposon Mutant Library (NTML), a sequence-defined transposon mutant library covering the non-essential genome of S. aureus US A300 (Fey, Endres et al. 2013), against spermine at l / 8thand l / 16ththe wild-type MIC. ,sy>cG::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, / cz / A:Tn, c7.s::Tn, prmCA n, and / ?yr ::Tn, were also sensitized to spermine (Figure 1C). Dose-response assays showed an 8-fold reduction in spermine MIC in / ?eG::Tn, and a decrease of the other four mutants at U 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; SspeG 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 uracilxanthine permease. The molecular basis linking these determinants to polyamine response warrants further investigation, which falls beyond the scope of this study. Notably, ZwTUTn, 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, SsbnB mutant was not sensitized to spermine (Supplementary Fig. 3). Whole-genome 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.

[0168] 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 II- 1 and OES1-1087, and OES1-0507 was lost or reduced in the SspeG mutant (Figure 1 E), suggesting that SpeG is the putative target of these compounds.

[0169] We performed checkerboard assays with spermine and II- 1 or OESl-1087 against other speG+and speG~ S. aureus strains. The tested strains included another US A300 strain CMRSA-10 that encodes speG as well as two strains that do not encode speG 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 II- 1 or OES1-1087, phenocopying the USA300 AspeG mutant (Figure 2 C-E). For both II- 1 and OES1-1087, CMRSA-10 displayed a similar synergistic phenotype to that observed in the other wild-type USA300 strain, JE2 (Figure 2 A and B). These results further suggest that II- 1 and OES 1-1087 are inhibiting SpeG activity, and that strains must encode speG to observe the spermine synergistic phenotype.SpeG as the target of OES 1-1087 and II- 1

[0170] 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 isproportional 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 Kmand S0.5, respectively. The SpeG Kmwith spermine was 1312 ± 348.1 pM and S0.5 with spermidine was 8669 ± 3031 pM (Figure 3). Catalytic efficiency of SpeG with spermine and spermidine as substrates were 5.58E+03 and 2.51E+02 M^s’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).

[0171] Next, we confirmed that 11-1 and OES1-1087 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 checkerboardstyle format estimating IC50 and Ki, respectively. The IC50 of II- 1 against SpeG was 34.82 ± 6.810 pM at ~ 1 x Kmof spermine and the Ki was 20.20 ± 3.446 pM when varying spermine concentrations both at a fixed saturating AcCoA concentration (Figure 4 O and P). The IC50 of OES1-1087 was 5508 ± 1890 pM (at ~1 x Kmof AcCoA and a spermine ~ 1 x Kmconcentration. OES1-1087 did not exhibit inhibitory activity under a fixed AcCoA concentration and variable spermine concentrations; however, at a fixed spermine concentration and variable AcCoA, OES 1-1087 inhibited SpeG with an estimated Ki of 5153 ± 2126 pM. 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. 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-HCl) 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 II- 1 and OES 1-1087 inhibit SpeG with II- 1 being more potent as a SpeG inhibitor, matching with the whole-cell assay results (Figure 4 O and P).Uncovering parallel polyamine acetyltransferase mechanisms in USA 300

[0172] Checkerboard assays with II- 1 and OES 1-1087 revealed only a partial loss of synergy in the HvspeG mutant with the polyamine spermidine, suggesting other potential mechanisms may also be at play in the spermidine synergy and that these may also be targeted by the inhibitors. SpeG preferentially acetylates spermine while it acetylates spermidine with a lower affinity (Figure 3) (Li, Maezato et al. 2019, Tsimbalyuk, Shomikov et al. 2023). The lower affinity may contribute to a decreased ability to detoxify spermidine, possibly requiring other detoxification mechanisms that function in parallel to SpeG. Some bacteria encode SSATs distinct from SpeG, such as B. subtilis encoding two enzymes paiA and bltD (Woolridge, Martinez et al. 1999, Forouhar, Lee et al. 2005) and Enterococcus faecalis encoding pmvE a homolog of B. subtilis paiA (Woolridge, Martinez et al. 1999, Forouhar, Lee et al. 2005, Martini, Michaux et al. 2015). The presence of both paiA and bltD in B. subtilis provides evidence for the existence of parallel polyamine detoxification pathways. Further, a recent report observed an accumulation of Macetyl putrescine in the MSSA strain ATCC 25923, which does not encode SpeG; however, they did not identify the enzyme responsible for its production (Seravalli, Portugal et al. 2023). This further suggests that S. aureus may encode alternative polyamine acetyltransferases and that they may have activity towards different polyamines. We identified potential additional polyamine acetyltransferases in S. aureus by performing protein blast of the B. subtilis 168 strain sequences of BltD (Accession NP 390537.1) and PaiA (Accession NP 391095.1) against S. aureus to identify potential homologs. Blastp results for BltD revealed two hypothetical proteins of S. aureus USA300 FPR3757; SAUSA300_0441 with 29.76 percent identity (query coverage 54%, E value of 2e'O9) and SAUSA300_2083 with 27.41 percent identity (query coverage 86%, E value of 5e'O9). PaiA blastp results identified a homolog of S. aureus USA300 FPR3757; SAUSA300_2316 (denoted PaiAsa herein), with 40.35 percent identity (99% query coverage, E value of 3 e'39). These S. aureus proteins have not been previously characterized functionally; all three are annotated as ribosomal-protein-alanine acetyltransferases whereas SAUSA300 2083 and SAUSA300 2316 (PaiAsa) are also annotated as spermine / spermidine acetyltransferase bit and PaiA, respectively.

[0173] To confirm or rule out SSAT activity of SAUSA300_2083, SAUSA300_0441, and PaiAsa, we overexpressed and purified these enzymes to assess their activity in vitro. We observed acetyltransferase activity of PaiAsa when spermine and spermidine are used as substrates but no activity with putrescine under the test conditions (Figure 8A-C). PaiAsa substrate affinity appears higher for spermine than spermidine (KM = 2144 ± 443.3 pM and 3996 ± 670.8 pM, respectively).Further, the catalytic efficiency of PaiAsa with spermine as a substrate is ~7-fold higher than with spermidine (Kcat / KM = 2.41E+03 and 3.47E+02 M^s’1, respectively; Figure 8A-C). Thus, these results suggest that PaiAsa is better at acetylating spermine at lower substrate concentrations than spermidine. Notably, SAUSA300_2083 and SAUSA300_0441 did not exhibit actetyltransferase activity against the polyamines spermine, spermidine or putrescine under the tested conditions. These data suggest that the PaiAsa is likely to function as a spermine / spermidine acetyltransferase. Next, we tested the ability of II- 1 to inhibit PaiAsa spermine acetylation; the low spermidine acetylation signal precluded testing II- 1 PaiAsa inhibition with spermidine as a substrate. II-l inhibited PaiAsa activity exhibiting an IC50 value of 52.17 ± 5.897 pM and estimated Ki of 43.83 ± 6.693 (Figure 8C). Next, we performed whole-cell spermine - II-l mini-checkerboard assays against a paiA::Tn mutant, compared to the wild-type USA300, revealing no loss of synergy in the paiAsamutant (Figure 8 E and F). SpeG, hypothesized to be the primary spermine / spermidine detoxification enzyme in S. aureus, may have compensated for the absence of PaiAsa, masking any phenotypic changes in the paiAsa mutant; a speG paiAsa double mutant may be required to assess the contribution of PaiAsa in whole cells. Together, our results indicate that polyamine acetylation in S. aureus may occur through PaiAsa in addition to SpeG, serving as complementary acetyltransferase enzymes, and that the SSAT function of both enzymes is inhibited by II-l.Uncovering the mechanism of growth inhibitory effects of II-l.

