US 20090264.342A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0264342 A1 Cottarel et al. (43) Pub. Date: Oct. 22, 2009

(54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data ANTIBOTC POTENTATION AND DRUG (60) Provisional application No. 60/772,648, filed on Feb. DISCOVERY 13, 2006, provisional application No. 60/835,596, (75) Inventors: Guillaume Cottarel, Mountain filed on Aug. 4, 2006. View, CA (US); Jamey Publication Classification Wierzbowski, Stoneham, MA (US); Kolol Pal, Needham, MA (US); (51) Int. Cl. Michael Kohanski, Allston, MA A638/00 (2006.01) (US); Daniel Dwyer, Watertown, CI2O I/68 (2006.01) MA (US); James Collins, Newton, CI2N 5/02 (2006.01) MA (US); Michael Almstetter, A63L/70 (2006.01) Grasbrunn (DE); Michael A63L/435 (2006.01) Thormann, Martinsried (DE); A63L/352 (2006.01) Andreas Treml, Bodenmais (DE) (52) U.S. Cl...... 514/9:435/6: 435/375; 514/42: 514/277; 514/456 Correspondence Address: (57) ABSTRACT CHOATE, HALL & STEWART LLP TWO INTERNATIONAL PLACE The present invention provides methods for identifying target BOSTON, MA 02110 (US) genes whose partial or complete functional inactivation potentiates the activity of an antibiotic agent, e.g., a quinolone (73) Assignees: TRUSTEES OF BOSTON antibiotic. The invention further provides methods for iden UNIVERSITY, Boston, MA (US); tifying agents that modulate expression of target genes or that PURETECH VENTURES, modulate activity of expression products of target genes. Boston, MA (US) Agents identified according to various methods of the inven tion potentiate the activity of antibiotics such as quinolones, (21) Appl. No.: 12/279,237 aminoglycosides, peptide antibiotics and B-lactams. Also provided are agents that suppress and/or retard resistance to (22) PCT Filed: Feb. 13, 2007 antibiotics. The inventive methods provide potentiating agents and compositions comprising potentiating agents and (86). PCT No.: PCT/US07/03698 antibiotics. Such agents and compositions can be used for inhibiting growth or survival of a microbial cellor of treating S371 (c)(1), a subject suffering from or susceptible to a microbial infec (2), (4) Date: May 21, 2009 tion. Patent Application Publication Oct. 22, 2009 Sheet 1 of 50 US 2009/0264.342 A1

Figure Patent Application Publication Oct. 22, 2009 Sheet 2 of 50 US 2009/0264.342 A1

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O. : O alt O O S 4. 4. 13 O r is OH N12 7SNNN-2 3 8 4-oxo-4-dihydroquinoline 4-oxo-1,4-dihydronaphthyridine

Figure 9

4-Quirolone 2-Pyridone

O O O O S is OH 72°NN 3 OH 7 N12 BS1S-12 S 9 4-oxo-1,4-dihydroquinoline 4H-4-oxoquinoltzine O O S R. O f 5 s 7s 2 'on 2S1'sCroitN \, 18 4-oxo-1,4-dihydronaphthyridine 6H-6-oxo-pyrido1,2-apyiridise

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benzobnaphthyidones O O F 21 21 O

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y-df CC N y-/ O e-N R

Diberizoi,5naphthyridiones

ad C, OH HN R N OR F Rio 1-Hydroxy-7-amino-s-acidones

Pyrrolo[1,2-aquinclinesO , and isDindolo2,1-aquinolines (7 NH A-B O O. N -d R AN R AN Pyi Toiloquinolones and Pyrazoloquinolones O O F CN, N- OH R 2-4-(4-Fluorophenyl)piperzin-1-yl)-4,7-dihydro-7-to-1,2,4-triaepio 5-epyrimidine-6- carboxylic Acids "" O R NS OH r1N1 R p-Quinone ethicies

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Patent Application Publication Oct. 22, 2009 Sheet 24 of 50 US 2009/0264342 A1

Database Chemical Percent Structure D Name Inhibition ICso (ig/mL) CC lg/mL COC

FO2 BO9 Armentoflavone 109 OO5

Aos SORHAMNETN 1 OO C C CO OC on

IF Ot

G05 NOKFAVONE 1001

AO 88 OOO

FO2 B08 Maclurin 1 OO15

or CO P

FO2 sco, EO7 heafavin 501-5

F02 A06 KAEMPFEROL 7O 1 OO15 Figure 14 (page 1 of 2) Patent Application Publication Oct. 22, 2009 Sheet 25 of 50 US 2009/0264.342 A1

Structure Database Chemical Percent -Cio- D Name Inhibition ug/nl. IC50 (ug/mL)

CD 01 G08 Cuercetin dihydrate 67 2OO

AO8

3,7,4'- RHYDROXYFLAV A ONE 63. DO10

CO3 6, 200100

E03

ic? AO3 QUERCETAGETIN 1 OO16

FD1- APIGENIN-7-O- AO2 GLUCOSDE 48 OO-1 Figure 14 (page 2 of 2) Patent Application Publication Oct. 22, 2009 Sheet 26 of 50 US 2009/0264.342 A1

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Gene O Description

ace O114 ara 96

b3.

atpH 837356T

bO401 cydB b0734 cytochromed terminal oxidase polypeptide subunit degF

DNA biosynthesis; primosomal protein i

fepD b0590 ferric enterobactin (enterochein) transport

guab TTE250B IMP dehydrogenase T scS b2530 putative aminotransferase JWS360 lipoamide dehydrogenase (NADH); component of 2-oxodehydrogenase and pyruvate complexes; L-protein of glycine cleavage complex

nuoB 52287 NADH dehydrogenase chain B

osme b1283 osmotically inducible lipoprotein

priA 53935 primosomal protein NE factor Y(putative helicase) pris b420 prinosomal replication protein N recA DNA strand exchange and renaturation, DNA-dependent ATPase, DNA- and ATP dependent coprotease fad ADP---glycero-D-mannoheptose-6-epimerase rpm 53299 50S ribosoma subunit protein 36 T rpoo b3057 RNA polymerase, sigma(70) factor regulation of proteins induced at high ta

seda boss negative modulator of initiation of replication TT setA ...... sucB 50727 2-oxoglutarate dehydrogenase (dihydrolipoyltranssuccinase E2 component)

at . . . . T. O...... taurine ATP-binding component of a transport system tnut bill b2907 2-octaprenyl-6-methoxyphenol-2-octaprenyl-6-methoxy-4-benzoquinone T site-specific recombinase, acts on cer sequence of ColE1, effects chromosome segregation at cell division

yaa bOO45 putative transport protein yciNT b1105 Torf, hypothetical protein outative ATP-binding component of a transport system orf, hypothetical protein EP orf, hypothetical protein hel.

yhew yheN b3345 orf, hypothetical protein yidyice b4130b3646 Torf,putative hypothetical peptide transparterprotein O. yet b1176 off, hypothetical protein yid b4337 putative transport protein T. yy ba402 ori, hypothetical protein Figure 29 Patent Application Publication Oct. 22, 2009 Sheet 42 of 50 US 2009/0264.342 A1

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Gene BLATED

putative N-acetylgalactosamine-6-phosphate deacetylase membrane-bound ATP synthase, F1 sector, alpha-subunit membrane-bound ATP synthase, FO Sector, Subunit b membrane-bound ATP synthase, F1 sector, delta-subunit PTS system beta-glucosides, enzyme, Cryptic erine acetyltransferase cysl b2763 suite reductase, alpha subunit TP-binding component of ferric enterobactin transport erric enterobactin (enterochelin) transport putative frv operon regulatory protein quaA GMP synthetase (glutamine-hydrolyzing MP dehydrogenase hit >1 hoff 3. 3 2 Outative general protein Secretion protein S 34 Specificity determinant for hsdM and hsdr b 2 5 3 O Outative aminotransferase JW4016 5075 5227 JW5257 JW5360 etodeoxydiuconokinase ipoate synthesis, sulfur insertion? diaminopimelate decarboxylase

nei b0714 endonuclease VI and DNAN-glycosylase with an AP lyase activity Inmpc b0553 outer membrane porin protein; locus of qst prophage nudh

required for NMN transport Outrescine transport protein

Figure 32 (page 1 of 2) Patent Application Publication Oct. 22, 2009 Sheet 45 of 50 US 2009/0264342 A1

Gene BLATED Description

sbp b3917 periplasmic sulfate-binding protein

bO727 islateComponent dehydrogenase (hydroipolitansuccinate E2 sugE b4148 suppresses groSL may be chaperone less less outer membrane channel; specific tolerance to Colicin E1, segregation tdcG b3O35 of daughter chromosomes

- 2-octaprenyl-6-methoxy-1,4-benzoquinone --> 2-octaprenyl-3-methyl XA b3833 6-methoxy-1,4-benzoquinone 2-octaprenyl-6-methoxyphenol--> 2-octaprenyl-6-methoxy-1,4- b2907 benzoquinone ubi b2311 outer membrane channel; specific tolerance to colicin E1; segregation ubiX b3035 of daughter chromosomes putative transport ybby b1105 orf, hypothetical protein

ygaAb0513 putative transport ygiz b2898 ori, hypothetical protein putative transport system permease protein orf, hypothetical protein

orf, hypothetical protein yPyaeC -- yai b3047 ... putative membrane protein orf, hypothetical protein

Figure 32 (page 2 of 2) Patent Application Publication Oct. 22, 2009 Sheet 46 of 50 US 2009/0264.342 A1

colistin mefloquine synergy (MIC of mefloquine >200ug/ml)

e d 9 O Colistin MC 9. s

1 OO ug ?ml Mefloquine

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Patent Application Publication Oct. 22, 2009 Sheet 49 of 50 US 2009/0264.342 A1

Norfloxacin

mowt mhnhof (wt)+ dipyrilyl for wt)--thiourea mem. AeS. 2 3 o 10 Ampicillin EFEFF8 to: E ------al O LL U

Hemp wt) Amp (wt) + dipyridyl Ampwt)+thiourea m. Emp S.

10

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500 SOS induction 40 O

3 OO

2 O O

OO

g5.

-A-Nor (Areca) -O Amp (Areca)

Time (Hours) Post Treatment

Figure 37 US 2009/0264.342 A1 Oct. 22, 2009

COMPOSITIONS AND METHODS FOR prolongation of the QT interval, which may predispose to ANTIBOTC POTENTIATION AND DRUG serious arrhythmias, is also a concern. DISCOVERY 0006. The adverse effects of many potent quinolones has hindered efforts to develop them as therapeutic agents. Gemi RELATED APPLICATIONS floxacin, for example, has an enhanced affinity for its target relative to many other quinolones and displays potent activity 0001. The present application claims priority from U.S. against most Gram positive cocci, particularly against Strep Provisional Application No. 60/772,648 filed on Feb. 13, tomyces pneumoniae. Gemifloxacin is 30 fold more active 2006 and entitled “Compositions and Methods for Antibiotic than ciprofloxacin against this pathogen. Unfortunately, the Potentiation and Drug Discovery, and from U.S. Provisional side effects of the compound were significant enough that the Application No. 60/835,596 filed on Aug. 4, 2006 and entitled further development by the pharmaceutical company was "Rec A Inhibition”. Each of the provisional applications is terminated. incorporated herein by reference in its entirety. 0007. In addition, the rapid development of resistance to a number of quinolones is a cause for alarm and has led to a BACKGROUND OF THE INVENTION number of treatment failures. The spread of resistance has 0002 The emergence of resistance to antibacterial agents motivated calls for the development of policies that would is a growing problem for human and animal health, and new restrict unnecessary use of these agents. Resistance can drugs to treat infections due to microorganisms that display develop in a variety of ways including mutations in the genes resistance to currently used antibiotics are urgently needed. encoding either DNA gyrase or topoisomerase IV and over Efforts to overcome the growing problem of resistance have expression of efflux pumps. included modification of known antibiotics, classical screen 0008 Quinolones are only one example of antibiotic ing of new compound libraries and natural product libraries, agents that exhibit side effects and/or to which resistance has and genomic efforts to identify novel targets to which no cross been developed. Thus, there is clearly a need in the art both for resistance with existing antibiotics would be anticipated. new agents to combat microbial infection and for new Even with this significant antibiotic discovery effort, only a approaches to antibiotic drug discovery. There is also a need few agents that represent new chemical classes of antibiotic in the art for new approaches to improving the safety profile agents have been approved by regulatory agencies in recent of certain antibiotics. years. In addition, few antibiotics that are effective against bacterial that have developed resistance to currently used SUMMARY OF THE INVENTION antibiotics are in clinical development. Furthermore, a num ber of potent antibiotic agents are too toxic for clinical use or 0009. The present invention provides compositions and have significant side effects that limit their therapeutic utility. methods for potentiating the activity of antibiotic agents and 0003 Quinolones are one of the most widely used classes for discovering new agents of use in treating bacterial infec of antimicrobial agents worldwide and serve to exemplify the tion. promise and the problems associated with antibiotic develop 0010. In one aspect, the invention provides a method of ment and use. Their therapeutic indications in humans have inhibiting growth or Survival of a microbial cell comprising: evolved from urinary tract infections to infections of almost (a) contacting the microbial cell with a antibiotic (e.g., an all body compartments. Various members of the class are also quinolone); and (b) contacting the cell with an agent that widely administered for veterinary purposes. potentiates activity of the antibiotic. 0004 Quinolones exert their antimicrobial effect by tar 0011. In another aspect the invention provides a method of geting bacterial type II topoisomerases, namely gyrase and treating a subject in need thereof comprising: administering topoisomerase IV, essential enzymes that catalyze breakage an antibiotic to the Subject in combination with an agent that and rejoining of DNA strands during normal cell growth. potentiates activity of the antibiotic. In certain embodiments Quinolones form a ternary complex between DNA and either of either of the foregoing methods, the agent preferably inhib gyrase or topoisomerase IV, thereby blocking DNA replica its or interferes with activity of an expression product of an tion and leading to events such as double-stranded DNA antibiotic potentiator target gene. In either of the methods the breaks that are rapidly lethal to the cell. As a class, quinolones agent and the antibiotic may be administered individually or have a broad spectrum of activity against Gram positive and as components of a single composition. The antibiotic may be Gram negative species, including both aerobic and anaerobic used at a concentration below its MIC, or may be adminis microorganisms. Activity against Gram negative microorgan tered at a dose below that at which it would be effective as a isms often depends strongly upon activity against DNA single agent. gyrase, while topoisomerase IV is often the primary target of 0012. In another aspect, the invention provides a method quinolones that display activity against Gram positive spe of identifying an antibiotic potentiator target gene comprising cies. A number of therapeutically useful quinolones display steps of: (a) contacting a microbial cell with an antibiotic, activity towards both of these targets. wherein the microbial cell has a genetic alteration that sig 0005 Quinolones, like most other antibiotics, can have nificantly reduces or eliminates expression of a gene; (b) adverse effects with the potential to affect virtually every comparing growth or survival of the microbial cell with major system in the body. The most frequent side effects growth or Survival of a microbial cell having higher expres associated with fluoroquinolone use involve the GI tract, skin, sion of the gene than the cell having the genetic alteration; and and/or central nervous system (CNS). Fluoroquinolones can (c) determining that the gene is an antibiotic potentiator target also cause hypersensitivity reactions. A mild transient gene if the growth, survival, or both of the microbial cell increase in liver function is seen in 2-3% of the patients who having the genetic alteration is lower than the growth, Sur are treated with a fluoroquinolone, and severe liver toxicity is vival, or both of the microbial cell having higher expression of a rare side effect of quinolone use. Cardiac side effects such as the gene. US 2009/0264.342 A1 Oct. 22, 2009

0013. In another aspect the invention provides a set of product of a quinolone potentiator target gene. In certain target genes whose functional inactivation potentiates the embodiments of the invention the potentiating compound is activity of antibiotics. The set comprises dapF, fabH, fis, fliN. active by itself as an antibiotic. In other embodiments of the jw 5303, priA, recA, recB, recC, recC, resA, ruVA, ruvB, invention the potentiating compound is not active by itself as ruvC, ruvC, uVrD, yCS, ydfL, yhf TydgC, aceE, ara.J. asp.A, an antibiotic (at doses that can be tolerated inaccordance with atpA, atpF, atph, brnO, cydB. degP. dnaT, fepD, folP. guaB, Sound medical practice). The agent modulates expression of Hfq, iscS, JW53600, lip A, lpdA, nuoB, nuol, nuoL, osmB. an antibiotic potentiator target gene or activity of an expres pdxH, pnuC, priA, priB, rbfA, recA, rfal), rpm.J. rpoD, res.A. sion product of the target gene. The agent may, for example, ruvC, SdhC, seqA, setA, sucB, SurA, tar, tatE, tauB, trimu, (i) inhibit or interfere with expression of a microbial antibi ubiH, ubiX, XerC., yaal J, yefM, yeO, ygfz, ygiH, yheL, otic potentiator target gene; or (ii) inhibit or interfere with yheM, yheN, yicG, yidLyjeT, yiO, yY, agaAk, atp A, atpF. activity of an expression product of a microbial antibiotic atpH, bglF, cysE, cysI, fepC, fepD. frvR, guaA, guaB, hoff, potentiator target gene. In certain embodiments of the inven hsdS, iscS, JW4016, JW5075, JW5227, JW 5257, JW5360, tion the antibiotic potentiator target gene is a quinolone kdgK, lip A, IySA, malG, mbh A, mdo.G, Nei, nmpC, nudH, potentiator target gene. pdxH, phn B. phn L. phnO, pnuC, potE, pshM, pts.A, rhaT, 0020. The invention provides compositions comprising rpiA, resA, Sbp, speA, sucB, SugE, tdcE, tdcG, tolC, trx A, the inventive agents, e.g., compositions comprising a phar ubiE, ubiH, ubiX, Xni, ybby, yefM, yole J, yeeY, yfeTyga A, maceutically acceptable carrier, excipient, vehicle, etc. In ygf/, yhdX,yheLyheM, yiaY, yidK, yihV.ybn, yjcR, yjcz. certain embodiments of the invention the composition is a ynjD, ydeC., ydiH, and yrfA. Sustained release formulation, e.g., for oral administration. 0014. In another aspect the invention provides an assay 0021. The invention further provides compositions com system comprising: (a) a test agent; and (b) components Suit prising one or more antibiotic potentiating agents and an able for performing an assay that detects expression of an antibiotic whose activity is potentiated by the compound antibiotic potentiator target gene or detects activity of an and/or whose resistance it Suppresses or retards. expression product of an antibiotic potentiator target gene, 0022. The invention further comprises a method of treat wherein the gene is from the set described above. ing a subject in need thereof comprising the step of adminis 0015 The invention further provides methods of identify tering any of the inventive agents or compositions of the ing an agent that modulates expression and/or activity of an invention to the subject. Preferably an antibiotic potentiating antibiotic potentiator target gene or its expression product. agent is administered to a Subject who also receives an anti 0016 One such method comprises (a) providing an assay biotic whose activity the compound potentiates. The antibi system comprising a test compound, wherein the assay sys otic and the potentiating agent may be delivered together in a tem is Suitable for performing an assay to detect expression of single composition or separately. They may be delivered by an antibiotic potentiator target gene or to detect activity of an the same route of administration or different routes. expression product of an antibiotic potentiator target gene; 0023. Unless otherwise indicated the present invention and (b) determining whether the test compound inhibits utilizes well known methods of molecular biology, bacteriol expression of the antibiotic potentiator target gene or inhibits ogy, cell culture, etc., as described in, for example, Current or interferes with activity of an expression product of the Protocols in Molecular Biology, and Current Protocols in gene; and (c) identifying the test compound as a potentiator of Cell Biology, all John Wiley & Sons, N.Y., edition as of July the antibiotic if the test agent inhibits expression of the poten 2002; Sambrook, Russell, and Sambrook, Molecular Clon tiator target gene or inhibits or interferes with activity of an ing: A Laboratory Manual, 3" ed., Cold Spring Harbor Labo expression product of the potentiator target gene. ratory Press, Cold Spring Harbor, 2001; Woodford, N. and 0017. A variety of assays suitable for identifying agents Johnson, Alan, Molecular Bacteriology. Protocols and Clini that inhibit polypeptides encoded by specific target genes, cal Applications (Humana, 1998); and Gerhardt et al., Meth e.g., RecA, are provided. Assays provided include binding ods for General and Molecular Microbiology (American assays, reporter-based assays, and enzymatic assays. Also Society for Microbiology, 1994); each of which is incorpo provided are collections of bacterial strains suitable for per rated herein by reference. forming the target gene identification methods of the inven tion and/or for identifying agents that modulate expression of BRIEF DESCRIPTION OF THE DRAWING a target gene or modulate activity of an expression product of a target gene. 0024 FIG. 1 is a schematic diagrams showing functional 0018. In another aspect, the invention provides a method modules of the E. coli RecA protein. Amino acids numbers of identifying an agent comprising steps of: (a) computation bracketing modules associated with particular functional ally analyzing interaction of each of a plurality of test agents activities are shown. These modules are highly conserved with a protein comprising a polypeptide encoded by an anti among bacteria. The figure is taken from Karlin and Brocch biotic potentiator target gene with each of a plurality of test ieri, 1996, and is not drawn to scale. agents; and (b) identifying at least one test agent having 0025 FIG. 2 shows results of a survival assay comparing favorable interactions with the active site. The agent may be the viability of wild type bacteria or various deletion strains further tested, e.g., to determine whether it potentiates activ following exposure to a lethal concentration of norfloxacin ity of an antibiotic against bacteria. (right panel). Untreated control cells are shown in the left 0019. In another aspect the invention provides a composi panel. tion comprising an agent (potentiating agent) that inhibits 0026 FIG. 3 is a graph comparing survival of four E. coli activity of an expression product of an antibiotic potentiator strains following exposure to a Sublethal concentration of target gene. In another aspect the invention provides a com norfloxacin. position comprising an inhibitor of a microbial type II topoi 0027 FIG. 4 presents graphs showing the effect of dele Somerase and an agent that inhibits activity of an expression tion of either recA or tolC on bacterial growth in the presence US 2009/0264.342 A1 Oct. 22, 2009

of norfloxacin (left panel) and survival following a period of 0046 FIG. 23 summarizes the results of hinokiflavone exposure to norfloxacin (right panel). docking study predictions. 0028 FIG. 5 is a graph showing that deletion of recA 0047 FIG. 24 highlights particular RecA residues dramatically decreases the ability of bacteria to survive expo involved in the hinoki flavone binding site. Sure to norfloxacin. 0048 FIG. 25 illustrates the positioning of the hinokifla 0029 FIG. 6 shows results of a colony formation assay vone binding site in the context of a RecA filament. comparing the ability of strains that carry a recA allele in a 0049 FIG. 26 represents a cross-section of a RecA fila quinolone resistant (grlA542) and nonresistant strain back ment, with the hinokiflavone binding site indicated. ground to Survive exposure to a lethal concentration of nor 0050 FIG. 27 illustrates attractive and repulsive interac floxacin. tions of hinokiflavone with its binding site. 0030 FIG. 7 is a bar graph comparing fluorescence inten 0051 FIG. 28 illustrates the cross-species conservation of sity detected from wild type bacteria containing a recA:GFP the hinokiflavone binding site. reporter construct (left bars in each group) with fluorescence 0.052 FIG. 29 lists the names, accession numbers and intensity detected from either recA" bacteria (middle bars in descriptions of certain aminoglycoside potentiator target each group) or recA bacteria containing the recA:GFP genes. reporter construct (right bars in each group) in the presence of 0053 FIG. 30 shows results of a survival assay comparing norfloxacin. The figure shows that norfloxacin causes the viability of S. aureus and recA defective S. aureus follow increased transcription of the reporter gene, reflecting ing a 4 hour-exposure of different concentrations of gentami increased RecA activity, validating the use of this system to C1. detect inhibitors of RecA. 0054 FIG. 31 presents two graphs showing that Meflo 0031 FIG. 8 shows is a bar graph comparing luciferase quine (an ATPase synthase inhibitor) potentiates Gentamicin luminescence as described in Example 7. (an aminoglycoside antibiotic). 0032 FIG. 9 shows the core structures of classical qui 0055 FIG. 32 lists the names, accession numbers and nolone antibiotics (4-quinolone and 4-naphthyridine sys descriptions of certain antibiotic cyclic peptide potentiator tems). target genes. 0033 FIG. 10 shows the structural formulae and number 0056 FIG. 33 presents a graph showing that Mefloquine ing systems of the 4-quinolone and 4-naphthyridine systems (an ATPase synthase inhibitor) potentiates colistin (an anti with the corresponding 2-quinolone and 2-maphthyridine sys biotic cyclic peptide). tems and also shows a benzobnaphthyridone structure. 0057 FIG. 34 is a scheme showing overlapping target 0034 FIG. 11A shows core structures of benzobnaph genes for quinolones (citoflaxin), aminoglycosides (gentami thyridones and some examples of this compound class. FIG. cin) and antibiotic cyclic peptides (colistin). 11B shows pyrroloquinolones, pyrazoloquinolones, and 0.058 FIG. 35 is a set of 4 graphs illustrating hydroxyl Some core structures structurally related to quinolones. radical production in E. coli by antibiotics. a.c, Log change in 0035 FIG. 12 is Table 1, which presents the names and colony forming units per mL (CFU/mL, mean+/-standard accession numbers of certain quinolone potentiator target deviation); filled squares represent a no-drug control. b.d. genes. generation of hydroxyl radicals. Representative measure 0036 FIG. 13 shows the amino acid sequence of E. coli ments are shown and were taken 3 hours following addition of RecA protein and homologs in two other bacterial species. drug. Diamonds represent time-Zero baseline measurements. 0037 FIG. 14 presents chemical structures and ICso data a, Survival and b, hydroxyl radicals generation following for 14 compounds that showed at least 50% inhibition of exposure to bactericidal antibiotics. c. Survival and d. RecA ATPase activity in the luciferase assay described in hydroxyl radical generation following exposure to bacterio Example 23. static drugs (see Example 39). 0038 FIG. 15 presents chemical structures of potential 0059 FIG.36 is a set of 3 graphs showing the effect of iron RecA inhibitors that are structurally related to hinokiflavone. chelation, hydroxyl radical quenching of iron-sulfur synthe sis on the filling efficiency of bactericidal antibiotics. a.c.e, 0039 FIG. 16 presents chemical structures of potential Log change in colony forming units per mL (CFU/mL, RecA inhibitors that are electronically related to hinoki fla mean+/-standard deviation). Effect of addition of 500 uM WO. 2,2'-dipyridyl or 150 mMthiourea to wildtype E. coli, and on 0040 FIG. 17 is a graph showing inhibition of RecA an iron-sulfur cluster synthesis mutant, AiscS, on bactericidal ATPase activity by hinokiflavone. drug efficacy and hydroxyl radical formation following expo 0041 FIG. 18 is a photograph of a survival assay plate Sure to a, 250 ng/mL norfloxacin (Nor), c. 5ug/mL amplicillin assessing the ability of various RecA inhibitors to potentiate (Amp), or e. 5ug/mL. kanamycin (Kan). ciprofloxacin. 0060 FIG. 37 is a set of two graphs showing SOS induc 0042 FIG. 19 is a photograph of a survival assay plate tion in E. coli by bactericidal antibiotics. Activation of the assessing the ability of hinoki flavone to kill cells, and to SOS response was monitored using an engineered sensor potentiate ciprofloxacin. construct that employs the Lex A repressor for control of 0043 FIG. 20 is a graph comparing the ability of hinoki Green Fluorescent Protein (GFP) expression. Shown are rep flavone to potentiate ciprofloxacin’s activity in a survival resentative GFP histogram measurements taken 3 hours after assay against a RecA+ (S3) and recA- (S4) strain. addition of a, bactericidal antibiotics (5ug/mL Amp, 5ug/mL 0044 FIG. 21 illustrates the method by which the docking Kan, 25 ng/mL Nor). b. Log change in CFU/mL of Areca E. site(s) of hinoki flavone on RecA were predicted, and also coli following exposure to 5 ug/mL Kanamycin (open dia illustrates two potential sites. monds), 25 ng/mL Norfloxacin (open triangles), and 5ug/mL 0045 FIG.22 is a close-up view illustrating the predicted Ampicillin (open circles). For comparison, CFU/mL are also potential hinokiflavone docking sites. shown for widltype E. coli exposed to 5 Lig/mL Kanamycin US 2009/0264.342 A1 Oct. 22, 2009

(diamonds), 25 ng/mL Norfloxacin (triangles), and 5 g/mL tion” are mutually exclusive. Deletion is one among a number Ampicillin (circles), as well as a lower concentration of nor of different means by which a gene can be functionally inac floxacin in these experiments to highlight the significant tivated. increase in killing in a recA knockout. 0.066 Approximately. The terms “approximately” or “about in reference to a number generally include numbers DEFINITIONS that fall within a range of 5% in either direction (greater than 0061 Active agent. An “active agent” refers to an agent or less than) of the number unless otherwise stated or other that either possesses antimicrobial activity, i.e., inhibits wise evident from the context (except where such number growth and/or Survival of a microorganism, or that potentiates would exceed 100% of a possible value). an agent that possesses antimicrobial activity. 0062 Aliphatic: The term “aliphatic' or “aliphatic 0067 Biochemical or biological pathway. The terms “bio group', as used herein, denotes a hydrocarbon moiety that chemical pathway' or “biological pathway” refer to a con may be straight-chain (i.e., unbranched), branched, or cyclic nected series of biochemical reactions and/or molecular inter (including fused, bridging, and Spiro-fused polycyclic) and actions normally occurring in a cell, or more broadly, a may be completely saturated or may contain one or more units cellular event or process such as cell division or DNA repli of unsaturation, but which is not aromatic. Unless otherwise cation. Typically, the steps of a biochemical or biological specified, aliphatic groups contain 1-20 carbon atoms. In pathway occur in a coordinated fashion to produce a specific Some embodiments, aliphatic groups contain 1-10 carbon product or products or to produce a particular biochemical atoms. In other embodiments, aliphatic groups contain 1-8 action, effect, or event. A biochemical or biological pathway carbon atoms. In still other embodiments, aliphatic groups requires the expression product of a gene (e.g., the gene is contain 1-6 carbon atoms, and in yet other embodiments essential for that pathway) if the absence of that expression aliphatic groups contain 1-4 carbon atoms. Suitable aliphatic product either directly or indirectly prevents the completion groups include, but are not limited to, linear or branched, of one or more steps in that pathway, thereby preventing or alkyl, alkenyl, and alkynyl groups, and hybrids thereof Such significantly reducing the production of one or more normal as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alk products, actions, events, or effects of that pathway. Thus, an enyl. agent specifically inhibits a biological or biochemical path 0063 Agent. Agent” refers to any compound or chemical way requiring the expression product of a particular gene, if entity and includes, without limitation, Small molecules, the presence of the agent stops or Substantially reduces the polypeptides, nucleic acids, and carbohydrates. completion of the series of steps in that pathway. The agent, 0064 Antibiotic agent. Antibiotic”, “antibiotic agent’, or may, but does not necessarily, act directly on the expression 'antimicrobial agent’ as used herein, refers to any agent that product of that particular gene. A biochemical or biological inhibits and/or stops growth and/or proliferation of one or pathway may involve but not require the expression products more species of microorganism (e.g., bacteria or fungus). The of one or more genes, in which case the absence of that antibiotic may display inhibitory activity in vitro (e.g., when expression product may either directly or indirectly reduce contacted with cells in cell culture), in vivo (e.g., when the extent to which one or more steps in that pathway occurs administered to a Subject at risk of or Suffering from an and may thereby reduce the production of one or more normal infection), or both. The terms include bactericidal and bacte products, actions, events, or effects of that pathway. While the riostatic agents. "Bactericidal' means that the agent kills present invention does not specifically differentiate between bacteria. A bactericidal agent may inhibit or stop growth or use of the terms “biological pathway” and “biochemical path proliferation of the bacteria before killing them. “Bacterio way', it may be more appropriate to utilize the former where static' means that the agent Substantially inhibits or stops the focus is on broader cellular events or processes and to growth or proliferation of bacteria but does not kill them. utilize the latter where the focus is on specific cellular mol 0065 Antibiotic potentiator target gene. As used herein, ecules and the chemical reactions that they undergo. the term “antibiotic potentiator target gene' refers to a gene 006.8 Biological or assay system. “Biological system” or characterized in that the growth and/or viability (i.e., ability 'assay system’ refers to any system containing at least one to survive) of microbial cells having a deletion or functional biological component, e.g., a biological macromolecule Such inactivation of the gene is/are substantially reduced by expo as a protein or nucleic acid, Suitable for performing an assay sure to an antibiotic relative to the growth and/or viability of of a biological or biochemical function or activity. The term otherwise genetically identical microbial cells not having a includes cell-free systems, cells, collections of cells, animals, deletion or functional inactivation of the gene under the same etc. conditions. The term includes any gene identified according 0069 Conventional dose. A “conventional dose” of an to the gene identification methods of the invention. The expo antibiotic agent means a dose that is (i) in the case of humans sure to the antibiotic can be either transientor continuous. The or animals, recommended on the package insert; (ii) in the antibiotic concentration may be a Sublethal concentration case of humans, recommended in Goodman and Gilman, (e.g., a non-inhibitory concentration) or a lethal concentra Supra; Katzung, Supra, and/or The Merck Manual of Diagno tion. The antibiotic may be any antibiotic known in the art or sis and Therapy, 17" ed. (1999), or the 18' ed (2006) follow discovered in the future. It will be appreciated that the growth ing its publication, Mark H. Beers and Robert Berkow (eds.), and/or viability will typically be measured with respect to a Merck Publishing Group; (iii) in the case of animals, recom population of microbial cells and refers, for example, to the mended in The Merck Veterinary Manual, 9" ed., Kahn, C.A. overall growth of the population or the percentage of cells that (ed.), Merck Publishing Group, 2005. It will be appreciated remain viable. Deletion or functional inactivation of the gene that a conventional dose may be modified appropriately for an may potentiate one or more antibiotics. It is noted that the individual Subject taking into account, for example, factors phrase "deletion or functional inactivation of a gene is not Such as the Subject's age, diet, renal and/or hepatic function, intended to imply that “deletion' and “functional inactiva other medications, other diseases or conditions (i.e., diseases US 2009/0264.342 A1 Oct. 22, 2009

