(12) United States Patent (10) Patent No.: US 9.260,481 B2 Gualtieri Et Al

USOO926.0481 B2

(12) United States Patent (10) Patent No.: US 9.260,481 B2 Gualtieri et al. (45) Date of Patent: Feb. 16, 2016

(54) PEPTIDE DERIVATIVES AS ANTIBIOTICS A61K 45/06 (2013.01); C12P 21/02 (2013.01); CI2R I/01 (2013.01); A61 K38/00 (2013.01) (71) Applicants: Maxime Gualtieri, Gallargues le (58) Field of Classification Search Montueux (FR); Philippe None Villain-Guillot, Montpellier (FR); Alain See application file for complete search history. Givaudan, Saint Mathieu de Treviers (FR); Sylvie Pages, Saint Clement de (56) References Cited Riviere (FR) U.S. PATENT DOCUMENTS

(72) Inventors: Maxime Gualtieri, Gallargues le 2011/0033389 A1 2/2011 Chen ...... CO7K 16,087 Montueux (FR); Philippe 424/9.6 Villain-Guillot, Montpellier (FR); Alain Givaudan, Saint Mathieu de Treviers FOREIGN PATENT DOCUMENTS (FR); Sylvie Pages, Saint Clement de GB 2207433 A 2, 1989 Riviere (FR) WO O2O55545 A1 T 2002 (73) Assignees: Nosopharm (FR); Institut National de WO 2010.136532 A1 12/2010 la Recherche Agronomique (FR) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Wang et al., Enhanced antibiotic activity of Xenorhabdus patent is extended or adjusted under 35 nematophila by medium optimization, Bioresource Tech, 99 (2008) 1708-1715. U.S.C. 154(b) by 35 days. GenBank: AAM02072.1, RNA-binding protein involved in rRNA processing, of Methanopyrus kandleri AV 19, Jan. 2014.* (21) Appl. No.: 14/342,446 Identification of a new antimicrobial lysine-rich cyclolipopeptide family from Xenorhabdus nematophila, Gualtieri et al., Jl. Of Anti (22) PCT Filed: Sep. 28, 2012 biotics, (2009) 62,295-302.* Berdy Janos, et al., “Bioactive Microbial Metabolites.” The Journal (86). PCT No.: PCT/EP2012/069166 of Antibiotics, 2005, pp. 1-26, vol. 58, No. 1. Bodanszky, M., The Practice of Peptide Synthesis, Reactivity and S371 (c)(1), Structure Concepts in Organic Chemistry, Springer Laboratory, (2) Date: Mar. 3, 2014 1994, 16 pages. Forst, S., et al., Molecular Biology of the Symbiotic-Pathogenic Bacteria Xenorhabdus spp. and Photorhabdus spp., American Soci (87) PCT Pub. No.: WO2013/045600 ety for Microbiology, Mar. 1, 1996, pp. 21-43. PCT Pub. Date: Apr. 4, 2013 Marion, D., et al., Rapid Recording of 2D NMR Spectra without Phase Cycling. Application to the Study of Hydrogen Exchange in (65) Prior Publication Data Proteins, Journal of Magnetic Resonance, Jul. 25, 1989, pp.393-399. Nolden, S., et al., Analysis of RegA, a Pathway-Specific Regulator of US 2015/O175659 A1 Jun. 25, 2015 the Friulimincin Biosynthesis in Actinoplanes Friuliensis, Journal of Biotechnology, Mar. 10, 2009, vol. 140, No. 1-2, pp. 99-106. Piotto, M., et al., Gradient-Tailored Excitation for Single-Quantum Related U.S. Application Data NMR Spectroscopy of Aqueous Solutions, Journal of Biomolecular (60) Provisional application No. 61/540,085, filed on Sep. NMR, Oct. 15, 1992, pp. 661-665. 28, 2011. * cited by examiner (30) Foreign Application Priority Data Primary Examiner — Karlheinz, R Skowronek Sep. 28, 2011 (EP) ...... 11183O34 Assistant Examiner — Joseph Fischer (74) Attorney, Agent, or Firm — Banner & Witcoff, Ltd. (51) Int. Cl. C07K 7/06 (2006.01) (57) ABSTRACT A6 IK38/08 (2006.01) The present invention relates to antibiotic compounds, meth CI2P 21/02 (2006.01) ods for producing said compounds, pharmaceutical compo CI2R I/OI (2006.01) sitions comprising said compounds, and methods of treat A6 IK 45/06 (2006.01) ment comprising administering said compounds and/or A61 K38/00 (2006.01) compositions comprising said compounds. (52) U.S. Cl. CPC. C07K 7/06 (2013.01); A61K38/08 (2013.01); 9 Claims, 4 Drawing Sheets U.S. Patent Feb. 16, 2016 Sheet 1 of 4 US 9.260,481 B2

ppm

I.0 -

1.5 -

2.0.

2.5 al 0 3.0 - 6

3.5 -

4.0 -

4.5 - d

5.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 ppm U.S. Patent Feb. 16, 2016 Sheet 2 of 4 US 9.260,481 B2

ppm s s' so a is

r 40

45 s 50

4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm FIG 2 U.S. Patent Feb. 16, 2016 Sheet 3 of 4 US 9.260,481 B2

100E+10 100E+09 100E+08 - --Control 1,00E+07 - 1,00E+06 --Vancomycin (6.25 ug/mL) 100E+05 --Odilomycin A (3.12 ug/mL) 100E+04 100E+03 100E+02 100E+01 1,00E+00 U.S. Patent Feb. 16, 2016 Sheet 4 of 4 US 9.260,481 B2

(IE E. - IES - E- x is , EC6 t Sax a see Citri 1,00E+05Ei ". --Fly Eryxin (.36x s ::s girl. & . , i. W, * Cristoraycin& A. (12.3: ;8. S: girl L) IEE+03 W.: (GE--03 IEE+01 W (DE431 s s fire it

FIG.4 US 9,260,481 B2 1. 2 PEPTIDE DERVATIVES AS ANTIBIOTICS SUMMARY OF THE INVENTION

RELATED APPLICATION DATA In one aspect, the present invention is related to a com

5 pound of formula (I): This application is a National Stage Application under 35 (I) U.S.C. 371 of co-pending PCT application PCT/EP2012/ wherein 069166 designating the United States and filed Sep. 28, 2012: Xaa, Xaa, Xaa, Xaas, Xaa, Xaas and Xaao are indepen which claims the benefit of EP application number 10 dently selected from the group consisting of lysine, 3-hy 11183034.5 and filed Sep. 28, 2011 each of which are hereby droxylysine, 4-hydroxylysine, 5-hydroxylysine, 3,4-dihy incorporated by reference in their entireties. droxylysine, 3,5-dihydroxylysine, 4,5-dihydroxylysine, ornithine, 3-hydroxyornithine, 4-hydroxyornithine, 3,4- 15 dihydroxyornithine, 2,4-diaminobutanoic acid, 3-hy FIELD OF THE INVENTION droxy-2,4-diaminobutanoic acid, arginine, histidine, serine, and threonine; The present invention relates to new antibiotic compounds Xaa is glycine, 3-aminopropanoic acid, or 4-aminobutanoic and compositions comprising the same, to a strain of 20 acid; Xaa is proline, 3-hydroxyproline, 4-hydroxyproline, aziri Xenorhabdus nematophila capable of producing said antibi dine-2-carboxylic acid, aZetidine-2-carboxylic acid, pipe otic compounds, and to the use of Such compounds and com colic acid, 4-oxaproline, 3-thiaproline, 4-thiaproline, 3,4- positions thereof in the treatment of microbial disease. dehydroproline, 4-aminoproline, 4-fluoroproline, 25 C.-methylproline, or C-allylproline; Xaa is arginine, 2.3-dehydroarginine, citrulline, 2.3-dehy BACKGROUND OF THE INVENTION drocitrulline, canavanine, or 2,3-dehydrocanavanine; n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and Antimicrobial resistance is a major public health problem 30 R is -OH, -NH, or -COOH. with a significant impact on morbidity, mortality and health In one embodiment, Xaa is lysine; Xaa, and Xaas are each care-associated costs. The problem has been worsened by the 3-hydroxy-2,4-diaminobutanoic acid; Xaa is glycine; Xaas restriction of antibiotic drug discovery and development pro is ornithine; Xaa is proline; Xaa, is histidine; and Xaa, is grams. Nowadays, the most relevant multiresistant bacterial is 2,3-dehydroarginine. pathogens are methicillin-resistant Staphylococcus aureus In another embodiment, Xaa is lysine; Xaa, and Xaa, are (MRSA), Vancomycin-resistant enterococci (VRE), each 3-hydroxy-2,4-diaminobutanoic acid; Xaa, is glycine; extended spectrum B-lactamase formers (ESBL), multiresis Xaas is ornithine; Xaa is proline: Xaa, is histidine; Xaas is tant Pseudomonas and Acinetobacter species. For these bac lysine or 5-hydroxylysine; Xaa, is 2.3-dehydroarginine; and teria, only a few of the existing antibiotics are efficient. There Xaao is lysine or 5-hydroxylysine. is an urgent need for new antibacterial compounds to ensure In another embodiment, Xaa is lysine; Xaa, and Xaa, are that bacterial infections can be effectively treated in the each 3-hydroxy-2,4-diaminobutanoic acid; Xaa, is glycine; future. Living organisms have proven to be a reliable source Xaas is ornithine; Xaa is proline; Xaa, is histidine, Xaas is of bioactive chemicals with antimicrobial activity (Berdy J. 45 lysine or 5-hydroxylysine; Xaa, is 2.3-dehydroarginine; J. Antibiot. 58, 1-26 (2005)). Environmental microbes con Xaao is lysine or 5-hydroxylysine; n is 4; and R is NH2. tinue to be promising resources for the identification of new In a preferred embodiment, the present invention is related molecules. to a compound of formula (Ia):

NH2 NH2 "N, NH OH NH O O HN1\ H H N N N N S. HN N N O O H H H H O O O OH O N N N N N 1N1N1 NH2 H H H NH2 O O

HO HO US 9,260,481 B2 3 4 In another preferred embodiment, the invention is related droxylysine, 4-hydroxylysine, 5-hydroxylysine, 3,4-dihy to a compound of formula (Ib): droxylysine, 3,5-dihydroxylysine, 4,5-dihydroxylysine,

NH2 HN Y NH OH NH O O HN1\ H H N N N N S. HN N N O O H H H H O O OH O N N N N 1N1N1 NH2 H H O O

HO NH2 NH2

In another preferred embodiment, the invention is related ornithine, 3-hydroxyornithine, 4-hydroxyornithine, 3,4- to a compound of formula (Ic): dihydroxyornithine, 2,4-diaminobutanoic acid, 3-hy