[0174] II-l and OES1-1087 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 II-l 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 ofll-l, spanning1 / 4th-l / 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 II-l relative to the wild-type, suggesting the absence of an essential protein target.Next, we hypothesized that the growth-inhibitory activity of II-l is due to an effect on membrane integrity in the absence of an essential protein target. Using a DiSCs(5) membrane integrity assay (Farha, Verschoor et al. 2013), we found that II-l permeabilized the membrane of USA300 atconcentrations starting from its MIC (> 20 pM) in a concentration-dependent manner (Figure 6 A and B), suggesting membrane activity at high concentrations as the mechanism of growth inhibitory effects of II-l. Notably, a DiSCs(5) assay with OES1-1087 did not have an observable effect on membrane integrity.Next, we sought to check whether membrane disruption contributed to the observed synergy between II-l and spermine. We conducted a checkerboard-style DiSCs(5) assay with a combination of spermine and II-l. II-l concentrations that correspond to the range of synergy (2.5-10 pM) resulted in no observable permeabilization (Figure 6 A and B). Like II-l, 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 II-l and spermine at concentrations where synergy is observed (Figure 6 B), suggesting that membrane permeabilization does not contribute to the observed synergy. Taken together, we revealed a dual mode of action of II-l 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.Broad-spectrum activity of II-l on clinically-r elevant bacteria.

[0175] Given that SpeG has been implicated in virulence phenotypes in multiple bacteria (Thurlow, Joshi et al. 2013, Fang, Huang et al. 2017, Hu, Filippova et al. 2018), we tested II-l being the most potent identified SpeG inhibitor against a broad range of Gram-positive and Gram-negative bacteria and the pathogenic yeast Candida albicans in checkerboard assays with spermine. The combination of II-l and spermine exhibited a synergistic interaction or an increase in spermine susceptibility in B. subtilis, E. faecalis, E. faecium, S. epidermidis, M. smegmatis, B. cenocepacia, E. coli, K. pneumoniae, S. Typhimurium, and C. albicans (Figure 7 A-K). In A. baumannii the polyamine acetyltransferase Dpa preferentially acetylates 1,3 -diaminopropane (Armalyte, Cepauskas et al. 2023), which is a diamine; therefore, we tested the diamine putrescine and II-l in combination. II-l synergized with putrescine against A. baumannii (Figure 7 L). As in S. aureus, II-l also inhibited the growth of all tested bacteria and C. albicans at the micromolar range (Figure 7). Next, we tested the combination of spermine and II-l against the respective SSAT mutants of E. coli BW25113, K.pneumoniae MKP103, S. enterica sv. Typhimurium 14028s, and B. subtilis 168 (Figure 7 M-P). II-l no longer potentiated the effects of spermine in the SSAT mutants as opposed to their respective wildtype strains (Figure 7 M-P), suggesting II- 1 mediates its broad-spectrum spermine synergy through SSAT inhibition. Together, II- 1 exhibited broad-spectrum dual activity - polyamine synergy and growth inhibition - against Gram-positive and Gram-negative bacteria and fungi, including clinically relevant World Health Organization priority pathogens (2024).Polyamine-mediated altered antibiotic susceptibility and the potential ofll-l as an antibiotic adjuvant.

[0176] Checkerboard assays of polyamines and representative antibiotics from seven classes with diverse cellular targets revealed various polyamine-mediated altered antibiotic responses. The three tested polyamines synergized with the macrolide azithromycin while spermine synergized with the P-lactam cefuroxime. The spermine P-lactam synergy matches previous reports of the phenotype in S. aureus (Kwon and Lu 2007, Yao and Lu 2012). Similarly, polyamine synergy with macrolides has been reported in the Gram-negative pathogens K. pneumoniae (Adams, Moulding et al. 2024) and E. coli (Kwon and Lu 2007). In K. pneumoniae the synergistic interaction between azithromycin and putrescine is mediated through the combined effects of putrescine-induced membrane perturbation and protein synthesis inhibition via interaction at the ribosome (Adams, Moulding et al. 2024). The effects of polyamines on ribosome function is well documented (Dever and Ivanov 2018) and polyamines are known to interact with biological macromolecules, including DNA, RNA, proteins, and phospholipids (Igarashi and Kashiwagi 2000, Igarashi and Kashiwagi 2010). In contrast, the three polyamines antagonized rifampicin and kanamycin whereas spermine antagonized with vancomycin, defined as a FICI value > 4. The mechanisms of the observed polyamine-mediated alterations to antibiotic susceptibility warrant further investigation. On the other hand, we observed no alterations in daptomycin or ciprofloxacin susceptibility in combination with the tested polyamines.

[0177] Interestingly, the MIC of vancomycin is increased 4-fold when combined with spermine (FICI = 4.69 ± 1.57), but the interaction is lost in a lspeG mutant (FICI = 2.17 ± 0.26), suggesting that S. aureus requires a functional SpeG to use exogenous polyamines to resist vancomycin (Figure 9 A and B). Supplementing a sub-inhibitory concentration of II- 1 (2.5 pM) to a vancomycin-spermine checkerboard against S. aureus USA300 phenocopied the loss of antagonism observed against the lspeG mutant (FICI = 1.59 ± 0.62; Figure 9 C). This phenotype is not due to a synergistic interaction between II-l and vancomycin. Vancomycin is one of the first-line antibiotics for the treatment of severe MRSA infections (Liu, Chen et al. 2021), suggesting the antagonismbetween polyamines and vancomycin may be detrimental to MRSA treatment. Similarly, the polyamine antagonistic interactions with rifampicin and kanamycin in USA300 were lost in the speG mutant and with supplementation of a sub-inhibitory concentration of II- 1 to the US A300 checkerboard (Figure 9 D-I). The loss of antagonism was not due to synergy between II- 1 and kanamycin or rifampicin. While kanamycin and rifampicin are not considered first-line monotherapies for MRSA infections, they are often used in conjunction with vancomycin (Choo and Chambers 2016, Ma, Cheng et al. 2020); polyamines may reduce bacterial susceptibility to these antibiotics. We also identified a synergistic interaction between II-l and cefuroxime, where II-l lowered the MIC of the antibiotic to <16 pg / mL from 1024 pg / mL (FICI = 0.35 ± 0.13; Figure 9 J). II-l and azithromycin also exhibited a slight potentiation effect against USA300 (FICI = 0.72 ± 0.21; Figure 9 K). A similar potentiation effect was observed, where azithromycin synergized with II-l against K. pneumoniae (Adams, Moulding et al. 2024). These results suggest that II-l may serve as an antibiotic adjuvant by preventing S. aureus from utilizing exogenous polyamines to resist antibiotics (e.g., glycopeptides, aminoglycosides, and rifamycins) or by potentiating the effects of antibiotics phenocopying the polyamine-mediated sensitization to certain antibiotics (e.g., macrolides and P-lactams).Evaluation of II-l and OES1-1087 potential eukaryotic activity.

[0178] Given the membrane activity of II-l against S. aureus (Figure 6 A and B), we sought to evaluate its potential effects on mammalian cell membranes. To that end, we assessed the hemolytic activity of II-l against sheep red blood cells. II-l exhibited hemolytic effects, which are first observed at 160 pM with an increase at 640 pM reaching an absorbance of roughly U that of the positive control (Triton X100) (Figure 12 H). The hemolytic effects do occur at a concentration significantly higher than what is necessary 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, OES 1-1087 did not exhibit hemolytic effects at the concentrations tested.

[0179] Next, we assessed the activity of II-l and OES 1-1087 against the human spermine / spermidine acetyltransferase, SATE 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^s'1; Figure 11 A and B). Our results are inagreement 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 II- 1 and OES1-1087 to inhibit SAT1. The IC50 of 11-1 against SAT1 was 26.39 ± 6.411 pM (Figure 11 C), whereas OES1- 1087 did not inhibit SAT1 under any of the tested conditions. Although, II- 1 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).Small-scale structure activity relationship of 11-1 suggests the structural components necessary for activity.