or conditions other than the infection for which an antibiotic 0076 Gene product or expression product. A "gene prod agent is administered), past experience with the antibiotic uct” or “expression product of a gene is an RNA transcribed agent, etc. from the gene (e.g., either pre- or post-processing) or a 0070 Docking. “Docking is used herein consistently polypeptide encoded by an RNA transcribed from the gene with its use in the art to refer to the application of any of a (e.g., either pre- or post-modification). variety of computational methods to predict or identify a 0077 Growth and Proliferation. “Growth refers to an preferred structure of a complex formed by a receptor and increase in microbial biomass. "Proliferation” refers to an ligand. Docking typically involves computational explora increase in microbial number. Since bacterial proliferation, tion of the possible binding modes of a ligand to a receptor rather than mere increase in cell mass without cell division, is and identification of low energy binding modes. The receptor usually of primary concern, and since under most circum can be, e.g., a macromolecule Such as a polypeptide, protein, stances of interest herein proliferation is accompanied by an or nucleic acid. The ligand can be, e.g., a Small molecule. increase in microbial biomass, the term 'growth' is generally (0071. Effective amount. An “effective amount of an understood to mean “proliferation', and the two terms are agent refers to the amount of the agent Sufficient to elicit a used interchangeably herein although it is recognized that desired biological response. As will be appreciated by those different assays may measure either or both of these param of ordinary skill in this art, the absolute amount of a particular eters. For example, optical density reflects biomass and does agent that is effective may vary depending on Such factors as not specifically reflect cell number, whereas an assay based the desired biological endpoint, the agent to be delivered, the on detecting colonies formed from individual cells reflects target tissue, etc. Those of ordinary skill in the art will further cell number rather than biomass. understand that an “effective amount may be administered in 0078 Heteroatom: The term "heteroatom' means one or a single dose, or may be achieved by administration of mul more of oxygen, Sulfur, nitrogen, phosphorus, or silicon. This tiple doses. For example, an effective amount may be an includes any oxidized form of nitrogen, Sulfur, phosphorus, or amount sufficient to achieve one or more of the following: (i) silicon; the quaternized form of any basic nitrogen, or, a inhibit microbial growth in culture or in vivo; (ii) reduce the Substitutable nitrogen of a heterocyclic ring including=N- severity of or prevent one or more symptoms or signs of an as in 3,4-dihydro-2H-pyrrolyl. —NH as in pyrrolidinyl, or infection; (iii) significantly reduce the risk of recurrence of an =N(R)—as in N-substituted pyrrolidinyl. infection; (iv) significantly reduce the risk of a clinically 0079 High throughput screening. “High throughput significant infection in a subject who has been exposed to an screening as used herein refers to an assay that allows for infectious agent, etc. multiple candidate agents or samples to be screened substan 0072 Comparably, an effective amount of a potentiating tially simultaneously. Such assays typically entail the use of agent may be an amount Sufficient to achieve the same level of microtiter (microwell) plates (e.g., plates having 96, 384, or antibiotic activity with a particular antibiotic agent as is 1596 wells) which are particularly convenient because a large achieved when that antibiotic agent is administered at its number of assays can be carried out simultaneously, using conventional dose, in circumstances where the antibiotic Small amounts of reagents and samples. Such assays may also agent is administered at a reduced dose as compared with its advantageously minimize the number of steps such as wash conventional dose. ing cells, removing culture medium, and/or pipetting 0073 For example, an effective amount of a RecA inhibi reagents. tor according to the present invention may be, for example, (i) 0080 Hypersensitivity. “Hypersensitive”, “hypersensitiv an amount Sufficient to act as an antibiotic agent; (ii) an ity”, “hypersusceptible”, “hyper-susceptibility” and like amount sufficient to inhibit one or more activities of RecA (or terms refer to a cell that exhibits increased sensitivity to a relevant homolog); (iii) an amount Sufficient to potentiate inhibition of growth and/or survival by an antibiotic as a activity of one or more antibiotic agents (e.g., with which the consequence of a gene being underexpressed or as a conse RecA inhibitor is administered in combination); and/or (iv) quence of a gene product being inhibited or interfered with by an amount sufficient to reduce or retard the incidence of an agent (i.e., a potentiating agent), relative to the sensitivity resistance developed to another antibiotic agent (e.g., with that the cell would exhibit to that antibiotic if the gene was not which the RecA is administered in combination). underexpressed or the gene product not inhibited or interfered 0074 Essential gene. An "essential gene is one whose with. Typically the minimum inhibitory concentration (MIC) deletion or functional inactivation renders a microorganism of that antibiotic will have been reduced in a cell that is lacking the gene incapable of growth or proliferation and/or hypersensitive to the antibiotic. causes the cell to die. It will be appreciated that whether or not 0081. In combination. “In combination” as used herein a particular gene is essential depends at least in part on the with respect to administration of first and second agents is environmental conditions to which the cell is subjected. administration performed Such that (i) a dose of the second 0075 Gene. The term “gene' refers to a nucleic acid com agent is administered before more than 90% of the most prising a nucleotide sequence that encodes a polypeptide or a recently administered dose of the first agent has been metabo biologically active ribonucleic acid (RNA) such as a tRNA. lized to an inactive form or excreted from the body; or (ii) The nucleic acid can further include regulatory elements doses of the first and second agents are administered within (e.g., expression control sequences such as promoters, 48 hours of each other, or (iii) the agents are administered enhancers, etc.) and/or introns. A gene that encodes a during overlapping time periods (e.g., by continuous or inter polypeptide contains one or more coding portions (open read mittent infusion); or (iv) any combination of the foregoing. ing frames), by which is meant a series of nucleotide triplets The agents may, but need not, be administered together as coding foramino acids uninterrupted by termination codons, components of a single composition. The agents may be wherein the sequence can be translated into a polypeptide administered individually at substantially the same time (by using the codon usage information appropriate for a particu which is meant within less than 10 minutes of one another). lar cell or multicellular organism. The agents may be administered individually within a short US 2009/0264.342 A1 Oct. 22, 2009

time of one another (by which is meant less than 1 hour apart). 0084. Microbe. “Microbe’, “microbial, and like terms, as The agents may, but need not, be administered by the same used herein, refers to microscopic organisms, e.g., bacteria or route of administration. When administered in combination fungi, particular bacteria. For example, when used in refer with a second agent, the effective concentration of a first ence to quinolone antibiotics, “microbe' and like terms typi agent needed to elicit a particular biological response may be cally refer to bacteria, although it can encompass any micro less than the effective concentration of the first agent when organism against which quinolone antibiotics display administered in the absence of the second agent, thereby inhibitory activity. allowing a reduction in the dose of the first agent relative to 0085 Microbial infection. The term “microbial infection the dose that would be needed if the first agent was adminis refers to the invasion of the host organism, whether the organ tered in the absence of the second agent. The effects of mul ism is a vertebrate, invertebrate, fish, plant, bird, or mammal, tiple agents may, but need not be, additive or synergistic. The by pathogenic microbes, e.g., bacteria, fingi, and protists. This includes the excessive growth of microbes that are nor agents may be administered multiple times. In some embodi mally present in or on the body of a mammal or other organ ments of the invention, a lower amount of first agent (e.g., ism. More generally, a microbial infection can be any situa antibiotic agent) is required in the presence of the second tion in which the presence of a microbial population(s) is agent (e.g., inventive RecA inhibitor). The effects of multiple damaging to a host organism. Thus, an organism is “suffer agents may, but need not be, additive or synergistic. One or ing from a microbial infection when excessive numbers of a more of the agents may be administered multiple times. microbial population are present in or on the organism's body, I0082 Identity. “Identity” refers to the extent to which the or when the effects of the presence of a microbial population sequence of two or more nucleic acids or polypeptides is the (s) is damaging the cells or other tissue of an organism. The same. The percent identity between a sequence of interestand agents and compositions of certain embodiments of the a second sequence over a window of evaluation, e.g., over the invention are also useful in treating microbial growth or con length of the sequence of interest, may be computed by align tamination of cell cultures or other media, or inanimate Sur ing the sequences, determining the number of residues faces or objects, and nothing herein should limit the invention (nucleotides or amino acids) within the window of evaluation to treatment of higher organisms, except when explicitly so that are opposite an identical residue allowing the introduc specified in the claims. tion of gaps to maximize identity, dividing by the total num I0086 Minimal inhibitory concentration (MIC): The terms ber of residues of the sequence of interest or the second “minimal inhibitory concentration’ (MIC) and “minimal bac sequence (whichever is greater) that fall within the window, tericidal concentration” (MBC) are used herein consistently and multiplying by 100. When computing the number of with their use in the art, i.e., to indicate the concentration of an identical residues needed to achieve a particular percent iden agent that will inhibit bacterial proliferation (growth) (MIC) tity, fractions are to be rounded to the nearest whole number. or kill bacteria (MBC). MIC values may be for example, the Percent identity can be calculated with the use of a variety of concentration of agent that inhibits visible growth or may be computer programs known in the art. For example, computer expressed as MICso, MICoo or MIC values i.e., the concen programs such as BLAST2, BLASTN, BLASTP. Gapped tration of an agent that reduces bacterial proliferation to 50% BLAST, etc., generate alignments and provide percent iden or less, 10% or less, or 1% or less, respectively, of the control tity between a sequence of interest and sequences in any of a value that would occur in the absence of the agent. As is well variety of public databases. The algorithm of Karlin and known in the art, MIC and MBC can be measured by a variety Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, of methods, including automated and non-automated meth 1990, 87: 22264-2268) modified as in Karlin and Altschul, ods. Suitable methods are described in publications of the Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877 is incorpo Clinical Laboratory Standards Institute (CLSI), formerly the rated into the NBLAST and XBLAST programs of Altschul National Committee for Clinical Laboratory Standards (NC et al. (Altschulet al., J. Mol. Biol., 1990, 215: 403-410). To CLS), as set forth in NCCLS: Performance Standards docu obtain gapped alignments for comparison purposes, Gapped ments referenced below. BLAST is utilized as described in Altschul et al. (Altschu et I0087 Modulation. To “modulate” means to change or al., Nucleic Acids Res., 1997.25:3389-3402). When utilizing alter, e.g., to cause an increase or decrease. Thus an agent is BLAST and Gapped BLAST programs, the default param said modulate an amount or activity if it causes the amount or eters of the respective programs are used. A PAM250 or activity to change or alter, e.g., to increase or decrease. An BLOSUM62 matrix may be used. See the web page having agent that inhibits or activates expression of a gene or activity URL www.ncbi.nlm.nih.gov at the National Center for Bio of a gene product is said to modulate expression of the gene or technology Information website for these programs. In a spe modulate activity of the gene product, respectively. cific embodiment, percent identity of a sequence of interest I0088 Naturally-occurring. As used herein, a “naturally and a second sequence is calculated using BLAST2 with occurring nucleic acid molecule refers to an RNA or DNA default parameters. molecule having a nucleotide sequence that occurs in nature 0083) Isolated. The term "isolated means 1) separated (e.g., encodes a natural polypeptide). A “naturally occurring from at least Some of the macromolecular components with polypeptide is a polypeptide having an amino acid sequence which it is usually associated in nature; 2) prepared or purified that occurs in nature. by a process that involves the hand of man; and/or 3) not I0089 Operably linked or associated. “Operably linked” or occurring in nature. For example, an "isolated bacterial cell “operably associated refers to a functional relationship is a cell that is not located in or on a multicellular organism, between two nucleic acids, wherein the expression, activity, e.g., the cell is maintained in culture. An "isolated polypep localization, etc., of one of the sequences is controlled by, tide' is one that is not present in a cell or at the surface of a cell directed by, regulated by, modulated by, etc., the other nucleic in which the polypeptide was synthesized. The isolated acid. The two nucleic acids are said to be operably linked or polypeptide may be present in a cell-free solution. operably associated. “Operably linked' or “operably associ US 2009/0264.342 A1 Oct. 22, 2009

ated can also refers to a relationship between two polypep detail in J. Pharmaceutical Sciences, 66:1, 1977, incorpo tides wherein the expression of one of the polypeptides is rated herein by reference. Pharmaceutically acceptable salts controlled by, directed by, regulated by, modulated by, etc., of the compounds of this invention include those derived from the other polypeptide. The two polypeptides are said to be Suitable inorganic and organic acids and bases. operably linked or operably associated. For example, tran 0094. Examples of pharmaceutically acceptable, nontoxic scription of a nucleic acid is directed by an operably linked acid addition salts are salts of an amino group formed with promoter, post-transcriptional processing of a nucleic acid is inorganic acids such as hydrochloric acid, hydrobromic acid, directed by an operably linked processing sequence; transla phosphoric acid, Sulfuric acid and perchloric acid or with tion of a nucleic acid is directed by an operably linked trans organic acids such as acetic acid, oxalic acid, maleic acid, lational regulatory sequence Such as a translation initiation tartaric acid, citric acid, Succinic acid or malonic acid or by sequence; transport, stability, or localization of a nucleic acid using other methods used in the art Such as ion exchange. or polypeptide is directed by an operably linked transport or Other pharmaceutically acceptable salts include adipate, algi localization sequence Such as a secretion signal sequence; nate, ascorbate, aspartate, benzenesulfonate, benzoate, bisul and post-translational processing of a polypeptide is directed fate, borate, butyrate, camphorate, camphorsulfonate, citrate, by an operably linked processing sequence. Preferably a first cyclopentanepropionate, digluconate, dodecylsulfate, nucleic acid sequence that is operably linked to a second ethanesulfonate, formate, fumarate, glucoheptonate, glycero nucleic acid sequence, or a first polypeptide that is operatively phosphate, gluconate, hemisulfate, heptanoate, hexanoate, linked to a second polypeptide, is covalently linked, either hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lac directly or indirectly, to Such a sequence, although any effec tate, laurate, lauryl Sulfate, malate, maleate, malonate, meth tive three-dimensional association is acceptable. One of ordi anesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, nary skill in the art will appreciate that multiple nucleic acids, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, or multiple polypeptides, may be operably linked or associ 3-phenylpropionate, phosphate, picrate, pivalate, propionate, ated with one another. Stearate. Succinate, Sulfate, tartrate, thiocyanate, p-toluene 0090. Overexpression. “Overexpressed” or “overexpres Sulfonate, undecanoate, Valerate salts, and the like. sion” means expression of a gene at levels above the wildtype 0.095 Examples of pharmaceutically acceptable salts levels observed for the gene in organisms of that type under derived from appropriate bases include alkali metal, alkaline the same conditions, i.e., expression of the gene is increased earth metal, ammonium and N(C. alkyl) salts. relative to wild type levels. For example, the value of the 0096 Representative pharmaceutically acceptable alkali steady state or average rate of synthesis or absolute amount of or alkaline earth metal salts include sodium, lithium, potas an expression product of the gene (e.g., a polypeptide) may be sium, calcium, magnesium, and the like. at greater than the wild type value by at least a factor of 1.5, at 0097. Further pharmaceutically acceptable salts include, least a factor of 2, at least a factor of 5, at least a factor of 10, when appropriate, nontoxic ammonium, quaternary ammo etc. nium, and amine cations, for example formed using counte 0091 Pharmaceutically acceptable derivative: According rions such as halide, hydroxide, carboxylate, Sulfate, phos to the present invention, a pharmaceutically acceptable phate, nitrate, lower alkyl Sulfonate and aryl Sulfonate. derivative of a particular chemical compound includes, but is 0.098 Physiologically acceptable carrier or excipient: As not limited to, pharmaceutically acceptable salts, esters, salts used herein, the term "physiologically acceptable carrier or of such esters, or any other adduct or derivative which upon excipient” refers to a carrier medium or excipient which does administration to a patient in need is capable of providing, not interfere with the effectiveness of the biological activity directly or indirectly, a compound as otherwise described of the active ingredients and which is not excessively toxic to herein, or a metabolite or residue thereof. Thus, pharmaceu the host at the concentrations at which it is administered. The tically acceptable derivatives can include Salts, prodrugs, and/ term includes solvents, dispersion media, coatings, antibac or metabolites of relevant compounds. The phrase “pharma terial and antifungal agents, isotonic agents, absorption ceutically acceptable derivative' may also encompass delaying agents, and the like. The use of Such media and quaternization of any basic nitrogen-containing groups of the agents for the formulation of pharmaceutically active Sub compounds disclosed herein. Water or oil-soluble or dispers stances is well-known in the art (see, for example, "Reming able products may be obtained by Such quaternization. ton's Pharmaceutical Sciences”, E. W. Martin, 18" Ed., 0092 Pharmaceutically acceptable salt: As used herein, 1990, Mack Publishing Co.: Easton, Pa., which is incorpo the term “pharmaceutically acceptable salt” refers to those rated herein by reference in its entirety). salts which are, within the scope of sound medical judgment, (0099 Polynucleotide. The terms “polynucleotide' and Suitable for use in contact with the tissues of humans and “nucleic acid are used interchangeably herein to refer to a lower animals without undue toxicity, irritation, allergic polymer of nucleotides, e.g., DNA or RNA. The polymer may response and the like, and which are commensurate with a comprise or natural nucleosides found in DNA or RNA, other reasonable benefit/risk ratio. A “pharmaceutically acceptable nucleosides or nucleoside analogs, nucleosides containing salt' means any non-toxic salt or salt of an ester of a com chemically modified bases and/or biologically modified pound of this invention that, upon administration to a recipi bases (e.g., methylated bases), modified Sugars, modified ent, is capable of providing, either directly or indirectly, a backbone linkages, etc. The nucleic acid molecule can be compound of this invention oran inhibitorally active metabo single-stranded or double-stranded or can contain single lite or residue thereof. As used herein, the term “inhibitorally Stranded regions and double-stranded regions. The term active metabolite or residue thereof means that a metabolite "polynucleotide sequence' or “nucleic acid sequence’ as or residue thereof acts as a RecA inhibitor. used herein can refer to the nucleic acid material itself and/or 0093. A wide variety of appropriate pharmaceutically to the sequence information (i.e. the Succession of letters acceptable salts are well known in the art. For example, S. M. chosen among the five base letters A, G, C, T, or U) that Berge et al., describe pharmaceutically acceptable salts in biochemically characterizes a specific nucleic acid, e.g., a US 2009/0264.342 A1 Oct. 22, 2009

DNA or RNA molecule. A nucleic acid sequence is presented as a nucleic acid or polypeptide is considered pure when it is in the 5' to 3' direction herein unless otherwise indicated. removed from substantially all other compounds or entities 0100 Polypeptide. “Polypeptide', as used herein, refers to other than a solvent and any ions contained in the solvent, i.e., a chain of amino acids connected by peptide bonds. A protein it constitutes at least about 90%, more preferably at least is a molecule composed of one or more polypeptides. Each about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or polypeptide in a protein may be referred to as a “subunit of greater than 99% of the dry weight of the composition. A the protein. A peptide is a relatively short polypeptide, typi partially or Substantially purified compound or entity Such as cally between about 2 and 60 amino acids in length. The terms a nucleic acid or polypeptide may be removed from at least “protein', 'polypeptide', and "peptide' may be used inter 50%, at least 60%, at least 70%, or at least 80% of the material changeably herein unless indicated otherwise. Polypeptides with which it is naturally found, e.g., cellular material Such as as used herein typically contain amino acids such as those that cellular proteins and/or nucleic acids. In certain embodiments are naturally found in proteins. However, amino acids that are of the invention a purified nucleic acid, polypeptide, or pro not naturally found in proteins (e.g., amino acids that do not tein constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, occur in nature or that do occur in nature but are not naturally 70%, 80%, 90%, 95%, 99% or even more, by dry weight, of found in proteins and that can be incorporated into a polypep the total nucleic acid, polypeptide, or protein, respectively, in tide chain), and/or amino acid analogs can also or alterna a composition. Methods for purifying nucleic acids, polypep tively be used in polypeptides that are employed in the screen tides, and proteins and for assessing purity are known in the ing assays of the invention. One or more of the amino acids in art and include spectophotometric methods, chromatro a polypeptide may be modified, for example, by the addition graphic methods, immunological methods, electrophoretic of one or more chemical entities such as a carbohydrate methods, etc. group, a phosphate group, a lipid group, etc. Specific 0104 Sensitive/susceptible and resistant. A microorgan examples include a farnesyl group, an isofarnesyl group, a ism is “sensitive' or “susceptible' to an agent if the agent fatty acid group, a glycosyl group, an acetyl group, etc. inhibits proliferation of the microorganism and/or kills the Polypeptides may contain a linker for conjugation, function microorganism when contacted with the agent at a particular alization, or other modification, etc. The term “polypeptide concentration. Sensitivity may be assessed using any of a sequence' or "amino acid sequence' as used herein can refer variety of methods known in the art. A microorganism that is to the polypeptide material itself and is not restricted to the not “sensitive' is considered “resistant”, i.e., the microorgan sequence information (i.e. the Succession of letters or three ism can Survive and proliferate in the presence of the agent. letter codes chosen among the letters and codes used as abbre Methods for assessing sensitivity typically involve determin viations for amino acid names) that biochemically character ing the MIC by methods such as the broth microdilution izes a polypeptide. A polypeptide sequence presented herein method, agar dilution, and the agar disk diffusion method. is presented in an N-terminal to C-terminal direction unless The MIC may then be compared with a predefined “break otherwise indicated. point', wherein a MIC greater than the breakpoint indicates 0101 Potentiate: The term “potentiate', as used herein, that the microorganism is resistant to the agent and a MIC means to enhance or increase at least one biological effect or equal to or below the breakpoint indicates that the microor activity of a biologically and/or pharmacologically active ganism is sensitive to the agent. Sensitivity and/or resistance agent so that either (i) a given concentration or amount of the may be assessed according to the guidelines and methods agent results in a greater biological effect or activity when the established by the Clinical Laboratory Standards Institute agent is potentiated than the biological effect or activity that (CLSI), formerly the National Committee for Clinical Labo would result from the same concentration or amount of the ratory Standards (NCCLS), as set forth in NCCLS: Perfor agent when not potentiated; or (ii) a lower concentration or mance Standards for Antimicrobial Susceptibility Testing: amount of the agent is required to achieve a particular bio Fourteenth Informational Supplement. NCCLS document logical effect or activity when the agent is potentiated than M100-S14. Wayne, Pa...: NCCLS 2004; NCCLS: Perfor when the agent is not potentiated; or (iii) both (i) and (ii). The mance Standards for Antimicrobial Disk Susceptibility Tests: biological effect or activity may be, for example, the ability to Approved Standard-Eighth Edition. NCCLS document catalyze or inhibit one or more chemical reactions, the ability M2-A8. Wayne, Pa...: NCCLS 2003; or NCCLS: Methods for to activate or inhibit a biological or biochemical pathway, the Dilution Antimicrobial Susceptibility Tests for Bacteria that ability to reduce or inhibit microbial proliferation, the ability Grow Aerobically: Approved Standard-Sixth Edition. to kill a microorganism, etc. An agent whose presence poten NCCLS document M7-A6. Wayne, Pa. NCCLS 2003. tiates another agent may be referred to as a “potentiating “Intrinsic resistance” means that a bacterial species is inher agent'. A potentiating agent may show biological activity by ently resistant to the effects of an antibacterial agent. itself, or may exhibit biological activity only when used in “Acquired resistance' means that a bacterial species, Subtype, combination with a biologically and/or pharmacologically or strain has acquired a mechanism of resistance since the active agent. introduction of the antibacterial agent into use. Resistance 0102 Protist. The term “protist” refers to any member of a may, for example, be acquired by mutation of a target gene, by diverse group of organisms, comprising those eukaryotes that overexpression of an efflux pump, etc. A Subpopulation of a are not animals, plants or fungi. Protists can be unicellular or bacterial species that has acquired resistance may be consid multicellular. Protists are group in three Subcategories: ani ered a distinct strain of that species. mal-like protists, fungus-like protists, and plant-like protists. 0105 RecA Inhibitor. According to the present invention, 0103 Purified. “Purified’, as used herein, means that an an agent is a RecA inhibitor if one or more RecA activities is entity or Substance is separated from one or more other enti reduced in the agent's presence as compared with its absence, ties or substances with which it was previously found before or if the level or amount of RecA protein or gene product is being purified. An entity or Substance may be partially puri reduced in the agent's presence as compared with its absence. fied, Substantially purified, or pure. A substance or entity Such In certain embodiments, RecA inhibitors act directly on RecA US 2009/0264.342 A1 Oct. 22, 2009

in that they physically interact with RecA. In other embodi significant animals or companion animals (e.g., cows, sheep, ments, inhibitors act indirectly on RecA. goats, horses, Swine, dogs, cats, rabbits, birds, fish, etc.), 0106 Small Molecule. In general, a small molecule is laboratory animals (e.g., mice, rats) primates, or humans. understood in the art to be an organic molecule that is less than 0110. Sublethal. A “sublethal’ concentration of an antibi about kilodaltons (KDa) in size. In some embodiments, the otic refers to a concentration that is less than the MIC of the small molecule is less than about 3 KDa, 2 KDa, or 1 KDa. In antibiotic. In certain embodiments of the invention a sublethal some embodiments, the small molecule is less than about 800 concentration is not sufficient to significantly reduce the daltons (Da), 600 Da, 500 Da. 400 Da,300 Da,200 Da, or 100 growth rate (proliferation) of a microbial cell, e.g., the growth Da. In some embodiments, Small molecules are non-poly rate is reduced by less than 20%, preferably less than 10%. meric. In some embodiments, Small molecules are not amino Such a concentration is referred to herein as a “non-inhibiting acids. In some embodiments, Small molecules are not nucle concentration'. A "lethal’ concentration of an antibiotic is otides. In some embodiments, Small molecules are not sac one that is equal to or greater than the MIC and would ulti charides. mately result in microbial death and complete or essentially 0107 Specific binding. “Specific binding generally complete sterilization of a culture medium containing the refers to a physical association between a target polypeptide microbe if continued indefinitely assuming that no resistant (or, more generally, a target molecule) and a binding molecule strains arise during the incubation period. Such as an antibody or ligand. The association is typically 0111 Substituted. As described herein, compounds of the dependent upon the presence of a particular structural feature invention may contain “optionally substituted moieties. In of the target Such as an antigenic determinant or epitope general, the term “substituted, whether preceded by the term recognized by the binding molecule. For example, if an anti “optionally or not, means that one or more hydrogens of the body is specific for epitope A, the presence of a polypeptide designated moiety are replaced with a suitable Substituent. containing epitope A or the presence of free unlabeled A in a Unless otherwise indicated, an “optionally substituted group reaction containing both free labeled A and the binding mol may have a suitable substituent at each substitutable position ecule that binds thereto, will reduce the amount of labeled A of the group, and when more than one position in any given that binds to the binding molecule. It is to be understood that structure may be substituted with more than one substituent specificity need not be absolute but generally refers to the selected from a specified group, the Substituent may be either context in which the binding occurs. For example, it is well the same or different at every position. Combinations of sub known in the art that numerous antibodies cross-react with stituents envisioned by this invention are preferably those that other epitopes in addition to those present in the target mol result in the formation of stable or chemically feasible com ecule. Such cross-reactivity may be acceptable depending pounds. The term “stable', as used herein, refers to com upon the application for which the antibody is to be used. One pounds that are not substantially altered when subjected to of ordinary skill in the art will be able to select antibodies or conditions to allow for their production, detection, and, in ligands having a Sufficient degree of specificity to perform certain embodiments, their recovery, purification, and use for appropriately in any given application (e.g., for detection or one or more of the purposes disclosed herein. inhibition of a target molecule, for therapeutic purposes, etc). 0112 Suitable monovalent substituents on a substitutable It is also to be understood that specificity may be evaluated in carbon atom of an “optionally Substituted’ group are inde the context of additional factors such as the affinity of the pendently halogen; —(CH) R. —(CH), OR: —O— binding molecule for the target versus the affinity of the (CH2)C(O)OR: —(CH2)CH(OR); —(CH). SR; binding molecule for other targets, e.g., competitors. If a —(CH), Ph, which may be substituted with R: —(CH). binding molecule exhibits a high affinity for a target molecule 4(CH). Ph which may be substituted with R: —CH that it is desired to detect and low affinity for nontarget mol CHPh, which may be substituted with R: NO; –CN: ecules, the antibody will likely be an acceptable reagent. —N: —(CH2)N(R), —(CH), N(R)C(O)R: Once the specificity of a binding molecule is established in N(R)C(S)R;-(CH), N(R)C(O)NR: N(R)C(S) one or more contexts, it may be employed in other, preferably NR: -(CH.)N(R)C(O)OR: N(R)N(R)C(O)R; similar, contexts without necessarily re-evaluating its speci N(R)N(R)C(O)NR; N(R)N(R)C(O)OR; ficity. Binding of two or more entities (e.g., a receptor and a —(CH),C(O)R’; C(S)R: —(CH),C(O)CR: ligand, an antibody and an antigen, etc.) may be considered —(CH),C(O)SR: —(CH2)C(O)OSiR —(CH). specific if the equilibrium dissociation constant (Kd) of a 4OC(O)R’: —OC(O)(CH). SR-, SC(S)SR: -(CH). complex formed by the two or more entities is 10 Morless, 4SC(O)R’;-(CH), C(O)NR: C(S)NR:—C(S)SR: preferably 10 Morless, e.g., 10M or less, 107M or less, SC(S)SR, -(CH)OC(O)NR: C(O)N(OR)R; 10M or less, 10 M or less, or 10' M or less under the C(O)C(O)R: C(O)CHC(O)R: C(NOR)R; conditions tested. —(CH2)SSR: —(CH2)S(O).R. —(CH2)S(O) 0108 Strain. A “strain” is a genetic variant or subtype of a OR: —(CH)OS(O).R.: S(O)NR; —(CH), S(O) type or species or microorganism, e.g., an isolate of a micro R; N(R)S(O)NR: N(R)S(O).R: N(OR)R: organism that possesses the major properties that define the C(NH)NR: P(O).R: P(O)R: OP(O)R: OP species or type but differs from many or most other members (O)(OR); SiR —(C straight or branched alkylene)O— of the species or type in one or more other properties. The N(R); or —(C straight or branched alkylene)C(O)O N term “strain” can refer to a bacterium that harbors a particular (R), wherein each R may be substituted as defined below episome or contains a particular mutation in a gene that is not and is independently hydrogen, Caliphatic, —CH2Ph. found in many other Subtypes or strains of the species. —O(CH). Ph, or a 5-6-membered saturated, partially 0109 Subject. “Subject', as used herein, refers to an indi unsaturated, or aryl ring having 0-4 heteroatoms indepen vidual to whom an agent is to be delivered, e.g., for experi dently selected from nitrogen, oxygen, or Sulfur, or, notwith mental, diagnostic, and/or therapeutic purposes. Subjects of standing the definition above, two independent occurrences interest herein include animals, particularly agriculturally of R, taken together with their intervening atom(s), form a US 2009/0264.342 A1 Oct. 22, 2009

3-12-membered Saturated, partially unsaturated, or aryl - O(haloR), CN, C(O)OH, -C(O)OR, NH, mono- or bicyclic ring having 0-4 heteroatoms independently —NHR, —NR, or—NO, wherein each R. is unsubstituted selected from nitrogen, oxygen, or Sulfur, which may be or where preceded by “halo' is substituted only with one or substituted as defined below. more halogens, and is independently C aliphatic, 0113 Suitable monovalent substituents on R (or the ring —CHPh, —O(CH). Ph, or a 5-6-membered saturated, formed by taking two independent occurrences of R together partially unsaturated, or aryl ring having 0-4 heteroatoms with their intervening atoms), are independently halogen, independently selected from nitrogen, oxygen, or Sulfur. —(CH-)o-R, -(haloR), —(CH2)2OH, -(CH2)2OR. 0118. Survival. The term “survival', as used herein, refers —(CH)-CH(OR); —O(haloR), —CN, N'-(CH). to an ability of microbial cells to grow in the presence of one 2C(O)R. —(CH),C(O)OH, -(CH2)C(O)OR, or more antibiotic agent(s) present above the relevant mini —(CH). SR. —(CH), SH, -(CH2)NH2. —(CH). mum inhibitory concentration. In some embodiments, Sur 2NHR. —(CH)-NR, NO. —SiR —OSiR, vival is assessed at a concentration that is at or above a —C(O)SR. —(C straight or branched alkylene)C(O)OR, multiple of MIC (e.g., 2x, 4x, 5x, 6x, 8x. 10x, etc) or —SSR wherein each Ris unsubstituted or where preceded 0119 Target gene. The term “target gene' is used as under by “halo' is substituted only with one or more halogens, and stood in the art to refer to any gene, wherein modulating is independently selected from Caliphatic, —CHPh. expression of the gene or modulating activity of an expression —O(CH). Ph, or a 5-6-membered saturated, partially product of the gene would be expected to or does achieve a unsaturated, or aryl ring having 0-4 heteroatoms indepen biological effect. The term “target' is used to refer to either a dently selected from nitrogen, oxygen, or Sulfur. Suitable target gene or an expression product of a target gene, e.g., an divalent substituents on a saturated carbonatom of Rinclude RNA transcribed from the gene or more typically, a polypep —O and —S. tide encoded by the gene, or a protein of which the polypep 0114 Suitable divalent substituents on a saturated carbon tide is a subunit. The terms “target” and “target gene' are used atom of an “optionally substituted group include the follow for purposes of convenience and are not intended to be lim ing: —O, =S, —NNR* =NNHC(O)R*, =NNHC(O) iting. By way of example, any polypeptide or other molecule OR*, =NNHS(O).R*, —NR*, —NOR*, O(C(R*,)), or portion thereof whose synthesis by a microbial cell is 3O—, or —S(C(R)) S , wherein each independent needed in order for an antibiotic agent to inhibit growth occurrence of R is selected from hydrogen, Caliphatic and/or Survival of the cell may be a target molecule, and any which may be substituted as defined below, or an unsubsti gene that encodes such a polypeptide may be a target gene. tuted 5-6-membered saturated, partially unsaturated, or aryl I0120 Toxicity. "Toxicity” refers to any adverse and/or ring having 0 4 heteroatoms independently selected from undesired effect of a composition on the metabolism or func nitrogen, oxygen, or Sulfur. Suitable divalent Substituents that tioning of a cell, tissue, organ or body part, or subject. The are bound to vicinal substitutable carbons of an “optionally amount of toxicity associated with a composition may vary substituted” group include:—O(CR*). O—, wherein each with several conditions including, but not limited to, the independent occurrence of R* is selected from hydrogen, amount of composition present, the components present in Caliphatic which may be substituted as defined below, or the composition, the formulation of the composition, the an unsubstituted 5-6-membered Saturated, partially unsatur environmental conditions and physiological state of the cell, ated, or aryl ring having 0-4 heteroatoms independently tissue, organ or body part, or Subject, etc. selected from nitrogen, oxygen, or Sulfur. I0121 Treatment. “Treating or “treatment of as used 0115 Suitable substituents on the aliphatic group of R* herein, refers to providing any type of medical or Surgical include halogen, —R, -(haloR), —OH, -OR. —O(ha management to a subject. Treating can include, but is not loR), —CN, -C(O)OH, -C(O)OR-, - NH, -NHR, limited to, administering a pharmaceutical composition to a —NR, or - NO, wherein each Ris unsubstituted or where Subject. Treating is typically undertaken in an effort to alter preceded by “halo' is substituted only with one or more the course of a disease, disorder, or undesirable condition in a halogens, and is independently Caliphatic, —CHPh. manner beneficial to the subject. The effect of treating can —O(CH), Ph, or a 5-6-membered saturated, partially generally include reversing, alleviating, reducing, inhibiting unsaturated, or aryl ring having 0-4 heteroatoms indepen the progression of and/or reducing the likelihood of occur dently selected from nitrogen, oxygen, or Sulfur. rence of the disease, disorder, or condition to which such term 0116 Suitable substituents on a substitutable nitrogen of applies, or one or more symptoms or manifestations of Such an “optionally substituted” group include R, NR, disease, disorder or condition. A composition of this inven -C(O)R, C(O)OR, C(O)C(O)R, C(O)CHC(O) tion can be administered to a Subject who has developed an R, S(O).Rf, S(O)NR, C(S)NR, C(NH)NR, infection or is at increased risk of developing an infection or - N(R)S(O).R. wherein each R is independently relative to a member of the general population. A composition hydrogen, Caliphatic which may be substituted as defined of this invention can be administered prophylactically, i.e., below, unsubstituted —OPh, or an unsubstituted 5-6-mem before development of any symptom or manifestation of a bered saturated, partially unsaturated, or aryl ring having 0-4 condition. Typically in this case the subject will be at risk of heteroatoms independently selected from nitrogen, oxygen, developing the condition. The composition can be adminis or sulfur, or, notwithstanding the definition above, two inde tered prior to exposure of the Subject to an infectious agent or pendent occurrences of R, taken together with their interven prior to the occurrence of a pathogenic event. ing atom(s) form an unsubstituted 3-12-membered Saturated, 0.122 Underexpression. “Underexpressed’ or “underex partially unsaturated, or aryl mono- or bicyclic ring having pression” means expression of a gene at levels below the wild 0-4 heteroatoms independently selected from nitrogen, oxy type levels observed for the gene in organisms of that type gen, or Sulfur. under the same conditions, i.e., expression of the gene is I0117 Suitable substituents on the aliphatic group of R are decreased relative to wild type levels. For example, the value independently halogen, —R, -(haloR), —OH, - OR, of the steady state or average rate of synthesis or absolute US 2009/0264.342 A1 Oct. 22, 2009 amount of an expression product of the gene (e.g., a polypep need thereof using the agents and compositions containing tide encoded by the gene), may be between 0% and 95% of the the agents. An agent is said to potentiate an antibiotic if, for wild type value. example, (i) the concentration of antibiotic needed to achieve 0123 Unit Dosage Form. A “unit dosage form', as that a given effect is lowered if the agent and the antibiotic are term is used herein, refers to physically discrete units Suited used in combination; (ii) the spectrum of microorganisms as unitary dosages for the Subject to be treated; each unit whose growth or survival can be inhibited is expanded if the containing a predetermined quantity of active agent(s) calcu agent and the antibiotic are used in combination; (iii) a micro lated to produce the desired therapeutic effect in association organism that would otherwise be resistant to the antibiotic is with the required pharmaceutical carrier sensitive to it (or to aparticular concentration of it) if the agent 0124 Unsaturated. The term “unsaturated’, as used and the antibiotic are used in combination. herein, means that a moiety has one or more units of unsat I0127. The antibiotic potentiating agent may work by any uration. of a variety of different mechanisms. It may or may not affect 0.125 Vector. “Vector is used herein to refer to a nucleic the same molecular target or biological or biochemical path acid or a virus or portion thereof (e.g., a viral capsid) capable way as the antibiotic(s) whose activity it potentiates. The of mediating entry of, e.g., transferring, transporting, etc., a potentiating agent may inhibit or alter metabolism of the nucleic acid molecule into a cell. Where the vector is a nucleic antibiotic by the microorganism. For example, the potentiat acid, the nucleic acid molecule to be transferred is generally ing agent may be an inhibitor (e.g., a competitive or noncom linked to, e.g., inserted into, the vector nucleic acid molecule. petitive inhibitor) of a microbial enzyme that degrades the A nucleic acid vector may include sequences that direct antibiotic. The agent and the antibiotic may exhibit a “syn autonomous replication within Suitable host cells (e.g., an thetic lethal' effect, i.e., the combination of the two is lethal origin of replication), or may include sequences sufficient to at concentrations at which neither is lethal by itself. Prefer allow integration of part of all of the nucleic acid into host cell ably the compound is substantially nontoxic to eukaryotic DNA. Useful nucleic acid vectors include, for example, DNA cells, e.g., mammalian cells, over a range of concentrations, or RNA plasmids, cosmids, and naturally occurring or modi including concentrations at which it effectively potentiates fied viral genomes orportions thereofor nucleic acids that can the activity of an antibiotic. Typically the potentiating agent be packaged into viral capsids. An 'expression vector” is a potentiates the antibiotic by a mechanism that does not Sub vector Suitable for expressing a nucleic acid or polypeptide in stantially involve alternating the absorption, metabolism, dis a host cell. The expression vector typically includes regula tribution, or excretion (ADME) of the antibiotic by the sub tory elements (e.g., promoters, enhancers, and/or other ject to which it is administered, i.e., the potentiating agent expression signals and, optionally, additional regulatory ele would potentiate the antibiotic regardless of any alterations in ments such as termination signals sufficient to direct tran ADME that the agent may cause. Scription of a nucleic acid segment to which they are operably 0128. In certain embodiments of the invention a combina linked. The expression vector may also comprise operably tion of the antibiotic and the potentiating agent is therapeuti linked sequences required for proper translation of the cally effective when delivered by a route of administration by nucleic acid segment. The nucleic acid segment may, but need which the antibiotic agent by itself would not be effective. For not be, a protein coding sequence. The nucleic acid segment example, including the potentiating compound in a therapeu may be chimeric, meaning that it includes more than one tic regimen may allow delivery of the first antibiotic by the sequence of distinct origin that are joined together by recom oral route rather than the intravenous route. In certain binant DNA techniques, resulting in a nucleotide sequence embodiments of the invention including the potentiating that does not occur naturally. The term “expression vector agent in a therapeutic regimen reduces the dosing interval of can refer to a vector either before or after insertion of the the first antibiotic needed to achieve a desired therapeutic operably linked nucleic acid segment that is to be transcribed. effect. For example, the antibiotic agent may be effective for Certain expression vectors allow the shuttling of DNA a longer period of time in the presence of the potentiating between hosts such as bacteria-yeast, or bacteria-animal agent. In certain embodiments of the invention including the cells, or bacteria-fungal cells, or bacteria-invertebrate cells. A potentiating agent in a therapeutic regimen allows the use of typical expression vector will contain an origin of replication an antibiotic that is highly potent but too toxic for therapeutic for autonomous replication in host cells, one or more select use, i.e., the potentiating agent allows a lower dose of the able markers, one or more (typically several) useful restric compound to be effective such that the antibiotic can be safely tion enzyme sites, frequently a potential for high copy num used without unacceptable side effects. ber, and one or more promoters. I0129. Inhibitors of microbial type II topoisomerases are exemplified herein as antibiotics for which antibiotic poten DETAILED DESCRIPTION OF CERTAIN tiator target genes and potentiating agents that modulate these EMBODIMENTS OF THE INVENTION target genes or their expression products can be identified. Quinolone antibiotics are of particular interest. The invention I. Overview provides compositions containing an inhibitor of a microbial 0126 Various aspects of the present invention are directed type II topoisomerase and an agent that potentiates activity of towards (i) methods of Screening to identify target genes for the inhibitor of a microbial type II topoisomerase. In certain the development of new agents to be used to combat bacterial embodiments the invention provides compositions compris infection, (ii) target genes identified according to the methods ing a quinolone antibiotic and an agent that potentiates activ and the expression products of these target genes, (iii) meth ity of the quinolone. ods, reagents, and assay systems for the identification of 0.130 However, the methods of the invention are appli agents that modulate expression and/or activity of the targets, cable to any antibiotic class or particular antibiotic of interest. (iv) agents that modulate, e.g., inhibit, expression and/or One of ordinary skill in the art will be able to select other activity of the targets, and (V) methods of treating a Subject in classes of antibiotics for which potentiator target genes and/ US 2009/0264.342 A1 Oct. 22, 2009