NH2 HN Y NH OH NH O O HN1\ H H N N N N S. HN N N O O H H H H O O OH O N N N N 1N1N1 NH2 H H O O

NH2 NH2

In another aspect, the present invention is related to a droxy-2,4-diaminobutanoic acid, arginine, histidine, compound wherein the compound is not (Ia), (Ib), or (Ic). serine, and threonine; Preferably, the compounds of the present invention are Xaa is glycine, 3-aminopropanoic acid, or 4-aminobutanoic isolated. Preferably, the compound is greater than about 90% 45 pure. More preferably, the compound is greater than about acid; 95% pure. Even more preferably, the compound is greater Xaa is proline, 3-hydroxyproline, 4-hydroxyproline, aziri than about 98% pure. Even more preferably, the compound is dine-2-carboxylic acid, aZetidine-2-carboxylic acid, pipe greater than about 99% pure. colic acid, 4-oxaproline, 3-thiaproline, 4-thiaproline, 3,4- The present invention is also directed to a pharmaceutical 50 dehydroproline, 4-aminoproline, 4-fluoroproline, composition comprising a compound as described above and C.-methylproline, or C-allylproline; a pharmaceutically acceptable carrier. Xaa is arginine, 2.3-dehydroarginine, citrulline, 2.3-dehy Preferably, the pharmaceutical composition further com prises a second antibiotic compound. Preferably, the second drocitrulline, canavanine, or 2,3-dehydrocanavanine; antibiotic compound is an aminoglycoside antibiotic. Prefer 55 n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and ably, the second antibiotic compound is kanamycin. Prefer R is —OH, -NH2 or —COOH, or a composition compris ably, the second antibiotic compound is gentamicin. ing a compound of formula (I) and a pharmaceutically The present invention is also directed to a method for acceptable carrier, to a Subject in need thereof. treating bacterial infection in a Subject comprising adminis In the methods of the present invention it is preferred that tration of atherapeutically effective amount of a compound of 60 Xaa is lysine; Xaa, and Xaas are each 3-hydroxy-2,4-diami formula (I): nobutanoic acid; Xaa is glycine; Xaas is ornithine; Xaa is proline; Xaa, is histidine; Xaas is lysine or 5-hydroxylysine; Xaa-NH-(CH2), R (I) Xaa is 2.3-dehydroarginine; Xaao is lysine or 5-hydroxyl wherein, 65 ysine; n is 4; and R is NH. Xaa, Xaa, Xaas, Xaas. Xaa-7, Xaas and Xaalo are indepen In the methods of the present invention it is preferred that dently selected from the group consisting of lysine, 3-hy the compound of formula (I) is: US 9,260,481 B2

(Ia) NH2 NH NH2 OH O O H H N N N HN N N H H O O O OH O N H NH

Preferably, the methods of the present invention comprise Xaa- is glycine, 3-aminopropanoic acid, or 4-aminobutanoic administration of a second antibiotic compound. Preferably, acid; the second antibiotic compound is an aminoglycoside antibi Xaa is proline, 3-hydroxyproline, 4-hydroxyproline, aziri otic. Preferably, the second antibiotic compound is kanamy dine-2-carboxylic acid, azetidine-2-carboxylic acid, pipe cin. Preferably, the second antibiotic compound is gentami colic acid, 4-oxaproline, 3-thiaproline, 4-thiaproline, 3,4- C1. 25 dehydroproline, 4-aminoproline, 4-fluoroproline, In the methods of the present invention, the subject is O-methylproline, or C-allylproline: preferably a mammal. Preferably, the subject is avian, swine, Xaa- is arginine, 2.3-dehydroarginine, citrulline, 2,3-dehy bovine or human. Most preferably, the subject is a human. drocitrulline, canavanine, or 2.3-dehydrocanavanine: In the methods of the present invention, the compound or n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and composition is preferably effective against multi-drug resis Ris-OH, -NH, or -COOH. tant clinical bacteria. 30 In one embodiment, the present invention is directed to a In the methods of the present invention, the compound or method for producing a compound of formula (I), wherein composition is preferably administered intravenously, Xaa is lysine; Xaa, and Xaa, are each 3-hydroxy-2,4-diami parenterally, orally and/or topically. nobutanoic acid; Xaa is glycine; Xaas is ornithine; Xaa, is Another object of the present invention is a method for proline; Xaa, is histidine; and Xaa, is 2,3-dehydroarginine. producing a compound of formula (I) comprising the follow 35 In another embodiment, the present invention is directed to ing steps: a method for producing a compound of formula (I), wherein growing Xenorhabdus nematophila strain CNCM I-4530 Xaa is lysine; Xaa, and Xaa, are each 3-hydroxy-2,4-diami in a liquid culture medium; and nobutanoic acid; Xaa, is glycine; Xaas is ornithine; Xaa, is purifying a compound according to formula (I): proline; Xaa, is histidine: Xaas is lysine or 5-hydroxylysine: Xaao is 2.3-dehydroarginine; and Xaao is lysine or 5-hy 40 droxylysine. (I) In another embodiment, the present invention is directed to Wherein a method for producing a compound of formula (I), wherein Xaa, Xaa2, Xaas, Xaas. Xaa-7, Xaas and Xaalo are indepen Xaa is lysine; Xaa, and Xaa are each 3-hydroxy-2,4-diami dently selected from the group consisting of lysine, 3-hy nobutanoic acid; Xaa is glycine; Xaas is ornithine; Xaa, is droxylysine, 4-hydroxylysine. 5-hydroxylysine, 3,4-dihy 45 proline; Xaa, is histidine; Xaas is lysine or 5-hydroxylysine: droxylysine, 3,5-dihydroxylysine, 4,5-dihydroxylysine, Xaa- is 2.3-dehydroarginine; Xaao is lysine or 5-hydroxyl ornithine, 3-hydroxyornithine, 4-hydroxyornithine, 3,4- ysine; n is 4; and R is NH. dihydroxyornithine, 2.4-diaminobutanoic acid, 3-hy In another embodiment, the present invention is directed to droxy-2,4-diaminobutanoic acid, arginine, histidine, a method for producing a compound of formula (I), wherein serine, and threonine: the compound of formula (I) is (Ia) NH

NH2 OH O O H H N N N N S. HN N N 2 H H H O O O N OH O N H NH

HO NH2 US 9,260,481 B2 7 8 In preferred embodiments, the purifying step comprises persion media, coatings and the like that are physiologically cation-exchange chromatography and/or reverse-phase chro compatible. Non-limiting examples of Such pharmaceutical matography. carriers include liquids. Such as water and oils, including Another object of the present invention is a Xenorhabdus those of petroleum, animal, vegetable or synthetic origin, nematophila strain deposited at CNCM on 21 Sep. 2011 Such as peanut oil, soybean oil, mineral oil, sesame oil and the having the accession number CNCM I-4530. like. The pharmaceutical carriers may also be saline, gum Another object of the present invention is a culture Super acacia, gelatin, starch paste, talc, keratin, colloidal silica, natant from the Xenorhabdus nematophila strain CNCM urea, and the like. In addition, auxiliary, stabilizing, thicken I-4530 having antibiotic activity. ing, lubricating and coloring agents may be used. Other 10 examples of suitable pharmaceutical carriers are described in BRIEF DESCRIPTION OF THE FIGURES Remington’s Pharmaceutical Sciences (Alfonso Gennaro ed., Krieger Publishing Company (1997); Remington's: The FIG. 1 shows NMRTOCSY spectrum of Odilomycin A Science and Practice of Pharmacy, 21 Ed. (Lippincot, Will FIG. 2 shows NMR HSQC spectrum of Odilomycin A iams & Wilkins (2005); and Modern Pharmaceutics, Vol. 121 FIG. 3 shows bactericidal effects of Odilomycin A on 15 (Gilbert Banker and Christopher Rhodes, CRC Press (2002). growing S. aureus ATCC13709. Abbreviations: ATCC (American Type Culture Collec FIG. 4 shows bactericidal effects of Odilomycin A on tion), CNCM (Collection Nationale de Cultures de Microor growing P. aeruginosa ATCC27853. ganismes), INRA (Institut National de la Recherche Agronomique), MRSA (methicillin-resistant Staphylococcus DETAILED DESCRIPTION OF THE INVENTION aureus), VRE (vancomycin-resistant enterococci), ESBL Definitions and Abbreviations (extended spectrum B-lactamase formers), NMR (nuclear The terms “antibiotic”, “antibiotic activity”, “antibacte magnetic resonance), MS-MS (mass spectroscopy-mass rial”, “antibacterial activity”, “antimicrobial, or “antimicro spectroscopy), LC-MS (liquid chromatography-mass spec bial activity” as used herein refer generally to an effect in troscopy), ESI (electrospray ionization), HPLC (high-pres which a reduction, inhibition or a halt in the growth of a 25 Sure liquid chromatography), LB (Luria-Bertani medium), microorganism is achieved. Antibiotic activity may be tested NBTA (Nutrient agar (Difco) 31 g/L, bromothymol blue 25 according to any known method Such as, for example, a mg/L and 2,3,5-triphenyl tetrazolium chloride 1% 40 mg/L), microdilution method. TFA (trifluoroacetic acid), UV (ultraviolet), MIC (minimal The term “ODILOMYCIN, “Odilomycin” or “odilomy inhibitory concentration), MHB (Mueller-Hinton broth), cin' as used herein refers a compound of formula (I). In some 30 MBCs (Minimal bactericidal concentrations) embodiments, the compound of formula (I) is a compound of Description formula (Ia), formula (Ib), and/or formula (Ic). The term is In one aspect, the present invention is related to Xenorhab intended to encompass all Stereoisomeric forms such as, for dus nematophila strain 108 deposited at CNCM (Collection example, tautomers, diastereomers (including cis/trans iso Nationale de Cultures de Microorganismes) in the name of mers) and enantiomers. 35 INRA (Institut National de la Recherche Agronomique) on 21 A“pharmaceutical composition” refers to a mixture of one Sep. 2011 having the accession number CNCMI-4530. or more of the compounds described herein, or pharmaceuti It has been found that Xenorhabdus nematophila strain cally acceptable salts thereof, with other chemical compo CNCMI-4530 produces compounds exhibiting antibiotic or nents, such as physiologically acceptable carriers and excipi antimicrobial activity. When Xenorhabdus nematophila ents. The purpose of a pharmaceutical composition is to strain CNCM I-4530 is grown in a liquid culture medium, facilitate administration of a compound to an organism or 40 antibiotic compounds are secreted into the culture Superna Subject. tant. As used herein the term “about is used herein to mean In another aspect, the present invention is related to a approximately, roughly, around, or in the region of. When the culture Supernatant from Xenorhabdus nematophila strain term “about is used in conjunction with a numerical range, it CNCMI-4530 exhibiting antibiotic or antibacterial activity. modifies that range by extending the boundaries above and 45 For the preparation of a culture Supernatant having antibiotic below the numerical values set forth. In general, the term activity Xenorhabdus nematophila strain CNCM I-4530 is “about is used herein to modify a numerical value above and grown in a liquid culture medium under standard conditions, below the stated value by a variance of 10 percent up or down the bacterial cells are removed and the Supernatant is recov (higher or lower). ered. The bacterial cells may, for example, be removed by An “effective amount”, “sufficient amount’ or “therapeu 50 centrifugation or filtration. tically effective amount’ as used herein is an amount of a In yet another aspect, the invention is related to extracts compound that is sufficient to effect beneficial or desired from Xenorhabdus nematophila strain CNCMI-4530 show therapeutic effect, including clinical results, commensurate ing antibiotic activity. Cell extracts from Xenorhabdus nem with a reasonable benefit/risk ratio, when administered in atophila may be prepared according to any appropriate accordance with a desired treatment regimen. As such, the method known to the skilled person. effective amount may be sufficient, for example, to reduce or 55 In some embodiments, the present invention also encom ameliorate the severity and/or duration of bacterial infection, passes culture Supernatant and extracts from Xenorhabdus afflictions related thereto, or one or more symptoms thereof; nematophila strain CNCMI-4530 for use as medicament. prevent the advancement of conditions or symptoms (includ In some embodiments, the present invention comprises ing prophylactic prevention) related to afflictions related to methods of treatment, Suppression and/or prevention of bac bacterial infection, afflictions related thereto, or one or more 60 terial infection comprising administration of extracts from symptoms thereof, or enhance or otherwise improve the pro Xenorhabdus nematophila strain CNCMI-4530. phylactic ortherapeutic effect(s) of another therapy. An effec In some embodiments, the present invention also encom tive amount also includes the amount of the compound that passes culture Supernatant and extracts from Xenorhabdus avoids or substantially attenuates undesirable side effects. nematophila strain CNCM I-4530 for use as an antibiotic The term “carrier refers to a diluent, adjuvant, excipient, or 65 agent. vehicle with which a compound is administered. Pharmaceu Xenorhabdus nematophila strain CNCM I-4530, culture tically acceptable carriers include any and all solvents, dis supernatant from this strain and cell extracts derived from this US 9,260,481 B2 10 strain exhibit antibiotic activity against different microorgan In some embodiments, Xaa is lysine. isms including, for example, human bacterial pathogens. In some embodiments, Xaa is 3-hydroxy-2,4-diaminobu More particularly, Xenorhabdus nematophila Strain tanoic acid. CNCM I-4530, culture supernatant from this strain and cell In some embodiments, Xaa- is 3-hydroxy-2,4-diaminobu extracts derived from this strain exhibit antibiotic activity 5 against Acinetobacter baumannii, Bacillus subtilis, tanoic acid. Burkholderia cepacia, Enterobacter clocae, Enterococcus In some embodiments, Xaa, is glycine. faecalis, Escherichia coli, Klebsiella pneumoniae, Staphyllo In some embodiments, Xaas is ornithine. coccus aureus, Staphylococcus epidermidis, Stenotrophomo nas maltophilia, Serratia marescens and Pseudomonas In some embodiments, Xaa is proline. aeruginosa. Preferably, Xenorhabdus nematophila strain 10 In some embodiments, Xaa, is histidine. CNCM I-4530, culture supernatant from this strain and cell In some embodiments, Xaas is lysine or 5-hydroxylysine. extracts derived from this strain exhibit antibiotic activity In some embodiments, Xaa, is 2.3-dehydroarginine. against Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Klebsiella pneumonia, Klebsiella Oxytoca, In some embodiments, Xaao is lysine or 5-hydroxylysine. Acinetobacter baumannii, Enterobacter cloacae, Escheri 15. In some embodiments, n=4. chia coli, Moraxella catarrhalis, Pseudomonas aeruginos In some embodiments, R is —NH. and Stenotrophomonas maltophilia. In some embodiments, Xaa is lysine, Xaa, and Xaas are Exemplary compounds having antibiotic activity as each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, described herein have been purified from the culture super natant of Xenorhabdus nematophila strain CNCMI-4530. Xaas is ornithine, Xaa is proline, Xaa, is histidine, Xaas is In yet another aspect, the present invention is related to a lysine or 5-hydroxylysine, Xaa, is 2,3-dehydroarginine, and compound of formula (I): Xaao is lysine or 5-hydroxylysine. In some embodiments, Xaa is lysine, Xaa, and Xaas are each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, Xaalo-NH-(CH2), R (I) as Xaas is ornithine, Xaag is proline, Xaa, is histidine, Xaas is wherein lysine or 5-hydroxylysine, Xaa, is 2.3-dehydroarginine, Xaa, Xaa, Xaas, Xaas. Xaa-7, Xaas and Xaalo are indepen dently selected from the group consisting of lysine, 3-hy Xaao is lysine or 5-hydroxylysine, n is 4 and R is NH. droxylysine, 4-hydroxylysine, 5-hydroxylysine, 3,4-dihy The configuration at the C-carbon atoms in the amino acid droxylysine, 3,5-dihydroxylysine, 4,5-dihydroxylysine, residues may be "D' or “L”, and may be independent of the ornithine, 3-hydroxyornithine, 4-hydroxyornithine, 3,4- 30 configurations of otheramino acid residues in the compounds dihydroxyornithine, 2,4-diaminobutanoic acid, 3-hy of formula (I). Thus, in Some embodiments, Xaa, Xaa, droxy-2,4-diaminobutanoic acid, arginine, histidine, Xaas, Xaa, Xaas, Xaa, Xaa-7, Xaas, and Xaalo each have the serine, and threonine; “L” configuration at the C-carbon of the amino acid residue. Xaa is glycine, 3-aminopropanoic acid, or 4-aminobutanoic In some embodiments, one or more of Xaa, Xaa, Xaa, acid; 35 Xaa, Xaas. Xaa, Xaa, Xaas, and Xaao have the 'D' con Xaa is proline, 3-hydroxyproline, 4-hydroxyproline, aziri figuration at the C-carbon of the amino acid residue. dine-2-carboxylic acid, aZetidine-2-carboxylic acid, pipe colic acid, 4-oxaproline, 3-thiaproline, 4-thiaproline, 3,4- The configuration at the hydroxyl groups in the amino acid dehydroproline, 4-aminoproline, 4-fluoroproline, side chains may be “R” or “S”, and may be independent of the C.-methylproline, or C-allylproline; Xaa, is arginine, 2.3- configurations of other hydroxyl groups in the compounds of dehydroarginine, citrulline, 2.3-dehydrocitrulline, canava 40 formula (I). Thus, in some embodiments, one or more nine, or 2.3-dehydrocanavanine; hydroxyl groups have the “R” configuration. In some n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; and embodiments, one or more hydroxyl groups have the “S” R is -OH, -NH, or -COOH. configuration. In some embodiments, each of the hydroxyl In some embodiments, Xaa is lysine, Xaa, and Xaa, are groups have the “R” configuration. In some embodiments, each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, 45 each of the hydroxyl groups have the “S” configuration. Xaas is ornithine, Xaa is proline, Xaa, is histidine, and Xaa, In some embodiments, the compound of formula (I) is the is 2.3-dehydroarginine. compound of formula (Ia):