[0180] We performed a small-scale structure activity relationship (SAR) study of II- 1 by testing analogs thereof to identify the active moiety of II- 1 and detect preferential inhibitory activity against either SpeG or SAT1. The analyses included compounds with varied length of the saturated alkyl chain Ri and one of three R2 (a proton, an aminoethyl group, or an aminopropyl group) attached on either side of an amine group (Table 1). First, we tested each of the compounds in whole-cell assays in combination with spermine to assess their growth inhibitory and polyamine synergy effects against S. aureus USA300. We observed a general reduction in the MIC of the compound as the length of Ri and R2 increased (Table 3). Spermine synergy appeared to be dependent on both the length of the alkyl chain and the addition of either the aminoethyl or aminopropyl group. The FICI value decreased (indicating a greater degree of synergy) as the length of the alkyl chain increased; for example, 1-1 with Ri as a proton exhibited no synergy (FICI = 0.88 ± 0.13) whereas 1-4 with Ri (CEEjsCEfc showed synergy (FICI = 0.50 ± 0). The synergistic interaction is further improved when the primary amine is changed to a secondary one with an aminoethyl or aminopropyl; this trend is exemplified in 1-2 (R2 = H; FICI = 0.58 ± 0.12), II-4 (R2= aminoethyl; FICI = 0.56 ± 0.05), and II-3 (R2= aminopropyl; FICI= 0.38 ± 0).

[0181] The analogs that synergized with spermine lost the synergy against the speG mutant (Table 3), suggesting their synergy is mediated through SpeG inhibition similar to II-l. The analogs that did not exhibit synergy in whole-cell assays are hypothesized to have no SpeG inhibitory activity. Therefore, we tested each compound in enzymatic assays, against both SpeG and SAT1 and estimated Ki values using equation 1 (materials and methods). II- 1 remains the most potent inhibitor of SpeG, Ki = 16.25 ± 3.27 pM with 1-4 being the second most potent, Ki = 74.6 ± 18.28 pM (Table 3 and Figure 4). 1-1 and 1-2 do not synergize in whole-cell assays and do not inhibit SpeG in vitro., thus, they serve as controls to validate the correlation between in vitro and whole-cell assays and that our lead compound is targeting and inhibiting SpeG.

[0182] II-l inhibited SAT1 with a Ki of 13.03 ± 4.69 pM, similar to that of SpeG, but the Ki of 1-4 is roughly 2-fold higher in SAT1 than SpeG (160.22 ± 50.45 pM and 74.6 ± 18.28 pM, respectively; Table 3). Similarly, 1-3 has almost a two-fold higher Ki against SAT1 than SpeG (743.34 ± 250.46 and 397.89 ± 113.0 pM, respectively). Further, two of the tested compounds only inhibited SpeG (II-4 at Ki = 592.08 ± 126.15 pM and II-2 at Ki = 559.42 ± 201.51 pM) and not SAT1, in contrast, SAT1 activity increased at the highest tested concentrations of these analogs (Figure 4 and 5). Notably, II- 3 which synergized with spermine in whole-cell assays (FICI = 0.38 ± 0 pM), suggesting SpeG inhibitory activity, showed no inhibition in the in vitro enzymatic assays; however, it inhibited SAT1 (Ki = 227.99 ± 49.73pM; Table 3 and Figure 4 and 5). These results showing differential activity of some analogs against both enzymes suggest a potential to improve specificity of an inhibitor towards SpeG.

[0183] Given that the compounds also exhibit growth-inhibitory effects, we simultaneously evaluated their hemolytic activity. Hemolytic activity aligned with the MIC data where the concentration required to lyse red blood cells was observed at lower concentrations for compounds with a longer alkyl chain (Ri = (CH2)sCH3 or (CH2)sCH3) and with an aminoethyl or aminopropyl group at R.2 (Table 3 and Figure 12). Hemolysis for the SpeG inhibitory compounds II-4 and 1-3 occurred at 1280 pM (~2-fold higher than their MICs), while hemolytic activity of II-2, and 1-4 was observed at ~ 4-fold higher than their MICs (640 and 320 pM, respectively; Table 3 and Figure 12). We observed no hemolytic activity of two analogs with high MIC values 1-1 and 1-2 (Table 3 and Figure 12). Given the promising results of 1-4 being more potent against SpeG than SAT1, we tested if this compound retains the potential adjuvant activity of II-l. 1-4 abolishes the protective effect of spermine towards vancomycin, kanamycin, and rifampicin, and potentiates the activity of cefuroxime(Figure 10). This analysis provides a foundation for a larger scale targeted lead optimization to improve the potency of SpeG inhibition, while reducing the hemolytic and SAT1 inhibitory activity.

[0184] Table 3. Small scale structure activity relationship study of II-l. SAR study compounds were tested in combination with spermine against S. aureus US A300 and speG. FICI values are calculated as the mean ± standard deviation of at least three replicates, unless otherwise specified. Inhibitory activity against both SpeG and SAT1 was evaluated in colorimetric enzymatic assays to determine Ki values estimated by the Cheng-Prusoff equation and reported with relative error. Hemolytic activity of each compound was tested against sheep red blood cells.Compound MIC FICI I K I Av / icG SpeG Ki SAT1 Ki Observed(pM) USA300 (pM) (pM) hemolysis (pM) i1-1 2500 0.88 ± 0.13 1.38 ± 0.63 N / A N / A None (n=2) (n=2)1-2 5000 0.58 ± 0.12 0.92 ± 0.12 N / A N / A None625 0.56 ± 0.05 1.0 ± 0 592.08 ± N / A 1280126.151250 0.38 ± 0 0.67 ± 0.12 N / A 227.99 ± 128049.73625 0.50 ± 0 0.83 ± 0.12 397.89 743.34 ± 1280±113.0 250.46156.25 0.42 ± 0.12 0.83 ± 0.12 559.42 ± N / A 640201.5162.5 0.50 ± 0 1.25 ± 0.54 74.60 ± 160.22 ± 32018.28 50.4520 0.33 ± 0.06 0.75 ± 0 16.25 ± 3.27 13.03 ± 4.69 160(n=H) (n=9)Conclusions

[0185] We undertook a chemical screen to identify inhibitors of poly amine detoxification, and uncovered two lead compounds, II- 1 and OES 1-1087, that show growth inhibition and synergy withspermine. A chemogenomic screen and in vitro enzymatic assays revealed that both compounds inhibit the spermine / spermidine acetyltransferase SpeG. The most potent of the two, II- 1 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 II- 1 in a broad-spectrum of Grampositive 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 II- 1 was not completely lost in a US A300 HvspeG mutant, suggesting other underlying polyamine detoxification targets, which prompted us to search for SSATs homologous to B. subtilis Pai A and BltD. We found PaiAsa, characterized its SSAT activity in vitro, and showed that II-l inhibited its activity. Further, II- 1 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). II- 1 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 II-2 and 1-4. 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 polyamine- mediated resistance to other antibiotics in S. aureus suggest II-l may also be used as an antibiotic adjuvant.

[0186] 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 II-l 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 II-l, 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 polyamineanalogs 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.

[0187] 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 II- 1 caused hemolytic activity at 4X MIC against S. aureus US A300, SpeG-inhibitory activity of the compound was observed as low as l / 32ndthe MIC, suggesting a concentration range within which II- 1 may remain efficacious while avoiding potential adverse effects. Future studies will further assess the potential toxicity of II- 1 and its analogs and their interaction with other polyamine-related enzymes using natural polyamines as substrate.

[0188] We also identified OES 1-1087 (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 II- 1, and exhibits growthinhibitory activity. Importantly, it did not show detectable activity against the human SAT1 and no hemolytic activity.

[0189] 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 II- 1 inhibits speG, it will likely reduce the ability of bacteria to withstand host poly amines produced at the site of infection, suggesting it may allow these host compounds to help clear infection serving as antivirulence agent.

[0190] In vitro enzymatic assays suggest that II-l 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 II-l, II-4 and II-2, that showed inhibitory activity against SpeG but not SAT1 and another two, 1-3 and 1-4, which were more potent inhibitors of SpeG than SAT1. Although these analogs have lower inhibitory activity than II-l, they and other analogs from our analysis can guide future SAR studies to optimize the inhibition of SpeG while limiting activity against SAT1.