or potentiating agents can be discovered using the inventive from the cell. Mutations in genes that encode certain of these screening methods described herein. For example, the meth proteins can also contribute to resistance in some cases. A ods may be employed to identify genes whose deletion or number of Such pumps, transporters, and porins are known, functional inactivation potentiates the effects of antibiotics and their ability to mediate resistance to various antibiotics that are protein synthesis inhibitors, nucleic acid synthesis has been documented. A few examples of genes encoding inhibitors, antibiotics that prevent synthesis of cell wall com Subunits of such pumps, transporters, or porins are tolC, acrA, ponents, etc. The methods may also be employed to identify acrB, ompC, oprM, norA, mex A, and mexB. It has been agents that potentiate any such antibiotic agent. One of ordi postulated and in some cases demonstrated that agents that nary skill in the art can, for example, consult Goodman and block or interfere with Such pumps, transporters, or porins Gilman's The Pharmacological Basis of Therapeutics, 10" would reduce resistance and/or reduce the concentration of an Ed., McGraw Hill, 2001, and Katzung, B. (ed.) Basic and antibiotic needed to inhibit bacterial growth and/or survival Clinical Pharmacology, McGraw-Hill/Appleton & Lange; (See, e.g., A.Y. Coban et al., Chemotherapy, 2004, 50:22-26; 8th edition (Sep. 21, 2000), Merck Manual of Diagnosis and J. Kriengkauykiat et al. Antimicrob Agents Chemother. Therapy, 17" ed., Physician's Desk Reference, etc., to select 2005, 49: 565-570; see J. M. Pages et al., Trends Mol Med., other antibiotic agents for which potentiator target genes 2005, 11: 382-389, and B. Marquez, Biochimie, 2005, 87: and/or potentiating agents may be sought. Specific examples 1137-1147, for reviews) of antibiotic classes of interest include aminoglycosides, lac I0135) In certain embodiments of the invention a target tams (e.g., beta-lactams), and peptide antibiotics (e.g., cyclic gene identified according to the inventive methods does not peptides). encode a subunit of an efflux pump, transporter, or porin. In 0131 Considerable effort has been devoted to discovering certain embodiments of the invention an antibiotic potentiat bacterial targets for the development of novel antibiotic ing agent does not act solely or primarily by binding to, agents. However, attention has largely been focused on iden inhibiting, or interfering with expression or activity of a bac tifying essential bacterial genes and attempting to discover terial efflux pump, transporter, porin, or Subunit of any of agents that inhibit these genes or their expression products, these. The antibiotic potentiating agent may bind to or at least based on the premise that agents that target Such genes and/or partially inhibit with expression of a bacterial efflux pump, their expression products would effectively inhibit bacterial transporter, porin, or subunit thereof. However, in the sub growth. (see, e.g., Rational identification of new antibacterial stantial absence of the particular efflux pump, transporter, drug targets that are essential for viability using a genomics porin, or subunit, the antibiotic potentiating agent would still based approach. Pharmacol Ther. 2002, 95: 1-20, 2002: significantly potentiate the antibiotic, thereby indicating that DeVito et al., Nat. Biotechnol., 2002, 20: 478-483). the agent does not act solely or primarily by binding to, 0132) The present invention encompasses the recognition inhibiting, or interfering with expression or activity of a bac that this approach overlooks many potential target genes that terial efflux pump, transporter, porin, or Subunit of any of may not be essential, at least not under typically used standard these. Of course an inventive composition could contain an laboratory culture conditions. Among other advantages, the agent that binds to, inhibits, or interferes with expression or present invention identifies sets of microbial target genes that activity of a bacterial efflux pump, transporter, porin, or Sub may not be essential under standard laboratory culture con unit of any of these in addition to a potentiating agent that acts ditions but whose functional inactivation renders bacteria against a target other than a bacterial efflux pump, transporter, hypersensitive to an antibiotic, e.g., the bacteria in which the porin, or subunit. gene is inactivated or that otherwise underexpress the gene 0.136 An antibiotic for which antibiotic potentiator target are sensitive to the antibiotic at concentrations at which they genes and potentiating agents may be discovered using the would not otherwise be sensitive. In accordance with the methods of the present invention and/or that may be potenti invention, agents that inhibit with expression or activity of ated using an antibiotic potentiating agent of the invention is one or more of these target genes or their expression product typically a member of a class of agents, e.g., a class of chemi (s), potentiate the antibiotic. cal compounds having similar structural features. Exemplary 0133. The invention is based in part on the identification of classes of antibiotics wherein members of the class share one bacterial genes whose functional inactivation (or "knockout”) or more structural features include, but are not limited to, render the bacterium more sensitive to antibiotics (e.g., qui quinolones, B-lactams (e.g., penicillins or cephalosporins), nolones) than are otherwise identical bacteria in which the carbapenems, aminoglycosides, macrollides, ketolides, tetra gene is not inactivated. Inactivation of these genes therefore cyclines, glycycyclines, lincomycins, oxazolidinones, potentiates the activity of the antibiotics (e.g., quinolones). amphenicols, ansamycins, polymyxins, aminomethlycy These genes are targets for the development of agents that clines, lincosamides, streptogramins, 2,4-diamino-pyrim potentiate the activity of antibiotics and are therefore referred idines, nitrofurans, Sulfonamides, Sulfones, rifabutins, dap to herein as “antibiotic potentiator target genes'. The inven Sones, peptides, and glycopeptides. In accordance with the tion provides methods for identifying antibiotic (e.g., qui invention, an agent that potentiates the antibiotic may be used nolone) potentiator target gene. The invention further pro to potentiate other antibiotics in the same structural class. For vides methods for identifying agents that modulate (e.g., example, the antibacterial agent may be a quinolone antibi activate or inhibit) expression of an antibiotic potentiator otic. Thus, in a nonlimiting example, an agent that potentiates target gene and methods for identifying agents that increase a first quinolone antibiotic may be used to potentiate a second or inhibit an expression product of an antibiotic potentiator quinolone antibiotic. More specifically, an agent that poten target gene. tiates a first fluoroquinolone such as norfloxacin may be used 0134 One common means by which bacteria are intrinsi to potentiate a second fluoroquinolone such as ciprofloxacin. cally resistant to or acquire resistance to an antibiotic is by Of course it will be appreciated that an agent that potentiates expressing or overexpressing an efflux pump, transporter, or any particular antibiotic in a class of agents need not poten porin that actively removes or allows removal of the antibiotic tiate all, most, or any other members of that class, although in US 2009/0264.342 A1 Oct. 22, 2009

certain embodiments of the invention the agent does potenti agent that potentiates a first antibiotic that binds to and/or ate at least one, many, most, or all other members of that class. inhibits or interferes with expression or activity of a bacterial 0.137 The antibiotic may have a known mechanism of type II topoisomerase may be used to potentiate a second antimicrobial action and/or may bind to and/or inhibit one or antibiotic that binds to and/or inhibits or interferes with more bacterial target molecules or macromolecular com expression or activity of the same or a different type II topoi plexes containing a bacterial target molecule. Mechanisms of Somerase. More specifically, an agent that potentiates a qui action may include inhibiting or interfering with a biological nolone antibiotic whose primary target is DNA gyrase may or biochemical pathway of the bacterium. Exemplary path potentiate an antibiotic whose target is topoisomerase IV. Of ways include, but are not limited to, protein synthesis, cell course it will be appreciated that an agent that potentiates an wall synthesis, DNA replication, transcription, cell division. antibiotic that has a particular molecular target need not It will be appreciated that biological and biochemical path potentiate all, most, or any otherantibiotics having that target, ways are not mutually exclusive and that some biological or a structurally similar target, or a target that participates in the biochemical pathways may be considered to be subsets or same biological or biochemical pathway, although in certain Sub-pathways of other biological or biochemical pathways. embodiments of the invention the agent does potentiate at Mechanisms of action include, but are not limited to, inhib least one, many, most, or all other antibiotics having that iting protein synthesis (e.g., by binding ribosomal RNA or target, a structurally similar target, or a target that participates proteins, blocking tRNA binding to ribosome-mRNA com plex, inhibiting peptidyltransferase), inhibiting or interfering in the same biological or biochemical pathway. with synthesis of a cell wall component (e.g., inhibition of 0.139. The target genes of the invention may be found in peptidoglycan synthesis, disruption of peptidoglycan cross bacteria of any one or more types, e.g., Gram negative bacte linkage, disruption of movement of peptidoglycan precur ria, Gram positive bacteria, and/or acid fast bacteria. Any of sors, disruption of mycolic acid or arabinoglycan synthesis), these bacteria may therefore be used in the methods for iden cell membrane disruption, inhibiting or interfering with tifying a target gene described herein, and any target gene can nucleic acid synthesis or processing, acting as “antimetabo be used to identify agents that potentiate antibiotics (e.g., lites” and either inhibiting an essential bacterial enzyme or quinolone antibiotics). One of ordinary skill in the art will competing with a substrate of an essential bacterial enzyme, appreciate that it may be convenient to perform the target inhibiting or interfering with cell division. In accordance with gene identification methods using a bacterial species or strain the invention, an agent that potentiates the antibiotic may be that is commonly used in the laboratory and for which used to potentiate other antibiotics having the same or a reagents and methods for performing microbiological and/or similar mechanism of action. For example, the antibiotic may genetic manipulation are well developed and widely avail inhibit with bacterial protein synthesis. Thus, in a nonlimiting able. Many such strains are known to one of skill in the art. It example, an agent that potentiates a first antibiotic that inter may be desirable to utilize a non-pathogenic strain of bacteria feres with bacterial protein synthesis by binding to ribosomal to perform the target geneidentification methods, wherein the RNA may be used to potentiate a second antibiotic that inhib strain is a member of bacterial species or genus that contains its or interferes with protein synthesis by binding to riboso pathogenic members. Once a target gene is found in any mal RNA. Of course it will be appreciated that an agent that particular bacterial strain, species, or genus, homologs can be potentiates any particular antibiotic having a particular readily identified in one or more other bacterial strains, spe mechanism of action need not potentiate all, most, or any cies, or genuses, as discussed further below. Such homologs other members having that mechanism of action, although in can be used in the methods for identifying an antibiotic poten certain embodiments of the invention the agent does potenti tiating agent, e.g., a quinolone potentiating agent. Further ate at least one, many, most, or all otherantibiotics having that more, an antibiotic potentiating agent identified according to mechanism of action. a method that involves a target gene from a particular bacte 0138 Molecules or macromolecular complexes contain rial genus, species, or strain, is of use to inhibit homologous ing them that may be targets of an antibiotic include, but are target genes in other bacterial genuses, species, or strains, and not limited to, peptidoglycans, penicillin binding proteins, is therefore of use to potentiate the antibiotic when used to lipopolysaccharides, ribosomes or ribosomal Subunits or inhibit growth and/or Survival of bacteria of a genus, species, RNA or protein components thereof (23S rRNA, 16S rRNA, or strain that contains the homologous target gene. 5S rRNA, proteins of the 30S or 50S subunit), DNA-depen 0140) Suitable organisms from which target genes can be dent DNA polymerase, DNA-dependent RNA polymerase, identified and/or against which potentiating agents can be microbial type I topoisomerase, microbial type II topoi used can include, but are not limited to members of the fol Somerase (e.g., topoisomerase IV or gyrase), enzymes lowing genuses: Actinomyces, Staphylococcus, Streptococ involved in synthesis of essential cofactors or nucleic acid cus, Enterococcus, Erysipelothrix, Neisseria, Branhamella, precursors (e.g., nucleotides) such as dihydrofolate reduc Listeria, Bacillus, Corynbacterium, Erysipelothrix, Gardner tase, enzymes involved in cell division Such as FtsZ. etc. In ella, Mycobacterium, Nocardia, Enterobacteriaceae, accordance with the invention, an agent that potentiates the Escherichia, Salmonella, Shigella, Yersinia, Enterobacter, antibiotic may be used to potentiate other antibiotics that bind Klebsiella, Citrobacter; Serratia, Providencia, Proteus, Mor to and/or inhibit expression or activity of the same or a struc ganella, Edwardsiella, Erwinia, Vibrio, Aeromonas, Helico turally similar molecular target or macromolecular complex bacter; Campylobacter, Eikenella, Pasteurella, Pseudomo or may be used to potentiate other antibiotics that bind to nas, Burkholderia, Stenotrophomonas, Acinetobacter, and/or inhibit expression or activity of a molecular target that Ralstonia, Alcaligenes, Moraxella, Mycoplasma, Legionella, participates in the same biological or biochemical pathway. Francisella, Brucella, Haemophilus, Bordetella, For example, the antibacterial agent may be an inhibitor of a Clostridium, Bacteroides, Porphyromonas, PrevOtella, Fuso bacterial type II topoisomerase, e.g., an inhibitor of topoi bacterium, Borrelia, Chlamydia, Rickettsia, Ehrlichia, Bar Somerase IV. gyrase, or both. In a nonlimiting example, an tonella, Trichomonas, and Treponema. US 2009/0264.342 A1 Oct. 22, 2009

0141. In particular embodiments of the invention the bac classes of antibiotics include, but are not limited to, ami teria are species that are causative agents of disease in humans noglycosides, aminomethylcyclines, amphenicols, ansamy and/or animals. Examples include, but are not limited to, cins, B-lactams (e.g., penicillins or cephalosporins), carbap Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, enems, dapsones, 2,4-diaminopyrimidines, glycopeptides, Enterobacter cloacae, Campylobacter jejuni, Haemophilus glycycyclines, ketolides, lincomycins, lincosamides, mac influenzae, Klebsiella pneumoniae, Klebsiella Oxytoca, rolides, nitrofurans, oxazolidinones, peptides, polymyxins, Legionella pneumophila, Pasteurella multocida, Proteus mirabilis, Proteus vulgaris, Morganella morgani, Helico quinolones, rifabutins, streptogramins, Sulfonamides, Sul bacter pylori, Neisseria gonorrhoeae, Pseudomonas aerugi fones, tetracyclines, and combinations thereof. A few nosa, Salmonella enterica, Salmonella typhimurium, Staphy examples of classes of antibiotics are described below. lococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus Quinolone Antibiotics and Other Bacterial Type II Topoi agalactiae. somerase Inhibitors 0142. In certain embodiments of the invention the target genes are found in a bacterial species that is sensitive to a 0146 Quinolone antibiotics are compounds that contain a bacterial type II topoisomerase inhibitor Such as a quinolone quinolone or a naphthyridine nucleus with any of a variety of antibiotic. In certain embodiments of the invention the target different side chains and substituents. Numerous modifica genes are found in a bacterial species or strain that displays tions of the originally identified core structures have been intrinsic or acquired resistance to a bacterial type II topoi made resulting in a large number of compounds with activity Somerase II inhibitor, Such as a quinolone antibiotic. In cer against differing groups of bacteria. Quinolone antibiotics are tain embodiments of the invention the target genes are found described, e.g., in Ronald, A. R. and Low, D. E., (eds.), in a bacterial species that is sensitive to an antibiotic that binds “Fluoroquinolone Antibiotics'. Birkhäuser Verlag, Basel, to the bacterial 30S ribosomal Subunit Such as an aminogly 2003; DaSilva, AD, et al., “Biological activity and synthetic coside antibiotic. In certain embodiments of the invention, the methodologies for the preparation of fluoroquinolones, a target genes are found in a bacterial species or strain that class of potent antibacterial agents. Curr Med Chem 10(1): displays intrinsic or acquired resistance to an antiobiotic that 21-39, 2003; Van Bambeke, F., et al., “Quinolones in 2005: an binds to the bacterial 30S ribosomal subunit such as an ami update', Clin Microbiol. Infect., 11: 256-280, 2005: U.S. Pat. noglycoside antibiotic. Nos. 3,669,965); 4,563,459; 4,620,007; 4,382,892: 4,985, 0143. The invention provides methods and reagents that 5575,053.407; and 5,142,046). FIG.9 depicts the core struc may be used to identify agents that either directly or indirectly tures and numbering system of classical quinolone antibiotics modulate expression and/or activity of a target gene. The (4-quinolone and 4-naphthyridine systems). It is noted that methods include cell-free assays, cell-based assays, and com the numbering system shown herein is used for purposes of putational methods. convenience and is not intended to be limiting. The invention 0144. The invention also provides agents that modulate, encompasses quinolone compounds in which an alternative e.g., inhibit, expression of a antibiotic potentiator target gene. numbering system is used. The invention provides agents that modulate activity of an expression product of an antibiotic potentiator target gene, 0147 Quinolone antibiotics include, but are not limited to, e.g., that inhibit or interfere with activity of an expression any of the antibacterial agents disclosed in the foregoing product of an microbial antibiotic potentiator target gene. An references including, but not limited to, ciprofloxacin, oxo agent that modulates activity of an expression product of linic acid, cinoxacin, flumequine, miloxacin, roSoxacin, pipe target gene can do so directly, i.e., by a mechanism that midic acid, norfloxacin, enoxacin, moxifloxacin, gatifloxa involves a physical interaction of the agent with the expres cin, ofloxacin, lomefloxacin, temafloxacin, fleroxacin, sion product. Alternatively or additionally, an agent can pefloxacin, amifloxacin, sparfloxacin, levofloxacin, clina modulate activity of an expression product of a target gene floxacin, nalidixic acid, enoxacin, grepafloxacin, levofloxa indirectly, i.e., by a mechanism that does not involve a physi cin, lomefloxacin norfloxacin, ofloxacin, trovafloxacin, ola cal interaction with the expression product of the target gene. mufloxacin, cadrofloxacin, alatrofloxacin, gatifloxacin, For example, the agent can directly or indirectly modulate an rufloxacin, irloxacin, prulifloxacin, paZufloxacin, gemifloxa enzyme that is at least in part responsible for synthesizing a cin, sitafloxacin, tosulfloxacin, amifloxacin, nitrosoxacin-A, substrate of or cofactor for the expression product or is DX-619, and ABT-492. Quinolone antibiotics include fluo responsible for modifying or activating the expression prod roquinolones (e.g., having a fluorine Substituent at the C-6 uct. In a nonlimiting example, if a first and second polypep position), and non-fluoroquinolones. Also included within tide are subunits of a protein, then an agent that binds to the the scope of quinolone antibiotics are derivatives in which a second polypeptide may prevent or hinder interaction of the quinolone is conjugated with, e.g., covalently bound to, two polypeptides, and may thus indirectly modulate activity another core structure. For example, U.S. Pub. No. 2004 of the first polypeptide. Thus, antibiotic potentiating agents of 0215017 discloses compounds in which an oxazolidinone, the present invention are not limited to agents that modulate isoxazolinone, or isoxazoline is covalently bonded to a qui expression of a target gene or physically interact with an nolone. expression product of a target gene but encompass any agent 0.148. Included within the scope of quinolone antibiotics that director indirectly interferes with expression of the target that can be potentiated by inactivation of the quinolone poten gene and/or interferes with activity of an expression product tiator target genes or their expression products are com of the gene. pounds that have a core structure related to the 4-oxo-1,4- dihydroquinoline and 4-oxo-14 dihydronapthyridine II. Antibiotics systems, e.g., 2-pyridones, 2-naphthyridinones, and benzob 0145 A large variety of classes of antibiotics can be used napthyridones. 2-pyridones are potent inhibitors of bacterial according to the present invention. Exemplary structural type II topoisomerases (Shen, L.L., et al., Curr: Pharm. Des., US 2009/0264.342 A1 Oct. 22, 2009

3:169-176; Saiki, A Y C, et al., Antimicrob. Agents in either the same or different type II topoisomerase subunits. Chemother, 43: 1574-1577, 1999). The core structures are The invention contemplates use of a quinolone potentiating depicted in FIG. 9. agent in combination with a quinolone against bacteria hav 0149. Also included within the scope of quinolone antibi ing one or more mutations that confer quinolone resistance. otics that can be potentiated are compounds that have core structures related to the quinolone core structures depicted in Aminoglycosides FIG. 9 or 10. Certain of these core structures are shown in 0153 Aminoglycosides are a group of antibiotics that are FIGS. 11A and 11B, and references thereto are provided in effective against certain types of bacteria. Examples of ami Ronald, Supra. The invention encompasses the use of qui noglycosides include amikacin, gentamicin, kanamycin, neo nolone potentiating agents identified as described herein to mycin, netilmicin, paromomycin, Streptomycin, tobramycin, potentiate these antibiotics. adapramycin. Those which are derived from Streptomyces 0150. In addition to the quinolone antibiotics, a variety of genus are named with the Suffix -mycin, while those which agents are known in the art that inhibit one or more bacterial are derived from micromonospora are named with the suffix type II topoisomerase inhibitors. Some of which are structur -micin. ally related to quinolones. Exemplary inhibitors that bind to 0154 Aminoglycosides work by binding to the bacterial GyrB include the coumarins, novobiocin and coumermycin A 30S ribosomal subunit (some work by binding to the 50s 1, cyclothialidine, cinodine, and clerocidin. Additional com subunit), inhibiting the translocation of the peptidyl-tRNA pounds that are reported to bind to and/or inhibit gyrase, from the A-site to the P-site and also causing misreading of topoisomerase IV, or both, are disclosed in U.S. Pat. Nos. mRNA, leaving the bacterium unable to synthesize proteins 6,608,087 and 6,632,809 and in U.S. Pub. Nos. 2004.0043989 vital to its growth. However, the exact mechanism of action of and 2005.0054697. The present invention encompasses the aminoglycosides in not fully known. use of quinolone potentiating agents to potentiate any of these 0155 Aminoglycosides are useful primarily in infections agents, e.g., for use in combination with any of these com involving aerobic, Gram-negative bacteria, Such as pounds or any compound that inhibits one or more microbial Pseudomonas, Acinetobacter, and Enterobacter. In addition, type II topoisomerases. Some mycobacteria, including the bacteria that cause tuber 0151. Quinolones exert their antibacterial effects at least culosis, are susceptible to aminoglycosides. The most fre in part by targeting bacterial type II topoisomerases, namely quent use of aminoglycosides is empiric therapy for serious gyrase and topoisomerase IV, enzymes that catalyze breakage infections such as septicemia, complicated intra-abdominal and rejoining of DNA strands during normal cell growth. infections, complicated urinary tract infections, and nosoco DNA gyrase exists as an A.B. heterotetramer, encoded by the mial respiratory tract infections. Usually, once cultures of the gyra and gyrB genes. Topoisomerase IV exists as a CE causal organism are grown and their susceptibilities tested, heterotetramer, encoded by the parC and parE genes. It is aminoglycosides are discontinued in favor of less toxic anti noted that the foregoing nomenclature, while applicable to E. biotics. coli and many other microorganisms, is not universal. For 0156 Streptomycin was the first effective drug in the treat example, the parC and parE are sometimes referred to as grlA ment of tuberculosis, though the role of aminoglycosides and grlB respectively in Gram positive bacteria. These Such as streptomycin and amikacin has been eclipsed (be enzymes are conserved among Gram negative and positive cause of their toxicity and inconvenient route of administra bacteria and are essential for viability. DNA gyrase intro tion) except for multiple drug resistant strains. duces negative coils into DNA and can remove both negative 015.7 Infections caused by Gram-positive bacteria can and positive Supercoils. The main function of the topoi also be treated with aminoglycosides, but other types of anti somerase IV is the decatenation of interlinked replicated biotics are more potent and less damaging to the host. In the chromosomes, allowing the partitioning daughter chromo past, the aminoglycosides have been used in conjunction with somes following DNA replication. beta-lactam antibiotics in streptococcal infections for their 0152 Quinolones act by forming a ternary complex with synergistic effects, particularly in endocarditis. One of the the topoisomerase enzymes and DNA. The lethal effect may most frequent combinations is Ampicillin (a beta-lactam or result due to enhancement of DNA cleavage and/or by block penicillin-related antibiotic) and Gentamicin. ing DNA religation following cleavage by the topoisomerase 0158 Because of their potential for ototoxicity and neph rather than primarily from the inhibition of DNA replication. rotoxixity (kidney toxicity), aminoglycosides are adminis Quinolones increase the intracellular concentrations of the tered in doses based on body weight. Blood drug levels are cleavage complexes that are intermediates in the topoi used to estimate how well the kidneys are functioning and as Somerase-mediated reactions. The accumulation of perma a marker for kidney damage caused by these drugs. nent double-stranded DNA breaks eventually leads to bacte rial death. The affinity of many quinolone antibiotics and Peptide Antibiotics derivatives differs towards DNA gyrase and DNA topoi somerase IV (see, e.g., Drlica and Hooper, 2003). Resistance 0159. Over 400 natural antimicrobial peptides have been to quinolones arises primarily due to a variety of mutations isolated and characterized. Based on chemical structure, which make the enzymes less sensitive to quinolones or these peptides may be classified into two main groups: linear which affect microbial efflux pumps and decrease cellular and cyclic (R. E. Hancocket al., Adv. Microb. Physiol., 1995, accumulation of the drug. Amino acids at which mutations are 37: 135-137; H. Kleinkaufetal., Criti. Rev. Biotechnol., 198, particularly likely to result in resistance have been identified. 8: 1-32: D. Perlman and M. Bodansky, Annu. Rev. Biochem. Portions of the enzyme containing Such amino acids have 1971, 40: 449-464. The mode of action for the majority of been denoted quinolone resistance determining regions. See, these peptides (both linear and cyclic) is believed to involve e.g., Ronald, A L. Supra. Quinolone resistance can arise in a membrane disruption, leading to cell leakage (A. Mor, Drug step-wise fashion as bacteria accumulate multiple mutations Develop. Res., 2000, 50: 440-447). The linear peptides, such US 2009/0264.342 A1 Oct. 22, 2009 as magainins and melitting, exist mainly as a-helical amphi of the gene. Alternately, antibiotic sensitivity of cells that pathic structures (containing segregated hydrophobic and overexpress a particular gene can be compared with sensitiv hydrophilic moieties), or as B-helices as found in gramicidin ity of cells that express the gene at wild type levels or that A (GA). Cyclic peptides, which mainly adopt an amphipatic underexpress the gene. It will be appreciated that the com B-sheet structures can be further divided into two subgroups: parison can be performed in a number of ways, and that it is those containing disulfide bonds, such as tachyplesin, and not necessary to Subject cells displaying different levels of those that do not. Such as gramicidin S (D. Audreu and L. functional activity of a gene to the antibiotic at the same time Rivas, Biopolymers, 1998, 47: 415-433). or in the same experiment. For example, once the sensitivity 0160 Peptide antibiotics also fall into two classes: non ribosomally synthesized peptides, such as the gramicicins, of cells exhibiting any particular level of functional activity of polymyxins, bacitracins, glycopeptides, etc., and ribosomally a gene product (e.g., wild type cells) is determined, this infor synthesized (natural) peptides. The former are often drasti mation can be used as a basis for comparison in future experi cally modified and are largely produced by bacteria, whereas mentS. the latter are produced by all species of life (including bacte 0.165 Any of a variety of specific methods may be used to ria) as a major component of the natural host defense mol determine whether functional inactivation of a gene confers ecules of these species. hypersensitivity, and any Such method is within the scope of 0161 In certain embodiments, the peptide antibiotic is a the invention. One method of identifying an antibiotic poten lipopeptide antibiotic Such as colistin, daptomycin, Surfactin, tiator target gene comprises steps of: (a) contacting a first friulimicin, aculeacin A, iturin A, and tsushimycin. microbial cell with an antibiotic, wherein the microbial cell 0162 Colistin (also called Colimycin) is a polymixin anti lacks or has significantly reduced functional activity of an biotic discovered more than 50 years ago. It is a cyclic expression product of a gene relative to a second microbial lipopeptide antibiotic which penetrates the cell wall of Gram cell; and (b) determining that the gene is an antibiotic poten negative bacteria by self-induced mechanism by chelating tiator target gene if the growth or survival of the first microbial divalentions. Colistin destabilizes the wall and can insinuate cell is detectably lower than the growth or survival of the into it. Colistin basically perforates the cell wall, causing second microbial cell. It will be appreciated that the method distortion of this structure and the release of intracellular typically involves comparing growth or Survival that the first constituents. Increasing multidrug resistance in Gram-nega and second microbial cells would exhibit under substantially tive bacteria, in particular Pseudomonas aeruginosa, Acine equivalent conditions, particularly with respect to the concen tobacter baumannii, and Klebsiella pneumoniae, presents a tration of the antibiotic and the time of exposure. However, critical problem. Limited therapeutic options have forced Substantially equivalent conditions need not actually be infectious disease clinicians and microbiologists to reap employed in performing the method, provided that the growth praise the clinical application of Colistin. Colistin is associ or survival results for the first and second microbial cells can ated with neurotoxicity and nephrotoxicity. Dosage regimen be correlated with what would be expected to occur under and novel formulation may be an answer to address the tox Substantially equivalent conditions. icity issue. 0166 Substantially equivalent conditions of antibiotic III. Target Genes and Methods for their Identification exposure with respect to concentration of the antibiotic typi cally means that the concentration of antibiotic to which the 0163 The invention provides methods for identifying an first and second microbial cells are exposed are within a factor antibiotic potentiator target gene. Certain of the methods of 2-fold of one another, or that the concentrations would be involve determining whether complete or partial functional expected to have substantially the same effect on identical inactivation of a gene, e.g., by deletion or other genetic alter cells (for example, two different concentrations that are both ation that eliminates or greatly reduces expression of the gene much larger than the MIC would be expected to have substan or results in a gene product that Substantially lacks functional tially the same inhibitory effect even if the absolute concen activity (e.g., displays less than about 10% of the activity that trations varied by more than a factor of 2, and two different would be exhibited in the absence of the genetic alteration, concentrations that are both much smaller than the MIC preferably less than about 5% of the activity) or by inhibiting would be expected to have substantially no effect even if the an expression product of the gene, renders a microbial cell absolute concentrations varied by more than a factor of 2). more sensitive to the antibiotic relative to cells in which the Preferably, substantially equivalent antibiotic exposure is expression product of the gene has greater activity. Other exposure at concentrations that differ by no more than a factor methods involve determining whether overexpression of a of 2. The concentrations may be identical within experimen gene, e.g., relative to wildtype levels, confers increased resis tal error, or the larger concentration may be 110% or less, tance to the effect of an antibiotic. 120% or less, 130% or less, 140% or less, or 150% or less of 0164. A major concept of the invention is to compare the the Smaller concentration. Alternately, the concentrations antibiotic sensitivity of microbial cells that have differing may differ by 10% or less, 20% or less, 30% or less, 40% or levels of functional activity of a gene product and to either less, or 50% or less of the MIC for the antibiotic. With respect qualitatively or quantitatively determine whether sensitivity to time during which exposure occurs, Substantially equiva to the antibiotic agent depends at least in part on the level of lent conditions would typically mean that the length of expo functional activity of the gene product. Within this conceptual sure differs by no more than a factor of 2 and may, for framework, a variety of different approaches may be example, differ by 10% or less, 20% or less, 30% or less, 40% employed. For example, antibiotic sensitivity of cells in or less, or 50% or less of the shorter time of exposure or be which a gene is partly or completely functionally inactivated substantially identical (i.e., identical to within 2% of the (e.g., as a result of a genetic alteration Such as partial or shorter time of exposure). Substantially equivalent conditions complete deletion of the gene) can be compared with sensi may also entail use of the same growth medium, temperature, tivity of cells that display higher levels of functional activity etc., for cells whose growth or Survival is to be compared. US 2009/0264.342 A1 Oct. 22, 2009