(Ia)

NH HN1\ N S. O O H H N N N N 1N1N1 NH2 H H O O

HO HO NH2 NH2 US 9,260,481 B2 11 12 The compound of formula (Ia) is also defined as Lys-(3- In some embodiments, the Odilomycins are greater than hydroxy-2,4-diaminobutanoic acid)-(3-hydroxy-2,4-diami about 50% pure. In some embodiments, the Odilomycins are nobutanoic acid)-Gly-Ornithine-Pro-His-(5-hydroxylysine)- greater than about 60% pure. In some embodiments, the (2,3-dehydroarginine)-(5-hydroxylysine)-(1,4- Odilomycins are greater than about 70% pure. In some diaminobutane). embodiments, the Odilomycins are greater than about 80% The term "Odilomycin A' refers to the compound of for pure. In some embodiments, the Odilomycins are greater than mula (Ia). about 85% pure. In some embodiments, the Odilomycins are In some embodiments, the compound of formula (I) is the greater than about 90% pure. In some embodiments, the compound of formula (Ib): Odilomycins are greater than about 95% pure. In some

(Ib)

The compound of formula (IIb) is also defined as Lys-(3- 30 embodiments, the Odilomycins are greater than about 98% hydroxy-2,4-diaminobutanoic acid)-(3-hydroxy-2,4-diami pure. In some embodiments, the Odilomycins are greater than nobutanoic acid)-Gly-Ornithine-Pro-His-Lys-(2,3-dehy about 99% pure. In some embodiments, any stated purity droarginine)-(5-hydroxylysine)-(1,4-diaminobutane). values can form a lower and/or upper endpoint of a purity The term "Odilomycin B” refers to the compound of for range as appropriate or where any of the lower limits can be mula (Ib). 35 combined with any of the upper limits. In some embodiments, the compound of formula (I) is the In some embodiments, Odilomycins and/or compositions compound of formula (Ic): comprising the same are useful as a medicament, an antibiotic