[0191] S. aureus was not previously known to have an SSAT except for strains that acquired SpeG, such as USA300. Notably, the USA300 speG 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 US A300, suggesting a potential for anotherdetoxification 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 MpeG mutant than / / / dsa::Tn; the contribution of PaiAsa to polyamine resistance in whole cells requires assessment in a speG paiAsadouble mutant in USA300 or a single paiAsa mutant in a speG~ S. aureus strain. We further showed that II- 1 can inhibit PaiAsa in vitro.

[0192] This work provides insights into understudied aspects of chemically mediated host-pathogen interactions. We discovered two novel inhibitors of spermine / spermidine N- acetyltransferase. The discovered inhibitors have potential application as stand-alone antimicrobials, antivirulence agents, or antibiotic adjuvants. This work provides antimicrobial compounds with a novel mechanism for the fight against multidrug-resistant priority pathogens.Example 2: The effects of polyamine analogs on infections caused by Gram-negative bacteria Methods'.Antimicrobial susceptibility testing

[0193] All checkerboard assays were conducted following the CLSI method for MIC testing by broth microdilution (Clinical and Laboratory standards Institute, 2012). The MIC polystyrene 96- well plates were prepared with an eleven series 2-fold dilution. The checkerboard assay plates were prepared with a polyamine 2-fold dilution series on the y axis against an antibiotic 2-fold dilution series on the x axis. Checkerboard assays contained a bacterial suspension in Mueller-Hinton broth (MHB) media with 100 mM Tris-HCl buffer (pH 7.4) at a 0.001 optical density 600nm reading (ODeoo) and were added to each well for a combined total volume with the antibiotics and / or polyamines to be 100 pL. Plates were placed in a shaking incubator at 37°C (600 RPM); growth of the bacterial culture was determined by ODeoo readings at the end of 24 hours. To assess the presence of synergism or antagonism, fractional inhibitory concentration indices (FICI) were calculated as FICI = A / MICA + B / MICB. A and B represent the MIC of the drugs used in combination to inhibit bacterial growth. MICA and MICB represent the individual MIC of the polyamine and antibiotic when in the bacterial suspension by themselves. Checkerboard assays that returned FICI values less than or equal to 0.5 were considered synergism, values greater than 4 were considered antagonism and values between 0.5 and 4 were considered to have no interaction.Arrayed transposon Klebsiella pneumoniae KPNIH1 library screen

[0194] Overnight cultures of the KPNIH1 library were performed by pinning the library into 384 well-density plates containing MHB with 150 pg / mL of chloramphenicol and incubated at 37°C for 24 hours (Ramage, Erolin et al. 2017). Subsequently, the entire library was pinned into MHB with azithromycin at 32 pg / mL and without azithromycin. The plates were incubated at 37°C, and ODeoo was read after 24 hours.Gene Ontology Enrichment analyses

[0195] Gene ontology enrichment was performed on the primary screen results by Biocyc (Karp, Billington et al. 2019).N-phenyl-l-napthylamine (NPN) assays

[0196] The outer membrane permeabilization of K. pneumoniae via exogenous putrescine was measured using NPN uptake assays as previously described (Hancock and Wong 1984). Briefly, bacteria were sub cultured in MHB until mid-late log phase, washed and resuspended in buffer (lOOmM Tris-HCl buffer, 20mM glucose). NPN was added to the washed cells to give a final concentration of 20 pM and dispensed into black 96 well plates with their respective concentrations of putrescine for a total volume of 200 pl. Putrescine treated cells were placed in pH 7.4 buffer and a control of non-treated cells at various pH’s was used as a control to account for any change in NPN uptake caused by change in the pH of the suspensions due to the putrescine concentration. Cells were treated with 8 ug / mL of colistin as a positive control. Fluorescence was read kinetically (excitation l=355nm, emission l=420nm) every 5 minutes for 215 minutes. Data was processed by first subtracting any fluorescence that was caused by the pH of the suspension from the putrescine treated cells. The %NPN uptake was calculated given the equation: %NPN uptake = (F0bs-Fo) / (Fioo-Fo) x 100, where Fobs is the observed fluorescence minus the effect of pH at a given concentration of putrescine, Fo is the fluorescence in non treated cells, and Fioo is the fluorescence of NPN in colistin treated cells. fl-lactamase assay

[0197] The outer membrane integrity of K. pneumoniae and iiw / / )::Tn were tested by measuring b-lactamase activity with nitrocefin hydrolysis as previously described (Farha, El-Halfawy et al. 2020). An overnight sample of K. pneumoniae pBR322, and K. pneumoniae waaD. '.ln pBR322 was diluted 100-fold and sub-cultured into trypticase soy broth (TSB) and incubated at 37°C until late log phase (ODeoo of 1.8). A sample of the sub-culture was centrifuged and washed with phosphate- buffered saline (PBS) two times then resuspended in 100 mM Tris-HCl buffer (pH 7.4) at a final ODeoo of 0.01. The bacterial suspensions were placed into 96 well plates as triplicates and 30 pM of nitrocefinwas placed into each well, and the bacterial suspension was added until the final volume of each well was 100 pL. Checkerboards were incubated at 37°C while kinetically monitored at 492 nM every 10 minutes for 8 hours.B-galactosidase assay

[0198] The inner membrane integrity of E. coli ML-35 pBR322 was tested by measuring b- galactosidase activity with o-nitrophenyl- P-D-galactopyranoside (ONPG) hydrolysis as previously described (Farha, El-Halfawy et al. 2020). An overnight sample of A. coli ML-35 pBR322 was diluted 100-fold and sub-cultured into TSB and incubated at 37°C until late log phase (ODeoo of 1.8). A sample of the sub-culture was centrifuged and washed with PBS two times then resuspended in 100 mM Tris- HC1 buffer (pH 7.4) at a final ODeoo of 0.01. The bacterial suspensions were placed into 96 well plates as triplicates and the bacterial suspensions containing 1.5 mM ONPG was added until a final volume of 100 pL. Checkerboards were incubated at 37°C while kinetically monitored at 405 nM every 2 minutes for 6 hours.Cell free protein expression

[0199] A NEBExpressRCell-free E. coli protein Synthesis System was utilized for in vitro protein expression. Transcription and translation were measured fluorometrically (excitation / emission wavelengths at 610 / 650 nm MGapt, 485 / 525 nm deGFP) using an engineered plasmid that encodes for deGFP with a malachite green RNA aptamer (Siegal -Gaskins, Tuza et al. 2014). Reagents of the cell-free protein synthesis system were combined following the New England Biolabs protocol and the mixture was dispensed as 5 pl aliquots into black 384 well plates. Putrescine (80 mM), azithromycin (16 ug / mL), dH2O, 267 ng / pl of plasmid, 200mM pH 7.4 Hepes buffer, and 10 pM malachite green dye were added into each reaction for a combined volume of 11 pl.Polyamine analog library screen

[0200] K. pneumoniae MKP103 was inoculated in MHB media with 100 mM Tris-HCl buffer (pH 7.4) at a 0.001 ODeoo and was added to a each well for a combined total volume with the polyamine analogs and / or azithromycin to be 100 pL. Polyamine analogs were screened at 2.5 mM alone and in combination with 32 pg / mL azithromycin. Plates were placed in a shaking incubator at 37°C (600 RPM); growth of the bacterial culture was determined by ODeoo readings at the end of 24 hours. Statistical analysis

[0201] All statistical analyses were calculated using Graphpad Prism 9.Results:Putrescine synergizes with azithromycin