0167. The particular antibiotic concentration selected will involves adding a single reagent (BacTiter-GloTM Reagent) depend on a variety of parameters including the bacterial directly to bacterial cells in medium and measuring lumines species or strains used, whether growth or survival (viability cence. Many additional assays Suitable for assessing bacterial following a period of exposure to the antibiotic) is to be viability are described in “Handbook of Fluorescent Probes assessed, etc. Typically, the concentration will be sublethal and Research Products” (Molecular Probes, 9th edition; for a growth assay. The concentration may be one that does 2002) and “The Handbook A Guide to Fluorescent Probes not significantly reduce bacterial growth but is Sufficient to and Labeling Technologies” (Invitrogen, 10th edition, avail cause at least some alterations in bacterial physiology. For able at the Invitrogen web site). In certain embodiments of the example, the concentration may be one that causes detectable invention the assay is performed in a high throughput format. alterations in expression of one or more genes. In certain 0169. The methods of the invention advantageously make embodiments of the invention the concentration selected for a use of a collections of bacterial strains in which a different screen employing a growth assay is between 1% and 5% of gene is altered in each strain. Typically the strains will be the MIC, between 5% and 10% of the MIC, between 10% and members of a bacterial species, e.g., E. coli or S. aureus and 25% of the MIC, between 25% and 50% of the MIC, between 50% and 75% of the MIC, between 75% and 95% of the MIC, will be genetically identical except for the genetic alteration. or any specific Subrange or value within a foregoing range. In The collection of strains will advantageously include mem certain embodiments of the invention the concentration bers with alteration or deletion of substantially all non-essen selected for a screen employing a growth assay reduces tial genes of the bacterium, with each member having an growth of wild type bacteria (not having functional inactiva alteration or deletion of a different gene. The alteration may, tion of a gene) to between 5% and 10%, between 10% and for example, involve deletion of all or part of the gene, so that 25%, between 25% and 50%, between 50% and 75%, or either (i) no functional gene product is synthesized; (ii) the between 75% and 95% of the growth in the absence of the amount of functional gene product is Substantially reduced; antibiotic, or any specific Subrange or value within a forego or (iii) the gene product has Substantially reduced or no activ ing range. In certain embodiments of the invention the con ity. The availability of complete genome sequences for a centration selected for a screen employing a Survival assay is variety of different bacteria has facilitated the development of between 1 and 2 times the MIC, between 2 and 5 times the Such strain collections. Deletion or functional inactivation MIC, or between 5 and 10 times the MIC. can beachieved using a variety of different methods known in 0168 Growth or survival can be assessed using cells grow the art. ing in liquid media or on solid or semi-solid media. Any (0170. In other embodiments a collection of bacterial method known in the art can be used to determine whetheran strains in which a different gene is overexpressed in each agent inhibits growth, proliferation, and/or Survival. strain is used. Overexpression can be achieved, e.g., by intro Examples include measuring optical density in liquid culture, ducing an expression vector containing the relevant gene (or measuring colony formation, or measuring bacterial viability. the coding portion thereof) into the cell. Either the naturally Bacterial viability can be assessed based on metabolic char occurring promoter or a heterologous promoter can be used. acteristics such as oxidation/reduction state, ability to The vector can be a multicopy plasmid, thereby providing metabolize particular Substrate(s) or produce particular many copies of the gene. Alternately or additionally, a strong metabolite(s), or based on membrane integrity, which can be promoter functional in bacterial cells could be used to express detected by evaluating ability of a bacterial cell to exclude a the gene at high levels. A recombinant nucleic acid construct particular substance Such as a detectable molecule (e.g., a encoding the gene can be integrated into the bacterial chro fluorescent or luminescent molecule) from the cell interior. In mosome. It will be appreciated that the sequence of the gene one embodiment, a commercially available assay Such as the introduced into the cells may differ from the wild type LIVE/DEAD BacLight Bacterial Viability assay (Molecular sequence but will typically have a high degree of nucleotide Probes, now owned by Invitrogen, Carlsbad, Calif.) is used. similarity to it (e.g., at least 80%, preferably at least 90%, and This assay utilizes mixtures of SYTOR 9 green fluorescent preferably close to 100% identity). In some embodiments a nucleic acid stain and the red fluorescent nucleic acid stain, gene derived from one bacterial species is introduced into a propidium iodide. These stains differ both in their spectral different bacterial species. characteristics and in their ability to penetrate healthy bacte (0171 In some embodiments of the invention the members rial cells. When used alone, the SYTO 9 stain labels bacteria of the strain collection express genes under the control of a with both intact and damaged membranes. Propidium iodide, regulatable promoter. A regulatable promoteris one for which however, penetrates only bacteria with damaged membranes, transcription of an operatively linked nucleic acid sequence is competing with the SYTO 9 stain for nucleic acid binding induced by an agent Such as an inducer, or repressed by an sites when both dyes are present. When mixed in recom agent such as a repressor, or induced or repressed by a con mended proportions, SYTO 9 stain and propidium iodide dition Such as heat. A variety of regulatable promoters that produce green fluorescent staining of bacteria with intact cell function in bacterial cells are known in the art. Typical induc membranes and red fluorescent staining of bacteria with dam ers and repressors include Small molecules such as arabinose, aged membranes. The background remains virtually nonfluo isopropyl-beta-thiogalactopyranoside (IPTG), tetracycline rescent. The ratio of green to red fluorescence intensities and Xylose. An exemplary regulatable promoter is the arabi therefore provides a quantitative index of bacterial viability. nose-regulated P. promoter (L-M. Guzman, et al., J. Bac A fluorometer can be used to detect the fluorescence intensi teriology, 177: 4121-4130, 1995), Other inducible/repress ties. Another suitable assay for determining the number of ible single or multi-plasmid bacterial expression systems are viable bacterial cells in culture is based on quantitation of the based on the lac promoter, hybrid lac promoter, or the tetra ATP present. ATP is an indicator of metabolically active cells. cycline response element, and variants thereof. Examples of The BacTiter-GloTM Assay (Promega, Madison, Wis.) is a such expression systems include the PLtetO-1 (tetracycline commercially available assay based on this principle that inducible) system & PLlacO-1 (IPTG-inducible) system (R. US 2009/0264.342 A1 Oct. 22, 2009

Lutz & H. Bujard, Nucleic Acids Research, 25: 1203-1210, 0180. The invention provides a first set of target genes 1997). See also U.S. Pat. Nos. 4,952,496 and 6,436,694. consisting of genes whose functional inactivation resulted in 0172 Methods for generating strains suitable for use in the strains that either (i) failed to grow in the presence of a methods of the invention are known in the art. For example, Sublethal, non-inhibitory concentration of a quinolone anti recombinant nucleic acid constructs can be made and intro biotic; (ii) failed to survive a period of exposure to a lethal duced into bacterial cells as described, e.g., in Current Pro concentration of a quinolone antibiotic; or (iii) both failed to tocols in Molecular Biology, Supra, and Sambrook, Supra. grow in the presence of a Sublethal, non-inhibitory concen Methods for performing gene disruption in various bacteria tration of a quinolone antibiotic and failed to Survive a period Such as E. coli and S. pneumoniae are described, e.g., in exposure to a lethal concentration of a quinolone antibiotic. (Datsenko et al., 2000, Proc. Natl. Acad. Sci. USA97: 6640 The set of target genes includes recA, recB, recC, recC, recN, 5: Murphy, 1998, J. Bacteriol. 180: 2063-71; and Winans et ruVA, ruvB, ruvC, uvrD. xerD, XseA, XseB, pinO, dnaT, priA. al., J. Bacteriol 161: 1219–21; Link et al., 1997, J. Bacteriol. polA, thy A, b1440, fabH, tolC, acrA, acrB, ompC, dkSA, fis, 179: 6228-37; Arigoni et al., 1998, Nat. Biotechnol. 16(9): dapF, fliN, jws303, yhfT, yogC, yeS, res.A, dapF, ygoC, 851-6: Akerley et al., Proc. Natl. Acad. Sci USA95: 8927-32: rimK, and yicn. The invention further provides subsets of Lee et al., 1999 Appl. Environ. Microbiol. 65: 1883-90 and these genes. A first Subset consists of genes whose deletion Lee et al., 1998, Appl. Environ Microbiol. 64: 4796-802). resulted in cells that both failed to grow in the presence of a Methods for obtaining chromosomal integration of nucleic Sublethal, non-inhibitory concentration of a quinolone anti acid constructs are also known, and have been described, for biotic and failed to survive a period exposure to a lethal example in, Gerhardt et al., supra; Link et al., 1997, J. Bac concentration of a quinolone antibiotic. A second Subset con teriol. 179: 6228-37; and Metcalf et al., 1996, Plasmid 35: sists of genes whose deletion resulted in cells that failed to 1-13. grow in the presence of a Sublethal, non-inhibitory concen tration of each of two different quinolone antibiotics. A third Quinolone Potentiator Target Genes Subset consists of genes for which homologs exist in repre sentative Gram positive and Gram negative organisms (e.g., 0173 As described in Example 1, a first set of target genes E. coli and S. aureus). The invention specifically includes was identified by exposing members of a collection of bacte each possible Subset of the aforementioned target genes, rial Strains to a Sublethal concentration of a quinolone. Each including Subsets in which any one or more of the target genes strain had a genetic alteration resulting in functional inacti is explicitly excluded. In some embodiments, genes that vation of a different gene. The sublethal concentration was encode a subunit of an efflux pump, transporter, or porin are selected so that it did not significantly impair bacterial growth excluded. In some embodiments, genes that encode a tran but was sufficient to cause alterations in gene expression, as determined by microarray analysis, thereby confirming that scription factor or subunit thereofare excluded. the concentration was sufficient to cause alterations in bacte 0181. The present invention provides a subset of qui rial physiology. Bacterial Strains that failed to grow in the nolone potentiator target genes including dapF, fabH, fis, fliN. presence of the quinolone were identified. A second screen jw$303, priA, recA, recB, recC, recC, resA, ruVA, ruvB, was performed (Example 2), in which the ability of the strains ruvC, ruvC, uvrD, yCS, ydfL, yhfT and ydgC. to Survive following exposure to a lethal concentration of a 0182. In addition to the quinolone potentiator target genes quinolone antibiotic was assessed. identified in the screens, the invention also contemplates that 0.174. In certain embodiments of the invention the qui other genes functioning in the same biological or biochemical nolone potentiator target gene encodes a DNA metabolizing pathway as any one or more of these genes are also potential enzyme, by which is meant any polypeptide that chemically targets for quinolone potentiation. Specific quinolone poten modifies, synthesizes, degrades, cleaves, processes, and/or tiator target genes and biological pathways of particular inter unwinds or otherwise alters the topology of DNA. Examples est are described further below. include DNA-dependent DNA polymerases, endonucleases, 0183 In certain embodiments of the invention the qui exonucleases, helicases, primases, recombinases, etc. nolone potentiator target gene encodes a helicase, helicase 0.175. In embodiments of particular interest the quinolone Subunit, or helicase-like polypeptide. Examples of such genes potentiator target gene encodes a helicase, helicase subunit, identified in the inventive screen include recA, recB, recC. recC, ruVA, ruvB, ruvC, priA. dnaT, pinO and uvrD. Heli or a helicase-like polypeptide. cases are a group of enzymes that catalyze the separation of 0176). In certain embodiments of the invention the qui duplex nucleic acids into single strands in an ATP dependent nolone potentiator target gene encodes an enzyme that plays process, hence allowing other critical cellular processes to a role in the microbial fatty acid synthesis pathway. take place. These enzymes have been classified into 5 major 0177. In certain embodiments of the invention the qui groups that share a conserved “helicase' structural module. nolone potentiator target gene encodes a transcription factor. This module is related to the structure of the bacterial RecA In other embodiments of the invention the quinolone poten protein and is recognized in the art. It contains five contiguous tiator target gene does not encode a transcription factor. For parallel B strands and their tandem C. helices. Within this example, in certain embodiments of the invention the gene is module there are a number of motifs recognized in the art as not dkSA or a homolog thereof. characteristic of helicases including the Walker A and B 0178. In certain embodiments of the invention functional motifs, which are involved in nucleotide interactions, e.g., inactivation of the quinolone potentiator target gene reduces with ATP. See, e.g., Story and Steitz, 1995; Caruthers, 2002, the MIC of a quinolone antibiotic by at least a factor of 5, 10, and references therein. For purposes of this invention any 50, 100, 500, 10, 5x10, 10, 5x10, or more. polypeptide that possesses the characteristic RecA-like core 0179 A target gene of the present invention may have one structure as described in Caruthers, 2002, and catalyzes the or more of the features described herein, provided that such separation of duplex nucleic acids into single strands in an features are not mutually inconsistent. ATP-dependent manner will be termed “helicases” while US 2009/0264.342 A1 Oct. 22, 2009

polypeptides that possess the characteristic RecA-like struc decrease the ability of mutant cells ability to survive in ture but that do not possess the aforementioned activity will response to UV radiation treatment. A domain in RecA that be termed “helicase-like'. likely forms part of the co-protease Substrate binding site has 0184 The RecA protein is a key sensor and activator in also been identified (Nastri et al., 1997). According to the response to DNA damage and plays a major role in inducing invention this information is used to select portions of RecA the SOS response pathway following Such damage. It is for computational screening against Small molecule libraries. known that RecA is also involved in other cellular processes 0187. Known inhibitors of RecA that are of use in the in addition to recombination and DNA damage repair. A invention include a variety of synthetic analogs of ATP and fundamental event in both homologous recombination and ADP as well as a variety of transition metals, e.g., Zinc(II)and SOS response induction is the formation of a RecA-ssDNA copper(II) (Lee, A M and Singleton, F., 2004). ATP nucleoprotein filament. In this conformation RecA acts as both a recombinase and co-protease. In the latter function 0188 The RecBCD enzyme is a complex helicase and it activates the SOS response by cleaving the Lex A repressor nuclease (referred to as exonuclease V in E. coli) involved in protein, which results in the induction of genes that are major pathways of homologous recombination and DNA repressed by Lex A under normal conditions. Over 30 SOS repair; it is also involved in loading RecA onto the DNA end genes, and Umul), a Sub-unit of polymerase IV, also involved (Singleton et al., 2004). It had been shown that survival after in the SOS response are induced (Courcelle and Hanawalt, double stranded DNA breakage depends on RecA and 2003; Sutton et al., 2000). Another fundamental role of RecA RecBCD, and that subsequent events can proceed via either of is to maintain the integrity of the genetic material. The bind two pathways, one dependent on the RuvABC Holliday junc ing of RecA to single-stranded DNA regions that block rep tion resolvase and the other on RecC helicase (from Med lication forks serves as the sensor that replication is blocked dows et al., 2004). Reversed forks are reset by the action of and maintains the integrity of the replication fork itself until RecBC on the DNA double-strand end, and in the absence of replication can resume (Courcelle, 2003). Thus one biologi RecBC chromosomes are linearized by the Holliday junction cal pathway of particular interest herein is recovery of resolvase RuvABC. arrested DNA replication forks. The invention implicates (0189 The UvrD helicase may be essential for RuvABC genes whose expression products play a role in this pathway dependent chromosome linearization in E. coli polymerase as quinolone potentiator target genes. The invention impli III mutants. UvrD also participates in replication fork reversal cates genes whose expression and/or activity is upregulated in E. coli (Flores et al., 2004). UvrD is involved in DNA lesion by RecA as quinolone potentiator target genes. and mismatch repair. Mutations in urVD can cause an increase 0185. The RecA protein is highly conserved and is cross in the frequency of homologous recombination. UVrD can species functional. For example, recA homologs from Yers destroy recombination intermediates made by RecA and can inia pestis, Bacillus anthracis and M. tuberculosis have been dislodge RecA from ssDNA (Morel et al., 1993; Veaute et al., shown to complement the E. coli recA- mutation (Suchkov 2005). The removal of RecA or RecA- made structure by and Mishankin, 1989: Ko et al., 2002: Nair and Steyn, 1991). UvrD hence allows replication fork reversal and proper rep Thus inhibitors of RecA identified using the RecA protein lication restart (Flores et al., 2005; Veaute et al., 2005). from one species would be expected to potentiate quinolone 0190. Fis is a 98 kD homodimeric protein that interacts activity in a wide variety of bacteria. with DNA, being involved in activities such as bending, loop 0186 The RecA protein has many functional features that ing and condensation of DNA substrates. Fis is involved in present points of intervention to inhibit its activity in accor DNA recombination, transcription control as either a repres dance with the present invention. Multiple alignment of the sor or activator, in DNA replication, chromosome replication sequence reveals a canonical structure of RecA-like proteins and cell division. Fis as a repressor notably negatively regu consisting of distinct segments or motifs (FIG. 1). These lates the promoter responsible for its transcription as well as segments or modules are highly conserved and have been those of the gyra and gyrB genes (Walker et al., 1999; assigned functional roles based on genetic, biochemical and Schneider et al., 1999). structural studies. Such modules are involved in DNA dam 0191 Exonucleases are enzymes that cleave nucleotides age recognition and binding, monomer interaction, filament sequentially from the free ends of a nucleic acid (DNA or formation, helicase motifs, ATP binding and hydrolysis, RNA). They have diverse modes of action and are involved in recombination, replication and co-protease activity. Muta many DNA metabolism processes. This is reflected by the tional studies have identified residues that are critical to these presence of multiple exonuclease encoding genes in the processes. For example the Gly 157 change resulted in a con genome of most organisms. The exonuclease VII of E. coli stitutive co-protease form of RecA and results in a lower degrades single-stranded DNA bidirectionally and proces Survival in response to UV treatment, a phenotype itself asso sively. It is composed of two subunits encoded by the XseA ciated to a low recombination competent form of RecA. The and xseB genes (Chase and Richardson, 1977; Chase et al., present invention makes use of this information to precisely 1986). It requires a single-stranded DNA terminus to initiate map regions of RecA to be targeted for compound discovery, hydrolysis, this unique Substrate specificity makes it an ideal e.g., using computational approaches (see below). For agent for various modifications of DNA termini. example, RecX (also called OraA) is an inhibitor of RecA for (0192. The role of RecN, an ATPase and member of the both recombinase and co-protease activities (Stohl et al., SMC (structural maintenance of chromosome) protein fam 2003). RecX appears to inhibit the ATPase activity of RecA. ily, is unclear. A recent study in Bacillus proposes that both Genetic and biochemical evidence identifies sites of interac RecA and RecN constitute or are part of a protein assembly tion between RecA and Lex A, suggesting that amino acids at that accepts incoming ssDNA at a cell pole, where it is pre positions 67, 154-157, 229 and 243 are responsible at least in pared for recombination with the chromosome (Kidane and part for the binding to Lex A (VanLook et al., 2003a). Amino Graumannn, 2005). The same authors showed that RecN, like acid changes at positions 122-123 and 150-161 dramatically RecA, is an ATP dependent ssDNA binding protein. US 2009/0264.342 A1 Oct. 22, 2009 20

0193 Beta-ketoacyl-acyl carrier protein (ACP) synthase of the E. coli K-12 polypeptide and its homolog in a second III (KAS III, also called acetoacetyl-ACP synthase), encoded bacterial species. Column 7 (E VALUE) presents the expec by the fabH gene is thought to catalyze the first elongation tation value for a match between the E. coli K-12 polypeptide reaction of type II fatty acid synthesis in bacteria and plant and its homolog in a second bacterial species. Column 8 plastids. FabH is believed to conduct the major condensation (BIT SCORE) presents the score for a match between the E. reaction in the initiation of type II fatty acid biosynthesis in coli K-12 polypeptide and its homolog in a second bacterial both Gram-positive and Gram-negative bacteria” (Lai and species. Cronan, 2003). Components of this biosynthetic pathway (0195 One of ordinary skill in the art will readily be able to have been identified as attractive targets for the development locate the nucleotide sequence of the genes that encode these of new antibacterial agents. FabH, beta-ketoacyl-ACP syn proteins using publicly available databases. Although the tar thase III, is a particularly attractive target, since it is central to get genes identified herein were identified in a laboratory the initiation of fatty acid biosynthesis and is highly con strain of E. coli, the invention encompasses corresponding served among Gram positive and negative bacteria. Small genes (homologs) in other bacterial species and/or strains. molecules that inhibit FabH enzymatic activity have the The expression products of the homologs preferably have the potential to be candidates within a novel class of selective, same or similar functional activity and will typically display nontoxic, broad-spectrum antibacterials. Using crystallo a degree of identity with the E. coli polypeptides. graphic structural information on these highly conserved 0196) Given the availability of accession numbers and active sites and structure based drug design principles, a public databases, it is considered unnecessary to provide benzoylaminobenzoic acid series of compounds was devel sequences for each of the target genes or their encoded oped as potent inhibitors of FabH. This inhibitor class dem polypeptides herein. Furthermore, it will be appreciated that onstrates strong antibacterial activity against Gram-positive minor differences in sequence of the same gene and, in some and selected Gram-negative organisms. (Nie et al., 2005). cases, the protein, may exist in different bacterial isolates, However, the utility of inhibitors of FabH (or inhibitors of even within a single species or strain. The invention encom other enzymes in the fatty acid synthesis pathway) as poten passes all such variants. FIG. 13 displays amino acid tiators of quinolone antibiotics has not heretofore been rec sequences and accession numbers for the RecA protein from ognized. The present invention encompasses the use of an 3 bacterial isolates. The E. coli RecA sequence was used as a inhibitor of bacterial fatty acid synthesis, e.g., an inhibitor of query sequence to identify the homologs in the otherbacteria. FabH, in combination with a quinolone antibiotic. The inhibi 0197) A variety of methods to identify homologs of target tor offatty acid synthesis may be employed at a concentration genes are known in the art and include both database search or dose below that which would be employed were it to be ing using either the nucleotide sequence of a target gene or the used as a single agent or may be employed against bacteria amino acid sequence of an encoded protein. Since many that are resistant to the effects of the inhibitor at clinically bacterial genomes have been fully sequenced and are publicly tolerated doses. available, the E. coli sequence can be used to generate a 0.194. A set of target genes provided by the present inven database query to identify homologs. Either general data tion is listed in Table 1 (FIG. 12). Both the E. coli gene and bases or organism-specific databases can be searched. The homologs in a variety of other bacterial species are listed. complete genome sequence of hundreds of microbes are Each E. coli gene is identified by its Gene ID (left column). available at the web site www.ncbi.nlm.nih.gov/genomes/ Each polypeptide encoded by a target gene is identified by the lproks.cgi, which provides links to the sequences. Homologs GenBank GI number and an accession number of the amino can be readily identified by one of ordinary skill in the art acid sequence of the polypeptide encoded by the gene. Most using programs such as BLASTN, BLASTP gapped BLAST, of the accession numbers are from GenBank (gb); some are etc., which are available on the web site of the NCBI. Default from the UniProt database at /www.ebi.uniprot.org (emb1). parameters can be used to perform the search. The overall In the case of Corynebacterium, the accession number used is percent identity may be as low as 20%. For purposes of the the one assigned by the Sanger Center, which provides the C. present invention any bacterial gene that encodes a bacterial diptheriae sequence at www.sanger.ac.uk/Projects/C diph polypeptide that is at least 20% identical to a polypeptide theriae/). The contents of the columns in Table 1 are as fol encoded by a target gene identified herein (e.g., a target gene lows: Column 1 (GENE ID ECOLI) presents the Gene ID of identified in Table 1) over the entire length of either polypep an E. coli target gene. Column 2 (ECOCYC ID ECOLI) tide is considered to also be a target gene, and the polypeptide presents the Ecocyc ID of the E. coli target gene. Ecocyc is considered to be a target polypeptide. However, polypep (Encyclopedia of Escherichia coli K12 Genes and Metabo tides having a lower percent identity are also encompassed. lism) is a bioinformatics database that describes the genome Furthermore, a target gene may display homology only or and the biochemical machinery of E. coli K-12 MG 1655 and primarily over one or more portions of the sequence. For is found at ecocyc.org/background.shtml. Column 3 (K12 example, a homolog may be at least 50%, 60%, 70%, 80%, or GENE) presents the name of the gene in E. coli. Column 4 90% identical across one or more portions having a length of (SUBJECT ORGANISM) presents the name of the bacte about 25, 50, 100, 150, or 200 amino acids. A homolog may rium whose genome was searched to identify a homolog of be highly conserved or conform to a consensus sequence the E. coli gene. Column 5 (SUBJECT ID) presents the GI across a plurality of relatively short motifs (e.g., about 10-30 number and accession number of a gene that was identified as amino acids). A sequence alignment program may generate a being homologous to the E. coli gene named in the same score S and an “expectation value” (E value) for each column. Note that this column provides the GI number and sequence identified in a comparison. The E value represents accession numbers of the E. coli genes themselves, as found the number of different alignments with scores equivalent to in Strain K-12. For example, the GI number and accession or better than Sthat are expected to occur in a database search number for E. coli K-12 AcrB are 1786667 and AAC73564.1, by chance. The lower the E value, the more significant the respectively. Column 6 (% IDENT) presents the '% identity score. In general, if the likelihood that a polypeptide would be US 2009/0264.342 A1 Oct. 22, 2009

identified as a “hit' when a polypeptide encoded by a target rial strains that failed to grow and/or survive in the presence of gene identified herein is used to query a database is less than the cyclic peptide were identified. The set of target genes or equal to 10, or preferably less than or equal to 10, the includes agaAk, atp A, atpF, atpH, bglF, cysE, cysI, fepC, gene that encodes the polypeptide is considered a target gene, fepD, frvR, guaA, guaB, hof F, hsdS, iscS, JW4016, JW5075, and the polypeptide is a target polypeptide. JW5227, JW5257, JW5360, kdgK, lip A, lysA, malG, mbh A, mdoG, Nei, nmpC, nudH, pdxH, phn B. phn L. phnO, pnuC, Aminoglycoside Potentiator Target Genes potE, pshM, ptsA, rhaT. rpiA, resA, Sbp, speA. Such3, SugE, tdcE, tdcG, tolC, trXA, ubiE, ubiH, ubiX, Xni, ybby, yefM, 0198 Using the same method as described in the case of yde J, yeeY, yfeT, ygaA, ygf7, yhdX, yhell, yheM, yiaY. quinolones, the present Applicants have identified a set of yidK, yihV, ybn, yick, yjcz, yn D, ydeC., yaiH, and yrfA. target genes by exposing members of a collection of bacterial strains to a Sublethal concentration of an aminoglycoside (see Example 34). Bacterial strains that failed to grow and/or PotentiatorTarget Genes Common to Several Classes of Anti survive in the presence of the aminoglycoside were identified. biotics The set of target genes includes aceE, ara, asp.A, atp A, atpF. 0204. In certain embodiments, the present invention pro atpH, brnO, cydB. degP. dnaT, fepD, folP. guaB, Hfq, iscS, vides Subsets of target genes that are potentiator target genes JW5360, lip A, lpdA, nuoB, nuo.J., nuoL, osmB, pdxH. pnuC, for more than one class of antibiotics (e.g., 2 or more than 2 priA, priB, rbfA, recA, rfal), rpm.J. rpoD, res.A, ruvC, schC, classes of antibiotics). In certain embodiments, the present seqA, setA, sucB, surA, tar, tatE, tauB, trimu, ubiH, ubiX. invention provides Subsets of target genes that are potentiator xerC., yaal J, yefM, yeO, ygf/, ygiH, yhe, yheM, yheN, target genes for at least two antibiotics (from the same class of yicG, yidL, yieT, yiO, and yi.Y. antibiotics or from different classes of antibiotics). 0199 The present invention also provides subset of ami 0205 For example, one of such subsets comprises priA, noglycoside potentiator target genes. A Subset of target genes recA, res.A, and ruvC, which have been found to be common is provided that includes recA seqA, and atp A, atpF, atpH. to the classes of aminoglycosides and quinolones. Another 0200. The seqA gene negatively modulates replicationini example of Subset of target genes comprises such3., ubiH and tiation at the E. coli origin, oriC. seqA is also essential for ubiX, yefM, ygf7, yheL and yheM, atpA, atpF, and atpH, sequestration, which acts at oriC and the dnaA promoter to iscS, JW5360, guaB, lip.A, pdxH, res.A and pnuC, which have ensure that replication initiation occurs exactly once per chro been found to be common to the classes of aminoglycosides mosome per cell cycle. Known secA inhibitors include and cyclic peptide antibiotics. Another Subset of target genes sodium azide, cerulenin, and other compounds such as those comprises ResA and RecA. described in L. E. Alksne et al., Antimicrob. Agents Chemother, 2000, 44: 1418-1427. Other Methods 0201 An ATP synthase is a general term for an enzyme that can synthesize adenosine triphosphate (ATP) from 0206. Other methods for identifying target genes in other adenosine diphosphate (ADP) and inorganic phosphate by bacterial species include functional complementation and utilizing some form of energy. ATP is formed by proton low stringency nucleic acid hybridization using the E. coli conducting, membrane-bound ATPase. ATPase is a multi gene sequence or a portion thereof (or using the nucleic acid component enzyme complex consisting of two main compo sequence that encodes a homolog in a different bacterial nents: F1 and F0. 1 is on the inner surface of the membrane species). Functional complementation could be performed, and is the catalytic center; F1 consists of nine polypeptide for example, by transforming a first bacterial strain in which chain subunits of five different types. F0 is embedded within a gene is functionally inactivated with a library of expression the membrane and forms the membrane proton channel. In E. vectors containing genes from a second bacterial species or coli, FO consists of three subunits (A=atpB, B atpF, and strain in which it is desired to identify a homolog. Transfor C=atpE); F1 consists of five subunits (alpha=atpA. mants are selected and are placed under conditions in which beta=atpl), gamma=atpG, delta-atpH, epsilon=atpC). the function of the gene product is required for survival. For example, the transformants may be cultured in the presence of 0202 Known inhibitors of ATP synthase include meflo a quinolone antibiotic at a concentration previously deter quine. Mefloquine (also called mefloquine hydrochloride or mined to be lethal in the absence of a functional gene but Lariam R. Roche Pharmaceuticals) is 4-quinolinemethanol sublethal if the gene is functionally active. The expression derivative. The exact mechanism of action of mefloquine is vector is isolated and the gene it encodes is identified, e.g., unknown, although atpA as been reported to be its target. The sequenced. Low Stringency hybridization can be performed antibiotics venturicidin, oligomycin and ossamycin have been investigated as potential inhibitors of the E. coli H+AT using genomic DNA or cDNA from a bacterium in which it is Pase. It was found that venturicidin strongly inhibited ATP desired to identify a homolog of a target gene using methods driven proton transport and ATP hydrolysis, while oligomy described, e.g., in Sambrook, Supra and well known in the art. cin weakly inhibited there functions. Inhibition of hite 0207 As noted above, the target gene products used in the H+ATPase by venturicidin and oligomycin was correlated methods of the invention include those gene products (e.g. with inhibition of FO-mediated proton transport. RNA or proteins) that are encoded by the target genes iden tified in FIG. 12, FIG. 29, FIG.32 and any homologs thereof that exist in other bacterial species or strains. Alternately or Peptide Antibiotic Potentiator Target Genes additionally, the methods of the invention also use and 0203 Using the same method as described in the case of encompass proteins and polypeptides that represent function quinolones and aminoglycosides, the present Applicants have ally equivalent gene products. For example, Such functionally identified a set of target genes by exposing members of a equivalent gene products include, but are not limited to, natu collection of bacterial strains to a sublethal concentration of a ral variants of the polypeptides having an amino acid cyclic peptide antibiotic (colistin) (see Example 36). Bacte sequences identified in FIG. 13. Functionally equivalent tar US 2009/0264.342 A1 Oct. 22, 2009 22 get gene expression products can contain, e.g., deletions, are dispensed into individual vessels, e.g., wells in a multi additions or substitutions of amino acid residues within the well plate. One or more test agents is added to each well, and amino acid sequences encoded by the target gene sequences information about one or more parameters, e.g., cell growth, described above, but which result in a silent change, thus is gathered. Plate readers can be used to detect signals such as producing a functionally equivalent target gene product. optical density, colorimetric or fluorescent readouts, etc. The Alternately or additionally, amino acid Substitutions can be readout may be indicative of growth of the cells (e.g., cell made on the basis of similarity in polarity, charge, solubility, density). However, a variety of other indicators may be hydrophobicity, hydrophilicity and/or the amphipathic nature assessed. of the residues involved. For example, nonpolar (i.e., hydro 0211. In general, the assays will make use of a biological phobic) amino acid residues can include alanine (Ala or A), system, which may be a cell-free or cell based system. Cell leucine (Leu or L), isoleucine (Ile or I), valine (Val or V), free assays refer to assays that are performed using a biologi proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp cal system that does not include intact cells but includes one or W) and methionine (Met or M); polar neutral amino acid or more biological macromolecules, e.g., protein(s) and/or residues can include glycine (Gly or G), serine (Ser or S), nucleic acid(s). Cell-based assays employ intact cells. The threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), cells may be maintained in liquid culture or on a solid or asparagine (ASn or N) and glutamine (Gin or Q); positively semi-solid culture medium. The biological system may com charged (i.e., basic) amino acid residues can include arginine prise purified or partially purified nucleic acids and/or pro (Arg or R), lysine (Lys or K) and histidine (His or H); and teins, cell lysates, cellular fractions such as cell membrane negatively charged (i.e., acidic) amino acid residues can preparations, cells, etc. The biological macromolecules may include aspartic acid (Asp or D) and glutamic acid (Glu or E). be chemically synthesized, produced by recombinant DNA 0208 A functionally equivalent polypeptide, as used technology, obtained from natural Sources (e.g., from cells, herein, refers to a polypeptide that exhibits a substantially etc.) similar in vivo activity as the polypeptide to which it is func 0212 Briefly, a “recombinant protein’ refers to a protein tionally equivalent. When used in an assay Such as those which is produced by recombinant DNA techniques, wherein described below, the term “functionally equivalent encom generally, DNA encoding a polypeptide is inserted into a passes peptides or polypeptides that are capable of interacting suitable expression vector which is in turn introduced into a with other cellular or extracellular molecules in a manner host cell to produce the recombinant protein. Vectors may be Substantially similar to the way in which the corresponding introduced into cells using any of a variety of suitable meth portion of the target gene product would interact with Such ods. Methods for introducing nucleic acids into cells are well other molecules. Functionally equivalent target gene prod known in the art. One of ordinary skill will be able to select ucts are typically about the same size oridentical in size to the appropriate cells for expression and an appropriate method polypeptide with which they are functionally equivalent and (e.g. calcium phosphate or lipid-mediated transfection, elec will typically have similar physicochemical properties Such troporation, bacterial or fungal transformation, etc.) for intro as pl. The invention explicitly includes any polypeptide hav ducing a nucleic acid into the cells, taking into consideration ing at least 80% identity, e.g., 90% identity, or more to any of the cell type, etc. Suitable host cells for producing recombi the polypeptides identified by accession number in FIG. 12, nant proteins include bacteria, yeast, insect cells, mammalian FIG. 29, FIG. 32 and nucleic acids encoding such polypep cells, etc. The biological macromolecules typically include tides, whether or not such polypeptides or nucleic acids are an expression product of a target gene or a functional equiva found in nature. lent thereof. Alternately the biological macromolecule can be a variant of a polypeptide encoded by a target gene. Certain IV. Screening Assays to Identify Agents that variants are specifically engineered, e.g., by point mutation, Potentiate Antibiotic Activity deletion, truncation, etc., to alter a biological activity of a 0209. The invention provides screens that may be used to protein. For example, constitutively active forms can be pro identify agents that modulate the expression of any antibiotic duced. Inactive forms can be produced, which may act potentiator target gene or that modulate the activity of an antagonistically to a naturally occurring protein, e.g., by com expression product of an antibiotic potentiator target gene. peting for binding to another component in a pathway. In Certain preferred screens identify agents that inhibit expres certain embodiments an active fragment or domain of a pro sion of the gene or inhibit one or more activities of a polypep tein is used. Typically the active fragment or domain com tide encoded by the gene. “Inhibit, when used in connection prises at least 50 amino acids of the complete polypeptide. with an agent that inhibits an activity of a protein, e.g., enzy Certain fragments retain at least one biological activity of the matic activity, means to directly or indirectly reduce the activ complete polypeptide, e.g., enzymatic activity, inhibitory ity of the protein. Preferably an agent that inhibits a protein activity, binding activity to a substrate or to an interacting causes a decrease in maximum activity of at least 50% at a polypeptide, etc. Additional preferred biological macromol concentration of approximately 500 uM or less, preferably at ecules that may be included in a biological assay system a concentration of 200 uM or less, more preferably at a include Substrates for a target polypeptide. concentration of 100 uM or less, “Inhibit or interfere with 0213. The biological macromolecules may be modified can encompass mechanisms such as causing a reduction in for use in the assays. For example, a polypeptide may incor the amount of a cofactor or Substrate, etc., porate a heterologous sequence, e.g., an epitope tag such as a 0210. The screens may advantageously be performed GST, Myc, HA, FLAGTTM, maltose-binding domain, 6x-His using a high throughput format. High throughput screens or other metal binding moiety, etc. Fusion proteins in which a typically make use of microwell plates (e.g., 96-well, 384 detectable polypeptide is fused to a polypeptide encoded by a well, 1596-well, etc.) and may employ robotics for various target gene are also of use. Detectable polypeptides include steps such as liquid handling, compound dispensing, plate fluorescent proteins such as green fluorescent protein (GFP) manipulation, etc. For example cells, or populations of cells, and variants thereof. A number of enhanced versions of GFP US 2009/0264.342 A1 Oct. 22, 2009