(Ic) NH2 NH2 HN NH NH2 OH NH O O H H N N N S. HN N N O O H H H H O O O OH O N N N N N 1N1N1 NH2. H H H NH2 O O

NH2 NH2

The compound of formula (Ic) is also defined as Lys-(3- agent, an antimicrobial agent, or in the treatment of microbial hydroxy-2,4-diaminobutanoic acid)-(3-hydroxy-2,4-diami 60 disease, in particular of bacterial infection caused, for nobutanoic acid)-Gly-Ornithine-Pro-His-Lys-(2,3-dehy example, by pathogenic bacteria. droarginine)-Lys-(1,4-diaminobutane). In some embodiments, Odilomycins and/or compositions The term "Odilomycin C refers to the compound of for comprising the same are useful in the treatment of a bacterial mula (Ic). infection, for example in the treatment of a hospital-acquired In some embodiments, Odilomycins do not include the 65 infection or a nosocomial bacterial infection. compounds of formulas (Ia), (Ib), and (Ic). In some embodiments, the present invention provides a In some embodiments, the Odilomycins are isolated. method for treating a subject Suffering from hospital-ac US 9,260,481 B2 13 14 quired bacterial infection comprising administering to said Photorhabdus, Porphyromonas, Prevotella, Propionibacte Subject an effective amount of a compound of formula (I). rium, Proteus, Pseudomonas, Rickettsia, Salmonella, Serra In some embodiments, the present invention provides a tia, Shigella, Sphaerophorus, Spirillum, Staphylococcus, method for treating a Subject Suffering from nosocomial bac Stenotrophomonas, Streptobacillus, Streptococcus, Tre terial infection comprising administering to said subject an 5 ponema, Tropheryma, Ureaplasma, Vibrio, or Yersinia fami effective amount of a compound of formula (I). lies. In one aspect, the invention is also related to the use of In some embodiments, the bacterial or microbial infection compounds of formula (I) for the manufacture of a medica is an infection caused in whole or in part by Acinetobacter ment for treatment of microbial infection or microbial dis baumannii, Bacillus subtilis, Burkholderia cepacia, Entero CaSC. 10 bacter clocae, Enterococcus faecalis, Escherichia coli, Kleb In another aspect, the invention is also related to the use of siella pneumoniae, Staphylococcus aureus, Staphylococcus compounds of formula (I) for the manufacture of an antibiotic epidermidis, Stenotrophomonas maltophilia, Serratia mare composition. scens or Pseudomonas aeruginosa. Preferably, Xenorhabdus In yet another aspect, the present invention is also directed nematophila strain CNCMI-4530, culture supernatant from to methods of treatment comprising administration of a com 15 this strain and cell extracts derived from this strain exhibit pound of formula (I), or pharmaceutical compositions com antibiotic activity against Staphylococcus aureus, Staphyllo prising a compound of formula (I), to a subject in need coccus epidermidis, Bacillus subtilis, Klebsiella pneumonia, thereof. The Odilomycins and/or compositions comprising Klebsiella Oxytoca, Acinetobacter baumannii, Enterobacter the same may be useful, for example, in treatment, Suppres cloacae, Escherichia coli, Moraxella catarrhalis, Pseudomo Sion, and/or prevention of bacterial infection and/or disease. nas aeruginos and Stenotrophomonas maltophilia. In some embodiments, the invention provides a method of In some embodiments, the disclosure provides for the use treating, preventing, and/or Suppressing bacterial infection of pharmaceutical compositions and/or medicaments com comprising administering to a Subject in need thereofathera prised of the compound of formula (I) in a method of treating peutically effective amount of a compound of formula (I) or a a bacterial infection, and/or disease state, and/or condition composition comprising a compound of formula (I). 25 caused by or related to such bacterial infection. In some embodiments, the present invention provides a In some embodiments, the methods comprise administer method for treating a subject Suffering from a bacterial infec ing to the Subject an effective amount of a compound of tion comprising administering to said subject a compound of formula (I) alone or in combination with a second antibiotic formula (I). compound, or a composition comprising a compound of for In some embodiments, the present invention provides a 30 mula (I) alone or in combination with a second antibiotic method for Suppressing a bacterial infection in a Subject com compound and a pharmaceutically acceptable carrier. The prising administering to the subject a compound of formula pharmaceutically acceptable carriers are well-known to those (I). skilled in the art, and include, for example, adjuvants, dilu In still Some embodiments, the present invention provides ents, excipients, fillers, lubricants and vehicles. Often, the a method for treating a Subject Suffering from multi-drug 35 pharmaceutically acceptable carrier is chemically inert resistant bacterial infection comprising administering to said toward the active compounds and is non-toxic under the con Subject an effective amount of a compound of formula (I). ditions of use. Examples of pharmaceutically acceptable car In some embodiments, methods of the present invention riers may include, for example, water or saline Solution, poly provide for inhibition of bacteria, or infection related thereto, mers such as polyethylene glycol, carbohydrates and that are resistant to other drugs or antibiotics. In some 40 derivatives thereof, oils, fatty acids, or alcohols. embodiments, the methods provide for treatment, Suppres In some embodiments, the method of treatment, prevention Sion, and/or prevention of infection from multi-drug resistant and/or Suppression of a condition related to bacterial infec bacteria. tion comprises the steps of: (i) identifying a subject in need of In some embodiments, the bacterial infection is multi-drug Such treatment; (ii) providing a compound of formula (I) resistant. In some embodiments, the bacterial Strain is hospi 45 alone or in combination with a second antibiotic compound, tal-acquired. In some embodiments, the bacterial Strain is ora composition comprising a compound of formula (I) alone nosocomial. or in combination with a second antibiotic compound and a In some embodiments, the bacterial infection comprises pharmaceutically acceptable carrier; and (iii) administering infection from Gram-negative bacteria. In some embodi said compound(s) or composition in a therapeutically effec ments, the bacterial infection comprises infection from 50 tive amount to treat, prevent and/or suppress bacterial infec Gram-positive bacteria. In some embodiments, the bacterial tion in a subject in need of Such treatment. infection comprises infection by more than one bacterial In some embodiments, the methods comprise administer strain. ing to the Subject an effective amount of a compound of In some embodiments, the bacterial or microbial infection formula (I); or a composition comprising a compound of is an infection caused in whole or in part by bacteria of the 55 formula (I) and a pharmaceutically acceptable carrier. Achronobacter; Actinobacillus, Actinomyces, Acinetobacter; In some embodiments, the method of treatment, prevention Aeromonas, Anaplasma, Bacillus, Bacteroides, Bartonella, and/or Suppression of a condition related to bacterial infec Bdellovibrio, Bifidobacterium, Bordetella, Borrelia, Bru tion comprises the steps of: (i) identifying a subject in need of cella, Burkholderia, Campylobacter; Capnocytophaga, Car Such treatment; (ii) providing a compound of formula (I) or a diobacterium, Chlamydia, Chlamydophila, Chromobacte 60 composition comprising a compound of formula (I) a phar rium, Citrobacter, Clostridium, Corynebacterium, Coxiella, maceutically acceptable carrier, and (iii) administering said Ehrlichia, Enterobacter, Enterococcus, Erysipelothrix, compound or composition in a therapeutically effective Escherichia, Francisella, Fusobacterium, Haemophilus, amount to treat, prevent and/or Suppress the disease state or Helicobacter; Hemobartonella, Klebsiella, Lactobacillus, condition related to bacterial infection in a subject in need of Legionella, Leptospira, Listeria, Mannheimia, Moraxella, 65 Such treatment. Morganella, Mycobacterium, Mycoplasma, Neisseria, In some embodiments, treatment refers generally to treat Neorickettsia, Nocardia, Pasteurella, Peptostreptococcus, ment and therapy, whether of a human or an animal (e.g., in US 9,260,481 B2 15 16 Veterinary applications), in which some desired therapeutic bacterial is kanamycin. In some embodiments, the additional effect is achieved, for example, the inhibition of the progress antibiotic is an aminoglycoside antibiotic. of the condition. Treatment may also include, but is not lim In some embodiments, the present invention provides a ited to, a reduction in the rate of progress, a halt in the rate of method for treating, Suppressing and/or preventing bacterial progress, amelioration of the condition, cure of the condition 5 infection by combined use of the compound of formula (I) a stabilized (i.e., not worsening) state of disease or affliction, and a second antibiotic compound in Subjects wherein the use preventing spread of disease or affliction, delay or slowing of of either the compound of formula (I) or the second antibiotic disease or affliction progression, amelioration or palliation of compound alone does not provide the desired therapeutic the disease or affliction state and remission (whether partial or effect. Surprisingly, it has been found that when other antibi total), whether detectable or undetectable. In some embodi 10 otic compounds are used together with a compound of for ments, treatment can also mean prolonging Survival as com pared to expected Survival if not receiving treatment. In some mula (I), statistically significant increases in antibiotic effects embodiments, treatment as a prophylactic measure (i.e., pro are observed. In some embodiments, there is a synergistic phylaxis) is also included. For example, use with Subjects effect between a second antibiotic compound. Such as kana who have not yet developed the condition, but who are at risk 15 mycin, and the compound of formula (I). Thus, in some of developing the condition, is encompassed by the term embodiments, kanamycin and the compound of formula (I) “treatment.’ are administered in amounts that exhibit synergistic lowering In some embodiments, treatment comprises combination of bacterial levels. In some embodiments, a kanamycin and treatments and therapies, in which two or more treatments or the compound of formula (I) are administered in amounts that therapies are combined, for example, sequentially or simul exhibit synergistic treatment, Suppression and/or prevention taneously. For example, the active agents of the present inven of bacterial infection. tion may also be used in further combination therapies, e.g., in Another aspect of the present invention is a pharmaceutical conjunction with other agents, for example, other antimicro composition comprising an effective amount of a compound bial or antibiotic agents, etc. of formula (I). In some embodiments, Xaa is lysine. The Subjects are in vitro and in vivo systems, including, for 25 In some embodiments, Xaa is 3-hydroxy-2,4-diaminobu example, isolated or cultured cells or tissues, non-cellular in tanoic acid. vitro assay systems and animals (e.g., an amphibian, a bird, a In some embodiments, Xaa- is 3-hydroxy-2,4-diaminobu fish, a mammal, a marsupial, a human, a domestic animal tanoic acid. Such as, for example, a cat, dog, monkey, mouse or rat; or a In some embodiments, Xaa is glycine. commercial animal such as, for example, a horse, bovine 30 (such as a cow), turkey, chicken or pig)). In some embodiments, Xaas is ornithine. In some embodiments, the subject is a mammal. In some In some embodiments, Xaa is proline. embodiments, the Subject is avian, Swine, a bovine or a In some embodiments, Xaa, is histidine. human. In some embodiments, the Subject is a human. In In some embodiments, Xaas is lysine or 5-hydroxylysine. Some embodiments, the Subject is avian. In some embodi 35 In some embodiments, Xaa, is 2.3-dehydroarginine. ments, the Subject is a Swine or pig. In some embodiments, the In some embodiments, Xaao is lysine or 5-hydroxylysine. Subject is a turkey or chicken. In some embodiments, in 4. In addition, the compositions or methods may further com In some embodiments, R is —NH. prise one or more additional antibacterial or antibiotic com In some embodiments, Xaa is lysine, Xaa, and Xaa are pounds in combination with a compound of formula (I) alone. 40 each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, Examples of such compounds include, but are not limited to, Xaas is ornithine, Xaa is proline, Xaa, is histidine, and Xaao daptomycin, oxacillin, piperacillin/taZobactam, ticaricillin/ is 2.3-dehydroarginine. clavulanic acid, amoxicillin/clavulanic acid, erythromycin, In some embodiments, Xaa is lysine, Xaa, and Xaas are cefepim, clindamycin, imipenem, gentamicin, ciprofloxacin, each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, aztreonam, Vancomycin, lineZolid, rifampicin, kanamycin, 45 Xaas is ornithine, Xaa is proline, Xaa, is histidine, Xaas is ampicillin, tetracycline, and the like. lysine or 5-hydroxylysine, Xaa, is 2,3-dehydroarginine, and In some embodiments, the additional (or second) antibiotic Xaao is lysine or 5-hydroxylysine. is an aminoglycoside antibiotic. Aminoglycoside antibiotics In some embodiments, Xaa is lysine, Xaa, and Xaas are are antibiotic compounds in which a portion of a molecule each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, contains an amino-modified Sugar. Examples of aminoglyco 50 Xaas is ornithine, Xaa is proline, Xaa, is histidine, Xaas is side antibiotics include, but are not limited to amikacin, apra lysine or 5-hydroxylysine, Xaa, is 2.3-dehydroarginine, mycin, arbekacin, astromicin, bekanamycin, capreomycin, Xaao is lysine or 5-hydroxylysine, n is 4 and R is NH2. dibekacin, dihydrostreptomycin, elsamitrucin, G418, gen In some embodiments, the present invention provides for tamicin, hygromycin B, isepamicin, kanamycin, kasugamy the use of pharmaceutical compositions and/or medicaments cin, micronomicin, neomycin, netilmicin, paromomycin Sul 55 comprised of a compound of formula (I), in a method of fate, ribostamycin, Sisomicin, Streptoduocin, Streptomycin, treating, Suppressing and/or preventing a disease state, and/or tobramycin and Verdamicin. condition caused by or related to bacterial infection. Thus, in Some embodiments the methods and/or composi In some embodiments, the method of treatment comprises tions further comprise one or more additional antibacterial the steps of: (i) identifying a subject in need of such treatment; compounds in combination with a compound of formula (I). 60 (ii) providing a compound of formula (I); and (iii) adminis In Some embodiments, the additional antibacterial compound tering said compound of formula (I) in a therapeutically effec is selected from the group consisting of daptomycin, oxacil tive amount to treat, Suppress and/or prevent the disease state lin, piperacillin/taZobactam, ticaricillin.clavulanic acid, or condition in a Subject in need of Such treatment. amoxicillin/clavulanic acid, erythromycin, cefepim, clinda In some embodiments, the method of treatment comprises mycin, imipenem, gentamicin, ciprofloxacin, aztreonam, 65 the steps of: (i) identifying a subject in need of such treatment; Vancomycin, lineZolid, rifampicin, kanamycin, ampicillin, (ii) providing a composition comprising a compound of for and tetracycline. In some embodiments, the additional anti mula (I); and (iii) administering said composition in a thera US 9,260,481 B2 17 18 peutically effective amount to treat, Suppress and/or prevent and/or composition is administered Subcutaneously. In some the disease state or condition in a subject in need of Such embodiments, the compound and/or composition is adminis treatment. tered intravenously. In some embodiments, the compound In some embodiments, the Odilomycins are formulated and/or composition is administered intramuscularly. In some into pharmaceutical compositions for administration to Sub embodiments, the compound and/or composition is adminis jects in a biologically compatible form Suitable for adminis tered topically. In some embodiments, the compound and/or tration in vivo. According to another aspect, the present composition is administered parenterally. invention provides a pharmaceutical composition comprising In Some embodiments, the composition is in unit dose form a compound of formula (I) in admixture with a pharmaceuti Such as a tablet, capsule or single-dose vial. Suitable unit cally acceptable diluent and/or carrier. The pharmaceutically 10 acceptable carrier is “acceptable' in the sense of being com doses, i.e., therapeutically effective amounts, may be deter patible with the other ingredients of the composition and not mined during clinical trials designed appropriately for each of deleterious to the recipient thereof. The pharmaceutically the conditions for which administration of a chosen com acceptable carriers employed herein may be selected from pound is indicated and will, of course, vary depending on the various organic or inorganic materials that are used as mate 15 desired clinical endpoint. rials for pharmaceutical formulations and which are incorpo In some embodiments, the pharmaceutical compositions rated as analgesic agents, buffers, binders, disintegrants, dilu for oral use comprise an Odilomycin together with the usual ents, emulsifiers, excipients, extenders, glidants, solubilizers, excipients as diluting agents such as mannitol, lactose and stabilizers, Suspending agents, tonicity agents, vehicles and Sorbitol; binding agents such as Starches, gelatins, Sugars, Viscosity-increasing agents. Pharmaceutical additives, such cellulose derivatives, natural gums and polyvinylpyrrolidone; as antioxidants, aromatics, colorants, flavor-improving lubricating agents such as talc, Stearates, hydrogenated veg