[0202] The effect of the poly amine putrescine on the susceptibility of K. pneumoniae MKP 103 to the macrolide azithromycin was tested in a checkerboard assay. K. pneumoniae MKP 103 is derived from the KPNIH1 clinical outbreak isolate with a carbapenemase-encoding gene removed (Ramage, Erolin et al. 2017). Putrescine synergized with azithromycin with a fractional inhibitory concentration index (FICI) of 0.47±0.01 (Fig 13A); FICI values below 0.5 denote synergism, values between 0.5 and 4 are additive, and values greater than 4 represent antagonistic compounds. This synergy matches previously reported polyamine-induced macrolide sensitivity in another Gram-negative organism, E. coli (Kwon and Lu 2007); the mechanism of such interaction is unknown. The putrescineazithromycin synergy reversed macrolide resistance of K. pneumoniae MKP103, bringing the MIC of azithromycin below the CLSI breakpoint when combined with putrescine at 20 mM (Fig. 13 A). Azithromycin synergy with other natural polyamines (spermine and spermidine, Fig. 13B, C) was also observed. Polyamine physiological levels vary but are generally within the mM range and are further increased 2-fold at the infection site upon infection (Thurlow, Joshi et al. 2013, Sagar, Tarafdar et al. 2021). Next, it was tested whether this synergistic phenotype was maintained across diverse clinical isolates or was strain-specific. To this end, putrescine-azithromycin checkerboard assays against a '. pneumoniae diversity panel were conducted. This panel comprise a collection of 100 clinical isolates gathered from 63 facilities in 19 countries with an array of genotypic and phenotypic differences that span from pan-sensitive to pan-resistant strains and contains hypervirulent lineages (Martin, Stribling et al. 2023). The putrescine-azithromycin synergy was observed in 78 isolates (Fig 13D), in 98.7% of which, putrescine lowered azithromycin MIC below the CLSI breakpoint (Examples in Fig 13E). Notably, such synergy was conserved across pansensitive to pan-drug-resistant isolates, including those encoding P-lactamases (including CTXM P-lactamases and NDM and OXA carbapenemases), Rmt(B,F,H) methyltransferases, MCR-1, Tet(X), and ErmB, which drive resistance against P-lactams (including carbapenems), aminoglycosides, colistin, tetracyclines, and erythromycin, respectively. On the other hand, the putrescine-azithromycin combination showed additive effects in 22 isolates, which harbour genes encoding the macrolide phosphotransferases Mph(A), Mph(B), and Mph(E).

[0203] Gram-negative bacteria are less resistant to macrolide antibiotics in the presence of the physiological buffer, bicarbonate (Farha, French et al. 2018). Hence, it was tested whether polyamines would further sensitize K. pneumoniae to azithromycin in bicarbonate. The exposure to the physiological bicarbonate concentration (25 mM) resulted in 4-fold reduction in azithromycin MICagainst K. pneumoniae (Fig. 13F), in agreement with a previous report (Farha, French et al. 2018). Putrescine further sensitized K. pneumoniae to azithromycin (further 16-fold reduction in MIC) in the presence of bicarbonate (Fig. 13F). These observations suggest that multiple physiol ogically-relevant factors may act in tandem to alter bacterial responses to antibiotics.Genomic screens identify a dual mechanism of the polyamine-azithromycin synergy.

[0204] Next, the mechanism of polyamine-azithromycin synergy (Fig. 14A) was identified. It was hypothesized that a mutant in the gene encoding the putrescine synergy determinant will show an increased susceptibility to azithromycin and a loss in synergy with polyamines. First, a comprehensive arrayed transposon mutant library which includes an average of 2.5 mutants per gene in the non- essential genome of K. pneumoniae MKP 103 was used to identify mutants susceptible to azithromycin (Fig 14A). The library at two sub-inhibitory concentrations, 32 and 64 pg / mL, of azithromycin was screened. The screen revealed 65 mutants with increased susceptibility to azithromycin representing 60 genes (Fig 14B). These genes include those whose products are involved in stress induced mutagenesis (recC, recB), ribosomal function (rpsO, rpsR, ybeY, rsmB, rlmH . and membrane integrity (waaD, waaC, waaE, dsbA, acrB, bamB, knockouts thereof in A. coli or K. pneumoniae have been previously identified to have increased sensitivity to macrolides, aminoglycosides, or penicillin’s (Izquierdo, Coderch et al. 2003, Liu, Tran et al. 2010, Furniss, Kaderabkova et al. 2022).

[0205] Next, putrescine-azithromycin checkerboard assays against the 65 mutants were conducted. Azithromycin sensitivity (MIC < 32pg / mL) and lack of synergy (FICI >0.5), suggested mutants of genetic determinants of the putrescine-azithromycin synergy; mutants with disruption in four genes (waaD, waaC, vpsO, and rpsR) fit these criteria (Fig 14C-F). Knockouts of waaD and waaC lead to the same deep rough lipopolysaccharide (LPS) phenotype (Regue, Climent et al. 2001, Kneidinger, Marolda et al. 2002, Wang, Ma et al. 2014). WaaD is necessary for the production of ADP-L-glycero-D-mannoheptose which WaaC incorporates into the LPS structure; loss of these enzymes results in a truncated LPS without an outer core and most of the inner core exposing Kdo2- lipid A (Wang, Ma et al. 2014). Notably, the waaD: :Tn was hypersensitive to novobiocin as expected for LPS deep rough mutants (Yethon, Heinrichs et al. 1998, Wang, Ma et al. 2014). These results match previous reports of E. coli with a compromised LPS structure being more sensitive to azithromycin (Delcour 2009, Liu, Tran et al. 2010).

[0206] When less stringent azithromycin susceptibility cutoffs are set (MIC < 64pg / mL), the mutants AdsbA, AbamB, and AKPNIH1_2556O (predicted waaE)(Regue et al., 2001), exhibit partialphenotypes. Loss of DsbA leads to inactive accumulation of LptD (a lipopolysaccharide assembly protein), BamB is a lipoprotein essential for assembly and insertion of P-barrel proteins into the LPS, and WaaE is responsible for the transfer of the branch P-D-glucopyranose (Glc) to L-glycero-D- manno-heptose I (Heptose I) (Meehan et al., 2017; Onufryk et al., 2005; Regue et al., 2001). Loss of these genes have been previously shown to exhibit macrolide or novobiocin sensitivity inE. coli o K. pneumoniae (Meehan et al., 2017; Onufryk et al., 2005; Regue et al., 2001).

[0207] Other mutants involved within the production of LPS were tested to determine the extent of LPS truncation necessary for loss of putrescine-azithromycin synergy. WaaF and WaaQ are enzymes responsible for the sequential addition of heptose II and heptose III (respectively) to complete the LPS inner core (Regue, Climent et al. 2001). Neither the knockout of waaQ or waaF led to loss of putrescine-azithromycin synergy nor did the MIC of azithromycin change, suggesting a deep rough LPS phenotype is necessary to confer loss of putrescine-azithromycin synergy.

[0208] The ribosomal proteins RpsO and RpsR are covalently linked within the platform domain of the 30S subunit. Both rpsO . Tn and rpsR xi mutants exhibit an azithromycin MIC of 32 ug / mL and an FICI value of 0.70 ± 0.04 and 0.60 ± 0.04 respectively (Fig. 14E, F). The protein RpsO is the sole primary binding protein attached to the central domain of the 16S rRNA (Bubunenko, Korepanov et al. 2006). The deletion of rpsO has been reported to influence the functional capacity of 30S subunits, affecting ribosomal biogenesis and decreasing the amount of 70S complexes present (Bubunenko, Korepanov et al. 2006). Further, the checkerboard of the mutant z B::Tn, the translation initiation factor for rpsO, exhibits a partial reduction in synergy and a 4-fold decrease in azithromycin MIC to that of the wildtype .

[0209] Taking these results together, it was hypothesized that polyamines synergize with azithromycin via a dual mechanism of action: (1) increasing membrane permeability allowing azithromycin to cross this barrier, and (2) acting on the 30S ribosomal subunit compounding the effects of azithromycin in suppressing protein synthesis.Putrescine increases azithromycin ’s permeability across the outer and inner membranes.