(eGFP) have been derived by making alterations such as 0217 Reporter constructs are also of use to identify com conservative substitutions in the GFP coding sequence. Other pounds that inhibit the activity of a target gene that encodes a readily detectable markers that produce a fluorescent signal transcription factor or subunit thereof. A reporter construct include red, blue, yellow, cyan, and Sapphire fluorescent pro containing a coding sequence for a detectable marker can be teins, reef coral fluorescent protein, etc. A wide variety of fused to a regulatory region (e.g., a promoter) of gene known such markers is available commercially, e.g., from BD Bio to be regulated by the transcription factor. Activity of the Sciences (Clontech). Enzymatic markers include, e.g., B-ga transcription factor is assessed based on the detectable signal lactosidase, chloramphenical acetyltransferSase, alkaline generated by the marker. The ability of a test compound to phosphatase, horseradish peroxidase, etc. Additional readily inhibit transcription is assessed. Note that the precise bound detectable markers preferred in certain embodiments of the aries of a regulatory region need not be defined in order for it invention include luciferase derived from the firefly (Photinus to be usable in a reporter construct, provided that it contains pyralis) or the sea pansy (Renilla reniformis). Methods for Sufficient sequences to render transcription of the reporter detecting expression of any of these markers are well known responsive to the presence, absence, or amount of a particular in the art. For example, expression off-galactosidase can be transcription factor, repressor, inducer, or condition of inter quantified using a biochemical colorimetric detection assay est, etc. (Miller, 1972). Other markers are readily detectable based on 0218 Binding assays are ofuse to identify agents that bind their fluorescence or luminescence or based on their ability to directly to target molecules such as those encoded by the produce a fluorescent or luminescent signal upon cleaving a target genes of the invention. Screening for compounds that substrate. bind to a polypeptide of interest has traditionally been per 0214. Other reporters include enzymes that cleave a sub formed using a variety of in vitro techniques such as radioli strate, wherein the Substrate has a fluorescent moiety and a gand binding assays, photocrosslinking, and affinity chroma fluorescence quencher attached thereto. Cleavage separates tography. These assays are based on the principles of a the fluorescent moiety from the quencher, resulting in a competitive binding assay in which a radiolabeled ligand detectable increase in the fluorescent signal. The biological competes with an unlabeled ligand for binding to a target system may comprise one or more antibodies, agonists, polypeptide. The ability of a test agent to compete with a antagonists, etc., for any of the afore-mentioned proteins, known ligand is assessed by measuring the binding of the inhibitors of transcription, inhibitors of translation, etc. labeled known ligand in the presence of a test agent. A 0215 Reporter constructs are also of use in certain cell decrease in the binding of the labeled known ligand indicates based assays that are designed to identify agents that modu that the test agent binds to the target molecule. Whether the late expression of a target gene. For example, a reporter test agent inhibits or activates the target is then determined construct can include the promoter of a target gene, operably using other methods, e.g., enzymatic assays, cell-based linked to a nucleic acid sequence that encodes a detectable assays, etc. When performing Such assays, any known ligand marker. An expression vector containing the reporter con or substrate for the various target polypeptide can be used. For struct is used to transform cells. Expression results in a detect example, ATP or an analog thereof could be used to identify able signal. Compounds are applied to the cells (in individual compounds that bind to an ATPase such as RecA or any of the vessels or wells), and expression is detected. Compounds helicases or helicase-like target polypeptides described whose presence results in altered expression are identified. herein. Methods for performing radioligand binding assays Optionally the screen can be performed in the presence of an are well known in the art, and a number of kits are commer antibiotic, e.g., a quinolone. cially available. See, e.g., Rahman, A., et al., Bioassay Tech 0216. In one embodiment the invention provides an assay niques for Drug Development, Supra. Examples of commer system comprising RecA protein, a reporter construct con cially available kits include the FlashPlateTM system taining a recA regulatory element (e.g., the recA promoter or (DuPont-NEN), the “Scintillation ProximityTM assay (Am a functional portion thereof) operably linked to a nucleic acid ersham), ScintiStripTM plates (Wallac), etc. Ligands labeled sequence that encodes a detectable marker, and a test com with nonradioactive detectable moieties, e.g., fluorescent pound. Typically the assay is a cell-based assay, and the moieties, may alternatively be used. reporter construct is introduced into cells that express RecA. 0219 Methods based on fluorescence polarization and However, cell-free transcription/translation systems could Surface plasmon resonance are increasingly employed to also be used. Transcription of the nucleic acid sequence is detect molecular interactions such as that between a polypep responsive to RecA. Such that higher transcription (and then tide and a potential modulator. The phenomenon of Surface translation) occurs in the presence of active RecA. Thus test plasmon resonance is used in Biacore systems (available from agents that inhibit RecA result in decreased fluorescence, Biacore International AB, Neuchatel, Switzerland). Such sys allowing the identification of RecA inhibitors. The invention tems can be used to detect interactions between a protein of further provides a method of identifying a compound com interest and a test agent. As described by Biacore, detection prising providing an assay system comprising RecA protein, using Surface plasmon resonance sensors works as follows: a reporter construct containing a recA regulatory element As molecules are immobilized on a sensor Surface the refrac (e.g., the recA promoter or a functional portion thereof) oper tive index at the interface between the surface and a solution ably linked to a nucleic acid sequence that encodes a detect flowing over the Surface changes, altering the angle at which able marker, and a test compound and determining whether reduced-intensity polarized light is reflected from a Support the compound inhibits expression of the detectable marker. ing glass plane. The change in angle, caused by binding or Optionally the assay is a cell-based assay, in which bacterial dissociation of molecules from the sensor Surface, is propor cells express RecA and contain the RecA responsive reporter tional to the mass of bound material and is recorded in a construct. Optionally the method includes a step of subjecting sensorgram. When sample is passed over the sensor Surface, the cells to a condition that induces RecA synthesis and/or the sensorgram shows an increasing response as molecules activation. interact. The response remains constant if the interaction US 2009/0264.342 A1 Oct. 22, 2009 24 reaches equilibrium. When sample is replaced by buffer, the transcriptional activator, e.g., one fusion protein contains a response decreases as the interaction partners dissociate. DNA binding domain and the other fusion protein contains a 0220 Many of the polypeptides encoded by target genes transactivation domain. Interaction between the fusion pro are part of multisubunit proteins or macromolecular com teins reconstitutes a transcriptional activator, leading to plexes. Agents that disrupt interactions between a target expression of a reporter construct that contains a binding site polypeptide and one or more of the polypeptides (or nucleic for the transcriptional activator fused to a sequence that acids) to which it binds in the bacterial cell effectively inhibit encodes a reporter protein. A variety of suitable reporter the activity of the polypeptide and are of use as antibiotic proteins are known in the art. Expression of the reporter potentiating agents. Formation of complexes between a pro construct is detected and provides an indication that interac tein of interestandone or more other proteins may be detected tion has occurred. The chimeric gene encoding the fusion using a number of methods well known in the art and can be protein containing the interacting protein can then be isolated, performed in either cell-free or cell-based systems. Methods allowing identification of the interacting protein. Numerous for detection include immunological methods, chromato variants and improvements on this method have been made graphic methods, etc. Frequently it will be desirable to detect since its initial description and can be employed in the present ably label the polypeptide of interest, e.g., with a fluorescent invention. For example, transcriptional repression domains or radioactive label, and/or to epitope tag the protein of inter could also be used, wherein the readout would be reduced est. Compounds are then screened to determine whether they expression of the reporter construct if an interaction occurs. prevent complex formation and/or disrupt complexes that 0223) The two-hybrid system can be used to identify have already formed. agents, e.g., Small molecules, that disrupt interaction of two 0221. In many of the assays, the protein of interest or a known polypeptides. In this approach, a transcriptional acti potential binding protein is immobilized, e.g., in a vessel Such vator is reconstituted as described above using first and sec as a microtiter plate or microfuge tube, to a chromatographic ond fusion proteins, each of which contains a domain of a matrix, etc. Immobilization may be accomplished using transcriptional activator and one of the known interacting crosslinking agents or antibodies or by biotinylating the pro polypeptides (or a portion thereof). Reconstitution of the tein and utilizing a vessel to which avidin is attached. In some transcriptional activator results in expression of a reporter embodiments a fusion protein comprising a protein of interest construct, which can be detected. A test agent is added to the and heterologous sequence comprising a binding domain biochemical system. If the test agent disrupts the interaction, (e.g., GST, 6x-His, maltose binding domain, etc.) is gener a decrease in expression of the reporter construct will be ated. After immobilizing the protein, the protein is contacted detected. with a potential interacting protein (either partially or fully 0224. Three hybrid screening assays are also of use in the purified, or in a cell lysate, etc.). After a period of incubation, present invention. These assays are useful for screening a wash is performed to remove unbound material. Complex chemical libraries, e.g., libraries of small molecules, to iden formation can be detected, e.g., using an antibody that binds tify agents that can bind to particular targets of interest. Meth to the potential interacting protein. The invention contem ods and reagents for performing three hybrid Screening plates use of antibodies that bind to a target polypeptide for assays are described in U.S. Pat. No. 5,928,868 and in Licitra use in the assays described herein. Methods for producing et al., Proc. Natl. Acad. Sci. USA 93: 12817-21 (1996). monoclonal or polyclonal antibodies are well known in the Briefly, the three hybrid assay involves the formation of a art. It is noted that the term “antibody' as used herein encom complex between a hybrid ligand and two hybrid proteins in passes antibody fragments, single chain antibodies, other pro which a portion of a component of the three hybrid complex teins that contain an antigen-binding domain, etc. Alternately, may be unknown. The unknown component can be either a the interacting protein may be detectably labeled (e.g., enzy small molecule that forms part of the hybrid ligand or forms matically, fluorescently, etc). The ability of an agent to pro part of one of the hybrid proteins. The three hybrid assay is mote or inhibit complex formation is assessed by allowing based on a similar concept to the two hybrid assay described complex formation to occur in the presence of the agent (or by above, i.e., formation of a complex (in this case a three com adding the agent following complex formation) and compar ponent complex) triggers the expression of a reporter gene. ing the extent of complex formation with that occurring in the Expression of the reporter gene is detected using a suitable absence of the agent. Proteins can also be subjected to various technique and indicates interaction of the members of the procedures that involve separation based on size. Complex complex. The unknown component is then identified. formation results in a detectable increase in size. Methods for 0225. In the context of the present invention the three detecting the increase in size include chromatography, gel hybrid assay can be used for any one or more of the following electrophoresis, etc. purposes: (i) determining the identity of a small molecule 0222. A widely used method for detecting protein-protein capable of direct binding to a known target polypeptide where interactions is the two-hybrid approach, which is described in the identified small molecule may be suitable as a modulator U.S. Pat. Nos. 5.283,173; 5.468,614; and 5,667,973. Briefly, of the activity of the target polypeptide or (ii) determining the the method is based on reconstituting a functional transcrip identity of a small molecule capable of binding competitively tional activator protein from two separate fusion proteins in a to a target polypeptide in the presence of a hybrid molecule so biological system, preferably in living cells in culture, as to inhibit the binding between the target and a second Small although in vitro biological systems other than intact cells, molecule that forms part of the hybrid molecule (e.g., the (optionally containing cellular constituents) could also be second Small molecule may be a known ligand for the target employed. This reconstitution makes use of chimeric genes molecule. which express fusion proteins. At least one of the fusion 0226 Cell-free assays based on the enzymatic activity of proteins contains a protein of interest or a portion thereof. The various target polypeptides of the present invention are also of other fusion protein contains a known or potential interacting use. For example, compounds can be screened to identify protein. Each fusion protein also contains a domain of a those that inhibit ATPase activity, helicase activity, exonu US 2009/0264.342 A1 Oct. 22, 2009 clease activity, etc. In one embodiment an assay system con Suboptimal Int protein activity can be enhanced by adding taining an isolated polypeptide having ATPase activity (e.g., increasing amount of Fis in the reaction. Compounds are RecA) is provided. The assay system also contains ATP and tested for their ability to prevent recombination when present reagents suitable for detecting ATP or for detecting products in an assay system containing Fis, Int, and an optionally formed by hydrolysis of ATP. A suitable assay is described in labeled nucleic acid substrate for recombination. For the Examples. In that assay, ATP remaining after a period of example, a labeled DNA substrate can be used, and substrate incubation of an ATPase (e.g., RecA) in the presence of ATP and products can be quantified, e.g., by separation using gel and, optionally, a test agent (potential inhibitor) is assessed. electrophoresis (Esposito and Gerard, 2003). The substrate ATP can be detected using a variety of methods known in the can be, e.g., fluorescently labeled, radioactively labeled, etc. art. In another embodimentapurified polypeptide (e.g., RecA The invention therefore encompasses an assay system com or any helicase or helicase-like polypeptide) is incubated with prising Fis, Int, a nucleic acid Substrate, and a test compound. 1 ug of DNA (Sigma D3287 or D8681) in 20 mM HEPES (pH The invention further encompasses a method of identifying a 7.6: 4-(2-hydroxyethyl)-1-piperazineethaneSulphonic acid), compound comprising providing an assay System containing 5 mM MgCl, 0.2-5 mM ATP 100 g of BSA (bovine serum albumin) per ml, 10% glycerol, 1 mM DTT (DL-dithiothrei Fis, Int, a nucleic acid substrate such as labeled DNA, and a tol) and, optionally, a test agent (potential inhibitor) in the test compound and assessing the ability of the compound to appropriate concentration at 37 degrees C. for 60 min. The inhibit recombination. A compound that inhibits recombina liberated inorganic phosphate is determined by colorimetry tion is identified as an inhibitor of Fis and is a candidate as described in "An improved assay for nanomole amounts of quinolone potentiating agent. inorganic phosphate’, 1979, P. A. Lanzetta, I.J. Alvarez, P. S. 0229. In another embodiment the ability of DNA binding Reinach and O. A. Candia, Analytical Biochemistry, 100:95 protein such as Fis or DkSA to bind DNA can be measured 97. The inhibition of the DNA-dependent ATPase activity using gel retardation assays (Esposito and Gerard, 2003; Choi may be calculated from the change in the absorption in the et al., 2005). The effect of a test compound on such binding is presence and absence of inhibitor. These ATPase assays are determined. A compound whose presence inhibits binding of only two of the numerous options which are available to the the DNA binding protein to DNA is identified as an inhibitor skilled person and are detailed, for example, in “High of binding and as a candidate quinolone potentiating agent. Throughput Screening Assay for Helicase Enzymes, 1998, 0230. In another embodiment a reporter assay is used to M. Sivaraja, H. Giordano and M. G. Peterson, Analytical identify compounds that inhibit Fis or DkSA. The invention Biochemistry, 265, 22-27; Berger MD, et al., Biochem Bio therefore encompasses an assay System comprising Fis or phys Res Commun. 2001; 286:1195-203, and Morrical SW, DkSA and a reporter construct operatively linked to a tran et al. Biochemistry. 1986; 25:1482-94. In certain embodi Scriptional regulatory sequence (e.g., a promoter) of a gene ments of the invention an agent identified using the inventive whose transcription is regulated at least in part by Fis or DkSA method inhibits ATPase activity of RecA but not of the related (e.g., an rRNA gene), and a test compound. The invention mammalian protein RadS1 (reviewed in Kawabata et al., further encompasses a method of identifying a compound 2005). comprising providing an assay system containing Fis or 0227. In another embodiment an assay to measure exonu DkSA, a reporter construct containing a regulatory region of a clease activity is used. Many methods for detecting and gene whose transcription is regulated at least in part by Fis or optionally measuring exonuclease activity are known in the DkSA, and a test compound and assessing the ability of the art. The assay system typically includes an isolated exonu compound to inhibit expression of the reporter. A compound clease polypeptide, a nucleic acid Substrate, and any appro that inhibits expression is identified as an inhibitor of Fis or priate cofactors, salts, buffers, etc. The specific assay and DkSA, respectively, and is a candidate quinolone potentiating nucleic acid Substrate selected may depend at least in part on agent. The invention encompasses Such an assay system and whether the exonuclease degrades double or single-stranded method of compound identification for any transcription fac nucleic acid, whether it is specific for RNA or DNA, whether tor or subunit thereof identified as being encoded by an anti it degrades in a 5' to 3' or 3' to 5' direction, etc. For example, biotic potentiator target gene. exonuclease activity can be measured using nucleic acids that 0231. Cell-based assays to identify agents that inhibit a incorporate detectably labeled nucleotides (e.g., radiola target gene of interest can also involve exposing cells that beled, fluorescently labeled, etc). Alternately, a nucleic acid express different amounts of a target gene to a Sublethal can incorporate a fluorescent reporter dye and a fluorescent concentration of an antibiotic. The cells are contacted with a quencher dye. While both the reporter and the quencher are in testagent. An agent that inhibits growth or Survival of the cells proximity with one another, fluorescence is quenched. How that express lower levels of the target gene to a greater extent ever, when the nucleic acid is degraded, the reporter and than it inhibits growth or survival of cells that express a higher quencher are no longer in proximity, and fluorescence is amount of the target gene is a potential inhibitor of the detected. This is assay, based on a similar principle to the polypeptide encoded by the target gene or a potential inhibitor widely used Taqman(R) assay for detecting PCR amplification, of expression of the target gene. In an exemplary embodiment is highly suitable for high throughput format. In another the ability of wildtype cells to grow or survive in the presence embodiment, an exonuclease is incubated with a nucleic acid of a Sublethal concentration of a quinolone and a test agent is Substrate for a period of time. Following incubation, gel elec compared with the ability of cells that overexpress the target trophoresis is performed and the integrity of the nucleic acid gene to Survive in the presence of the same concentration of is assessed. The above assays are performed in the presence of the quinolone and the test agent. If the test agent has a greater a test agent and the ability of the testagent to inhibit cleavage inhibitory effect on the wild type cells, the test agent is iden is assessed. tified as a potential inhibitor of the target gene or an expres 0228. In another embodiment, an in vitro recombination sion product thereof and, accordingly, as a candidate qui assay using Fis and a Int proteins in conjunction is employed. nolone potentiating agent. Of course any agent identified in US 2009/0264.342 A1 Oct. 22, 2009 26 any of the inventive cell-free or cell-based assays may be structure and thereby identify more effective compounds. Subjected to additional testing either in culture or in animal Binding interactions between test agents and a site on a target models of bacterial infection. molecule (e.g., a polypeptide encoded by an antibiotic poten 0232 Compounds suitable for screening according to the tiator target gene of this invention) can be determined by inventive methods include Small molecules, natural products, molecular modeling programs that are known to those of peptides, nucleic acids, etc. Sources for compounds include ordinary skill in the art. These molecular modeling programs natural product extracts, collections of synthetic compounds, include QUANTA (Accelrys Inc., San Diego, Calif.) and the and compound libraries generated by combinatorial chemis SYBYL suite of computational informatics software (Tripos try. Libraries of compounds are well known in the art. One Associates, Inc., St. Louis, Mo.) representative example is known as DIVERSetTM, available from ChemBridge Corporation, 16981 Via Tazon, Suite G. 0235 Structures of a number of the targets described San Diego, Calif. 92127. DIVERSetTM contains between herein are known in the art. For example, the structure of 10,000 and 50,000 drug-like, hand-synthesized small mol RecA, in some cases bound to a substrate and/or to DNA has ecules. These compounds are pre-selected to form a “univer been reported. The structures have been established for bac sal library that covers the maximum pharmacophore diver teria such as E. coli (Rossbach et al., 2005; Story and Steitz, sity with the minimum number of compounds and is Suitable 1992; Story, Weber and Steitz, 1992; VanLoocket al., 2003b; for either high throughput or lower throughput screening. For Xing and Bell, 2004: Yu and Egelman, 1997); Mycobacterium descriptions of additional libraries, see, for example, Tan, et sp. (Datta et al., 2000, Datta et al., 2003), Proteus mirabilis al., “Stereoselective Synthesis of Over Two Million Com (Weber and Steitz, 1986) and in Archea (Ariza et al., 2005; pounds Having Structural Features Both Reminiscent of Wu et al., 2004). The human RecA homolog RadS1 structure Natural Products and Compatible with Miniaturized Cell has been also determined (Conway et al., 2004: Wu et al., Based Assays”, Am. ChemSoc. 120,8565.8566, 1998; Floyd 2005); this structure can allow filtering for compounds that CD, Leblanc C, Whittaker M, Prog Med Chem 36:91-168, specifically dock with bacterial RecA and not with RadS1 1999. Numerous libraries are commercially available, e.g., (Aihara et al., 1999; Shinohara and Ogawa, 1999: Yu et al., from Analyticon USA Inc., P.O. Box. 5926, Kingwood, Tex. 2001). 77325; 3-Dimensional Pharmaceuticals, Inc., 665 Stockton 0236 Software programs for performing computer-based Drive, Suite 104, Exton, Pa. 19341-1151; Tripos, Inc., 1699 screening are now in common use. Examples include DOCK, Hanley Rd., St. Louis, Mo. 63144-2913, etc. FlexXTM, FRED, GOLD, and ICM. Although the approach 0233 Methods of synthesizing and encoding combinato can vary, these programs typically involve two major phases: rial libraries are known in the art. Libraries of compounds are searching and scoring. During the first phase, the program screened to identify compounds that function as modulators, e.g., inhibitors of the target gene product. For example, librar automatically generates a set of candidate complexes of two ies of Small molecules may be generated using methods of molecules (test compound and target molecule) and deter combinatorial library synthesis well known in the art. Library mines the energy of interaction of the candidate complexes. compounds may be provided in solution or may be attached to The scoring phase assigns scores to the candidate complexes a Solid Support Such as a bead. In certain embodiments of the and selects a structure that displays favorable interactions invention the compounds to be tested are synthesized to con based at least in part on the energy. To perform virtual screen tain a common core structure. The core structure may be one ing, this process is repeated with a large number of test com that characterizes a compound shown to display activity pounds to identify those that display the most favorable inter against aparticular gene product (e.g., using a cell-free assay) actions with the target. A database of commercially available and/or predicted to display activity based on computational compounds has been constructed for such use: the ZINC approaches. Once a library of compounds is screened. Subse database consists of over 700,000 molecules, each with 3-di quent libraries may be generated using those chemical build mensional structure, which are readily and freely available ing blocks that possess the features shown in the first round of for this purpose (Irwin and Shoichet, 2005). Other databases screen to have activity against the target gene product. Using are known in the art. Genetic and biochemical data may be this approach, Subsequent iterations of candidate compounds employed to focus the in silico Screen. For example, struc will possess an increasing number of those structural and tures for screening can be selected based on known ligands functional features required to inhibit or otherwise modulate and/or chemical information can be used to actively guide the the function of the target, until a group of compounds with orientation of the ligand into the binding site. See, e.g., Frad high activity and, optionally, specificity for the target can be era, X. and Mestres, J., 2004. Computation-based approaches found. These compounds can then be further tested for their to drug discovery have been extensively reviewed. See, e.g., safety and efficacy for therapeutic use. Schneidman-Duhovny, D., et al., 2004; Brooijmans, N. and 0234 Computational methods of use to identify an agent Kuntz, I.D., 2003; Alvarez, J C, 2004. See also, Tollenaere, J., that modulates activity of a polypeptide encoded by a target et al. (eds.) Computational Medicinal Chemistry and Drug gene are also provided by the present invention. Molecular Discovery, New York: Marcel Dekker, 2004. modeling can be used to identify a pharmacophore for a 0237 Compounds which virtually bind to a target protein particular target site, e.g., the minimum functionality or set of in silico can be evaluated to determine whether they are actual desirable features that a molecule should have to possess inhibitors of activities of the target protein either in cell-free activity at that target site. Such modeling can be based, for or cell-based assays such as those described above. Recent example, on a predicted or known structure for the target (e.g., Successes confirm the utility of the virtual screening a two-dimensional or three-dimensional structure). Software approach, which is now a common technique in early stage programs for identifying Such potential lead compounds are discovery and has yielded considerable Success. Drugs such known in the art, and once a compound exhibiting activity is as Gleevec (oncology, Novartis), Relenza (influenza virus, identified, standard methods may be employed to refine the GlaxoSmithKkline), Tamil flu (influenza virus, Roche) and US 2009/0264.342 A1 Oct. 22, 2009 27

Agenerase (HIV infection, VertexPharma) are examples that ATPase inhibitors may even have an ICs well below 1 lug/ml, show the Successful contributions of rational design (Ng, or even below 500 ng/ml, 100 ng/ml, 50 ng/ml, 30 ng/ml, 25 2004, Shoichet, 2004). ng/ml, 20 ng/ml, 15 ng/ml, 10 ng/ml, 5 ng/ml, 1 ng/ml, or less. 0238. In addition, known ligands or ligands identified can 0243 In some embodiments of the present invention, be modified, and the effects of the modified compounds on RecA inhibitors are broad spectrum agents in that they inhibit protein activity and/orantibiotic potentiation can be assessed. RecA (or the relevant RecA homolog) from more than one Thus any of the above screening methods may be performed different microbial source. In other embodiments, RecA using a modified version of an agent identified using one of inhibitors have a narrow spectrum activity in that they inhibit the inventive screens or otherwise identified as having activity one or more activities of RecA (or its relevant homolog) from towards one of the targets mentioned herein. a specific family of organisms or from a specific organism. In 0239. The invention also provides a computer-readable certain preferred embodiments, RecA inhibitors inhibit one medium on which are stored results of a screen to identify an or more activities of RecA (or its relevant homolog) from a agent that potentiates activity of an antibiotic, e.g., a qui disease-causing organism (in particular an organism that nolone. The results may be stored in a database and can causes disease in a mammal, e.g., a human). In some embodi include any screening protocols, results obtained from the ments, however, the RecA inhibitors (which may be broad screen or from additional screens, and/or protocols of or spectrum with regard to microbes) do not inhibit RecA (or the results obtained from tests performed on compounds identi relevant RecA homolog) from one or more higher organisms fied in the screen (e.g., tests in animal models of infection). (e.g., mammals, humans). For example, in Some embodi 0240. The invention further provides a method of conduct ments, RecA inhibitors do not inhibit RAD51. ing abusiness to identify atherapeutic agent, i.e., a compound 0244. In some embodiments, the present invention pro that potentiates an antibiotic. The method involves perform vides RecA inhibitors that inhibit the RecA ATPase activity. ing any of the screens described herein, optionally to identify For example, the present invention demonstrates that a variety a compound that potentiates the activity of a marketed anti of compounds inhibit RecA ATPase activity in an in vitro biotic agent, a non-marketed agent known to have antibiotic luciferase assay (see, for example, Example 24). The present activity, an agent not known to have antibiotic activity, etc. invention specifically provides the compounds depicted in The screens can be performed on a contract basis, e.g., as a FIG. 14 as RecA inhibitors with the indicated ICsos in the in service, in which a customer requests that a screen be per vitro luciferase assay. formed to identify a compound that potentiates activity of an 0245. In some embodiments of the present invention, antibiotic agent Suggested by or provided by the customer. In RecA inhibitors that inhibit the RecA ATPase activity do not certain embodiments of this method the agent for which a inhibit certain other cellular ATPases. potentiating agent is desired is a quinolone antibiotic, an 0246. In some embodiments, the present invention pro aminoglycoside antibiotic, or a lactam antibiotic. vides RecA inhibitors that bind directly to RecA. In some embodiments, RecA inhibitors bind to the RecA ATP binding V. Antibiotic Potentiation and Resistance site. However, in some embodiments, inventive RecA inhibi Suppression via RecA Inhibition tors do not bind to the RecA ATP binding site (even though 0241. As already mentioned above, recA was found to be they may inhibit the RecA ATPase activity). In certain a common antibiotic potentiator target gene of quinolones, embodiments, inventive RecA inhibitors bind to two or more and aminoglycosides. Accordingly, the present invention pro different sites on the RecA protein. For example, Example 31 vides RecA inhibitors that potentiate the activity of other proposes two potential new binding sites for RecA inhibitors, antibiotic agents. When RecA inhibitors are administered in including those that inhibit ATPase activity. combination with one or more Such antibiotic agents whose 0247. In some embodiments, inventive RecA inhibitors activity they potentiate, the antibiotic agent(s) may often be that bind directly to RecA bind to a site comprised of amino utilized at a lower dose, and/or less frequent dosing regimen acid residues including R85, F270, Y271, K310, and/or R324 than their conventional dose and/or schedule. In some (see, for example, Example 31). According to the present embodiments, addition of one or more RecA inhibitors to an invention, this site can be found on the outer surface of RecA antibiotic therapy regimen significantly reduces the Survival as positioned in a filament. According to the present inven of bacteria at conventional therapeutic antibiotic agent dos tion, hinokiflavone may bind to this RecA site. Further ing. In some embodiments, inventive RecA inhibitors reduce according to the present invention, compounds that compete the incidence of resistance developed toward one or more with hinokiflavone for binding to RecA may be desirable antibiotic agents. In some embodiments, RecA inhibitors RecA inhibitors. As used herein, the term “competes with retard resistance developed toward one or more antibiotic hinoki flavone for binding to RecA', when used to character agents. ize a compound, refers to a compound that has binding prop 0242. In general, agents that inhibit one or more activities erties to RecA similar to (i.e., similar binding site(s) than) of RecA, and/or that inhibit RecA expression levels, may be hinoki flavone). useful in accordance with the present invention. Exemplary 0248. In certain embodiments of the present invention, RecA activities that may be inhibited include, but are not RecA inhibitors potentiate the activity of one or more antibi limited to, DNA binding, monomer interaction, helicase otic agents. For example, the present Applicants have found activity, filament formation, ATP binding and/or hydrolysis, that hinokiflavone potentiates activity of a quinolone antibi co-protease activity (e.g., toward Lex A and/or Umud), otic (ciprofloxacin). Moreover, this potentiation is only recombinase activity, replication function, and combinations observed against cells that express RecA. thereof. In some embodiments, inventive RecA inhibitors 0249. In some embodiments of the present invention, inhibit one or more such activities with an ICso below about RecA inhibitors reduce the incidence of resistance that devel 100 ug/ml, 50 ug/ml, 15ug/ml, 10 g/ml; 5ug/ml, 3 g/ml, or ops to one or more antibiotic agents. In some embodiments of 1 ug/ml. According to the present invention, desirable RecA the present invention, RecA inhibitors retard the emergence US 2009/0264.342 A1 Oct. 22, 2009 28 of resistance that develops to one or more antibiotic agents. monocyclic 3-8 membered saturated, partially unsatur For example, hinokiflavone was found to reduce the inci ated, or aryl ring having 0-4 heteroatoms independently dence of resistance to ciprofloxacin. The generality of hinoki selected from nitrogen, oxygen, or Sulfur, or a bicyclic 8-10 flavone's mechanism of action (inhibiting RecA) Suggests membered saturated, partially unsaturated, or aryl ring that this compound should also suppress or retard resistance having 0-5 heteroatoms independently selected from nitro to and/or potentiate activity of other antibiotic agents. gen, oxygen, or Sulfur, 0250 In certain embodiments of the present invention, 0259 Q is a valence bond or a bivalent, saturated or unsat RecA inhibitors show intrinsic antibiotic activity, even in the urated, straight or branched C. hydrocarbon chain, absence of any other antibiotic agent. However, antibiotic wherein 0-2 methylene units of Q are independently activity is not necessary for antibiotic potentiating and/or antibiotic resistance Suppression. 0251. In other embodiments, RecA inhibitors can actually NR-, or - NRC(O)O : protect cells from death. Without wishing to be bound by any 0260 each R is independently hydrogen or an optionally particular theory, it is noted that one possible explanation for Substituted aliphatic group; the present findings is that RecA inhibitors effective for use as 0261 R is R or OR; and antibiotic agents or potentiating agents according to the 0262 Ring A is an optionally substituted 3-8 membered present invention are those that can simultaneously bind to bivalent, Saturated, partially unsaturated, or aryl monocy two distinct sites on RecA for example to the ATP binding clic ring having 0-4 heteroatoms independently selected site and to another binding site, including for example one of from nitrogen, oxygen, or Sulfur, or an optionally Substi the sites identified in Example 20. Alternatively or addition tuted 8-10 membered bivalent saturated, partially unsatur ally, it may be the case that, although many agents can inhibit ated, oraryl bicyclic ring having 0-5 heteroatoms indepen the RecA ATPase, most such agents also inhibit other dently selected from nitrogen, oxygen, or Sulfur. ATPases within a cell, causing a variety of stresses and induc 0263. As defined generally above, the X group of formula ing protective mechanisms (e.g., shut down of DNA replica I is oxygen, sulfur, or N(R). In certain embodiments, the X tion) that allow the cells to avoid the effects of antibiotic group of formula I is oxygen. Accordingly, the present inven agents. tion provides RecA inhibitors of formula I-a: 0252. In some embodiments of the present invention, RecA inhibitors are Small molecule agents, typically having some cyclic character (e.g., including one or more aryl rings). I-a Certain RecA inhibitors according to the present invention are flavones; certain RecA inhibitors according to the present N o1 invention are bisflavones. In some embodiments of the inven (R:), H tion, the RecA inhibitor is or includes hinokiflavone. 21 0253) In some embodiments of the present invention, RecA inhibitors are small molecule compounds that have the O structure of formula I: or a pharmaceutically acceptable salt or derivative thereof, wherein each of Ring A, Q, n, R', and Rare as defined above and described herein. 0264. As defined generally above, the R' group of formula I is hydrogen, or an optionally Substituted group selected from a Caliphatic group, a monocyclic 3-8 membered saturated, partially unsaturated, or aryl ring having 0-4 het eroatoms independently selected from nitrogen, oxygen, or R sulfur, or a bicyclic 8-10 membered saturated, partially unsat urated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or Sulfur. 0265. In certain embodiments, the R' group of formula I is or a pharmaceutically acceptable salt or derivative thereof, a bicyclic 8-10 membered saturated, partially unsaturated, or wherein: aryl bicyclic ring having 0-5 heteroatoms independently 0254 X is oxygen, sulfur, or N(R): selected from nitrogen, oxygen, or Sulfur. In still other 0255 n is 0 to 4: embodiments, R is a bicyclic 10 membered partially unsat (0256) R' is hydrogen, or an optionally substituted group urated ring having one oxygen atom optionally substituted selected from a Caliphatic group, a monocyclic 3-8 with 1 to 3 substituents independently selected from halogen, membered saturated, partially unsaturated, or aryl ring —(CH2)R. —(CH2) OR. -(CH2). SR, —(CH2). having 0-4 heteroatoms independently selected from nitro 4Ph, optionally substituted with R or OR, —(CH)O gen, oxygen, or Sulfur, or a bicyclic 8-10 membered Satu (CH). Ph optionally substituted with R or OR, rated, partially unsaturated, oraryl ring having 0-5 heteroa -CH=CHPh, optionally substituted with R or OR, toms independently selected from nitrogen, oxygen, or —(CH)N(R), wherein each R may be substituted as Sulfur, defined herein and is independently hydrogen, Caliphatic, 0257 each R is independently halogen, R, OR, SR, —CHPh. —O(CH), Ph, or a 5-6-membered saturated, N(R), C(O)R, C(O)OR, NRC(O)R, C(O)NR, partially unsaturated, or aryl ring having 0-4 heteroatoms SOR, NRSO.R., SON(R): independently selected from nitrogen, oxygen, or Sulfur. 0258 each R is independently hydrogen or an optionally According to one embodiments, R is substituted with 1 to 3 Substituted group selected from a Caliphatic group, a groups independently selected from OH and OMe. US 2009/0264.342 A1 Oct. 22, 2009 29