agents, preservatives, and Sweeteners, may also be added. etable oils, polyethylenglycol and colloidal silicon dioxide; Examples of acceptable pharmaceutical carriers include car disintegrating agents such as starches, celluloses, alginates, boxymethyl cellulose, crystalline cellulose, glycerin, gum gums and reticulated polymers; and other coloring, flavoring arabic, lactose, magnesium Stearate, methyl cellulose, pow 25 and Sweetening agents. ders, Saline, Sodium alginate, Sucrose, starch, talc and water, In Some embodiments, the compositions comprise an among others. In some embodiments, the term “pharmaceu Odilomycin with carriers or excipients suitable for topical tically acceptable” means approved by a regulatory agency of administration. Any topical preparation may be used in the the Federal or a state government or listed in the U.S. Phar present invention, for instance ointments, pomades, creams, macopeia or other generally recognized pharmacopeia for use 30 gels and lotions. Exemplary compositions for topical admin in animals, and more particularly in humans. istration according to the invention include ointments, Surfactants such as, for example, detergents, are also suit pomades, creams, gels, and lotions. able for use in the formulations. The doses of an Odilomycin depend on the desired effect, When administered to a subject, the compound of the the duration of the treatment and the route of administration present invention and pharmaceutically acceptable carriers 35 used. can be sterile. Suitable pharmaceutical carriers may also In some embodiments, the pharmaceutical compositions include excipients such as starch, glucose, lactose. Sucrose, according to the present invention are for use as an antimi gelatin, malt, rice, flour, chalk, silica gel, Sodium Stearate, crobial agent, for use as antibiotics or for use in the treatment glycerol monostearate, talc, Sodium chloride, dried skim of microbial disease, in particular of microbial disease caused milk, glycerol, propylene, glycol, polyethylene glycol 300, 40 by bacteria. water, ethanol, polysorbate 20, and the like. The present com The pharmaceutical compositions of the present invention positions, if desired, may also contain minor amounts of are preferably for use in the treatment of bacterial infection wetting or emulsifying agents, or pH buffering agents. and in particular for use in the treatment of hospital-acquired The pharmaceutical compositions or formulations of the infections or nosocomial bacterial infections. present invention are prepared by methods well-known in the 45 The Odilomycins may be combined with other active com pharmaceutical arts. For example, the compounds of formula pounds exhibiting an antimicrobial/antibiotic activity. The (I) are brought into association with a carrier and/or diluent, pharmaceutical compositions encompassed by the present as a suspension or Solution. Optionally, one or more accessory invention may also comprise a further therapeutic agent for ingredients (e.g., buffers, flavoring agents, Surface active the treatment of bacterial disease or bacterial infection. agents, and the like) also are added. The choice of carrier is 50 In some embodiments, the methods comprise administra determined by the solubility and chemical nature of the com tion of a therapeutically effective dosage of an Odilomycin. pounds, chosen route of administration and Standard pharma The dosage administered can vary depending upon known ceutical practice. factors such as the pharmacodynamic characteristics of the Additionally, Odilomycins or compositions comprising active ingredient and its mode and route of administration; same are administered to a Subject by known procedures 55 time of administration of active ingredient, age, sex, health including, without limitation, oral administration, Sublingual and weight of the recipient; nature and extent of symptoms; or buccal administration, parenteral administration, topical kind of concurrent treatment, frequency of treatment and the administration, transdermal administration, via inhalation or effect desired; and rate of excretion. These are all readily intranasally, vaginally, rectally, and intramuscularly. The determined and may be used by the skilled artisan to adjust or compounds or compositions are administered parenterally, 60 titrate dosages and/or dosing regimens. The precise dose to be by epifascial, intracapsular, intracranial, intracutaneous, employed in the compositions will also depend on the route of intrathecal, intramuscular, intraorbital, intraperitoneal, administration, and should be decided according to the judg intraspinal, intrasternal, intravascular, intravenous, paren ment of the practitioner and each patient’s circumstances. chymatous, Subcutaneous or Sublingual injection, or by way Any of the compounds and/or compositions may be pro of catheter. 65 vided in a kit comprising the compounds and/or composi In some embodiments, the compound and/or composition tions. Thus, in Some embodiments, the compound and/or is administered orally. In some embodiments, the compound composition is provided in a kit. US 9,260,481 B2 19 20 Another aspect of the present invention is a method for It will further be recognized that any or all of the combi producing a compound of formula (I) comprising the follow nations, embodiments and aspects of the invention may be ing steps: combined in any fashion to provide other combinations, a) growing Xenorhabdus nematophila strain CNCM embodiments and aspects within the scope of the invention I-4530 in a liquid culture medium; and unless otherwise not possible. b) purifying a compound of formula (I). The invention is further described by the following non In some embodiments of the compound of formula (I), limiting Examples. Xaa is lysine. In some embodiments, Xaa, is 3-hydroxy-2, 4-diaminobutanoic acid. In some embodiments, Xaa is 3-hy EXAMPLES droxy-2,4-diaminobutanoic acid. In some embodiments, 10 Xaa is glycine. In some embodiments, Xaas is ornithine. In Example 1 Some embodiments, Xaa is proline. In some embodiments, Xaa, is histidine. In some embodiments, Xaas is lysine or Production and Fermentation 5-hydroxylysine. In some embodiments, Xaa, is 2.3-dehy 15 droarginine. In some embodiments, Xaao is lysine or 5-hy Producing Organism droxylysine. In some embodiments, n=4. In some embodi Xenorhabdus nematophila CNCM I-4530 (Diversité, ments, R is NH. genomes et interactions microorganisms-insectes’ collection) In some embodiments, Xaa is lysine, Xaa, and Xaas are was grown on Luria-Bertani medium (LB, composed of bac each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, totryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L) for Xaas is ornithine, Xaa is proline, Xaa, is histidine, Xaas is liquid culture and on LB-agar for Solid cultures. The phase lysine or 5-hydroxylysine, Xaag is 2,3-dehydroarginine, and status (I or II) of this strain was determined by culturing on Xaao is lysine or 5-hydroxylysine. NBTA (Nutrient agar (Difco) 31 g/L, bromothymol blue 25 In some embodiments, Xaa is lysine, Xaa, and Xaas are mg/L and 2,3,5-triphenyl tetrazolium chloride 1% 40 mg/L) each 3-hydroxy-2,4-diaminobutanoic acid, Xaa, is glycine, 25 and measuring antibacterial activity against Micrococcus Xaas is ornithine, Xaa is proline, Xaa, is histidine, Xaas is luteus. Xenorhabdus exhibit two colony forms or variants lysine or 5-hydroxylysine, Xaa, is 2.3-dehydroarginine, when cultured in vitro. Modifications of the outer membrane Xaao is lysine or 5-hydroxylysine, n is 4 and R is NH. induce differential adsorption of dyes by variants. Phase I In some embodiments, the compound of formula (I) is the variants absorb dyes and are blue on NBTA plates, while compound of formula (Ia). 30 phase II colonies are red. Phases I and II of strains are indi In some embodiments, the compound of formula (I) is the cated as Suffixes (71 and /2, respectively) attached to strain compound of formula (Ib). designations. This strain was maintained at 15° C. on NBTA In some embodiments, the compound of formula (I) is the medium. compound of formula (Ic). Fermentation The Odilomycins can be purified from the Xenorhabdus 35 Xenorhabdus nematophila CNCM I-4530 was cultivated nematophila cells of the present invention. Advantageously, for 72 h, at 28°C. with shaking in a 2 L Erlenmeyer flask the compounds may be purified from the culture Supernatant containing 500 mL of medium broth composed of bactopep after removal of the Xenorhabdus nematophila cells. For the tone 15 g/L, MgSO.7HO 2 g/L and glucose 2 g/L. The preparation of a culture Supernatant having antibacterial culture was inoculated with 0.1% (v/v) of a 24h pre-culture in activity Xenorhabdus nematophila strain CNCM I-4530 is 40 the same medium. The antibiotic production was monitored grown in a liquid culture medium under standard conditions, by analytical HPLC. the bacterial cells are removed and the Supernatant is recov ered. The bacterial cells may for example be removed by Example 2 centrifugation or filtration. Further purification of Odilomycins may be carried out by 45 Isolation any known method including cation-exchange chromatogra phy, reversed-phase chromatography and/or reverse phase Bacterial cells were removed by low-speed centrifugation HPLC. (6000xg, 10 min at 4°C.) and supernatant was sterilized onto In some embodiments, Odilomycins are purified from the 0.22 Lum pore size filter. Supernatant was added (1:1; V/v) to a culture Supernatant of Xenorhabdus nematophila strain 50 0.1 M NaCl-0.02 M Tris buffer (pH 7), and subjected to CNCMI-4530 by successive cation-exchange chromatogra cation-exchange chromatography on a Sep Pack CarboxyM phy, reversed-phase chromatography and reverse phase ethyl cartridge (Accell Plus CM, Waters). Unbound material HPLC. was removed by washes with a 0.1 MNaCl-0.02 MTris buffer Odilomycins can also be synthesized according to standard (pH 7) and the antibiotic actives eluted with 1 MNaCl-0.02M techniques in the art including, but not limited to, Solution 55 Tris buffer (pH 7). This eluate was acidified with 0.1% (v/v) phase organic synthesis and solid-phase organic synthesis. In trifluoroacetic acid (TFA) and was then subjected to reverse Some embodiments, Solid-phase organic synthesis comprises phase chromatography on a Sep Pack C18 cartridge (Sep-Pak synthesis via peptide synthesizer machinery. Such embodi Plus C18, Waters). Unbound material was removed by wash ments and execution thereof are well within the scope of the ing with HO-TFA 0.1% and the antibiotic pool was eluted ordinarily skilled artisan. Exemplary synthetic methods are 60 with acetonitrile. The eluate was freeze-dried then resus described in Bodanzky, et al. “The Practice of Peptide Syn pended in water (1:5; V/v). Pure compounds were isolated thesis.” Springer-Verlag (1994). from the crude extract by reverse phase HPLC using a C18 Those skilled in the art will recognize, or be able to ascer column (Waters; Symmetry Symmetry C18; 5 um; 4.6x150 tain using no more than routine experimentation, many mm), a linear gradient of HO/0.1%TFA-acetonitrile starting equivalents to the specific embodiments of the invention 65 from 0% to 30% in 30 min, a flow rate of 1 mL/minandan UV described herein. Such equivalents are intended to be within detection from 200 to 400 nm, yielding Odilomycins with the the scope of the present invention. following HPLC-retention times: Odilomycin A 14.16 min US 9,260,481 B2 21 22 (purity: 98% UV), Odilomycin B 14.44 min (purity: 95% NMR data were obtained in water and a set of experiments UV) and Odilomycin C 14.6 min (purity: 94% UV). including DQF-COSY, TOCSY, NOESY. H C HSQC and H C HSQC-TOCSY experiments were recorded Example 3 (FIGS. 1 and 2). The 1D spectrum revealed features of a peptidic compound with at least 6 amide signals spanning the Characterization and Physicochemical Properties 8.9-7.0 ppm chemical shift area, alpha proton signals in the 4.8-3.7 ppm area, and beta proton signals in the 3.7-1.1 ppm NMR and MS Analysis area. No methyl signal was observed in the high field area The purified compound was analyzed by Mass Spectros indicating the absence of Ala, Thr, Leu, Val and Ile residues. copy and NMR to determine its chemical structure. 10 In contrast, signals including the 9.60 ppm singlet and the The NMR study was carried out on a Bruker Avance spec 6.17 ppm triplet were observed suggesting the presence of trometer operating at 700 MHZ equipped with a cryoprobe. non-classical residues. In addition with homonuclear data, The sample (10 mM) was solubilized in water (95/5 HO/ the "H 'Cheteronuclear data were particularly helpful to DO V/v) and pH was set to 3.5 with hydrochloric acid. All 15 characterize the spin systems of the non-classical residues. data were recorded at 280°K. Protons chemical shifts are The combined analysis of all these data allowed identifi expressed with respect to sodium 4.4-dimethyl-silapentane cation of 11 spin systems including 4 types of non-classical 1-sulfonate, according to IUPAC recommendations. Double residues: an O.Y-diamino B-hydroxy butyric acid (Dab quantum filtered-correlated spectroscopy (DQF-COSY), (BOH)), an 8-hydroxylysine (Dhl), an O.B-dehydro arginine Z-filtered total-correlated spectroscopy (Z-TOCSY) and (Dha) and, an O.8-diaminobutane (Dbt). Neither the configu nuclear Overhauser effect spectroscopy (NOESY) spectra ration of the asymmetric carbons of these non-classical resi were acquired in the phase-sensitive mode, using the States dues, nor that of the classical residues was determined. The TPPI method (Marion D. Etal, J. Magn. Reson. 85,393-399 strong intensity of the OrnH-ProH NOE suggests that (1989)). Z-TOCSY spectra were obtained with a mixing time the Orn-Pro amide bond adopts the trans conformation. By of 80 ms and NOESY spectra with mixing times of 220 ms. 25 The H C HSQC and H C HSQC-TOCSY experi using sequential NOEs, the sequence of this pseudopeptide ments were carried out with the same sample. The water was identified as following: resonance set at the carrier frequency was Suppressed by the Lys-Dab(BOH)-Dab(BOH)-Gly-Orn-Pro-His7 WATERGATE method (Piotto M. Et al. J. Biomol. NMR2, Dhl-Dha-Dhl-Dbt 661-665 (1992)). All data were processed with the XWIN 30 NMR data are reported in Table 1 (Odilomycin A), Table 2 NMR software. The non-classical residues were identified (Odilomycin B), and Table 3 (Odilomycin C). from the analysis of the homo- and hetero-nuclear data. The sequential assignment was achieved using the general strat This sequence is in full agreement with the molecular egy described by Withrich (Wuthrich K. NMR of Proteins weight of 1297 Da measured by Mass Spectroscopy data and and Nucleic Acids, John Wiley & Sons, New York (1986)). 35 the non-classical residues confirmed by MS/MS fragmenta LC-MS was first performed in order to obtain the m/z value tion data. of the protonated molecules of all Odilomycin derivatives. MS-MS fragmentation was then carried out on Odilomycin TABLE 1 A, B and C. ESI-LC-MS data were obtained in the positive mode on a Waters alliance LC-MS system (Waters ZQ mass 40 Chemical shifts of Odilomycin A (water, 280K detector, Waters photodiode array detector 2696, Waters alli Spin system Group "H (ppm) C (ppm) ance HPLC systems 2790). The HPLC column used was a C18 column (Waters Symmetry C18 5 um 4.6x150 mm) Lys' HN maintained at 35° C. Solvents were (A) water+0.1%TFA, (B) CH 3.8 53.0 45 CH2 1.661.62 3O4 acetonitrile+0.1%TFA, and the flow rate was 1 mL/min. The CH2 1.2 21.1 mobile phase composition was 100% A at 0 min, ramped to CH2 1.45 26.3 30% B at 30 min. Samples were dissolved in solvent A (100 CH2 2.78 39.0 uL). Sample injection volume was 10 uL. UV-Visible detec Hydroxy Diamino HN 8.83 tion was by absorbance at 200-400 nm. Solvent flow to the butyric acid CH 4.28 56.3 MS was diverted to waste for the first 5 minto minimise salt 50 Dab(BOH)2 CH-OH 3.89 674 CH2 2.99.2.79 41.9 build-up. MS-MS fragmentation data were obtained on a NH, Waters Micromass Q-T of micro mass spectrometer. Hydroxy Diamino HN 8.63 Physico-Chemical Properties of Odilomycins butyric acid CH 4.33 56.3 Three compounds referred as Odilomycin A, B, and C were Dab(BOH) CH-OH 3.93 67.7 isolated, purified to homogeneity as a white powder and char 55 CH2 2.98.2.80 41.5 NH, acterized by mass spectrometry. ESI-MS experiments Gly HN 8.37 revealed the molecular weights of different Odilomycins. CH 3.8 42.1 Odilomycin A: White powder; UV: ) (MeOH)—214 nm: Ornithine HN 8.15 ESI-MS (m/z): 1297 M+H"; Orn CH 4.46 51.2 Odilomycin B: White powder; UV: (MeOH)—214 nm: 60 CPH2 1.581.42 27.5 CH2 1.491.4 23.2 ESI-MS (m/z): 1281 M+H"; CH2 2.74 39 Odilomycin C: White powder; UV: ) (MeOH)—214 nm: NH, ESI-MS (m/z): 1265 M+H". Pro CH 4.10 60.4 Chemical Structure Elucidation CH2 1.981.60 29.5 The chemical structure of Odilomycin A was established 65 CH2 1.72 24.3 from the combined analysis of NMR and mass spectrometry CH2 3.48, 3.38 47.7 data. US 9,260,481 B2 23 24 TABLE 1-continued TABLE 2-continued - Chenical shifts of Odilonycin A (water 280S- Chemical shifts of Odilomycin B (water, 280K) Spin system Group H (ppm) 3 C (ppm) 5 Spin system Group "H (ppm) C (ppm) His 7 HN 8.49 CH 4.46 S2.O Lys HN 841 CPH2 2.98-291 26.3 CH 4.12 53.8 s s A. CPH2 1.70 27.0 8 Hydroxy lysine HNel 2 841 10 CH2 1.361.26 3O.O Dh3 CH 4.12 53.8 CH2 1.53 26.4 CPH2 1.70 27.0 CH2 2.862.63 445 CH2 1.361.26 3O.O NH, CH-OH 3.62 67.0 dehydro arginine' HN 9.6 CH2 2.862.63 44.5 Dha C. NH, 15 CHCH2 6.17 132 dehydro arginine' HN 9.6 CH2 2.2O 26.5 Dha C. CHCH2 6.17 132 cin. NH y 39.5 CH2 2.2O 26.5 2 y - HN 3.10 39.5 C(NH2) = NH 7.0 2O 8 Hydroxy HN 8.OS lysine CH 4.06 54.0 8 Hydroxy HN 8.OS Dhillo CPH2 1.65 27.0 lysine CH 4.06 S4O CH2 1.27 3O.O Dhillo CH2 1.65 27.0 CH-OH 3.60 67.0 CH2 1.27 3O.O 25 CH 2.862.63 445 CH-OH 3.60 67.0 el 2 W. V. CH2 2.862.63 44.5 NH, NH, Diaminobutane' HN 8.OS Diaminobutane'' HN 8.OS Dbtil CH2 2.95 39.5 Dbtil CH2 2.95 39.5 CPH2 1.30 25.3 CBH2 1.30 25.3 CH, 140 24.O 30 CH2 140 24.0 CH2 2.78 39.0 CH2 2.78 39.0 NH, NH,