[0210] It has been previously shown that macrolides synergize with compounds that disrupt the outer membrane by increasing the intracellular concentration of azithromycin, allowing the antibiotic to gain access to its internal cellular target (Al-Marzooq, Ghazawi et al. 2022, Wesseling and Martin 2022). To study the effect of putrescine on the outer membrane, it was shown, in kinetic 1-N-phenylnaphthylamine (NPN) fluorometric assays, that polyamines disrupted the outer membraneof K. pneumoniae (Fig. 15 A). A disruption of the outer membrane occurs at 10 mM and 20 mM (P < 0.0001), which is within the synergistic region of putrescine and azithromycin.[0021 1 ] To confirm that deep rough mutations of the LPS contributes to increased sensitivity to azithromycin due to increased permeability waaD: :Tn was tested as a representative of the deep rough mutants and compared the outer membrane permeability to the wild-type K. pneumoniae strain. A kinetic NPN assay of both the parent and waaD :Tn mutant showed that waaD :Tn exhibits more than a 30% increase in NPN uptake compared to the wildtype (P < 0.005) (Fig. 15B). This result is in agreement with previous NPN assays conducted in S. enterica Serovar Typhimurium and E. coli that showed waaD deleted mutants exhibit a 2-5-fold increase in NPN uptake in comparison to the parent strain (Sporing, Feigner et al. 2018, Soh, Jang et al. 2020). To further confirm that iiw / / J::Tn has a perturbed membrane, the probe nitrocefin was utilized and a colorimetric assay was conducted. Both iiw / / J::Tn and the wild-type strain were transformed with the P-lactamase containing plasmid pBR322. The amount of hydrolyzed nitrocefin is correlated to the permeability of the outer membrane to nitrocefin or P-lactamase. The iiv / a / J::Tn mutant exhibited an increased absorbance signal in comparison to the wild type, supporting the conclusion that the outer membrane of waaD: :Tn mutants are more permeable (P < 0.001) (Fig. 15C). The increased P-lactamase activity in the waaD :Tn mutant is in agreement with previous reports conducted in K. pneumoniae (Frirdich, Bouwman et al. 2005).

[0212] Then, it was hypothesized that bacteria with a membrane that is chemically or genetically perturbed should exhibit increased susceptibility to azithromycin and exhibit a loss of synergy with putrescine. Polymyxin B nonapeptide (PMBN) is a cationic peptide that disrupts the outer membrane with little to no antimicrobial activity (Al-Marzooq, Ghazawi et al. 2022). It has been previously reported that PMBN synergizes with azithromycin in Gram-negatives due to disruption of the outer-membrane and leads to a similar increased sensitivity to hydrophobic antibiotics as mutants containing a deep rough LPS phenotype (Vaara 1992, Delcour 2009, Al-Marzooq, Ghazawi et al. 2022). A checkerboard assay conducted with putrescine and azithromycin in a concentration of 1 ug / mL of PMBN exhibited a 4-fold decreased MIC, and a 4-fold loss of synergy (FICI 0.83 ± 0.10) (Fig. 15D, E). Moreover, checkerboard assays of a genetically compromised outer membrane model in E. coli containing 9 transmembrane protein deletions, was compared to the parent E. coli strain (Rosner and Martin 2013) . The wild-type E. coli strain exhibited a synergistic phenotype (FICI = 0.41 ± 0.07), while the mutant strain exhibited an 8-fold decrease in azithromycin MIC and a 4-fold loss in synergy (FICI = 1.00 ± 0.00).

[0213] The inner membrane of Gram negatives is the second physical barrier for azithromycin. It was tested whether putrescine also exhibited an effect on the inner membrane by measuring the P- galactosidase activity of E. coli ML-35 with o-nitrophenyl-P-D-galactopyranoside (ONPG). E. coli ML-35 is an engineered strain that is lactose permease-deficient and has constitutive cytoplasmic P- galactosidase activity. Putrescine at 40 mM had a significant effect on the rate of ONPG hydrolyses (P < 0.0001) (Fig. 15F) suggesting that polyamines can also disrupt the inner membrane, potentially contributing to increasing intracellular azithromycin concentration.

[0214] Taken together it has been shown that putrescine, at physiologically relevant concentrations, acts on both the outer and inner membranes contributing to the synergistic combination of azithromycin and putrescine. Previously it has been reported that polyamines engage in electrostatic interactions with negatively charged phosphate groups in the LPS of A. coli (Li, Beuerman et al. 2020). Spermidine and spermine have been reported to confer resistance to some antibiotics in Pseudomonas aureus when tested at the pM range by decreasing the permeability of the antibiotics over the outer membrane (Johnson, Mulcahy et al. 2012). The activity of putrescine at the outer membrane in K. pneumoniae at the physiologically relevant mM range has not been previously reported. It is most likely that putrescine’s activity on the outer membrane is dose-dependent, and the interaction becomes unfavourable at higher concentrations.Polyamines act on the ribosome compounding the effect of azithromycin protein synthesis inhibition.

[0215] Next, the potential target site within the ribosome was determined through a combinatorial evaluation of antibiotics targeting different ribosomal sites and a control (rifampicin) acting outside the ribosome. It was hypothesized that in the wild-type, compounds with targets similar to those of putrescine within the ribosome will show similar effects when in combination with azithromycin; additionally, it was hypothesized that a compound that mirrors the phenotype of putrescine-azithromycin will show additive effects when in combination with putrescine. Seven antibiotics were tested in combination with each other and azithromycin to compare their FICI values (Fig. 16A). These antibiotics targeted the various locations of the ribosome: paromomycin and gentamicin bind to the aminoacyl site within the 30S ribosome leading to misreading of genetic code, tetracycline and doxycycline bind to the aminoacyl site of the 30S ribosome and prevent mRNA binding, chloramphenicol binds to the aminoacyl site on the 50S ribosome and prevents peptidyl transferase, azithromycin and erythromycin both block the 50S nascent peptide tunnel inhibiting transpeptidation; rifampicin, an RNA polymerase inhibitor, acted as a control. Out of the sevenantibiotics tested, only tetracyclines (tetracycline and doxycycline) synergized with azithromycin as putrescine did. Checkerboard assays of putrescine-azithromycin combinations were phenocopied by tetracycline-azithromycin combinations in the K. pneumoniae wild-type strain (Fig. 16B, C); tetracycline also showed additive effects with putrescine (Fig. 16D). Similarly the putrescineazithromycin phenotype was copied in tetracycline-azithromycin treated ribosomal mutants rpsO n and rpsR n (Fig. 16E-H). Additionally, the combination of tetracycline and azithromycin, in checkerboards of PMBN treated K. pneumoniae (FICI 0.50 ± 0.00) and the wv / a / )::Tn mutant still exhibited significant synergy (FICI 0.41 ± 0.07); these phenotypes confirm that putrescine acts via a dual mechanism. Together, these results provide support that putrescine acts on the 30S ribosomal subunit in a similar manner to tetracycline.

[0216] Tetracyclines are bacteriostatic inhibitors, targeting the 30S ribosomal acceptor site preventing the association of aminoacyl-tRNA with the ribosome complex (Chopra and Roberts 2001). The synergistic activity between the tetracycline doxycycline and azithromycin has been characterized before in Pseudomonas aeruginosa, but its mechanism has never been resolved (Saiman, Chen et al. 2002). Cross-linking studies show that ribosomes have >500 stably linked polyamines per ribosome, and it has been suggested polyamine binding happens in a stochastic manner (Amarantos, Zarkadis et al. 2002, Dever and Ivanov 2018). Previously, E. coli treated on serial passages of sub lethal putrescine concentrations elucidated mutants that developed a specific tolerance mechanism to putrescine. The evolved isolates had truncations of the S7 30S ribosomal protein, a protein that organizes the folding of the 3’ major domain of 16S RNA and crosslinks to the anticodon loop of tRNAs (Wimberly, White et al. 1997, Lennen, Lim et al. 2023). These isolates exhibited increased relative growth rates compared to the wild-type when grown with putrescine (Lennen, Lim et al. 2023). Adaptive resistance is likely to occur at the target for a compound, supporting the findings of this study that putrescine acts on the 30S ribosomal subunit. Polyamine cross-linking in the 30S subunit leads to 16S RNA structural changes (Amarantos, Zarkadis et al. 2002); similarly, tetracycline has been shown to have a regional effect in altering the crosslinking structure of 16S RNA (Noah, Dolan et al. 1999). Moreover, 30S subunits lacking RpsO or RpsR exhibit subtle changes in 16S RNA nucleotide reactivity (Svensson, Changchien et al. 1988, Bubunenko, Korepanov et al. 2006). Thus, without wishing to be limited by theory, it is probable that the mutants of RpsO and RpsR lead to an altered 16S RNA structure similar to that of poly amine and tetracycline binding on the 3 OS subunit.