0266. According to one embodiment of the invention, the R" group of formula I is selected from: -continued OH

O

MeO N

2 O wherein each wavy line indicates the point of attachment to Q. OH 0269. As defined generally above, the Q group of formula OH, or O I is a valence bond or a bivalent, Saturated or unsaturated, straight or branched C. hydrocarbon chain, wherein 0-2 HO methylene units of Q are independently replaced by —O—, 1N NR , S— —OC(O)— —C(O)O—, —C(O)—, SO-, -SO, , – NRSO, , -SONR-, - NRC 21 O (O) , —C(O)NR - OC(O)NR , or - NRC(O)O-. In certain embodiments, Q is a valence bond such that R is OH directly attached to Ring A. In other embodiments, Q is a O bivalent, saturated, and straight C. hydrocarbon chain, HO wherein 0-1 methylene units of Q is replaced by —O—, NR , S— —OC(O)— —C(O)O—, —C(O)—, SO-, -SO, , – NRSO, , -SONR-, - NRC (O) , —C(O)NR - OC(O)NR , or - NRC(O)O-. In O s still other embodiments, Q is —O— —NR— —S —OC OH (O)—, C(O)O , C(O)—, SO , SO , NRSO, , -SONR-, - NRC(O) , —C(O)NR-, —OC(O)NR , or NRC(O)C)—. According to another wherein each wavy line depicts the point of attachment to Q. embodiment, Q is —O—. 0267. In certain embodiments, the R' group of formula I is 0270. As defined generally above, the Ring A group of a monocyclic 3-8 membered saturated, partially unsaturated, formula I is an optionally substituted 3-8 membered bivalent, or aryl ring having 0-4 heteroatoms independently selected saturated, partially unsaturated, or aryl monocyclic ring hav from nitrogen, oxygen, or Sulfur. In other embodiments, the ing 0-4 heteroatoms independently selected from nitrogen, R" group of formula I is a monocyclic 5-6 membered aryl ring oxygen, or sulfur, oran optionally substituted 8-10 membered having 0-2 nitrogen atoms, wherein R' is optionally substi bivalent Saturated, partially unsaturated, or aryl bicyclic ring tuted with 1 to 3 substituents independently selected from having 0-5 heteroatoms independently selected from nitro halogen, —(CH2)R. —(CH) R. —(CH2). SR, gen, oxygen, or Sulfur. In certain embodiments, Ring A is an —(CH), Ph, optionally substituted with R or OR, optionally substituted 3-8 membered bivalent, saturated, par —(CH) O(CH). Ph optionally substituted with R or tially unsaturated, or aryl monocyclic ring having 0-4 het OR, -CH=CHPh, optionally substituted with R or OR, eroatoms independently selected from nitrogen, oxygen, or —(CH)N(R), wherein each R may be substituted as Sulfur. In other embodiments, Ring A is an optionally substi defined herein and is independently hydrogen, Caliphatic, tuted 5-6 membered bivalent aryl ring having O nitrogen —CHPh, —O(CH). Ph, or a 5-6-membered saturated, atoms. In still other embodiments, Ring A is phenylene partially unsaturated, or aryl ring having 0-4 heteroatoms optionally substituted with 1 to 4 groups independently independently selected from nitrogen, oxygen, or Sulfur. selected from halogen, —(CH2)R. —(CH)OR, According to one embodiment of the present invention, R' is —(CH2)oSR. —(CH2). Ph, optionally Substituted with phenyl substituted with 1 to 3 groups independently selected R or OR, —(CH), O(CH). Ph optionally substituted from halogen, —(CH) R, and —(CH), OR. Such with R or OR, -CH=CHPh, optionally substituted with groups include chloro, fluoro, OH, OMe, methyl, ethyl, pro R or OR, —(CH)N(R), wherein each R may be sub pyl, cyclopropyl, isopropyl, and the like. In some embodi stituted as defined herein and is independently hydrogen, C. ments, OH or OMe groups are present. aliphatic, —CHPh, —O(CH). Ph, or a 56-membered satu rated, partially unsaturated, or aryl ring having 0-4 heteroat 0268 According to another embodiment, the R' group of oms independently selected from nitrogen, oxygen, or Sulfur. formula I is selected from: According to another embodiment, Ring A is phenylene optionally Substituted with 1-2 groups independently selected from halogen, —(CH) R, and —(CH) R. OH Such groups include chloro, fluoro, OH, OMe, methyl, ethyl, OMe, OH, propyl, cyclopropyl, isopropyl, and the like. In some embodi ments, OH and/or OMe group(s) are present. (0271. As defined generally above, each R group of for mula I is independently halogen, R, OR, SR, N(R), C(O) R, C(O)OR, NRC(O)R, C(O)NR, SOR, NRSOR, SO.N(R), wherein each R is independently hydrogen oran US 2009/0264.342 A1 Oct. 22, 2009 30 optionally Substituted group selected from a Caliphatic Scope of formula I. The present invention also encompasses group, a monocyclic 3-8 membered Saturated, partially unsat the recognition that compounds that share structural (see, for urated, or aryl ring having 0-4 heteroatoms independently example, FIG. 15) and/or energetic (see, for example, FIG. selected from nitrogen, oxygen, or Sulfur, or a bicyclic 8-10 16) features of hinokiflavone may be useful as RecA inhibi membered Saturated, partially unsaturated, or aryl ring hav tors as described herein. Thus, according to the present inven ing 0-5 heteroatoms independently selected from nitrogen, tion, compounds having the structures set forth in FIG. 15 or oxygen, or sulfur. In certain embodiments, each R group is 16 and/or having the structure of formula II may be useful as independently halogen, R, OR, SR, or N(R), wherein RecA inhibitors as described herein: each R is as defined above. According to one embodiment, at least one R group is OH. According to another embodiment, one R group is R wherein R is an optionally substituted II bicyclic 8-10 membered saturated, partially unsaturated, or aryl ring having 0-5 heteroatoms independently selected from nitrogen, oxygen, or Sulfur. In still other embodiments, one R’ group is R wherein R is a bicyclic 10 membered partially unsaturated ring having one oxygen atom optionally Substi or a pharmaceutically acceptable salt or derivative thereof, tuted with 1 to 3 substituents independently selected from wherein: halogen, —(CH) R. —(CH)OR. -(CH2). SR, (0275 Cy' is a an optionally substituted 5-6 membered —(CH). Ph, optionally substituted with R or OR, aryl ring having 0-3 heteroatoms independently selected —(CH) O(CH). Ph optionally substituted with R or from nitrogen, oxygen, or Sulfur, OR, -CH=CHPh, optionally substituted with R or OR, —(CH) N(R), wherein each R may be substituted as (0276 L' is a valence bond, a C, bivalent saturated, defined herein and is independently hydrogen, Caliphatic, unsaturated, straight or branched hydrocarbon chain, —CHPh, —O(CH). Ph, or a 56-membered saturated, par - N(R)-, - N(R)SO. , N(R)SON(R) - N(R) tially unsaturated, or aryl ring having 0-4 heteroatoms inde C(O) = C(O)N(R) , or - N(R)C(O)N(R)–: pendently selected from nitrogen, oxygen, or Sulfur. Accord 0277 each R is independently hydrogen or an option ing to one embodiments, R is substituted with 1 to 3 groups ally substituted Caliphatic group: independently selected from OH and OMe and optionally 0278 Cy is an optionally substituted 6-membered aryl substituted phenyl. ring having 0-2 nitrogenatoms, an 8-10 membered bicy 0272 According to some embodiments of the invention, clic heteroaryl ring having 1-3 heteroatoms indepen the R group formula I is OH, OMe, or is selected from: dently selected from nitrogen, oxygen, or Sulfur, or an optionally substituted 5-membered heteroaryl ring hav ing 1-2 heteroatoms independently selected from nitro OH, gen, oxygen, or Sulfur, O 0279 L is a C bivalent saturated, unsaturated, MeO straight or branched hydrocarbon chain, —CH2CHC 1N (—W)N(R)N(R)C(=W) , N(R)C(=W)N(R)C (—W)C(R), W , C(—W)N(R)N(R)C(—W)N 2 O (R) - C(=W)N(R)N(R)C(=W)N(R)CH=CH, or - C(=W)N(R)C(=W)N(R) , wherein each W is OH independently oxygen or Sulfur, and OH, or (0280 Cy is an optionally substituted 6-membered aryl O ring having 0-2 nitrogen atoms. HO 0281. It should be understood that, unless otherwise 1N stated, chemical structures or formulae depicted herein are also meant to include all isomeric (e.g., enantiomeric, dias 2 O tereomeric, and geometric (or conformational)) forms of the OH structure; for example, the R and S configurations for each OH, asymmetric center, (Z) and (E) double bond isomers, and (Z) O and (E) conformational isomers. Therefore, single stere ochemical isomers as well as enantiomeric, diastereomeric, HO and geometric (or conformational) mixtures of depicted - N structures or formulae are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the structures 2 O or formulae of the invention are within the scope of the invention. Additionally, unless otherwise Stated, structures OH depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically wherein each wavy line depicts the point of attachment to the enriched atoms. For example, compounds having the phenyl group of formula I. depicted structures or formulae except for the replacement of 0273 Exemplary compounds of formula I are set forth in hydrogen by deuterium or tritium, or the replacement of a FIGS. 15 and 16. carbon by a C or 'C-enriched carbon are within the 0274 The present invention specifically identifies hinoki Scope of this invention. Such compounds may be useful, for flavone as a particularly desirable RecA inhibitor within the example, as analytical tools or probes in biological assays. US 2009/0264.342 A1 Oct. 22, 2009

0282. It should further be understood that the present unusual circumstances, establishes the likelihood of activity invention encompasses pharmaceutically acceptable deriva with regard to other fluoroquinolones, and indeed with regard tives, and in particular prodrugs, metabolites, and pharma to quinolones generally. In fact, such demonstration can even ceutically acceptable salts of the depicted compounds. establish the likelihood of activity with regard to other anti 0283. As discussed herein, inventive RecA inhibitors are biotic agents generally, certainly for others with a common desirably utilized in combination with one or more antibiotic mechanism of action (i.e., within the same mechanistic class). agents, and particularly in combination with one or more 0289. Of course, it will be appreciated that an agent that antibiotic agents whose activity is potentiated by inventive potentiates the activity of or Suppresses/retards resistance to RecA inhibitors and/or whose effectiveness can be reduced any particular antibiotic agent within a particular structural or by development of resistance, which development is Sup mechanistic class need not potentiate all, most, or any other pressed or retarded by inventive RecA inhibitors. members of that class, although in certain embodiments of the 0284 Exemplary structural classes of antibiotics for use in invention the agent does potentiate at least one, many, most, combination with RecA inhibitors according to the present or all other members of that class. invention include, but are not limited to, aminoglycosides, 0290 Exemplary quinolone antibiotics include, but are aminomethylcyclines, amphenicols, ansamycins, B-lactams not limited to, any of the antibacterial agents disclosed in the (e.g., penicillins or cephalosporins), carbapenems, dapsones, foregoing references including, but not limited to, ciprofloxa 2,4-diaminopyrimidines, glycopeptides, glycycyclines, cin, oxolinic acid, cinoxacin, flumequine, miloxacin, roSoxa ketolides, lincomycins, lincosamides, macrollides, nitro cin, pipemidic acid, norfloxacin, enoxacin, moxifloxacin, furans, oxazolidinones, peptides, polymyxins, quinolones, gatifloxacin, ofloxacin, lomefloxacin, temafloxacin, fleroxa rifabutins, streptogramins, Sulfonamides, Sulfones, tetracy cin, pefloxacin, amifloxacin, sparfloxacin, levofloxacin, clines, and combinations thereof. clinafloxacin, nalidixic acid, enoxacin, grepafloxacin, levof 0285 Exemplary mechanistic classes of antibiotics for use loxacin, lomefloxacin norfloxacin, ofloxacin, trovafloxacin, in combination with RecA inhibitors according to the present olamufloxacin, cadrofloxacin, alatrofloxacin, gatifloxacin, invention include, but are not limited to, those that inhibit rufloxacin, irloxacin, prulifloxacin, paZufloxacin, gemifloxa protein synthesis, cell wall synthesis, DNA replication, tran cin, sitafloxacin, tosulfloxacin, amifloxacin, nitrosoxacin-A, scription, and/or cell division. It will be appreciated that DX-619, and ABT-492. Quinolone antibiotics include fluo biological and biochemical pathways are not mutually exclu roquinolones (e.g., having a fluorine Substituent at the C-6 sive and that some biological or biochemical pathways may position), and non-fluoroquinolones. Also included within be considered to be subsets or sub-pathways of other biologi the scope of quinolone antibiotics are derivatives in which a cal or biochemical pathways. Mechanisms of action more quinolone is conjugated with, e.g., covalently bound to, specifically include, but are not limited to, inhibiting protein another core structure. For example, U.S. Pub. No. 2004 synthesis (e.g., by binding ribosomal RNA or proteins, block 0215017 discloses compounds in which an oxazolidinone, ing tRNA binding to ribosome-mRNA complex, inhibiting isoxazolinone, or isoxazoline is covalently bonded to a qui peptidyl transferase), inhibiting or interfering with synthesis nolone. of a cell wall component (e.g., inhibition of peptidoglycan 0291 Included within the scope of quinolone antibiotics synthesis, disruption of peptidoglycan cross-linkage, disrup that can be utilized in accordance with the present invention tion of movement of peptidoglycan precursors, disruption of are compounds that have a core structure related to the 4-oxo mycolic acid or arabinoglycan synthesis), cell membrane dis 1,4-dihydroquinoline and 4-oxo-1.4 dihydronapthyridine ruption, inhibiting or interfering with nucleic acid synthesis systems, e.g., 2-pyridones, 2-naphthyridinones, and benzob or processing, acting as “antimetabolites' and either inhibit napthyridones. 2-pyridones are potent inhibitors of bacterial ing an essential bacterial enzyme or competing with a Sub type II topoisomerases (Saiki et al., Antimicrob. Agents strate of an essential bacterial enzyme, inhibiting or interfer Chemother, 1999, 43: 1574). The core structures are depicted ing with cell division. in FIG. 9. 0286. It is understood by those of ordinary skill in the art 0292 Also included within the scope of quinolone antibi that antibiotic agents of a particular structural class typically otics that can be potentiated are compounds that have core (though not necessarily) fall within the same mechanistic structures related to the quinolone core structures depicted in class. FIG. 9 or 10. Certain of these core structures are shown in 0287. As established herein and in related patent applica FIG. 11A or 113B and references thereto are provided in tion Ser. No. 60/772,648, the entire contents of which are Ronald and Low (Eds.), “Fluoroquinolone Antibiotics”, incorporated herein by reference, RecA inhibitors can poten Birkhäuser Verlag, Basel, 2003: DaSilva et al., Curr. Med. tiate the activity of certain antibiotics. In particular, RecA Chem., 2003, 10: 21. inhibitors can potentiate the activity of quinolone antibiotics, 0293. The invention encompasses the use of RecA inhibi e.g., fluoroquinolones such as norfloxacin and ciprofloxacin. tors as described herein to potentiate the activity of and/or to RecA inhibitors also suppress or retard resistance to qui Suppress/retard the resistance to, these antibiotic agents. nolone antibiotics, e.g., fluoroquinolines Such as norfloxacin 0294 As noted above, RecA inhibitors that potentiate the and ciprofloxacin. activity of or Suppress/retard to an antibiotic agent within a 0288 Given the generality of the effect of RecA inhibition particular mechanistic class are likely to also potentiate the as it relates to the activity of and/or resistance to antibiotic activity of or suppress/retard resistance to other antibiotic agents, it is understood that RecA inhibitors that potentiate agents within the same mechanistic class. Thus, RecA inhibi the activity of or Suppress/retard resistance to a particular tors that potentiate the activity of and/or Suppress/retard resis antibiotic agent are likely to similarly potentiate the activity tance to particular quinolones are likely to also potentiate the of or Suppress/retard resistance to other agents of the same activity of and/or Suppress/retard resistance to certain other structural and/or mechanistic class. Thus, demonstration of topoisomerase II inhibitors, some of which are structurally activity with regard to a particular fluoroquinolone, absent related to quinolones. Exemplary topoisomerase II inhibitors US 2009/0264.342 A1 Oct. 22, 2009 32 that bind to GyrB include the coumarins, novobiocin and 0299 The antibiotic potentiating agents and compositions coumermycin A1, cyclothialidine, cinodine, and clerocidin. containing them can be used to inhibit bacterial growth and/or Additional agents that are reported to bind to and/or inhibit Survival in a variety of contexts. For example, they may be gyrase, topoisomerase IV, or both, are disclosed in U.S. Pat. employed to inhibit growth and/or survival of bacteria main Nos. 6,608,087 and 6,632.809 and in U.S. Pub. Nos. tained in cell culture or inhabiting locations in the environ 2004.0043989 and 2005.0054697. The present invention ment, e.g., inert Surfaces, clothing, towels, bedding, utensils, encompasses the use of RecA inhibitors to potentiate the etc. Of particular interest are fomites, i.e., inanimate objects activity of and/or to Suppress/retard resistance to any of these that may be contaminated with disease-causing microorgan agents. That is, the present invention provides RecA inhibi isms and may serve to transmit disease to a human or animal. tors for use in combination with any of these compounds or Such locations or objects can be contacted with a solution any compound or agent that inhibits one or more microbial containing the potentiating agent and an antibiotic that it type II topoisomerases. potentiates. The antibiotic potentiating agents, antibiotics that they potentiate, and/or compositions containing them can VI. Potentiating Cidal Antibiotics via RecA be added to food or water, particularly for the prevention of Inhibition bacterial disease in animals. 0300. An antibiotic agent and an agent that potentiates the 0295 Current antimicrobial therapies, which cover a wide antibiotic (e.g., a quinolone antibiotic and a quinolone poten array of targets (C. Walsh, Nature Rev. Microbiol., 2003, 1: tiating agent), may be administered in combination to a Sub 65-70), fall into two general categories: bactericidal drugs ject in need thereof, e.g., a human or animal Suffering from or which kill bacteria with an efficiency of >99.9% and bacte at risk of a bacterial infection. The antibiotic agent and the riostatic drugs which merely inhibitgrowth (G. A. Pankey and antibiotic potentiating agent, may be components of a single L. D. Sabath, Clin. Infect. Dis., 2004, 38: 864-870). Antibac pharmaceutical composition or may be administered as indi terial drug-target interactions are well-studied and predomi vidual pharmaceutical compositions. They may be adminis nantly fall into three classes: inhibition of DNA replication tered using the same route of administration or different and repair, inhibition of protein synthesis, and inhibition of routes of administration. In certain embodiments of the inven cell-wall turnover (C. Walsh, Nature, 2000, 406: 75-781). tion a unit dosage form containing a predetermined amount of However, our understanding of many of the bacterial an antibiotic and a predetermined amount of an antibiotic responses that occur as a consequence of the primary drug potentiating agent is administered. target interaction remains incomplete (A. Tomasz, Annu. Rev. 0301 A therapeutic regimen that includes an antibiotic Microbiol., 1979,33: 113-137: K. Drlica and X. Zhao, Micro and an antibiotic potentiating agent may (i) allow the use of a biol. Mol. Biol. Rev., 1997, 61:377-392: B. D. Davis, Micro reduced daily dose of the antibiotic without significantly biol. Rev., 1987, 51: 341-350; K. Lewis, Microbiol. Mol. reducing efficacy; (ii) allow the use of a shorter course of Biol. Rev., 2000, 64: 503-514). administration of the antibiotic without significantly reduc 0296. The present Applications have shown (see Example ing efficacy; (iii) be effective against a microorganism species 39) that the three major classes of bactericidal antibiotics, or strain that would otherwise be resistant to the antibiotic regardless of drug-target interaction, stimulate the production when used at clinically tolerated doses, e.g., conventional of highly deleterious hydroxyl radicals in gram-negative and doses. gram-positive bacteria, which ultimately contribute to cell 0302 Infections and infection-related conditions that can death. The Applicants have shown that, in contrast, bacterio be treated using an antibiotic potentiating agent and an anti static drugs do no produce hydroxyl radicals, and that the biotic include, but are not limited to, pneumonia, meningitis, mechanism of hydroxyl radical formation, induced by bacte sepsis, septic shock, sinusitis, otitis media, mastoiditis, con ricidal antibiotics, involves the tricarboxylic acid cycle and a junctivitis, keratitis, external otitis (e.g., necrotizing otitis transient depletion of NADH. The results obtained suggest externa and perichondritis), laryngeal infections (e.g., acute that all three major classes of bactericidal drugs can be poten epiglottitis, croup and tuberculous laryngitis), endocarditis, tiated by targeting bacterial systems that remediate hydroxyl infections of prosthetic valves, abscesses, peritonitis, infec radical damage, including proteins involved in triggering the tious diarrheal diseases, bacterial food poisoning, sexually DNA damage response, e.g., RecA. transmitted diseases and related conditions, urinary tract 0297 Accordingly, the present invention provides meth infections, pyelonephritis, infectious arthritis, osteomyelitis, ods that involve the use of RecA inhibitors to potentiate cidal infections of prosthetic joints, skin and Soft tissue infections, antibiotics, and/or any therapeutic agent that produces oral infections, dental infections, nocardiosis and actinomy hydroxyl radicals. Such methods are suitable for use with any cosis, mastitis, brucellosis, Q fever, anthrax, wound infec member of a large variety of classes of antibiotics mentioned tions, etc. herein, as long as such member produces hydroxyl radicals. 0303. In certain embodiments of the invention an antibi otic potentiating agent and an antibiotic that it potentiates are VII. Applications used to treat or prevent infection associated with an indwell ing device. Indwelling devices include Surgical implants, 0298 As noted above, the target genes of the invention prosthetic devices, and catheters, i.e., devices that are intro may be found in many organisms, including bacteria of any duced to the body of an individual and remain in position for one or more types, e.g., Gram negative bacteria, Gram posi an extended time. Such devices include, for example, artifi tive bacteria, and/or acid fast bacteria. Antibiotic potentiating cial joints, heart valves, pacemakers, defibrillators, vascular agents and compositions containing them may accordingly be grafts, vascular catheters, cerebrospinal fluid shunts, urinary used to inhibit growth of bacteria of a wide variety of types catheters, continuous ambulatory peritoneal dialysis (CAPD) including, but not limited to, members of any bacterial genus catheters, spinal rods, implantable pumps for medication or species mentioned above. delivery, etc. Potentiating agents identified by the methods of US 2009/0264.342 A1 Oct. 22, 2009

the invention can be applied to, coated on, imbedded in, or humans, and Such homologs may themselves be targets for otherwise combined with an indwelling device to prophylac treatment of disease in those organisms. Alternatively or addi tically prevent infections, optionally together with an antibi tionally, an agent identified according to a method of the otic. Alternatively, an agent of the invention may be admin invention may potentiate a therapeutic agent used in treating istered to a subject, e.g., by injection to achieve a systemic a disease other than a microbial infection. Agents that inhibit effect shortly before insertion of an indwelling device. The mammalian topoisomerases are of use for the treatment of a antibiotic to be potentiated could be applied to, coated on, variety of cancers. Exemplary agents include camptothecins imbedded in, or otherwise combined with an indwelling (e.g., irinotecan and topotecan) and edotecarin (which inhibit device or may also be delivered systemically. Of course local mammalian type I topoisomerase), and etoposide (a mamma delivery of the potentiating agent and/or antibiotic may also lian type II topoisomerase inhibitor). Without wishing to be be employed. Treatment may be continued after implantation bound by any theory, agents that potentiate a microbial topoi of the device during all or part of the time during which the Somerase inhibitor may also potentiate an agent that inhibits device remains in the body and, optionally, thereafter. Agents mammalian topoisomerase. Such agents may therefore be of of this invention may be used in combination with an antibi use in cancer chemotherapy regimens that employ a mamma otic prophylactically prior to dental treatment or Surgery. lian topoisomerase inhibitor. 0304 Alternatively, an agent of this invention and an anti biotic that it potentiates can be used to bathe an indwelling VIII. Pharmaceutical Compositions and Kits device immediately before insertion and/or to bathe wounds 0308 Suitable preparations, e.g., substantially pure prepa or sites of insertion. Exemplary concentrations useful for rations of the agents described herein may be combined with these purposes range between 1 Jug/ml to 10 g/ml for bathing pharmaceutically acceptable carriers, diluents, solvents, of wounds or indwelling devices. excipients, etc., to produce an appropriate pharmaceutical 0305 Diagnostic methods for determining whether a sub composition. The invention therefore provides a variety of ject is suffering from or at risk of a microbial infection are pharmaceutically acceptable compositions for administration well known in the art, and any Such method can be used to to a Subject comprising (i) an antibiotic potentiating agent; identify a suitable subject for administration of an antibiotic and (ii) a pharmaceutically acceptable carrier or excipient. and an agent that potentiates the antibiotic. Methods include The invention further provides a pharmaceutically acceptable clinical diagnosis based at least in part on symptoms, imaging composition comprising (i) an antibiotic potentiating agent; studies, immunodiagnostic assays, nucleic acid based diag (ii) an antibiotic whose activity is potentiated by the com nostics, and/or isolation and culture of potentially causative pound; and (iii) a pharmaceutically acceptable carrier or microorganisms from Samples such as blood, urine, sputum, excipient. The invention further provides a pharmaceutically synovial fluid, cerebrospinal fluid, pus, or any sample of body acceptable unit dosage form containing a predetermined fluid or tissue. The inventive methods can include a step of amount of an antibiotic and a predetermined amount of an identifying a Subject Suffering from or at risk of a microbial antibiotic potentiating agent, wherein the predetermined infection, a step of identifying a microorganism Suspected of amounts are selected so that the antibiotic potentiating agent causing the infection, a step of selecting a therapeutic regi potentiates the antibiotic when the unit dosage form is admin men based at least in part on the identity or Suspected identity istered to a subject. In certain embodiments, the antibiotic is of the microorganism and/or the location or characteristics of a quinolone antibiotic. In other embodiments, the antibiotic is the infection. In certain embodiments of the invention the an aminoglycoside. In still other embodiments, the antibiotic method includes determining that the Subject has a significant is a lactam (e.g., a B-lactam). In certain embodiments, the likelihood (e.g., at least 5%) of suffering from or being at risk antibiotic potentiating agent is a RecA inhibitor. of infection by a microorganism that is resistant to one or 0309. In certain embodiments of the invention the phar more antibiotics and that antibiotic potentiation is advisable. maceutical composition is a Sustained release formulation. A 0306 A subject is “at risk of an infection in any of a variety of methods are known in the art for achieving Sus variety of circumstances. At risk of implies at increased risk tained release, e.g., by prolonging residence time in the stom of relative to the risk such subject would have in the absence ach (such as through the use of Swellable polymers), provid of one or more circumstances, conditions, or attributes of that ing pH or enzyme-sensitive coatings, employing bioadhesive subject, and/or (in the case of humans) relative to the risk that coatings that Stick to the walls of the stomach or intestine, etc. an average, healthy member of the population would have. See, e.g., U.S. Pub. No. 2004-0024018 and references Specific examples of conditions that place a subject “at risk” therein. include, but are not limited to, immunodeficiencies (particu 0310. It is to be understood that the pharmaceutical com larly those affecting the humoral or non-specific (innate) positions of the invention, when administered to a Subject, are immune system), prior treatment with antibiotics that may preferably administered for a time and in an amount Sufficient have reduced or eliminated normal microbial flora, treatment to treat or prevent the disease or condition for whose treat with agents that Suppress the immune system (e.g., cancer ment or prevention they are administered, e.g., a bacterial chemotherapy, immunosuppressive agents), chronic diseases infection. Such as diabetes or cystic fibrosis, Surgery or other trauma, 0311. Further provided are pharmaceutically acceptable infancy or old age, occupations or living conditions that entail compositions comprising a pharmaceutically acceptable exposure to pathogenic microorganisms, etc. derivative (e.g., a prodrug) of any of the agents of the inven 0307 While it is anticipated that the antibiotic potentiat tion, by which is meant any non-toxic salt, ester, salt of an ing agent identified according to the inventive methods will ester or other derivative of a agent of this invention that, upon find particular use for inhibiting the growth and/or survival of administration to a recipient, is capable of providing, either microorganisms, they may also be used for other purposes. directly or indirectly, an agent of this invention or an inhibi For example, certain of the target genes identified herein may torily active metabolite or residue thereof. As used herein, the have homologs in multicellular organisms, e.g., animals or term “inhibitorily active metabolite or residue thereof means US 2009/0264.342 A1 Oct. 22, 2009 34 that a metabolite or residue thereof exhibits inhibitory activity intramuscular, intradermal, or Subcutaneous application can towards a protein or microorganism. The inhibitor of a bac include the following components: a sterile diluent Such as terial type II topoisomerase may also be provided as a pro water for injection, Saline Solution, fixed oils, polyethylene drug. glycols, glycerine, propylene glycol or other synthetic Sol 0312. In various embodiments of the invention an effec vents; antibacterial agents such as benzyl alcohol or methyl tive amount of the pharmaceutical composition is adminis parabens; antioxidants such as ascorbic acid or sodium tered to a subject by any suitable route of administration bisulfite; chelating agents such as ethylenediaminetetraacetic including, but not limited to, intravenous, intramuscular, by acid; buffers such as acetates, citrates or phosphates and inhalation (e.g., as an aerosol), by catheter, intraocularly, agents for the adjustment of tonicity Such as Sodium chloride orally, rectally, intradermally, by application to the skin, etc. or dextrose. pH can be adjusted with acids or bases, such as 0313 The term “pharmaceutically acceptable carrier, hydrochloric acid or sodium hydroxide. The parenteral prepa excipient, or vehicle' refers to a non-toxic carrier, excipient, ration can be enclosed in ampoules, disposable Syringes or or vehicle that does not destroy the pharmacological activity of the agent with which it is formulated. Pharmaceutically multiple dose vials made of glass or plastic. acceptable carriers, excipients, or vehicles that may be used in 0317 Pharmaceutical compositions suitable for injectable the compositions of this invention include, but are not limited use typically include sterile aqueous solutions (where water to, ion exchangers, alumina, aluminum Stearate, lecithin, soluble) or dispersions and sterile powders for the extempo serum proteins, such as human serum albumin, buffer Sub raneous preparation of sterile injectable solutions or disper stances such as phosphates, glycine, Sorbic acid, potassium Sion. For intravenous administration, Suitable carriers include Sorbate, partial glyceride mixtures of Saturated vegetable physiological saline, bacteriostatic water, Cremophor ELTM fatty acids, water, salts or electrolytes, such as protamine (BASF, Parsippany, N.J.), phosphate buffered saline (PBS), Sulfate, disodium hydrogen phosphate, potassium hydrogen or Ringer's Solution. phosphate, Sodium chloride, Zinc salts, colloidal silica, mag 0318 Sterile, fixed oils are conventionally employed as a nesium trisilicate, polyvinyl pyrrolidone, cellulose-based Solvent or Suspending medium. For this purpose, any bland Substances, polyethylene glycol, Sodium carboxymethylcel fixed oil may be employed including synthetic mono- or lulose, polyacrylates, waxes, polyethylene-polyoxypropy di-glycerides. Fatty acids, such as oleic acid and its glyceride lene-block polymers, polyethylene glycol and wool fat. Sol derivatives are useful in the preparation of injectables, as are vents, dispersion media, coatings, antibacterial and natural pharmaceutically-acceptable oils, such as olive oil or antifungal agents, isotonic and absorption delaying agents, castor oil, especially in their polyoxyethylated versions. and the like, compatible with pharmaceutical administration These oil solutions or Suspensions may also contain a long may be included. Supplementary active compounds, e.g., chain alcohol diluent or dispersant, such as carboxymethyl compounds independently active against the disease or clini cellulose or similar dispersing agents that are commonly used cal condition to be treated, or compounds that enhance activ in the formulation of pharmaceutically acceptable dosage ity of a compound, can also be incorporated into the compo forms including emulsions and Suspensions. Other com sitions. monly used surfactants, such as Tweens, Spans and other 0314 Pharmaceutically acceptable salts of the agents of emulsifying agents or bioavailability enhancers which are this invention include those derived from pharmaceutically commonly used in the manufacture of pharmaceutically acceptable inorganic and organic acids and bases. Examples acceptable solid, liquid, or other dosage forms may also be of suitable acid salts include acetate, adipate, alginate, aspar used for the purposes of formulation. tate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, 0319. In all cases, the composition should be sterile, if camphorate, camphorsulfonate, cyclopentanepropionate, possible, and should be fluid to the extent that easy syring digluconate, dodecylsulfate, ethanesulfonate, formate, fuma ability exists. rate, glucoheptanoate, glycerophosphate, glycolate, hemisul 0320 Preferred pharmaceutical formulations are stable fate, heptanoate, hexanoate, hydrochloride, hydrobromide, under the conditions of manufacture and storage and must be hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, preserved against the contaminating action of microorgan malonate, methanesulfonate, 2-naphthalenesulfonate, nicoti isms such as bacteria and fungi. In general, the relevant carrier nate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phe can be a solvent or dispersion medium containing, for nylpropionate, phosphate, picrate, pivalate, propionate, Sali example, water, ethanol, polyol (for example, glycerol, pro cylate. Succinate, Sulfate, tartrate, thiocyanate, tosylate and pylene glycol, and liquid polyetheylene glycol, and the like), undecanoate. Other acids, such as oxalic, while not in them and suitable mixtures thereof. The proper fluidity can be selves pharmaceutically acceptable, may be employed in the maintained, for example, by the use of a coating such as preparation of salts useful as intermediates in obtaining the lecithin, by the maintenance of the required particle size in the compounds of the invention and their pharmaceutically case of dispersion and by the use of surfactants. Prevention of acceptable acid addition salts. the action of microorganisms can be achieved by various 0315 Salts derived from appropriate bases include alkali antibacterial and antifungal agents, for example, parabens, metal (e.g., sodium and potassium), alkaline earth metal (e.g., chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. magnesium), ammonium and NNo(C1-4 alkyl)4 salts. This In many cases, it will be preferable to include isotonic agents, invention also envisions the quaternization of any basic nitro for example, Sugars, polyalcohols such as manitol, Sorbitol, gen-containing groups of the compounds disclosed herein. Sodium chloride in the composition. Prolonged absorption of Water or oil-soluble or dispersible products may be obtained injectable compositions can be brought about by including in by Such quaternization. the composition an agent which delays absorption, for 0316 A pharmaceutical composition is formulated to be example, aluminum monostearate and gelatin. Prolonged compatible with its intended route of administration. Solu absorption of oral compositions can be achieved by various tions or Suspensions used for parenteral (e.g., intravenous), means including encapsulation. US 2009/0264.342 A1 Oct. 22, 2009