35 TABLE 2 TABLE 3 Chemical shifts of Odilonycin B (water, 280K Chemical shifts of Odilonycin C (water, 280K Spin system Group "H (ppm) C (ppm) Spin system Group "H (ppm) C (ppm) Lys' HN 40 Lys' HN CH 3.8 53.0 CH 3.8 53.0 CH2 1.661.62 30.4 CH2 1.661.62 30.4 CH2 1.2 21.1 CH2 1.2 21.1 CH2 1.45 26.3 CH2 1.45 26.3 CH2 2.78 39.0 CH2 2.78 39.0 Hydroxy Diamino HN 8.83 45 Hydroxy Diamino HN 8.83 butyric acid” CH 4.28 56.3 butyric acid” CH 4.28 56.3 Dab(BOH)? CH-OH 3.89 67.4 Dab(BOH)? CH-OH 3.89 67.4 CH2 2.99.2.79 41.9 CH2 2.99.2.79 41.9 NH, NH, Hydroxy Diamino HN 8.63 Hydroxy Diamino HN 8.63 butyric acid CH 4.33 56.3 50 butyric acid CH 4.33 56.3 Dab(BOH) CH-OH 3.93 67.7 Dab(BOH) CH-OH 3.93 67.7 CH2 2.98.2.80 41.5 CH2 2.98.2.80 41.5 NH, NH, Gly HN 8.37 Gly HN 8.37 CH 3.8 42.1 CH 3.8 42.1 Ornithine HN 8.15 55 Ornithine HN 8.15 Orn CH 4.46 51.2 Orn CH 4.46 51.2 CPH2 1.581.42 27.5 CPH2 1.581.42 27.5 CH2 1.491.4 23.2 CH2 1.491.4 23.2 CH2 2.74 39 CH2 2.74 39 NH, NH, Pro® CH 4.10 60.4 60 Pro® CH 4.10 60.4 CH2 1.981.60 29.5 CH2 1981.60 29.5 CH2 1.72 24.3 CH2 1.72 24.3 CH2 3.48, 3.38 47.7 CH2 3.48, 3.38 47.7 His 7 HN 8.49 His 7 HN 8.49 CH 4.46 S2.O CH 4.46 S2.0 CPH2 2.98-291 26.3 CPH2 2.98-291 26.3 Cs2H 7.03 118 65 Cs2H 7.03 118 CH 8.33 134 CH 8.33 134 US 9,260,481 B2 25 26 TABLE 3-continued TABLE 5-continued Chemical shifts of Odilomycin C (water, 280K Key fragmentations of the 1281M + H ions of Odilomycin B Spin system Group "H (ppm) C (ppm) Lys HN 841 363 CH 4.12 53.8 380 CPH2 1.70 27.0 CH2 1.361.26 3O.O 406 CH2 1.53 26.4 S14 CH2 2.862.63 445 10 NH, 517 dehydro arginine' HN 9.6 Dha C. 533 CHCH2 6.17 132 534 CH2 2.2O 26.5 551 HN 3.10 39.5 15 C(NH2) = NH 7.0 667 Lysio HN 8.OS 688 CH 4.06 S4O 749 CPH2 1.65 27.0 804 CH2 1.27 3O.O CH-OH 1.58 26.3 920 CH2 2.862.63 44.5 948 NH, Diaminobutane'' HN 8.OS 1036 Dbt CH2 2.95 39.5 1048 CPH2 1.30 25.3 25 1281 CH2 140 24.O CH2 2.78 39.0 NH,

30 TABLE 6 TABLE 4 Key fragmentations of the 1264M + H ions of Odilomycin C Key fragmentations of the 1297M+H" ions of Odilomycin A 172 172 233 233 35 235 235 245 -- 245 282 266 299 361 361 3S4 370 363 379 Pro/His'/Dhill" 40 380 396 406 4O6 498 436 His7/Dhi/Dhat 517 530 533 533 534 Dhat 45 550 551 567 688 683 733 704 804 765 904 82O 50 932 936 102O 964 1048 1052 126S 1064 1297 55

Example 4 TABLE 5

Key fragmentations of the 1281M +H" ions of Odilomycin B 60 In Vitro Studies 172 Gly'Orn" 233 235 Antibacterial Susceptibility Testing Methods 245 266 His" Lys' The minimal inhibitory concentrations (MIC) were deter 361 65 mined according to the Clinical and Laboratory Standards 370 Institute (CLSI) conditions guidelines detailed in Table 7. Assays were performed in triplicate. US 9,260,481 B2 27 28 TABLE 7 MIC determination parameters Relevant CLSI Growth Media and Strain(s) guidelines Incubation Conditions Acinetobacter battmannii M07-A8. Methods for Mueller-Hinton Broth (MH) Bacilius subtiis Dilution Antimicrobial 37o C. Enterobacter cloacae Susceptibility Tests for aerobic Enterococci is faecalis Bacteria That Grow 18-24h Enterococci is faecium Aerobically; Approved Escherichia coi Standard-Eighth Edition Klebsiella pneumonia Klebsiella Oxytoca Pseudomonas aeruginosa Staphylococcusatiretts Staphylococci is epidermidis Stenotrophomonas maliophia Streptococci is pneumonia MO7-A8 MH plus 5% Lysed Horse Streptococci is pyogenes Blood (MHB) 37o C. aerobic 18-24h Haemophilus influenzae MO7-A8 Haemophilus Test Medium (HTM) 37o C. Aerobic 18-24 h. Moraxeiia caiarrhais M45-A2. Methods for MH Antimicrobial Dilution and 37o C. Disk Susceptibility Testing of aerobic Infrequently Isolated or 24h. Fastidious Bacteria: Approved Guideline Second Edition Pasteureia mitocida M45-A2 MHB 37o C. aerobic 24h. Mannheimia haemolytica None available, followed MHB MO7-A8 37o C. aerobic 24h Proprionibacterium acnes M11-A7. Methods for Schaedler Broth plus 1 g/ml Bacteroides fragilis Antimicrobial Susceptibility vitamin K1 and 5% lysed Testing of Anaerobic horse blood (SB) Bacteria: Approved Standard- 37° C. Seventh Edition anaerobic 48 h.