[0217] It was hypothesized that polyamines exhibit a synergistic interaction with azithromycin at the ribosome by inhibiting protein synthesis compounding the effect of azithromycin. To this end, we used a cell-free protein synthesis system, an extract-based transcription / translation system derived from E. coli designed to synthesize proteins under the control of T7 RNA Polymerase. We quantified the levels of a plasmid encoded destabilized enhanced Green Fluorescent Protein (deGFP) fluorometrically in the presence and absence of putrescine (Fig 17). Simultaneously, we measured the effects on transcriptional activity in real-time (Fig 17), since the plasmid also included a malachite green mRNA aptamer allowing for such measurement fluorometrically via binding of malachite green dye (Siegal-Gaskins, Tuza et al. 2014). At the higher tested concentration of putrescine, mRNA levels were reduced but protein synthesis was further inhibited. Similar reduction in gene expression has been previously observed in cell-free in vitro gene expression models at high spermine and spermidine concentrations (Kanemura, Yoshikawa et al. 2018). Thus, to determine the effect of putrescine on protein synthesis due to a ribosomal effect, the deGFP signal was normalized by the mRNA signal (Fig 17). The lower putrescine concentrations exhibited increased protein synthesis (in the first ~90 minutes of exposure, Fig 17), which is in agreement with previous reports that low concentrations of polyamines promote protein synthesis in cell free systems (Kanemura, Yoshikawa et al. 2018).

[0218] Polyamines are integral for translation, playing an essential role in ribosome biogenesis, tRNA positioning on the ribosome, and translation elongation speed (Agrawal, Penczek et al. 1999, Igarashi and Kashiwagi 2018, Winther, Sorensen et al. 2021). It has also been previously shown that the addition of spermine to ribosome complexes slows down protein expression, can inhibit peptidyl transferase activity, and may lead to altered ribosome conformation (Teraoka and Tanaka 1973, Kalpaxis and Drainas 1993). The results of this study are in agreement with the previous literature and suggest that putrescine at high concentrations alters ribosomal function leading to a decrease in protein synthesis.A screen of a polyamine analog library uncovers azithromycin synergists.

[0219] The natural polyamine-azithromycin synergy and the mode of action thereof were exploited to uncover an antibiotic adjuvant that may expand the spectrum of azithromycin to K. pneumoniae. It was hypothesized that a polyamine analog may have azithromycin synergy as displayed by endogenous polyamine and serve as a potential adjuvant treatment with azithromycin. To this end, a library of 86 polyamine analogs were screened against MKP103 alone and incombination with a sub-inhibitory concentration (l / 8thMIC) of azithromycin (Fig. 18A). Six compounds displayed growth inhibition alone or in combination with azithromycin. Follow up checkerboard assays of the six primary hits identified four compounds, II-3, 1-2, 1-5, and II-l, as azithromycin synergists with the respective FICI values of 0.1873, 0.3125, 0.3125, and 0.3748 (Fig. 18B-E). Similar to putrescine, the identified compounds showed growth inhibitory effects against K. pneumoniae MKP103; their MIC values (31.25 M, 2.5, 2.5, and 5 mM for II-l, II-3, 1-2, and 1-5, respectively) were substantially lower than that of putrescine (40 mM).

[0220] Next, the mechanism of these four compounds was identified, first by checking if they acted through a similar dual mechanism as that of putrescine. Therefore, checkerboard assays of each compound were conducted with azithromycin against the rpsO Tn, rpsR Tn, and iiw / / J::Tn K. pneumoniae mutants, against which putrescine lost its synergy with the macrolide antibiotic. Both II- 1 and 1-5 showed reduced or complete loss of azithromycin synergy against all three mutants, suggesting they act, at least in part, at the outer membrane and ribosomal level similar to putrescine. Conversely, II- 3 and 1-2 exhibited no loss of synergy against the mutants, potentially suggesting other mechanisms at play.

[0221] It was shown in NPN fluorometric kinetic assays that all four compounds II-3, 1-2, 1-5, and II-l, in descending order of magnitude, disrupt the outer membrane of K. pneumoniae in a dose dependent manner across the synergistic range of each compound (Fig. 19A-D). Further, the inner membrane activity of all four compounds was tested using the previously described colorimetric ONPG E. coli ML-35 assay. All four analogs exhibit inner membrane activity in a dose dependent manner across the synergistic range of each compound (Fig. 20A-D). Each polyamine analog exhibited disruption of the inner membrane to various extents, which could be a contributing factor for the synergy some analogs continue to display in the rpsO Tn, rpsR Tn, and iiw / / J::Tn backgrounds. Taken together these results suggest that the identified polyamine analogs have membrane activity similar to putrescine. They synergize with azithromycin at substantially lower concentrations than putrescine and thus may serve as an adjuvant treatment strategy.Polyamine analogs synergy with other antibiotics

[0222] An additional combination was further tested. Interestingly, 1-1 and 1-5 showed substantial synergy with the cationic antimicrobial peptide, colistin, which serves as last resort antibiotic (Fig 21). In addition the fold shift increase in sensitivity of K. pneumoniae wild-type vs iiw / / J::Tn mutant for multiple antibiotics, including some that are typically active against Gram-positive bacteria only (Table 4) was shown. An increased antibiotic susceptibility in the waaD::Tn suggests that compounds that perturb the membrane, including polyamine analogs, may synergize with these antibiotics thus serving as antibiotic adjuvants that can restore antibiotic sensitivity in resistant bacteria.

[0223] Supplementary Table 4. MIC of gram-positive and gram-negative antibiotics in K. pneumoniae MKP103 wild-type vs waaD::Tn mutantConclusion

[0224] In the present study, a previously undescribed mechanism of alteration to azithromycin susceptibility in K. pneumoniae under the infection relevant compound putrescine was uncovered. The research found that putrescine synergizes with azithromycin in pan-drug-resistant backgrounds and hypervirulent lineages to a large enough extent to bring 78 of the 100 clinical isolates tested below the CLSI breakpoint. This alteration of macrolide susceptibility under physiological levels of polyamines is clinically relevant as broadening the spectrum of currently used antibiotics isa current potential strategy to combat AMR (Wesseling and Martin 2022). This study provides evidence towards a dual mechanism of action in the putrescine-azithromycin synergy phenotype. In mechanism 1) putrescine increases the permeability of azithromycin across the outer and inner membrane and in mechanism 2) putrescine compounds the protein synthesis inhibiting effects of azithromycin at the ribosome.

[0225] Further, it has been shown that analogs of polyamines possess increased potency against K. pneumoniae and synergy with various anti-Gram-negative and anti-Gram-positive antibiotics, such as azithromycin and others. The synergistic activity of these analogs may serve as a strategy to combat antimicrobial resistance by broadening the spectrum of azithromycin use to treat K. pneumoniae.

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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 I, II, and III or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula I, II, or III is as follows:(i); wherein:R1is selected from Ce-iealkyl; andR2and R3are independently selected from H and Ci-iealkyl, orR2and R3are linked together with the nitrogen atom to which they are attached to form a 3- to 6- membered heterocyclic ring, or one of R2and R3is H or Ci-4alkyl and the other is selected from C(NR4)NR5Ci-4alkyl, C(NR4)NR5C2- 4alkenyl and C(NR4)NR5C2-4alkynyl; andR4and R5are independently selected from H and Ci-ealkyl;R6R8'N-X1-N'R7'R9(II); wherein:R6, R7, R8and R9are independently selected from H and Ci-iealkyl, orR6and R7and / or R8and R9are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R6and R7and / or one of R8and R9is H or Ci-4alkyl and the other is selected from C(NR10)NR11Ci-4alkyl, C(NR10)NR11C2-4alkenyl and C(NR10)NR11C2-4alkynyl;R10and R11are independently selected from H and Ci-ealkyl;X1is selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R6, R7, R8and R9are all H, then X1is not n-butylene;wherein:R12, R13, R14, R15and R16are independently selected from H and Ci-isalkyl, orR12and R13and / or R15and R16are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R12and R13and / or one of R15and R16is H or Ci-4alkyl and the other is selected from C(NR17)NR18Ci-4alkyl, C(NR17)NR18C2.4alkenyl and C(NR17)NR18C2-4alkynyl;R17and R18are independently selected from H and Ci-ealkyl;X2and X3are independently selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R12, R13, R14, R15and R16are all H, then X2and X3are not both n-butylene; 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 selected from the compound of Formula I:R2R1-N" 3(i) or a pharmaceutically acceptable salt and / or solvate thereof.