0321 Sterile injectable solutions can be prepared by 0326. The pharmaceutically acceptable compositions of incorporating the active compound in the required amount in this invention may also be administered by nasal aerosol or an appropriate solvent with one or a combination of ingredi inhalation. Such compositions are prepared according to ents enumerated above, as required, followed by filtered ster techniques well-known in the art of pharmaceutical formula ilization. Preferably solutions for injection are free of endot tion and may be prepared as solutions in Saline, employing oxin. Generally, dispersions are prepared by incorporating benzyl alcohol or other suitable preservatives, absorption the active compound into a sterile vehicle which contains a promoters to enhance bioavailability, fluorocarbons, and/or basic dispersion medium and the required other ingredients other conventional Solubilizing or dispersing agents. from those enumerated above. In the case of sterile powders 0327 Systemic administration can also be by transmu for the preparation of sterile injectable solutions, the pre cosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be ferred methods of preparation are vacuum drying and freeze permeated are used in the formulation. Such penetrants are drying which yields a powder of the active ingredient plus any generally known in the art, and include, for example, for additional desired ingredient from a previously sterile-fil transmucosal administration, detergents, bile salts, and tered solution thereof. fusidic acid derivatives. Transmucosal administration can be 0322 Oral compositions generally include an inert diluent accomplished through the use of nasal sprays or Supposito or an edible carrier. For the purpose of oral therapeutic admin ries. For transdermal administration, the active compounds istration, the active compound can be incorporated with are formulated into ointments, salves, gels, or creams as gen excipients and used in the form of tablets, troches, or cap erally known in the art. Sules, e.g., gelatin capsules. Oral compositions can also be 0328. The compounds can also be prepared in the form of prepared using a fluid carrier for use as a mouthwash. Phar Suppositories (e.g., with conventional Suppository bases Such maceutically compatible binding agents, and/or adjuvant as cocoa butter and other glycerides) or retention enemas for materials can be included as part of the composition. The rectal delivery. tablets, pills, capsules, troches and the like can contain any of 0329. In addition to the agents described above, in certain the following ingredients, or compounds of a similar nature: embodiments of the invention, the active compounds are pre a binder Such as microcrystalline cellulose, gum tragacanthor pared with carriers that will protect the compound against gelatin; an excipient Such as starch or lactose, a disintegrating rapid elimination from the body, such as a controlled release agent such as alginic acid, Primogel, or corn starch; a lubri formulation, including implants and microencapsulated cant such as magnesium Stearate or Sterotes; a glidant Such as delivery systems. Biodegradable, biocompatible polymers colloidal silicon dioxide; a Sweetening agent Such as Sucrose can be used, such as ethylene vinyl acetate, polyanhydrides, or saccharin; or a flavoring agent Such as peppermint, methyl polyglycolic acid, collagen, polyorthoesters, polyethers, and salicylate, or orange flavoring. Formulations for oral delivery polylactic acid. Methods for preparation of such formulations may advantageously incorporate agents to improve stability will be apparent to those skilled in the art. Certain of the within the gastrointestinal tract and/or to enhance absorption. materials can also be obtained commercially from Alza Cor 0323 For administration by inhalation, the inventive com poration and Nova Pharmaceuticals, Inc. Liposomal Suspen positions are preferably delivered in the form of an aerosol sions can also be used as pharmaceutically acceptable carri spray from a pressured container or dispenser which contains ers. These can be prepared according to methods known to a suitable propellant, e.g., a gas such as carbon dioxide, or a those skilled in the art, for example, as described in U.S. Pat. nebulizer. Liquid or dry aerosol (e.g., dry powders, large No. 4.522,811 and other references listed herein. Liposomes, porous particles, etc.) can be used. The present invention also including targeted liposomes (e.g., antibody targeted lipo contemplates delivery of compositions using a nasal spray. Somes) and pegylated liposomes have been described 0324 For topical applications, the pharmaceutically (Hansen CB, et al., Biochim Biophy's Acta. 1239(2):133-44, acceptable compositions may be formulated in a Suitable 1995: Torchilin V P. et al., Biochim Biophy's Acta, 1511(2): ointment containing the active component Suspended or dis 397-411, 2001; Ishida T, et al., FEBS Lett. 460(1): 129-33, Solved in one or more carriers. Carriers for topical adminis 1999). One of ordinary skill in the art will appreciate that the tration of the compounds of this invention include, but are not materials and methods selected for preparation of a controlled limited to, mineral oil, liquid petrolatum, white petrolatum, release formulation, implant, etc., should be such as to retain propylene glycol, polyoxyethylene, polyoxypropylene com activity of the compound. For example, it may be desirable to pound, emulsifying wax and water. Alternatively, the phar avoid excessive heating of polypeptides, which could lead to maceutically acceptable compositions can be formulated in a denaturation and loss of activity. Suitable lotion or cream containing the active components 0330. It is typically advantageous to formulate oral or Suspended or dissolved in one or more pharmaceutically parenteral compositions in dosage unit form for ease of acceptable carriers. Suitable carriers include, but are not lim administration and uniformity of dosage. Dosage unit form as ited to, mineral oil, Sorbitan monostearate, polysorbate 60, used herein refers to physically discrete units Suited as unitary cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl dosages for the Subject to be treated; each unit containing a alcohol and water. predetermined quantity of active compound calculated to pro 0325 For local delivery to the eye, the pharmaceutically duce the desired therapeutic effect in association with the acceptable compositions may be formulated as micronized required pharmaceutical carrier. Suspensions in isotonic, pH adjusted Sterile Saline, or, prefer 0331 Toxicity and therapeutic efficacy of such com ably, as Solutions in isotonic, pHadjusted Sterile Saline, either pounds can be determined by standard pharmaceutical pro with or without a preservative such as benzylalkonium chlo cedures in cell cultures or experimental animals, e.g., for ride. Alternatively, for ophthalmic uses, the pharmaceutically determining the LDs (the dose lethal to 50% of the popula acceptable compositions may be formulated in an ointment tion) and the EDs (the dose therapeutically effective in 50% Such as petrolatum. of the population). The dose ratio between toxic and thera US 2009/0264.342 A1 Oct. 22, 2009 36 peutic effects is the therapeutic index and it can be expressed route of administration, the rate of excretion, any drug com as the ratio LDs/EDs. Compounds which exhibit high thera bination, and the degree of expression or activity to be modu peutic indices are preferred. While compounds that exhibit lated. toxic side effects can be used, care should be taken to design 0335 The invention further provides pharmaceutical a delivery system that targets such compounds to the site of compositions comprising two or more compounds of the affected tissue in order to minimize potential damage to unin invention and, optionally, one or more antibiotic agents. The fected cells and, thereby, reduce side effects. invention further provides pharmaceutical compositions comprising one or more compounds of the invention, option 0332 The data obtained from cell culture assays and ani ally one or more antibiotic agents, and an additional active mal studies can be used in formulating a range of dosage for agent. The additional active agent may be an antibiotic that use in humans. The dosage of such compounds lies preferably has a different mechanism of action to that of the antibiotic within a range of circulating concentrations that include the that is potentiated by the compound. EDs with little or no toxicity. The dosage can vary within this 0336. The present invention also provides pharmaceutical range depending upon the dosage form employed and the packs or kits comprising one or more containers (e.g., vials, route of administration utilized. For any compound used in ampoules, test tubes, flasks, or bottles) containing one or the method of the invention, the therapeutically effective dose more ingredients of the inventive pharmaceutical composi (e.g., dose that is therapeutically effective to achieve a desired tions, for example, allowing for the simultaneous or sequen degree of antibiotic potentiation) can be estimated initially tial administration of the antibiotic potentiating agent and from cell culture assays. A dose can be formulated in animal antibiotic agent(s) it potentiates. Optionally associated with models to achieve a circulating plasma concentration range Such container(s) can be a notice in the form prescribed by a that includes the ICs (e.g., the concentration of the test com governmental agency regulating the manufacture, use or sale pound which achieves a half-maximal inhibition of symp of pharmaceutical products, which notice reflects approval by toms, half-maximal inhibition of growth or survival of an the agency of manufacture, use or sale for human administra infectious agent, etc.) as determined in cell culture. Such tion. Different ingredients may be supplied in Solid (e.g., information can be used to more accurately determine useful lyophilized) or liquid form. Each ingredient will generally be doses in humans. Levels in plasma can be measured, for Suitable as aliquoted in its respective container or provided in example, by high performance liquid chromatography. a concentrated form. Kits may also include media for the 0333 Atherapeutically effective amount of a pharmaceu reconstitution of lyophilized ingredients. The individual con tical composition typically ranges from about 0.001 to 100 tainers of the kit are preferably maintained in close confine mg/kg body weight, preferably about 0.01 to 25 mg/kg body ment for commercial sale. weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, Equivalents and Scope 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The 0337 Those skilled in the art will recognize, or be able to pharmaceutical composition can be administered at various ascertain using no more than routine experimentation, many intervals and over different periods of time as required, e.g., equivalents to the specific embodiments of the invention multiple times per day, daily, every other day, once a week for described herein. The scope of the present invention is not between about 1 to 10 weeks, between 2 to 8 weeks, between intended to be limited to the above Description and Examples about 3 to 7 weeks, about 4, 5, or 6 weeks, etc. The skilled below, but rather is as set forth in the appended claims. In the artisan will appreciate that certain factors can influence the claims and elsewhere in the specification, articles such as “a”, dosage and timing required to effectively treat a subject, “an and “the may mean one or more than one unless indi including but not limited to the severity of the disease or cated to the contrary or otherwise evident from the context. disorder, previous treatments, the general health and/orage of Claims or descriptions that include “or” between one or more the Subject, and other diseases present. Generally, treatment members of a group are considered satisfied if one, more than of a Subject with an inventive composition can include a one, or all of the group members are present in, employed in, single treatment or, in many cases, can include a series of or otherwise relevant to a given product or process unless treatments. It will be appreciated that a range of different indicated to the contrary or otherwise evident from the con dosage combinations (i.e., doses of the antibiotic and antibi text. The invention includes embodiments in which exactly otic potentiating agent) can be used. one member of the group is present in, employed in, or oth 0334 Exemplary doses include milligram or microgram erwise relevant to a given product or process. The invention amounts of the inventive compounds per kilogram of subject also includes embodiments in which more than one, or all of or sample weight (e.g., about 1 microgram per kilogram to the group members are present in, employed in, or otherwise about 500 milligrams per kilogram, about 100 micrograms relevant to a given product or process. Furthermore, it is to be per kilogram to about 5 milligrams per kilogram, or about 1 understood that the invention encompasses all variations, microgram per kilogram to about 50 micrograms per kilo combinations, and permutations in which one or more limi gram.) For local administration (e.g., intranasal), doses much tations, elements, clauses, descriptive terms, etc., from one or smaller than these may be used. It is furthermore understood more of the listed claims (or from the portion of the specifi that appropriate doses depend upon the potency of the agent, cation relevant to Such claim or claim element) is introduced and may optionally be tailored to the particular recipient, for into another claim. For example, and without limitation, any example, through administration of increasing doses until a claim that is dependent on another claim can be modified to preselected desired response is achieved. It is understood that include one or more elements or limitations found in any the specific dose level for any particular subject may depend other claim (or from the portion of the specification relevant upon a variety of factors including the activity of the specific to such claim or claim element) that is dependent on the same compound employed, the age, body weight, general health, base claim. Furthermore, where the claims or description gender, and diet of the Subject, the time of administration, the recite a composition, it is to be understood that methods of US 2009/0264.342 A1 Oct. 22, 2009 37 administering the composition according to any of the meth latter dose was shown to activate the SOS response using ods disclosed herein, and methods of using the composition microarray analysis (data not shown), it allowed bacterial for any of the purposes disclosed herein are included, and growth to 80% of wild type levels. The wild type E. coli was methods of making the composition according to any of the also shown to be able to grow in the presence of 0.1 ug/mL of methods of making disclosed herein are included, unless Cipro (a Sublethal concentration). otherwise indicated or unless it would be evident to one of 0343 0.05ug/mL of Nor and Cipro at 0.010 ug/mL were ordinary skill in the art that a contradiction or inconsistency used as Sublethal concentrations to screen a set of approxi would arise. mately 4.200 E. coli mutants, each having a deletion (knock 0338 Where elements are presented as lists, e.g., in out) of a single gene. Each deletion strain was constructed by Markush group format, it is to be understood that each sub replacing the chromosomal copy of the relevant gene in a wild group of the elements is also disclosed, and any element(s) type E. coli strain (K-12) with a selectable antibiotic resis can be removed from the group. It should it be understood tance gene, essentially as described, to create a precise gene that, in general, where the invention, or aspects of the inven deletion (Datsenko, K.A. and Wanner, B.L., “One-step inac tion, is/are referred to as comprising particular elements, tivation of chromosomal genes in Escherichia coli K-12 features, etc., certain embodiments of the invention or aspects using PCR products”, Proc Natl Acad Sci USA, 97, 6640 of the invention consist, or consist essentially of Such ele 6645, 2002); Baba, T., Ara, T., Okumura, Y., iki Hasegawa, ments, features, etc. For purposes of simplicity those embodi M., Takai, Y., Baba, M., Oshima, T., Tomita, M., Wanner, B. ments have not been specifically set forth in haec verba and Mori, H. (2003) Systematic construction of single gene herein. deletion mutants in Escherichia coli K-12 (in preparation) 0339. The inclusion of a “providing a subject... 'step in and see also the web site at ecoli.aist-nara.ac.jp/gb5/Re certain methods of the invention is intended to indicate that sources/deletion/deletion.html). The entire deletion set was the composition is administered to treat a bacterial infection. screened 3 times with Nor and 2 times with Cipro, with the Thus the subject will have or beat risk of a bacterial infection goal of identifying mutants unable to grow at Sublethal con and the composition is administered to treat the infection, centrations of either or both of these drugs. typically upon the Sound recommendation of a health care 0344) For each mutant, the ratio of growth of the mutant in provider, who may or may not be the same individual who the presence of a Sublethal concentration of quinolone to administers the composition. The invention includes embodi growth of the mutant in the absence of quinolone was calcu ments in which a step of providing is not explicitly included lated after 12 hours of growth at 37° C., based on ODoo and embodiments in which a step of providing is included. measurements. From these studies, 36 strains were identified 0340 Where ranges are given in the instant specification, as being completely unable to grow in the presence of Suble including the claims, endpoints are included. Furthermore, it thal concentrations of Nor and/or Cipro relative to their abil is to be understood that unless otherwise indicated or other ity to grow in the absence of the respective quinolone. Out of wise evident from the context and understanding of one of this screen tolC was identified as one of the genes whose ordinary skill in the art, values that are expressed as ranges deletion resulted in an inability to grow in the presence of a can assume any specific value or Subrange within the stated Sublethal concentration of quinolone. tolC encodes for an ranges in different embodiments of the invention, to the tenth efflux pump and its loss of function was shown to increase of the unit of the lower limit of the range, unless the context sensitivity to drugs (e.g., Husain et al., 2004; reviewed in clearly dictates otherwise. Koronakis, 2003). Therefore, this result was expected and 0341 Any particular embodiment of the invention (e.g., was a validation of the ability of the screen to successfully any antibacterial agent, any bacterial target, any compound, identify genes whose mutation results in increased sensitivity any method of treatment etc.) can be excluded from any one to quinolones. These genes are therefore targets for the devel or more claims, for any reason. In particular, and without opment of quinolone potentiating agents. Further details of limiting the preceding sentence, any embodiment of the the screen are provided below: present invention that falls within the prior art may be explic itly excluded from any one or more of the claims. Since such Protocol embodiments are deemed to be known to one of ordinary skill 0345 K12 E. coli whole genome deletion stocks. The in the art, they may be excluded even if the exclusion is not set whole genome deletion set was obtained as a frozen stock in forth explicitly herein. 90, 96 well plates containing LB, Kan 50 lug/mL. and 10% glycerol. The frozen stock was inoculated into new plates EXAMPLES containing 150 uL of LB Kan 50 ug/mL per well using a Example 1 96-pin replicator (VP scientific. VP407A). These plates were grown statically overnight at 37°C. Identification of Mutations that Potentiate Quinolone 0346 Preparing the assay plates. 90, 96-well flat bottom Activity. Using a Growth Assay polystyrene plates were filled with 150 uL of LB per well. 90 0342. In order to discover genetic targets for quinolone 96-well flat polystyrene plates were filled with 150 uL of LB, potentiating agents the present Applications have established Norfloxacin 25 ug/mL per well. 18096-well flat polystyrene a screen to identify E. coli mutants that would be largely plates were filled with 150 uL of sterile, deionized water. unable to grow in the presence of a concentration of qui 0347 Inoculating the assay plates. The optimal inocula nolone that would not allow the growth of wild type E. coli. tion is a 1000:1 dilution of an overnight growth culture. Each They decided to initially focus on two important members of pin holds ~15 uL of culture so the pin tool must be used to this compound class, i.e., norfloxacin (Nor) and ciprofloxacin serially dilute 3 times. The replicator was used to transfer the (Cipro). The growth of wild type E. coli was first shown to be overnight mutant growth plates to the sterile waterplate (dilu fully inhibited by 0.1 ug/mL of Nor (a lethal concentration) tion plate 1). The replicator was sterilized by washing in 70% but not by 0.05ug/mL (a sublethal concentration). While the ethanol, then 95% ethanol, then flamed. The replicator was US 2009/0264.342 A1 Oct. 22, 2009

again used to transfer from dilution plate 1 to a second (dilu ing. Nor (2 g/mL) was added to the cultures, and incubation tion plate 2). After sterilizing, the replicator was again used to was continued for 60 minutes. Samples of the cells were transfer from dilution plate 2 to an LB plate. The replicator collected and washed in PBS at 0 min (untreated) and 60 was again used to transfer from dilution plate 2 to an LB, minutes after adding Nor. The reduction in number of viable Norfloxacin 25 ng/mL plate). The OD600 was measured for a cells that occurred as a result of exposure to Nor was deter blank plate to determine OD600 for the plate and media mined by stamping identical 10-fold serial dilutions of the alone. The inoculated plates were grown statically overnight treated cells from each time point on LB agar as shown in at 37° C. FIG. 2. Columns 1 and 12 represent wild type bacteria. 0348 Resultanalysis. After overnight growth, the OD600 0352. A number of strains were identified that displayed was measured for every plate. The BLANKED OD600 was significantly reduced survival following exposure to Norrela obtained for every culture by subtracting the average OD600 tive to survival of the parental (wildtype) strain. For example, value from the blank plate. The RATIO of OD600 in the FIG. 2 shows that the recA null mutant, which was unable to presence of compound relative to the no drug control for that grow in the presence of a Sublethal concentration of Nor, was plate was calculated for every culture. (compound OD 600/no also unable to survive the Nortreatment. Thus in the left panel drug OD600). of FIG. 2, the recA deletion mutant (column 5) shows growth comparable to that of the wild type strain (columns 1 and 12) Overview 384 Well Plate whereas cells of this strain show very poor growth following (0349 12-384 well plates were filled with 50 uL of LB treatment with Nor (compare uppermost spot in column 5, left using the Beckman Multimek robot. The above plates were panel (untreated) with uppermost spot in column 5, right inoculated from the partially thawed glycerol stock plates panel). However, the tolC mutant (column 9 in FIG. 2) was using the VP384 replicator. The replicator is dipped in 70% shown not to have a significant decrease in Survival and ethanol for 10 seconds, 100% ethanol and then flamed in behaved more similarly to the wildtype strain. A subset of the between plates. The cultures are grown statically overnight in strains showing poor Survival, including the recA deletion the plates at 37°C. with a plastic wrapping to prevent desic strain, had also been identified using the growth assay cation. 24 dilution plates are filled with 50 uL of sterile dHO described in Example 1, thus providing further evidence of for dilution of the cultures before stamping. Before the drug is the significance of the deleted genes as targets for the identi added, a sample of the culture is taken and diluted into the fication of compounds that potentiate the activity of qui above water plates using the VP384 replicator. The replicator nolone. holds 0.2 LL of liquid. This corresponds to a 1:250 dilution. 0353. The recA null mutant was tested using the survival Using the Multimek, 5uL of LB Norfloxacin (10 ug/mL) was assay with Cipro rather than Norand the mutant was observed added to every well of every growth plate. While the drug to display reduced survival following exposure to a lethal treatment was incubating, the 0 drug time point dilution can concentration of Cipro (500 ng/mL). be stamped on 245 mm bioassay dishes using the VP384. (The plates must be sufficiently dry for this to work properly.) Example 3 Two plates stamped in duplicate will fit on one 245 mm assay dish. After a 3 hour incubation with Norfloxacin, another recA Mutant Response to Nor and Cipro sample can be diluted in HO as described above. The 3 hour time point can then be stamped on the agar plates as described 0354 As described in Examples 1 and 2, deletion of recA above. No washing is necessary because the cells are diluted in E. coli was shown to result in an inability to grow in the down to 4 ng/mL of Norfloxacin. This is less than 1/25" MIC. presence of sublethal concentrations of either Nor or Cipro (Example 1) and a reduced Survival following exposure to Example 2 lethal concentrations of Nor or Cipro (Example 2). The present Applicants decided to focus on recA as an exemplary Identification of Mutations that Potentiate Quinolone target gene. Loss of recA function was first confirmed not to Activity. Using a Survival Assay alter growth of E. coli (data not shown). The ability of Nor 0350 188 E. coli genes were identified for which a sensitive and Nor-resistant wildtype E. coli and recA deletion homolog existed in S. aureus (MRSA 252) by performing mutants to survive in the presence of Nor was then tested searches of publicly available sequence databases. Homologs using the Survival assay described in Example 2. were identified using the NCBI Genplot pairwise genome 0355 Nor-resistant E. coli strains were then constructed comparison of protein homologues. A 96 well plate format by introducing mutations into the gyra locus using a standard was then used to perform a Survival assay on a set of E. coli molecular genetics approach (Datsenko and Wanner, 2000) deletion strains, each of which had a deletion in one of these and selecting for cells able to form colonies on plates con highly conserved genes. The Survival assay measured the taining Nor. The transformants were plated on LB plus Nor at number of cells which form colonies (colony forming units, 0.1 g/mL. The individual colonies were streaked for single or CFU) after growth in the presence of a compound. This colonies on LB plus Nor at 0.1 lug/ml. The final selected assay was applied to identify deletion strains which cannot candidates could form colonies at 0.175ug/mL of Nor. Muta survive following a limited time period of exposure to a tions in Gyr A at position 83 and 87 are known to confer quinolone treatment at a lethal concentration as compared to resistance to quinolones (Everett et al., 1996: Komp Lindgren survival of the parent strain. The lethal concentration one that et al., 2003). A PCR product containing a portion of gyra and inhibits growth and would eventually be lethal if exposure harboring mutations that result in a serine to leucine change at was continued indefinitely. 2 ug/mL of Nor was selected as position 83 and an aspartic acid to asparagine change at the concentration to use. position 87 in the GyrA polypeptide was transformed into 0351. The assay was performed as follows: Cultures were MG1655 recA" (wild type strain) and MG1655 recA (recA grown to early log phase in LB medium at 37°C. with shak deletion strain) cells, which were then plated on medium US 2009/0264.342 A1 Oct. 22, 2009 39 containing Nor for selection. Transformants able to survive cating that loss of RecA function increases sensitivity to Nor. on this medium were selected and further characterized. MICs were determined as described above. 0356. The ability of these fours strains to survive exposure 0359 FIG. 4 summarizes the effect of deletion of either to Nor was then tested. The experiment was performed as recA or tolC on bacterial growth in the presence of Nor (left follows. Cultures were grown to early log phase in LB at 37° panel) and survival following a period of exposure to Nor C. with shaking. At the 0 time point Nor was added to the (right panel). Both recA and tolC strains showed a reduced cultures to a final concentration of 0.1 ug/mL. This concen MIC. However, only the recA strain showed a decrease in tration of Noris just below the MIC for strain MG 1655 under survival while the tolC strain has a survival response similar these growth conditions. Samples of the cells were collected to that of the parent strain. Without wishing to be bound by and washed in PBS at 0, 30,90 and 180 mintime points. The any theory, this result suggests that inhibitors of RecA or number of viable cells was determined by plating the cells other target polypeptides whose absence or inactivation from each time point on LB agar and counting colonies. The inhibits both growth and Survival may have advantages rela CFU counts were normalized based on the time 0 time point tive to inhibitors of bacterial efflux pumps. inoculum for each culture (i.e., the CFU count obtained from the 0 time point inoculum was taken to represent 100% sur Survival Assay in the RN4220 Background. vival) and the log % survival was plotted (FIG. 3). 0357 Both of the E. coli recA strains were observed to be 0360. The survival ability of RN4220 and RN4220 recA highly sensitive to quinolone treatment and showed very poor following exposure to Nor was then tested as described in viability following exposure to a sublethal concentration of Example 2 for E. coli strains. The recA null mutant and the drug. As shown in FIG.3, far fewer recA cells than recA parental strains (RN4220 recA and RN4220, respectively) cells were able to form colonies after treatment with Nor. The were grown to early log phase in LB at 37°C. with shaking. recA deletion caused a 10,000 fold increase in quinolone At the 0 time point Nor was added to the cultures to a final sensitivity in a wild type background after 3 hours of expo concentration of 0.5 lug/mL, which is just below the MIC for sure to Nor. In addition, deletion of recA in the strain back wildtype cells (RN4220). Samples of the cells were collected ground containing the mutation that confers Norresistance on and washed in PBS at 0, 30.90 and 180 minutes time points. wild type E. coli caused the strain to exhibit wild type levels The number of viable cells was determined by plating the of sensitivity to Nor, i.e., it rendered a strain that would cells on LB from each time point and counting colonies. The otherwise have been Nor-resistant sensitive to Nor. recA was CFU counts were normalized based on the time 0 time point concluded to be essential for survival in the presence of qui inoculum for each culture (i.e., the CFU count obtained from nolones in both wild-type and Nor resistant strain back the 0 time point inoculum was taken to represent 100% sur grounds. Based on these results, inhibiting the expression vival) and the log % survival was plotted (FIG. 5). and/or activity of E. coli RecA (or its homolog in other bac 0361) RN4220 recA was observed to be highly sensitive teria) is expected to increase the sensitivity of the bacteria to to quinolone treatment and could not recover following a quinolones to which they already display sensitivity (i.e., will period of exposure to Nor. It was concluded that recA is reduce the MIC), thereby allowing the use of a lower dose of essential for survival in the presence of quinolones (FIG. 5). the drug for treating Subjects suffering from or at risk of As in E. coli, recA deletion causes 10,000x increase in qui bacterial infection. In addition, inhibiting the expression and/ nolone sensitivity compared to the parental Strain after 3 or activity of E. coli RecA (or its homolog in other bacteria) hours of exposure to Nor. is expected to renderbacterial strains that have acquired resis 0362. Further details of the assay areas follows: Overnight tance to quinolones (e.g., due to mutation in one or more mutant MG 1655 or Staph cultures of clones were grown in 5 Subunits of the bacterial gyrase or topoisomerase IV and/or mL of LB with appropriate selective agents at 37° C. with due to increased copy number of a gene encoding one of these shaking. A morning culture was started in a 96 well plate with polypeptides) sensitive once again to these compounds. 16 uL of cells into 1.6 mL of LB and the appropriate plasmid selective agent. Cultures were grown at 37° C. for approxi Example 4 mately 3 hours to mid log phase A dilution series of Norfloxa cin or Cipro was made. A 50 lug/mL stock of Norfloxacin/ RecA Deletion Increases Sensitivity of Staphylococ Cipro was diluted in a 2-fold dilution series to the final cus aureus to Quinolones in Both Wild Type and concentrations below. Serial dilutions were done with 100 uL Quinolone Resistant Strain Backgrounds of drug into 100LL of H.O. (The 50 ug/mL Norfloxacin/cipro Growth Assay stock was made by the dilution of 40 uL of 1.25 mg/mL stock in 960 ul of HO). 20 uL of each log phase culture was put into 0358. The growth and response to quinolone of S. aureus a well of one row of a 96 well plate containing 2 LL of LB and strain RN4220 (wild type) and its recA derivative (RN4220 the appropriate selective agent. 4.5 uL of each drug concen recA) were first examined. These strains have been tration was arrayed into the 8 rows of a sterile 300 uL 96-well described in the literature (Bayles et al., 1994). The RN4220 plate. 250 uL of inoculated culture was added to each column recA null mutant (RN4220 recA) contains the recA569 of the sterile 300 uL 96-well plate with drug. The final plate allele and is referred to KB103 in Bayles, et al., 1994. has a different clone in each column and a different Norfloxa RN4220 recA was shown to be affected in genetic recombi cin concentration in each row. Plates were grown overnight at nation and DNA repair (Bayles et al., 1994). The present 37°C. with shaking. The OD600 of the plate was measured Applicants first showed that RN4220 recA and the parental after 16 hours. strain RN4220 have similar growth curves as measured by optical density (ODoo) or colony formation when grown in Survival Assay in a Quinolone Resistant Strain Background rich liquid medium in the absence of quinolone (not shown). They then determined that the MICs for Norfor RN4220 and 0363 The Applicants then compared the ability of strains RN4220 recA were 1.53 and 0.38 ug/mL, respectively, indi that carry a recA allele in a quinolone resistant (grlA542) US 2009/0264.342 A1 Oct. 22, 2009 40 and nonresistant strain background to Survive exposure to a lethal concentration of Nor (Trucksis M. Wolfson J S. Hooper DC.J. Bacteriol. 1991 September; 173(18):5854-60. A novel S. airetts locus conferring fluoroquinolone resistance in Staphylococ Norfloxacin Ciprofloxacin homolog cus aureus). “Lethal here refers to a concentration lethal to a wildtype strain. The grlA542 allele contains a mutation in the Gene Growth Survival Growth Survival identified grlA locus (also called parC), resulting in a serine to pheny recA ------lalanine change at position 80 in the GirlA subunit of S. aureus rec ------DNA topoisomerase IV that confers quinolone resistance on recC -- -- recC ------S. aureus strains that would otherwise be sensitive to quino recN -- -- lones (Fournier et al., 2000). Cultures were grown to early log ruvA ------phase in LB at 37° C. with shaking. Nor was added to the ruw8 ------ruvC -- -- cultures to a final concentration of 2 ug/mL. Samples of the uwrD ------cells were collected and washed in PBS at 0, 120 (2 hr) and xerD -- -- 180 (3hr) mintime points. The reduction in number of viable XseA -- -- XseB -- -- cells was determined by stamping identical 10-fold serial pinO -- -- dilutions of the treated cells from each time point on LB as dnaT -- shown in FIG. 6, where wt refers to wild type, recA" refers priA ------polA -- -- to the strain containing the recA null allele in an otherwise hyA -- wild type strain background, grlaš42 refers to the strain con acrA -- -- taining the grlA542 allele in an otherwise wild type strain acrE -- background, and grlA542recA" refers to the strain contain olC -- -- b1440 -- ing both the recA null allele and the grlA542 allele. The MIC ompC -- for strains wit, recA", grla542 and grlA542recA" were abH ------0.78, 0.19, 12.5 and 1.56 g/mL, respectively. dkSA -- is ------0364. At time 0, the number of cells for the parental and dapF -- recA strains were almost the same, while the strain harboring N -- -- the quinolone resistance mutation (grlA542) had more cells. wS303 -- -- After 2 and 3 hours of growth in presence of Nor, aliquots yhfT -- -- yggC -- -- were taken, washed then serial dilution were plated onto LB ycS ------containing plate without Nor. Colonies were observed only res.A -- -- for the grlA mutant strain after 3 hours of treatment, while the dapF ------glrA-recA-strain was barely able to form colonies (FIG. 6). ygoC -- It was concluded that the recA mutant S. aureus strains are rimK -- more sensitive to quinolone treatments in both wild type and yicN -- quinolone resistant background. 0365 Based on these results, inhibiting the expression and/or activity of S. aureus RecA is expected to increase the Example 5 sensitivity of S. aureus to quinolones to which they already recA Null Mutation Reduces Bacterial Viability in display sensitivity (i.e., will reduce the MIC), thereby allow Rats and Mice ing the use of a lower dose of the drug for treating Subjects Suffering from or at risk of S. aureus infection. In addition, 0367 The present Applicants studied the viability of recA inhibiting the expression and/or activity of S. aureus RecA is null mutants in a rat model. S. aureus RN4220 and RN4220 expected to render S. aureus strains that have acquired resis recA, strains (see Example 3) were grown to logarithmic tance to quinolones (e.g., due to mutation in one or more phase. Individual animals were inoculated with 10, 10 or Subunits of the bacterial gyrase or topoisomerase IV and/or 10° cells of either strain in a volume of 200 uL of PBS by due to increased copy number of a gene encoding one of these intraperitoneal injection. A control animal was injected with polypeptides) sensitive once again to these compounds. PBS only. At the end of day 1, the animal infected with the 0366. The table below summarizes results of the screens highest inoculum of RN4220 was showing signs of illness and homology searches. The left column of the table lists while the 6 other animals were apparently unaffected. On day quinolone potentiator target genes, i.e., genes whose deletion 4, the animals were sacrificed, and the spleens were collected resulted in significantly reduced growth or both significantly and processed to quantify the progression of the infection reduced growth and survival in either Nor, Cipro, or both. The using a colony formation assay. Briefly, a spleen tissue extract remaining columns from the left indicate whether the mutant was made, which was then diluted and plated on antibiotic having a deletion of the indicated gene was able to grow or containing plates to allow colony formation. For the recA survive in the presence of sublethal or lethal concentrations of strain, the numbers of colonies recovered were 8, 22, 104 for Nor. The fourth and fifth columns from the left indicate the animals inoculated with 10, 10 or 10° cells, respectively. whether the mutant having a deletion of the indicated gene For the recA strain, the numbers of colonies recovered were was able to grow or survive in the presence of a sublethal or 1, 0, 1 for the animals inoculated with 10, 10 or 10° cells, lethal concentration of Cipro, respectively. In each case, “+” respectively. As expected, we recovered no colonies from the indicates that the strain failed to grow or survive. The column control animal injected with PBS only. on the right indicates whethera homolog of the indicated gene 0368. A larger experiment was then performed in mice to was identified in S. aureus. assess the effect of a recA null mutation in a Nor resistant US 2009/0264.342 A1 Oct. 22, 2009