Preparation of Inocula. Five to 10 well-isolated colonies Minimal bactericidal concentrations (MBCs) were estab were picked and resuspended in 3 ml sterile saline. The inocu 45 lished by extending the MIC procedure to the evaluation of lum was re-suspended by vigorous shaking on a Vortex mixer bactericidal activity. After 24 hours, 10 uL were drawn from for 15 S. Turbidity was adjusted to McFarland standard 0.5 the wells, serially diluted and then spotted onto suitable agar (1-5x106 CFU/ml). The inoculum was further diluted in the appropriate media (Table 7) to give a final inoculum in each plates. The plates were incubated at 37° C. overnight. The well of -2-8x105 CFU/ml. MBC was read 18 h later as the lowest concentration of Addition of Test Article. Stock solutions of Odilomycin A 50 antibiotic which resulted in 0.1% survival in the subculture. or comparator were diluted in the appropriate media to give a All the experiments were done in triplicate. maximum starting concentration of 50 g/mL in the assay. 50 MBCs were also determined in presence of 50% and 95% LL of medium was dispensed into each well in columns two to (v/v) of human serum to assess protein binding. 12 of a 96 well plate. 100 uL of the appropriate test compound Bactericidal Effects of Odilomycin on Growing S. aureus Solution (100 ug/mL) was dispensed into each well in column 55 and P aeruginosa one. In the case of M. catarrhalis, M. haemolytica and P Bacterial killing curves were carried out by inoculating S. multocida these values were doubled (a final assay volume of aureus ATCC 13709 and P. aeruginosa ATCC 27853 with 200 uL was used). Serial two-fold dilutions were performed Odilomycin A concentration equal to four-fold the MIC. Van from column one to column 10 to give a concentration range comycin or Polymixin were used at four-fold their MIC. S. of 50 to 0.1 ug/mL of each compound in the assay. Columns 60 aureus and P. aeruginosa inoculi were prepared from colo 11 and 12 served as positive (no drug or test article, inoculum nies grown overnight in MHB. Antibiotics concentrations in added), and negative (no drug, testarticle, or inoculum added) the flask were adjusted in MHB according to the desired growth controls respectively. concentration. Culture tubes containing 10 mL were inocu Addition of Bacterial Strains. 50 uL of each inoculum lated with S. aureus or Paeruginosa at an approximate inocu Suspension was added to the appropriate wells, resulting in a 65 lum of 10 CFU/mL. Samples were drawn and bacteria were final volume of 100LL consisting of 50LL diluted compound counted at 0,1,2,3,4, 6 and 24h of incubation at 37°C. Thus, or diluents, and 50 uL of inoculum or broth alone. after vortexing the culture tubes, two 50 uL samples were US 9,260,481 B2 29 30 removed and serially diluted with MHB. After each dilution activities of Odilomycin A are strong, with MICs inferior to 1 step, 20 uL was plated onto LB agar plates, which were ug/mL against S. aureus, S epidermidis, and B. subtilis incubated for 24 h at 37° C. Afterwards the colonies were strains, including against Some multiresistant clinical isolates counted and back-extrapolated to the original Volume to (Table 9). Weak or no antibacterial activity was observed determine the initial concentration (CFU/mL). against E. faecalis, E. faecium, S. pneumoniae, and S. pyo In Vitro Biological Properties genes. With respect to Gram-negative bacteria, the antibacte Odilomycin A was tested for antimicrobial activity against rial activities of Odilomycin A is strong against K. pneumo a wide range of bacteria involved in nosocomial and animal niae and K. Oxytoca, and is good, with MICs inferior to 10 infection. It owns a wide spectrum of antibacterial activity ug/mL against A. baumannii, E. cloacae, E. coli, M. Catarrha against Gram-positive and Gram-negative pathogens (Table 10 lis, P. aeruginosa, and S. maltophilia strains, including some 8). With respect to Gram-positive bacteria, the antibacterial multiresistant clinical isolates (Table 8, Table 9). TABLE 8 Antibacterial activity of Odilomycins A, B, C

MIC mL Odilomycin Odilomycin Odilomycin Bacteria Strain A. B C Control Gram (-) A. battmanni ATCC 6.25 <0.1 BAA747 B. fragilis ATCC 1.56 0.39° 2S238 E. cloacae 11370 1.56 0.78 E. coi ATCC 3.13 0.39 25922 E. coi CTX- AEC7 3.13 0.39 M14: E. coi CTX- MEC23 3.13 0.39

E. coi TEM MEC12 1.56 0.39 K. pneumoniae ATCC O.78 0.39 43816 K. pneumoniae 2475 1.56 0.78 KPC: K. Oxytoca NEB9 O.39 0.39 M. haemolytica ATCC SO.O <0.16 3.3396 M. caiarrhais ATCC 1.56 <0.1 2S238 P. mitocida ATCC 2S.O <0.16 12945 Paeruginosa ATCC 3.13 3.13 6.25 0.39 27853 S. maltophilia ATCC 6.25 <0.16 H. influenzae ATCC 12.5 <0.1 49766 Gram (+) B. subtiis DSM347 <0.2O O.20 E. faecalis ATCC >SO.O 0.78 29212 E. faecium ATCC 2S.O >50.0 700221 S. pneumoniae ATCC >SO.O 0.39 496.19 S. pyogenes ATCC SO.O 0.20 12384 S. airetts ATCC O.39 O.39 O.78 0.78 13709 S. airetts ATCC O.39 O.39 O.78 O.78 25923 S. airetts ATCC O.78 O.78 1.56 0.78 USA300 BAA 1556 S. epidermidis ATCC <0.2O O.20 12228 P. acnes ATCC 3.13 0.39 6919 * clinical isolate from University Hospital of Nimes; 'ciprofloxacin; metronidazole; polymyxin; vancomycin US 9,260,481 B2 31 32 TABLE 9 Antibacterial activity of Odilomycin A against multiresistant clinical isolates F aeruginosa S. airetts

Antibacterial S618 4O1681 3517O 42162 S12232 23305 16666 2O681. 21840 20364 Pip. Taz. Ticar./CA Erythromycin R S S R Cefepim Cefoxitin I R S R Clindamycin R S S R Imipenem Gentamicin R S S S Ciprofloxacin R R S R Aztreonam Vancomycin S S S S Linezolid S S S S Odilomycin 6.25 25 >50 3.12 0.7 8 <0.19 1.56 O.39 O.78 As Piperacillin Tazobactam, Ticarcillin/Clavulanic acid, R: resistant, S: sensitive, I: intermediate.

MBCs of Odilomycins A-C were assessed on S. aureus and 25 modified Eagle's medium supplemented with 25 mM glucose P. aeruginosa. These molecules revealed to be bactericidal and 10% (v/v) fetal calf serum were inoculated into 96-well (Table 9). plates. The inoculating cell number was 800 cells per well for TABLE 10 HT29 and 2100 cells per well for MRC-5. The microplates 30 were incubated at 37° C. for 24 h with 5% of CO2. Bactericidal activity of Odilomycins A, B, C Twenty four hours later, Odilomycin A, B or C dissolved in S. attres ATCC 13709 P. aeruginosa ATCC 27853 water were added for 72 hat a final concentration from 0.78 ug/mL to 100 ug/mL in a fixed volume of water (1% final MIC MBC MIC MBC Molecule (ig/mL) (Ig/mL) (Lig/mL) (Lig/mL) concentration). The number of viable cells measured at 490 35 nm with the MTS reagent (Promega, Madison, Wis.) and Odilomycin A O.39 O.78 3.12 6.25 LDso was calculated as the concentration of compound elic Odilomycin B O.39 O.78 3.12 3.12 iting a 50% inhibition of cell proliferation. Experiments were Odilomycin C O.78 1.56 6.25 12.5 done in triplicate. The LDs was superior to 100 g/mL against both cell lines 40 for each of Odilomycin A, B and C. 100% of cell viability was Time-to-kill experiments were realized to assess the bac tericidal effects of Odilomycin A on growing S. aureus and P observed at a dose of up to 100 ug/mL. aeruginosa. At four-fold the MIC, Odilomycin Akills 100% Example 5 of P aeruginosa and S. aureus bacteria within 2 h and 3 h respectively, making this molecule a fast and powerful bac tericidal agent (FIGS. 3 and 4). 45 Studies on Synergy with Other Antimicrobials MBC of Odilomycin A was assessed in presence of 50% In vitro interactions of Odilomycin A with rifampicin, (v/v) and 95% (v/v) human serum (Table 10). The results ciprofloxacin, kanamycin, gentamicin, amplicillin, tetracy show that the antibacterial activity of the molecule is weakly affected by the presence of serum proteins. cline and Vancomycin were investigated by microdilution 50 checkerboard technique using 96-well microtiter plates for each combination. TABLE 11 Vancomycin, rifampicin, kanamycin, gentamicin, amplicil Effects of serum on the bactericidal activity of OdilonycinA lin, ciprofloxacin and tetracycline (Sigma-Aldrich) were pro vided as standard powders by the manufacturers. Serum 9% (v/v) MBC S. attres ATCC 13709 55 Serial two-fold dilutions of the antimicrobial agents in O O.78 MHB were placed alone or in combination in wells and inocu 50 1.56 lated with an appropriate S. aureus ATCC 25923 inoculum or 95 1.56 K. pneumoniae ATCC 43816 so that each well contained approximately 10 CFU/mL. After incubation at 37° C. for 16-20h, the MIC was considered as the well containing the Cytotoxicity Test 60 lowest concentrations of the two drugs in which no visible The cytotoxicity of the Odilomycins A-C was investigated growth was observed. Concentrations of each antimicrobial against Human colon adenocarcinoma (HT29) cells and which were tested in combination were between /8xMIC and Human normal lung fibroblasts (MRC-5) cells. 200 uL of the 2XMIC. cell Suspension of HT29 (human colorectal adenocarcinoma) Synergy with Other Antimicrobials prepared in RPMI 1640+1%L-Glutamine supplemented with 65 The interactions of Odilomycin A with other antibiotics 10% (v/v) fetal calf serum and 200 uL of the cell suspension from different classes were investigated against S. aureus of MRC5 (human lung fibroblast) prepared in Dulbecco's (Table 11). A synergistic interaction was observed with the US 9,260,481 B2 33 34 aminoglycoside antibiotics kanamycin and gentamicin. This of acute lethal infection. Mice were fully immunocompetent. synergistic interaction was also observed against K. pneumo Infection was induced by inoculating intraperitoneally in mice a bacterial suspension of S. aureus Smith ATCC 13709. niae (Table 12). TABLE 12 The bacterial challenges (ca 5 logo CFU/mouse) were given suspended in 0.5 mL of 20% hog mucin. Treatments were Synergistic interaction of Odilomycin A with antibiotics against S. aureus 5 administered once 1 hour after challenge via IV injection MIC (Ig/mL) Synergy with Combinations (ig/mL) (LineZolid was administered immediately after challenge) Antibiotic antibiotic alone Odilomycin A Odilomycin/antibiotic (Table 13). Mortality was tracked out to 5 days post chal lenge. Animals that remained Surviving at the end of the study Odilomycin A O.39 NA were humanely euthanized. Rifampicin O.O12 10 Kanamycin O.62 -- 0.190.31 O.19.0.15 TABLE 1.4 O.09.O.31 O.09.0.15 Challenge and dose schedule Gentamicin O.078 -- O.19,0.039 Ampicillin O.15 S. airetts Con Tetracycline O.78 15 Challenge centra Vancomycin O.78 (CFU/ tion Dosing Ciprofloxacin O.31 Group l mouse) Treatment (mg/kg) Route schedule 1 6 5 logo None NAA NAA NA TABLE 13 2 6 5 logo Linezolid 12.5 PO Ohr 3 6 5 logo Odilomycin A S.O IV 1 hr Synergistic interaction of Odilomycin A with 4 6 5 logo Odilomycin A 2.5 IV 1 hr aminoglycosides against K. pneumoniae 5 6 5 logo Odilomycin A 1.O IV 1 hr MIC (Ig/mL) Synergy with Combinations (ig/mL) Antibiotic antibiotic alone Odilomycin A Odilomycin/antibiotic Female BALB/c mice ordered from Harlan Laboratories Odilomycin A 25 NA 25 (Indianapolis, Ind.) weighing 19-21 g were used in experi Kanamycin 1.56 -- 6.25 O.39 ments of acute lethal infection. Mice were fully immunocom Gentamicin O.39 -- 12.S.O.09 3.12.19 petent. Infection was induced by inoculating intraperito neally in mice a bacterial Suspension of P aeruginosa ATCC 30 27853. The bacterial challenges (8.6 logo CFU/mouse) were Example 6 given Suspended in 0.1 mL sterile saline. Treatments were administered once 1 hour after challenge via IV injection In Vivo Studies (Tobramycin was administered via IP injection immediately In Vivo Biological Activity of Odilomycin A after challenge) (Table 13). Mortality was tracked out to 2 Female ICR mice ordered from Harlan Laboratories (In 35 days post challenge. Animals that remained Surviving at the dianapolis, Ind.) weighing 19-21 g were used in experiments end of the study were humanely euthanized. TABLE 1.5 Challenge and dose schedule Paeruginosa Challenge Concentration Dosing Group l (CFU/mouse) Treatment (mg/kg) Route schedule 1 6 8.6 logo None NA NA NA 2 6 8.6 logo Tobramycin O.39 IP Ohr 3 6 8.6 logo Odilomycin A 15 IV 1 hr 4 6 8.6 logo Odilomycin A 7.5 IV 1 hr