3. The method of claim 2, wherein the compound of Formula I is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.The method of claim 1, wherein the one or more compounds are selected from the compound of Formula II:or a pharmaceutically acceptable salt and / or solvate thereof.

5. The method of claim 4, wherein three of R6, R7, R8and R9are H and the remaining one is selected from Ci-iealkyl.

6. The method of claim 5, wherein X1is C2-salkylene.

7. The method of claim 4, wherein three of R6, R7, R8and R9are H and the remaining one is selected from Ce-iealkyl and X1is C2-salkylene.

8. The method of claim 7, wherein three of R6, R7, R8and R9are H and the remaining one is selected from Cio-ualkyl and X1is Csalkylene.9 The method of claim 4, wherein the compound of Formula II is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

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

11. The method of claim 10, wherein the compound of Formula III is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

12. 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.

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

15. 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.

16. The method of any one of claims 1 to 15, 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.

17. The method of claim 16, 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, cioxacillin, dicloxacillin, flucioxacillin, 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.

18. The method of claim 1, wherein the one or more compounds of claim 1 inhibit growth of a bacterium.

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

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

21. The method of claim 1, wherein the one or more compounds of claim 1 suppress virulence of fungus.

22. The method of claim 1, wherein the bacterial infection is caused by Gram-positive or Gramnegative bacterium or fungus.

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

24. The method of claim 22, wherein the Gram-positive bacterium is a species of Enterococcus, Mycobacterium, Bacillus or Staphylococcus, or a combination thereof.

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

26. The method of claim 22, wherein the fungus is a species of Candida or Saccharomyces, or a combination thereof.

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

28. The method of claim 27, wherein the one or more compounds is the compound of formula II- 1 :II-l, wherein said compound reduces or eliminates polyamine-mediated antibiotic resistance of the bacterium or fungus to the antibiotic.

29. The method of claim 28, wherein the bacterium is a drug-resistant Gram-positive bacterium.

30. The method of claim 29, wherein the bacterium is S. aureus USA300.

31. The method of any one of claims 1 to 26, wherein the one or more compounds are compounds of Formulae:1-5, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the bacterial infection is caused by Gram-negative bacterium.

32. The method of claim 31, the Gram -negative bacterium is K. pneumoniae.

33. The method of claim 31 or 32, wherein the antibiotic is macrolide antibiotic.

34. The method of claim 33, wherein the macrolide is azithromycin.

35. The method of any one of claims 1 to 26, wherein the one or more compounds is the compound of Formula 1-5:1-5, or a pharmaceutically acceptable salt and / or solvate thereof, wherein the bacterial infection caused by Gram-negative bacterium.

36. The method of claim 35, wherein the Gram-negative bacterium is K. pneumoniae.

37. The method of claims 31 or 35, wherein the antibiotic is anti-Gram-negative or anti-Gram- positive antibiotic.

38. The method of claim 37, wherein the antibiotic is azithromycin, novobiocin, rifampicin, doxycycline, tetracycline, vancomycin, oxacillin, erythromycin, tobramycin, gentamicin, ciprofloxacin, daptomycin or trimethoprim.

39. The method of claim 37, wherein the antibiotic is colistin.

40. 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 I, II, and III or a pharmaceutically acceptable salt and / or solvate thereof, wherein the compound of Formula I, II, or III is as follows:R2R1- f 3(i); wherein:R1is selected from Ce-iealkyl; andR2and R3are independently selected from H and Ci-iealkyl, orR2and R3are linked together with the nitrogen atom to which they are attached to form a 3- to 6- membered heterocyclic ring, or one of R2and R3is H or Ci-4alkyl and the other is selected from C(NR4)NR5Ci-4alkyl, C(NR4)NR5C2- 4alkenyl and C(NR4)NR5C2-4alkynyl; andR4and R5are independently selected from H and Ci-ealkyl;R6R8'N-X1-N'R7'R9(II); wherein:R6, R7, R8and R9are independently selected from H and Ci-iealkyl, orR6and R7and / or R8and R9are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R6and R7and / or one of R8and R9is H or Ci-4alkyl and the other is selected from C(NR10)NR11Ci-4alkyl, C(NR10)NR11C2-4alkenyl and C(NR10)NR11C2-4alkynyl;R10and R11are independently selected from H and Ci-ealkyl;X1is selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R6, R7, R8and R9are all H, then X1is not n-butylene;wherein:R12, R13, R14, R15and R16are independently selected from H and Ci-isalkyl, orR12and R13and / or R15and R16are linked together with the nitrogen atom to which they are attached to form a 3- to 6-membered heterocyclic ring, or one of R12and R13and / or one of R15and R16is H or Ci-4alkyl and the other is selected from C(NR17)NR18Ci-4alkyl, C(NR17)NR18C2.4alkenyl and C(NR17)NR18C2-4alkynyl;R17and R18are independently selected from H and Ci-ealkyl;X2and X3are independently selected from Ci-ioalkylene optionally interrupted by Cs-scycloalkylene and Cs-scycloalkylene; and provided that when R12, R13, R14, R15and R16are all H, then X2and X3are not both n-butylene; or a pharmaceutically acceptable salt and / or solvate of any of the above.

41. The method of claim 40, wherein the one or more compounds are selected from the compound of Formula I:R2R1-N" 3(i) or a pharmaceutically acceptable salt and / or solvate thereof.

42. The method of claim 41, wherein the compound of Formula I is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

43. The method of claim 40, wherein the one or more compounds are selected from the compound of Formula II:R6R8'N-X1-N'R7h®(ii) or a pharmaceutically acceptable salt and / or solvate thereof.

44. The method of claim 43, wherein three of R6, R7, R8and R9are H and the remaining one is selected from Ci-iealkyl.

45. The method of claim 44, wherein X1is C2-salkylene.

46. The method of claim 43, wherein three of R6, R7, R8and R9are H and the remaining one is selected from Ce-iealkyl and X1is C2-salkylene.

47. The method of claim 46, wherein three of R6, R7, R8and R9are H and the remaining one is selected from Cio-ualkyl and X1is Csalkylene.48 The method of claim 43, wherein the compound of Formula II is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

49. The method of claim 40, wherein the one or more compounds are selected from the compound of Formula III:or a pharmaceutically acceptable salt and / or solvate thereof.

50. The method of claim 49, wherein the compound of Formula III is selected from:or a pharmaceutically acceptable salt and / or solvate thereof.

51. The method of any one of claims 1 to 40, 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.

52. The method of any one of claims 1 to 50, 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.

53. The method of claims 51 or 52 , wherein the pharmaceutical composition further comprises a bicarbonate buffer.

54. A composition comprising: a) one or more compounds of Formula I, II, or III, or a pharmaceutically acceptable salt and / or solvate thereof; andb) a pharmaceutically acceptable carrier, wherein the composition is configured for administration in combination with an antibiotic for treating a bacterial or fungal infection.

55. A pharmaceutical composition comprising: a) an effective amount of one or more compounds selected from a compound of Formula I, II, or III, 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.

56. The composition of claim 55, wherein the bacterial infection is caused by a Gramnegative bacterium selected from Klebsiella pneumoniae, Escherichia coli, or Salmonella Typhimurium.

57. The composition of claim 55, wherein the bacterial infection is caused by a Gram-positive bacterium selected from Staphylococcus aureus o Enterococcus faecalis.

58. The composition of claim 55, wherein the one or more compounds are configured to inhibit bacterial polyamine detoxification enzyme activity.

59. A compound of Formula I, II, or III, 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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