so In Vivo Biological Properties Untreated animals died within 24–48 h after S. aureus infection. All mice treated with linezolid and Odilomycin A at doses of 5.0 mg/kg and 2.5 mg/kg Survived up to 5 days after infection (Table 14). One mouse died with Odilomycin Aata dose of 1.0 mg/kg 2 days after infection. TABLE 16

Survival of mice after S. aureus challenge and treatment

Dose Animal deaths

Group in Treatment (mg/kg) Day 0 Day 1 Day 2 Day 3 Day 4 Day 5

1 6 None NA O 6 O O O O 2 6 Linezolid 12.5 O O O O O O 3 6 Odilomycin A S.O O O O O O O US 9,260,481 B2 35 36 TABLE 16-continued Survival of nice after S. aureus challenge and treatment Dose Animal deaths Group in Treatment (mg/kg) Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 4 6 Odilomycin A 2.5 O O O O O O 5 6 Odilomycin A 1.O O O 1 O O O

Untreated animals died within 24–48 h after P aeruginosa TABLE 17-continued infection. All mice treated with Tobramycin survived up to 5 days after infection (Table 14). One mouse died at day 1 with Odilomycin A at a dose of 15 mg/kg, and 4 mice died at day Survival of mice after Paeruginosa challenge and treatment 1 with Odilomycin A at a dose of 7.5 mg/kg. 15 Dose Animal deaths TABLE 17

Survival of mice after P. aeruginosa challenge and treatment Group l Treatment (mg/kg) Day 0 Day 1 Day 2 2O Dose Animal deaths 3 6 Odilomycin A 15 Group in Treatment (mg/kg) Day O Day 1 Day 2 4 6 Odilomycin A 7.5 1 6 None NA O 6 O 2 6 Tobramycin O.39 O O O

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 3 <210s SEQ ID NO 1 &211s LENGTH: 10 212s. TYPE: PRT <213> ORGANISM: Xenorhabdus nematophilus 22 Os. FEATURE: <221s NAMEAKEY: SC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: 3-hydroxy-2, 4-diaminobutanoic acid 22 Os. FEATURE: <221s NAMEAKEY: SC FEATURE <222s. LOCATION: (3) . . (3) <223> OTHER INFORMATION: 3-hydroxy-2, 4-diaminobutanoic acid 22 Os. FEATURE: <221s NAMEAKEY: SC FEATURE <222s. LOCATION: (5) . . (5) 223 OTHER INFORMATION: Ornithine 22 Os. FEATURE: <221s NAMEAKEY: SC FEATURE <222s. LOCATION: (8) ... (8) <223> OTHER INFORMATION: 5-hydroxylysine 22 Os. FEATURE: <221s NAMEAKEY: SC FEATURE <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: 2, 3-dehydroarginine 22 Os. FEATURE: <221s NAMEAKEY: SC FEATURE <222s. LOCATION: (10) ... (10 <223> OTHER INFORMATION: 5-hydroxylysine

<4 OOs SEQUENCE: 1 Lys Xaa Xaa Gly Xaa Pro His Xaa Xaa Xaa 1.

SEO ID NO 2 LENGTH: 10 TYPE PRT ORGANISM: Xenorhabdus nematophilus FEATURE; NAME/KEY: MISC FEATURE LOCATION: (2) ... (2) OTHER INFORMATION: 3-hydroxy-2, 4-diaminobutanoic acid US 9,260,481 B2 37 38 - Continued

22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: 3-hydroxy-2,4-diaminobutanoic acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) 223s OTHER INFORMATION: Ornithine 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: 2, 3-dehydroarginine 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (10) ... (10) <223> OTHER INFORMATION: 5-hydroxylysine <4 OOs, SEQUENCE: 2 Lys Xaa Xala Gly Xaa Pro His Lys Xaa Xala 1.

<210s, SEQ ID NO 3 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Xenorhabdus nematophilus 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (2) ... (2) <223> OTHER INFORMATION: 3-hydroxy-2,4-diaminobutanoic acid 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (3) ... (3) <223> OTHER INFORMATION: 3-hydroxy-2,4-diaminobutanoic acid 22 Os. FEATURE: <221. NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) 223s OTHER INFORMATION: Ornithine 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (9) ... (9) 223 OTHER INFORMATION: 2,3-dehydroarginine

<4 OOs, SEQUENCE: 3 Lys Xaa Xala Gly Xaa Pro His Lys Xaa Lys 1.

The invention claimed is: n is 2, 3, 4, 5, 6, 7, 8, 9, or 10; 1. A pharmaceutical composition comprising a compound 45 R is -OH, -NH, or -COOH, and of formula (I): an aminoglycoside antibiotic. 2. The Pharmaceutical composition of claim 1, wherein Xaa-Xaa2-Xaa-Xaa1-Xaas-Xaa-Xaa17-Xaas-Xaag Xaa is lysine; Xaalo-NH-(CH2), R (I) Xaa, and Xaa, are each 3-hydroxy-2,4-diaminobutanoic wherein 50 acid; Xaa, Xaa, Xaas. Xaas, Xaa-7, Xaas and Xaalo are inde Xaa- is glycine; pendently selected from the group consisting of lysine, Xaas is ornithine; 3-hydroxylysine, 4-hydroxylysine, 5-hydroxylysine, Xaa is proline; 3,4-dihydroxylysine, 3,5-dihydroxylysine, 4,5-dihy Xaa, is histidine; and droxylysine, ornithine, 3-hydroxyornithine, 4-hydroxy 55 Xaa is 2,3-dehydroarginine. ornithine, 3,4-dihydroxyornithine, 2,4-diaminobutanoic 3. The pharmaceutical composition of claim 1, wherein acid, 3-hydroxy-2,4-diaminobutanoic acid, arginine, Xaa is lysine; histidine, serine, and threonine; Xaa, and Xaa, are each 3-hydroxy-2,4-diaminobutanoic Xaa is glycine, 3-aminopropanoic acid, or 4-aminobu acid; tanoic acid; 60 Xaa is proline, 3-hydroxyproline, 4-hydroxyproline, Xaa- is glycine; aziridine-2-carboxylic acid, azetidine-2-carboxylic Xaas is ornithine; acid, pipecolic acid, 4-oxaproline, 3-thiaproline, 4-thi Xaa is proline; aproline, 3,4-dehydroproline, 4-aminoproline, 4-fluoro Xaa, is histidine; proline, C.-methylproline, or C-allylproline; 65 Xaas is lysine or 5-hydroxylysine; Xaao is arginine, 2.3-dehydroarginine, citrulline, 2.3-dehy Xaao is 2,3-dehydroarginine; and drocitrulline, canavanine, or 2.3-dehydrocanavanine; Xaao is lysine or 5-hydroxylysine. US 9,260,481 B2 39 40 4. The pharmaceutical composition of claim 1, wherein Xaas is lysine or 5-hydroxylysine; Xaa is lysine; Xaa is 2,3-dehydroarginine; Xaa2. and Xaa- are each 3-hydroxy-2,4-diaminobutanoic Xaao is lysine or 5-hydroxylysine; XaaaC1d, is glycine; 5 n is 4;M. and Xaas is ornithine; R is NH. Xaa is proline; 5. The pharmaceutical composition of claim 1, wherein the Xaa, is histidine; compound is a compound of formula (Ia): (Ia) NH2

H O H NH O HN1\ N N S. HN SullO N O O NN H H O OH O O N N N N N 1N1N1 NH2. H H H NH2 O O

6. The pharmaceutical composition of claim 1, wherein the compound is a compound of formula (Ib): (Ib)

H O H NH O HN1\ N N S. HN ul N O O NN H H O OH O O N N N N N 1N1-1 NH2. H H H NH2 O O

HO NH2 NH2

7. The pharmaceutical composition of claim 1, wherein the compound is a compound of formula (Ic):

(Ic) NH2 NH2 HN NH NH2 Y NH O O HN 1\ N N S. HN N O O N H H H O OH O O O N N N N N 1n 1-NH2: H H H O O

NH2 NH2 US 9,260,481 B2 41 42 8. The pharmaceutical composition of claim 1 comprising a pharmaceutically acceptable carrier. 9. The pharmaceutical composition according to claim 1 wherein the aminoglycoside antibiotic compound is kanamy cin or gentamicin. 5