strain background using the bacterial strains described in detection of the amount of ATP remaining in a reaction mix Example 4. Fifteen female CD-1 mice (20-22 g) were inocu ture following incubation of RecA protein, DNA, and ATP. lated with about 5x107 CFU of bacterial strain grlA542 or 40-50 ug/mL of RecA (New England Biolabs) was combined grlA542 recA" (107 CFU) in 0.5 mL PBS via intraperitoneal with 20 uM ATP in a final volume of 50 uL PNK buffer 1x injection. Survival of the mice was monitored at 24 and 48 either with or without and 250 g/L of M13 DNA, and the hours after injection. By 48 hours after injection all of the reaction mixtures were incubated at 25°C. for 30 minutes. 20 mice inoculated with the recA strain (grlA542recA") sur uMATP-Gamma, a non-hydrolyzable form of ATP that inhib vived, while all but one of the mice inoculated with the recA its RecA, was added to some of the reactions 10 minutes after strain (grlA542) perished. In other words, only 6.6% of the the start of the incubation. mice inoculated with the recA" strain survived while 100% of 0373 The assay measures the amount of ATPase (adenos the mice inoculated with the recA- strain survived. These ine triphosphate (ATP)-adenosine diphosphate (ADP)) activ results show that S. aureus cells lacking a functional RecA are ity catalyzed by E. Coli RecA protein. In the assay, recA is notable to Survive in a rat and mice hosts, demonstrating that incubated in reaction buffer with DNA, ATP and compounds these models are useful for the identification and/or testing of of interest for a measured amount of time. The quantity of potential inhibitors or antagonists of RecA. ATP remaining after the RecA reaction is quantitated using a Subsequent luciferase assay. The amount of ATP remaining Example 6 can be compared to inhibited and fully active control samples. Development of a Cell-Based Assay to Identify Details of the assay are provided below. Inhibitors of RecA 0374. Materials. Kingase glo plus, adenosine triphosphate (ATP), Adenosine 5'-O-(3-thiotriphosphate), and 384 well 0369. As discussed above, the recA promoter is induced plates, white were obtained from Fisher; and M13 mp 18 when RecA becomes activated as a result of exposure to DNA Single-stranded DNA, RecA Protein and T4 Polynucleotide damaging agents. The present Applicants hypothesized that Kinase Reaction Buffer=PNK were obtained from New exposure to a quinolone may have a similar effect on the recA England Biolabs. PNK contained 70 mM Tris-CL pH 7.5, 10 promoter and that this effect could be exploited to develop a mM MgCl, and 5 mM DTT. cell-based assay for inhibitors of RecA. A low copy reporter 0375 Assay Procedure. Final reaction mixture contains: plasmid was used in which the recA promoter controls tran 0.5 LL of M13 single stranded DNA (250 ug/mL); 1.0 uL of Scription of the mRNA encoding green fluorescent protein 10xPHK buffer: 0.25 uL of RecA (2 mg/mL): 0.25 uL of 1 (GFP). The plasmid, referred to hereinas prec::GFP has been mMATP; 0.25uL of test compound in DMSO; and 12.0 uL of previously described (Ronen, 2002). See also Kuang, 2004. H.O. prec:GFP contains 2821707-2821893 of the E. coli MG 1655 0376 Test compounds are incubated at room temperature genome (numbering based on the sequenced E. coligenome in the presence of RecA protein and DNA prior to addition of as reported in Blattner, F. R., et al., Science, 277, pp. 1453 ATP. This can beachieved by the preparation of two mixes, as 1474, 1997) cloned upstream of a promoterless GFPmut3 indicated below. Desirably, both a no-DNA and a DNA/ gene in a low copy pSC101 origin plasmid as described DMSO-only (i.e., no test compound) control were included. (Kalir, S., et al., Science, 292, pp. 2080-2083, 2001). 0370. The prec::GFP plasmid was introduced into MG1655 and MG1655 recA null Strains and measured the fluorescence emitted by GFP in the presence or absence of Component Mix 1 (in ul) ATP Mix (in ul) quinolone as compared with background fluorescence M13 O.S O.O detected from cultures of MG 1655 lacking the plasmid. 1OXPNKBuffer 1.O O.25 Briefly cells of these three strains were grown to early log RecA O.25 O.O phase in LB at 37°C. with shaking. Various amounts of Nor 1 mM ATP O.O O.25 were added to 100 uL cultures of each strain to achieve final 1 mM ATP-G or Test compound O.S O.O concentrations ranging from 0 g/mL to 31.25 ug/mL. After HO 7.75 2.0 60 minutes, fluorescence due to GFP expression from the Total each 10 2.5 reporter plasmid was measured using a spectrophotometer. 0371. As shown in FIG. 7, fluorescence in a recA" wild type strain (red bars—middle bars in each group) was clearly 0377 The following steps are then performed: (1) Add detected while the fluorescence level in a recA null strain 7.75 uL of mix 1 to the well of a 384 well plate; (2) Add 0.5 (yellow bars—right bars in each group) was close to the LL of the compound of interest; (3) Incubate at room tem background level, i.e., approximately the same as that of the perature for 5 minutes; (4) Add 2.5 LL of the ATP mix. wild type strain lacking the plasmid (blue bars—left bars in Incubate at room temperature for 30 minutes; (5) Add 12.5 LIL each group). Based on these results it would be expected that of Promega Kinaseglo plus; (6) Incubate at room temperature a compound that inhibits expression of RecA or inhibits any for 10 minutes or more; and (7) Measure luminescence of the of a variety of functions of RecA will result in a decrease in reaction. GFP fluorescence (FIG. 7) in cells containing the reporter 0378 FIG. 8 shows results of the assay when performed plasmid. using four different reaction mixtures, as indicated on the figure beneath the bars showing luminescence (relative light Example 7 units). Bars C and Dillustrate the absence of RecA activity in reactions that contain ATP-gamma, which inhibits RecA. Development of an In Vitro Assay to Identify Inhibi Controls in which no RecA was added were also performed. tors of RecA A comparison between bar A and bars C and D shows that 0372. As discussed above, RecA is a DNA-dependent RecA activity in the presence of DNA can be readily detected ATPase. An assay was developed for RecA activity based on based on the reduced luminescence detected from reactions in US 2009/0264.342 A1 Oct. 22, 2009 42 which ATP has been consumed by active RecA. A comparison identified as RecA inhibitors. The extent of RecA inhibition between bars A and B demonstrates that RecA activity is was assessed by comparison with fluorescence in the wells DNA dependent in this assay. that contain ATP-gamma, which represents maximal inhibi tion of RecA (and thus approximately background levels of Example 8 fluorescence). Identification of Quinolone Potentiating Agents Example 10 Using a RecA Inhibition Assay Demonstrating Quinolone Potentiation in E. coli 0379 Test compounds used in this screen are housed in 0383. A growth assay was performed in order to demon wells of microwell plates in which 100 g of each compound strate potentiation of norfloxacin by candidate potentiating was resuspended in 50 uL of DMSO (2 ug/uL). The screen agents, e.g., compounds identified as RecA inhibitors using was performed using 384-well plates. A reaction mixture the assays described in Example 8 or 9. Samples of E. coli containing 2 mg/mL of RecA (New England Biolabs), 250 were cultured overnight in standard culture medium in the ng/uL of M13 DNA, and 1 mM ATP in a final volume of 50LL presence of Nor (control), and their growth was compared PNL buffer 1x was dispensed into each well. 5 uL of a with that of samples of E. coli that have been cultured in the different compound solution (10 ug of compound) was added presence of both Nor (0.05ug/mL) and a test compound (50 to each well, one compound per well. The plate included 4 ug/mL) over the same time period and under otherwise iden negative control wells in which no test compound was added tical conditions. The concentration of Nor used for the assay to the reaction mixture, and 4 positive control wells, in which was not sufficient to kill and/or inhibit growth of E. coli by the reaction mixture also contained 1 nM ATP-gamma to itself. The assay was performed in multiwell plates, e.g., maximally inhibit RecA and did not contain a test compound. microtiter plates, in which each well contained a different test The plates were incubated for 1 hour, following which 50 uL compound. Growth was quantified by measuring optical den of the Promega Kinase-GloTM Reagent (Promega) was added sity (OD) using a Tecam spectrophotometer at a wavelength to each well. Luminescence was measured. Test compounds of 600 nm. Growth in the well containing both norfloxacin in wells in which the luminescence was lower than the aver and a particular test compound was compared with growth in age luminescence detected in the negative control wells were a well containing Nor (0.05ug/mL) but not containing the test identified as RecA inhibitors. The extent of RecA inhibition compound and was also compared with growth in a well by any particular test compound was assessed by comparing containing the test compound but not containing Nor. Lack of the luminescence with that detected in the positive control growth or poor growth in the well containing both Nor and a well, which represent 100% inhibition of RecA. Compounds particular test compound relative to growth in a well contain that inhibit RecA ATPase activity by at least 80% were ing Nor but not containing a test compound indicates that the selected for further screening. test compound is either a potentiator of Nor activity or has 0380 For each test compound, the difference between the antibacterial activity by itself, or both. If, in addition, the OD600 for the well containing the test compound and Nor presence of the test compound does not result in lack of and the OD600 for the well containing the test compound but growth or poor growth in the absence of Nor, then the test no Nor was calculated. If the OD600 for the well containing compound is a potentiator of Noractivity but apparently lacks a particular test compound and Nor was significantly lower antibacterial activity by itself, at least under the conditions of than the OD600 for the well containing the same test com the assay, i.e., the compound has quinolone-dependent anti pound and not containing Nor, then the test compound was bacterial activity. identified as a quinolone potentiating compound. 0384 Compounds that were determined to have qui 0381 Compounds identified as RecA inhibitors using this nolone-dependent antibacterial activity were tested using the assay were further tested to determine whether they are qui same method at lower concentrations. nolone potentiating agents as described in Example 10. 0385 Compounds identified as quinolone potentiating compounds using this screen were tested to determine Example 9 whether their potentiating activity is specific to quinolones by examining their ability to inhibit bacterial growth in the pres Identification of Qainolone Potentiating Agents ence of Sublethal concentrations of other antibiotics, e.g., Using a Cell-Based RecA Reporter Assay macrollides, aminoglycosides, penicillins, cephalosporins, 0382 Test compounds used in this screen are housed in etc. Compounds that fail to inhibit bacterial growth in the wells in microwell plates in which 100 ug of each compound presence of sublethal concentrations of other antibiotics were is resuspended in 50 uL of DMSO (2 ug/uL). The screen was identified as being specific quinolone potentiating agents. performed using 384-well plates. 50 uL of an early log phase 0386 Compounds identified as quinolone potentiating culture of MG1655 containing the prec: GFP plasmid were agents were tested to determine whether they affected the dispensed into each well, and Nor was added to a final con growth of a variety of cell types at a range of concentrations. centration of 10 ug/mL. As shown in Example 6, this concen Compounds that have little or no effect on mammalian cell tration of Norresults in robustinduction of the recA promoter. growth at concentrations at which they display quinolone A different test compound was added to each well to a final potentiation were identified as being particularly promising concentration of 50 g/mL. Each plate contained 4 control for use in a therapeutic context. wells to which no test compound was added and 4 control Example 11 wells to which 50 nM ATP-gamma was added to inhibit RecA. The wells were incubated at 37° C. for 60 minutes, Demonstrating Quinolone Potentiation in Staphyllo following which GFP fluorescence was detected. Compounds COCCS CaliS in wells that display decreased fluorescence relative to the 0387 Example 10 was repeated except that Staphylococ control wells to which no test compound was added were cus aureus, a gram-positive coccus, was used instead of E. US 2009/0264.342 A1 Oct. 22, 2009 coli, in order to demonstrate that test compounds potentiate ing agent and a quinolone that is too toxic for routine clinical quinolone activity against a broad spectrum of microorgan use allows the use of a reduced amount of the quinolone. isms. Example 15 Example 12 RecA Activity Assay—DNA Binding Assay Demonstrating Quinolone Potentiation in Pseudomo 0392 This assay measures RecA DNA binding activity, nas aeruginosa and can be used to identify or characterize RecA inhibitors according to the present invention. 0388 Pseudomonas aeruginosa is an opportunistic bacte 0393. The plasmid puC19 contains 5 HpyCH4 IV sites, rial pathogen that, for example, causes chronic lung infec including one at position 374. A 60 mer oligonucleotide was tions in cystic fibrosis patients and is a major source of noso designed having complementarity to the region in puC19 that comial infections. Example 10 was repeated except that centers around the HpyCH4 IV site at position 374. The Pseudomonas aeruginosa, was used instead of E. coli, in following reaction components were combined in 40 L order to demonstrate that test compounds potentiate qui RecA Reaction Buffer: 1 g of puC19; 0.18 ug of 60 mer: 0.3 nolone activity against this important pathogen. mM ATP Y-S; and 4 ug of RecA, and were incubated at 37°C. for 10 minutes, so that a stable triple helix was formed. Unprotected sites were methylated using 8 units of SSSI Example 13 supplemented with 160 uMSAM for 10 minutes at 37°C. The reaction was stopped and the triple helix was disrupted by Demonstrating Quinolone Potentiation in Ciprof incubation at 65° C. for 15 minutes. The reaction was cooled loxacin-resistant Pseudomonas aeruginosa and 10 units of HpyCH4 IV were added. Digestion was allowed to proceed at 37° C. for 20 minutes. Greater than 90% 0389 Resistance of Paeruginosa to quinolones is becom of the product was single cut puC19. Thus, this assay can be ing an increasing problem. Example 10 was repeated except used to identify and/or characterize RecA inhibitors because, that a clinical isolate of ciprofloxacin-resistant Pseudomonas if they interfere with RecA DNA binding, then they will aeruginosa, was used. The strain has a MIC of about 16 ultimately reduce the amount of single cut product observed. ug/mL (determined as described in Lomholt, J A and Kilian, M. Br. J. Opthalmol., 87: 1238-1240, 2003). The strain exhib Example 16 its essentially no growth inhibition when cultured in vitro in medium containing 0.25 ug/mL ciprofloxacin. The bacteria RecA Activity Assay—Biochemical Co-Protease were also cultured in medium containing 0.25 ug/mL cipro Assay floxacin and 1 lug/mL of a quinolone potentiating agent iden 0394. In this assay, which is based on an assay developed tified as described above. The bacteria were unable to grow, by Dutreix et al., J. Bacteriol., 1989, 171: 2415), the Lex A demonstrating that the combination of a quinolone and a coding sequence will be fused to a HIS tag (Qiagen), and quinolone potentiating agent is effective against bacteria that expressed from the pGE vector under control of the T5 pro are resistant to the quinolone alone. moter. This vector allows for expression of C-terminally 6xHis-tagged proteins. The lexA ORF will be created by Example 14 introducing an NcoI restriction site at the ATG codon of the insert by PCR. Identity of the insert will be confirmed by Synergistic Effect of a Quinolone and a Quinolone DNA sequencing. A test expression experiment will be per Potentiating Agent Allows Reduction in Quinolone formed and the integrity of the Lex A protein will be checked Dose using an anti-HIS antibody as described in the Qiagen kit. The QIAexpress detection reagents include high-affinity, high 0390 Administration of certain quinolone antibiotics, specificity monoclonal Anti-His antibodies and antibody e.g., gatifloxacin, has been shown to result in a prolongation conjugates, and Ni-NTA conjugates. of the QT interval on the electrocardiogram, which can lead to 0395. The pGE-lexA plasmid will be transformed into a dangerous cardiac arrhythmias Such as torsades de pointes. recA+ and recA- strains to test the assay genetically. In a Certain of these agents are highly effective antibiotics. How recA+ background, Lex A protein is expected to be seen intact ever, concerns regarding their potential to cause arrhythmias unless DNA damage is induced, e.g., by exposure to an appro when administered at clinically effective doses has led to priate antibiotic agent (e.g., a quinolone). If DNA damage is termination of development of certain of these agents or their induced, either a cleaved (shorter) Lex A or no Lex A protein withdrawal from the market. It would be desirable to be able is expected to be observed. Lex A protein can be detected to administer these agents at a lower dose that would not have using a Western blot assay. In a recA-background, Lex A is proarrhythmic consequences while still providing a clinically expected to be seen intact, whether or not DNA damage is useful antibacterial effect. induced. 0391 E. coli are cultured at a concentration of gatifloxacin 0396 Once the assay is validated, it can be used to assess below its MIC and their growth is assessed as described in the ability of RecA inhibitors to interfere with the RecA Example 1. Bacterial growth is not significantly inhibited. co-protease activity. It is expected that addition of RecA However, when bacteria are cultured in the presence of a inhibitors, prior to or during DNA damage (i.e., exposure to quinolone potentiating agent and the same Sub-MIC concen quinolone antibiotic agent), will decrease the amount of tration of gatifloxacin, they fail to show significant growth, LeXAdegradation, and will increase the amount of full-length demonstrating that the combination of a quinolone potentiat LeXA. US 2009/0264.342 A1 Oct. 22, 2009 44

0397) Typically, assay reactions will be performed so that (generally well above, e.g., 4x or 8x) its MIC, and further in 50 ug/ml of test compound is utilized in the presence of 300 the presence or absence of a test agent (that may inhibit or ng/ml of Cipro. This amount of Cipro is enough to induce the enhance mutation). RecA response but is not enough to kill the cells. 0403. Every 24 hours, an aliquot is removed from the 0398. Typically, 2 ml of E. coli cells containing pCRE-lex.A culture and is diluted so that the antibiotic agent is no longer will be grown to an ODoo of 0.5, then exposed to Cipro or to present at a concentration above its MIC (e.g., so that the Cipro-test compound for 30-60 minutes. The cells will be antibiotic agent is present at 0.5xMIC or less). These ali harvested and disrupted; the protein extract will be applied on quoted Samples are then assayed to determine whether they to the Ni-NTA conjugate column, aliquots will be run onto a can grow in the presence of the antibiotic agent. Specifically, SDS-PAGE, and Lex A detection will be done using the anti when growth is observed in the presence of antibiotic agent at HIS antibodies. Compounds for which the Lex A protein a concentration that is 4xMIC, then resistance is said to have remains intact likely due to RecA inhibition will be further developed. Test compounds can therefore be assayed to examined. assess whether or not they can reduce the incidence of resis tance (e.g., by inhibiting mutation). Example 17 Example 19 Cell-Based Survival and Cell Growth Assays In Vivo Assay of RecA Inhibitor Activity in Rats 0399 Cell-based Survival Assay. This assay measures the 0404 An in vivo system has been developed for the iden number of cells that are able to form colonies (i.e., the number tification and/or characterization of RecA inhibitors in rats. of colony forming units, CFU) after a culture has been treated Specifically, the present Applicants have determined that with a particular antibiotic agent (e.g., norfloxacin or cipro). infectious disease can be established in rats by intraperitoneal Typically, the antibiotic agent is applied to an early log phase injection of an appropriate number of bacterial cells. They culture at a concentration that is above the minimum inhibi have further demonstrated that injection of the same number tory concentration (“MIC': the minimum concentration that of otherwise identical cells lacking RecA (i.e., recA-mutant will inhibit growth) for the relevant strain under the relevant cells) does not establish infection. Furthermore, by isolating growth conditions. For example, the antibiotic agent may be spleens from the infected rats and plating the colony forming applied at a level that is a multiple of the MIC, often 4x or units from within those spleens, they have established that 8xMIC. Samples of the cells are collected at various time recA- cells both (1) are avirulent; (2) and do not survive or points after addition of antibiotic agent (e.g., 0, 18, and 26 grow in the animal. hours). The samples are washed and plated (e.g., in serial 04.05 For example, each of 7 rats were injected with one of dilutions), typically on rich media, so that the number of CFU the following agents in 200ul PBS: no cells: 10, 10, or 10 can be counted. CFU are then normalized to the time 0 time CFU Staphylococcus aureus RN4220 (RecA+); 10, 10, or point, and can be plotted, for example, on a log graph. 10 CFU Staphylococcus aureus RN4220 recA-. On day 1, 0400 Such a cell-based survival assay can be used to the rat that received 10 CFU RN4220 (RecA+) was sick. On evaluate a test agent alone or in combination with another day 4, all rats were sacrificed and their spleens were recovered antibiotic agent. Often, a control assay is done with a known and processed. Cells were plated, and CFU present in the antibiotic agent, and in the absence of any test agent. Such a spleens were counted, with the following results: control assay can be compared with test agent alone and/or with known antibiotic--test agent. 04.01 Cell Growth Assay. This assay measures the ability Pathogen CFU Recovered from Spleen of cells to grow in the presence of a Sublethal dose (i.e., a dose No cells O cells O below the MIC) of an antibiotic agent (or test agent, or com RecA+ cells 10° cells 8 bination thereof). In general, cells are grown, typically to 10 cells 22 saturation, and are then diluted and inoculated onto rich 10° cells 104 medium and onto medium containing a Sublethal dose of recA- cells 10° cells 1 antibiotic agent. Plates are then grown overnight, and ODoo 10 cells O is measured after overnight growth, as compared with blank 10° cells 1 plate. If desired, a test agent can be added to the plate, either alone or in combination with the known antibiotic agent, and 0406. These results demonstrate that inactivation of RecA the ratio of ODoo in the presence of the test agent to ODoo in in bacterial cells reduces their ability to cause sickness, and the absence of the test agent, can be determined. Additional also reduces the number of CFU they can establish in rats. ratios that can be useful as controls include, for example, This system therefore can be used to identify and/or charac ODoo in the presence Vs absence of antibiotic agent; ODoo terize inventive RecA inhibitors. The recA deletion strain can in the absence of any agent VS in the presence of both antibi desirably be used as a positive control. otic agent and test agent, etc. Example 20 Example 18 In Vivo Assay of RecA Inhibitor Activity in Mice Mutation/Resistance Assay 0407. This Example describes uses an in vivo system for the identification and/or characterization of RecA inhibitors 0402. This assay measures the rate at which mutant cells in mice. Specifically, the present Applicants have determined arise in the presence of an antibiotic agent. Cells are grown in that infectious disease can be established in mice, and indeed the presence of antibiotic agent at a concentration above can kill the mice, by intraperitoneal injection of an appropri US 2009/0264.342 A1 Oct. 22, 2009 45 ate number of bacterial cells. They have further demonstrated Example 22 that all mice survive when the cells that are injected (same Serum Binding Assay number of otherwise identical cells) lack RecA (i.e., are recA- mutant cells). 0412. The present Applicants have found that it can be 0408 For example, each of 30 female CD-1 mice were desirable to assess the activity of RecA inhibitors in the injected with one of the following: >5x107 CFU Staphylococ presence of serum as an indicator of whether they are likely to cus aureus grlA542 (RecA+); or >5x107 CFU Staphylococ retain activity when administered in vivo. Any one or more of cus aureus grlA542recA" (recA-) in 0.5 ml PBS (see the activity/characterization assays described herein can be Fournier et al., Antimicrob. Agents Chemother, 2000, 44: performed so that a test agent/RecA inhibitor is exposed to 2160). The number of dead animals was assessed at 24 hours serum for a period of time before or during assessment of its and at 48 hours, with the following results: activity. Example 23 Pathogen Number of Surviving Mice Identification of RecA Inhibitors. Using. In Vitro Luciferase Assay At 48 hours: 0413 More than 2000 compounds were tested for their RecA+ cells 1/15 (6.6%) ability to inhibit RecA ATPase activity in the luciferase assay recA- cells 15/15 (100%) described in Example 7. Tested compounds were obtained from Boston University (1205 compounds from CMLD 04.09. These results indicate that loss of RecA function diversity library, available from Dr. Scott Schauss), IndoFine renders pathogens non-lethal in mice. These results also dem (66 compounds based on flavone structure), Chemdi V (48 onstrate that this system can be used to identify and/or char compounds known to be ATPase inhibitors and 720 com acterize inventive RecA inhibitors (which, when adminis pounds structurally related to CB101 (described, for tered prior to or during infection, should reduce lethality). example, in U.S. Ser. No. 60/662,038), and Asinex. Five (5) The recA deletion strain can desirably be used as a positive tested compounds were bisfiavones and 28 were predicted, control. based on structural modeling (see, for example, Example 31) to dock with Rec A. Many of the compounds are lipophilic, Example 21 natural-product-like compounds. Others are flavones, bisfla Vones, flavonoids, coumarins, and/or heterocyclic com In Vivo Assay of RecA Inhibitor Activity in Immu pounds. nocompromised Mice 0414. Of these compounds, 1 was found to have an ICs in this screen of below 100 g/ml, 11 were found to have an ICso 0410 This Example describes an in vivo system for the below 50 ug/ml, 9 were found to have an ICso below 15ug/ml; identification and/or characterization of RecA inhibitors in 2 were found to have an ICso below 10 ug/ml; 5 were found to immunocompromised mice. The experiment was similar to have an ICso below 5ug/ml, 3 were found to have an ICso that described above in Example 20 except that, 4 days prior below 3 ug/ml; and 1 was found to have an ICs below 1 to infection, mice were rendered immunocompromised by ug/ml. Structures of the 14 compounds that inhibited RecA treatment with 150 mg/kg cyclophosphamide in 0.5 ml sterile ATPase activity by more than 50% are presented in FIG. 14. water. One day prior to infection, the mice were treated again Percent inhibition is calculated according to the following with 100 mg/kg cyclophosphamide, again in O. ml sterile equation: water. Mice were then infected with RecA+ or recA- S. aureus as follows: 10, 10, or 10 CFU Staphylococcus aureus grlA542 (RecA+); or 10, 10, or 10 CFU Staphylo (RLU in presence of compound) - (RLU in DMSO) coccus aureus grlA542recA" (recA-) in 0.5 ml PBS (see (RLU in absence of DNA) - (RLU in DMSO) Fournier et al., Antimicrob. Agents Chemother, 2000, 44: 2160). The number of dead animals was assessed at 24 hours, A value of “-1 indicates higher than 50 lug/ml, therefore with the following results: undetermined. 0415 FIG. 14 presents representative structures of 14 par ticularly active compounds, including amentoflavone, apige Pathogen Number of Surviving Mice nin, apigenin-7-O-glucoside, fitsetin, hinokiflavone, 6-hy droxy-apigenin, isorhamnetin, kaempferol, maclurin, morin, RecA+ cells 10 cells 2 of S 10 cells 1 of S quercetagetin, quercetin dihydrate, 3.7,4'-trihydroxyflavone, 10 cells O of S fisetin, and theaflavin. Of these, hinokiflavone shows the low recA- cells 10 cells 5 of 5 est ICs (<1 g/ml). 10 cells 2 of S 10° cells 2 of S Example 24 Confirming RecA ATPase Inhibition Activity of 0411. These results indicate that loss of RecA function dramatically reduces pathogenicity even in immunocompro Hinokiflavone Using in Vitro Luciferase Assay mised mice. These results also demonstrate that this system 0416. In order to confirm the result obtained in the screen, can be used to identify and/or characterize inventive RecA the present Applicants retested hinoki flavone in the luciferase inhibitors. The recA deletion strain can desirably be used as a assay described in Example 15. Hinokiflavone was added at positive control. time t0 or at time t30 minutes, and several different con US 2009/0264.342 A1 Oct. 22, 2009 46 centrations were tested. FIG. 17 shows that hinoki flavone effectiveness of antibiotic compounds in the treatment of completely inhibited RecA ATPase activity at a concentration gram negative infections as compared with gram positive of 2.9 M. infections. Example 27 Example 25 Hinokiflavone Activity in Growth Assay RecA Inhibitors in Survival Assay 0423. The present Applicants have found that concentra tions of hinokiflavone that potentiate the antibiotic activity of 0417. Several RecA inhibitors that showed an ability to ciprofloxacin in a Survival assay (i.e., that potentiate cell inhibit RecA ATPase activity in the luciferase assay of killing by cirpofloxacin) do not increase the MIC of ciprof Example 23 were tested for their ability to potentiate ciprof loxacin in a growth assay. loxacin antibiotic activity in a Survival assay as described in Example 17. In particular, S. aureus strain S3 (ISP 794) was Example 28 grown in the presence of 1 ug/mL ciprofloxacin and in the Hinoki flavone Targets RecA; does not Potentiate presence or absence of 50 ug/mL of each compound for 16 Ciprofloxacin in the Absence of RecA hours. 0424 S. aureus strains S3 (RecA+) and S4 (recA-) were 0418 FIG. 18 shows a survival assay plate of 11 different used in a survival assay as described in Example 17. The RecA inhibitors. As can be seen, only one of the compounds strain was grown in the presence of the following agents: 1 (hinokiflavone) potentiated ciprofloxacin activity. Indeed, ug/mL ciprofloxacin (in DMSO) or 1 ug/mL ciprofloxacin-- each of the other compounds had a protective effect, allowing 25ug/mlhinokiflavone (in DMSO). FIG. 20 is a graph show more cells to survive ciprofloxacin treatment than were able ing that, in the absence of RecA (i.e., in the recA- strain S4), to do so in the absence of the RecA inhibitor. Without wishing hinoki flavone does not potentiate the activity of ciprofloxa to be bound by an particular theory, we note that such a cin. This finding confirms RecA as the target of hinokifla protective effect might be expected, for example, if these WO. compounds activate DNA repair, inhibit DNA gyrase, or gen 0425 The data presented in FIG. 20, in fact, show a mod erally have after effects in the cells. est protective effect of hinoki flavone in the absence of RecA. That is, in the recA- strain S4, more cells survived in the Example 26 presence of both ciprofloxacin and hinokiflavone than did in the presence of ciprofloxacin alone. One potential explana Hinoki flavone Activity in Survival Assay tion for this finding is that the culture grown in the presence of both agents had fewer CFU at time point t=0 than did the culture grown in the presence of ciprofloxacin alone, 0419 S. aureus strain S3 was used in a survival assay as although numbers were normalized for initial cell count. described in Example 17. The strain was grown in the pres Alternatively or additionally, it is possible that the RecA+ and ence of the following agents: DMSO alone; DMSO+1 ug/mL recA- strains have different growth rates. Thus, data points ciprofloxacin; DMSO+25ug/mL hinokiflavone; or DMSO+1 on the left hand sides of the graphs (e.g., up to about the 0.05 ug/mL ciprofloxacin-i-25 ug/mL hinokiflavone. ug/ml ciprofloxacin data points) may well be more reliable 0420 FIG. 19 shows a photo of the 16 hour growth time than other data points. point. As can be seen, hinokiflavone strongly potentiates ciprofloxacin antibiotic activity. Indeed, approximately 100 Example 29 fold (2 logs) fewer CFUs are observed in the presence of Stability of Hinokiflavone in Serum hinokiflavone as compared with in its absence. Although it is not immediately apparent from FIG. 19, several repeats of 0426. The present Applicants have assessed the ability of this experiment have also revealed modest antibiotic activity hinoki flavone to potentiate ciprofloxacin's antibiotic activity of hinokiflavone alone (i.e., in the absence of ciprofloxacin), in a survival assay in LB and in LB with 10% mouse serum, and found that hinokiflavone retained activity in the presence particularly at high concentrations. of mouse serum. 0421 Similar results were achieved when the experiment was performed with a different S. aureus strain (S7) that is Example 30 mildly resistant to ciprofloxacin. Specifically, hinoki flavone potentiated cirpofloxacin activity against this strain. Hinoki flavone Reduces the Incidence of Resistance to Ciprofloxacin 0422 Hinokiflavone was not observed to have antibiotic activity, or an ability to potentiate ciprofloxacin when the 0427. The present Applicants have found that the presence experiment was performed with an E. coli strain rather thanan of hinokiflavone can reduce the ability of S. aureus cells to S. aureus Strain. Given that E. coli are gram negative whereas develop resistance to the fluoroquinolone antibiotic known as S. aureus are gram positive, it is possible that this different ciprofloxacin. Specifically, S. aureus cells were grown in the result represents differential ability of the compound to enter presence of 4xMIC of ciprofloxacin, and further in the pres the cells (e.g., due to the stronger efflux pump and/or second ence or absence of hinokiflavone (25 g/mL). lipid membrane found ingram negative cells), although in our 0428 Aliquots were removed from the cultures every 24 hands, simple measures Such as using a strain with a defective hours, and were diluted Such that the concentration of cipro an efflux pump more permeable membranes were insufficient floxacin present in the culture was reduced to 0.5xMIC. to reveal activity. Those of ordinary skill in the art appreciate These aliquoted Samples were then assayed to determine that additional measures are commonly required to ensure whether they can grow in the presence of ciprofloxacin. Spe US 2009/0264.342 A1 Oct. 22, 2009 47 cifically, when growth is observed in the presence of ciprof both gram positive and gram negative RecA proteins. Inhibi loxacin at a concentration that is 4xMIC, then resistance is tors binding Such a site can act as broad-spectrum antibiotics, said to have developed. In the present experiment, the Appli useful in the treatment of infections caused by both gram cants counted the number of wells in which growth was positive and gram negative organisms. observed in the presence of ciprofloxacin at a concentration 0434 Work to prepare a co-crystal of RecA and hinokifla that is 4xMIC in order to assess the extent of resistance Vone is in progress. developed in our original culture. The following results were obtained: Example 33 Predicting Additional RecA Inhibitors 0435 Having identified hinoki flavone as a particularly Number of Wells in which Growth Observed in potent RecA inhibitor for use in accordance with the present Number of Days Culture Presence of Ciprofloxacin at 4x MIC invention, the present Applicants have assembled a collection of related compounds likely to share some or all of hinoki was Grown -Hinoki flavone +Hinoki flavone flavone's activities. For example, a variety of different fla O O O Vones are known to inhibit helicases (see, for example, Xu et 1 1 O 5 2 O al., Nuc. Acids Res., 2001, 29: 5058). Bisflavones are of 6 2 O particular interest, given their structural relationship to 7 3 3 hinoki flavone. 10 11 4 0436 FIG. 15 presents a variety of exemplary potential RecA inhibitors according to the present invention that show significant structural similarity to hinokiflavone. 0429. These results demonstrate that the presence of 0437. The present Applicants also considered the likeli hinokiflavone decreases the incidence of resistance to cipro hood that compounds sharing electronic characteristics with floxacin. Similar results would be expected to be obtained hinoki flavone may well have similar ability to interact with with other antibiotic agents, particularly with other quinolo RecA. FIG. 16 presents a variety of exemplary potential nes and/or aminoglycosides. RecA inhibitors according to the present invention that show Example 31 significant electronic similarity to hinokiflavone. Defining a Binding Site for Hinoki flavone on RecA Example 34 Identification of Mutations that Potentiate Aminogly 0430. The structure of RecA protein, in some cases bound cosides to a substrate and/or to DNA, has been reported. For example, structures have been established for RecA from bacteria such 0438. Each knockout strain was first pregrown in 50 as E. coli (Rossbach et al., BMC Struct. Biol., 2005, 20; 7: ug/mL of Kanamycin (an aminoglycoside antibiotic) in LB Story and Steitz, Nature, 1992,355:374; Story et al., Nature, broth, then regrown in LB broth without Kanamycin. A 1992, 355:37, Van Look et al., J. Mol. Biol., 2003, 333: 35: sample of these regrown cultures was then diluted 1/250 and Xing and Bell, Biochemistry, 2004, 43:1612; Yu and Edel stamped onto an LB agar plate. The remaining culture was man, Nat. Struct. Biol., 1997, 4: 101), Mycobacterium sp. then treated with 4 ug/mL of Gentamicin for 1.5 hours. After (Datta et al., J. Bacteriol., 2003, 185: 4280; Datta et al., Nuc. treatment, a second sample was taken and diluted 1/250 in Acid Res., 2000, 28: 4964), Proteus mirabilis (Weber and dHObefore being stamped onto an agar plate. After 24 hours Steitz, J. Mol. Biol., 1986, 188: 109), and for RecA from of growth at 37°C. the size and density of each KO strain to Archea (Ariza et al., Nuc. Acids Res., 2005,33:165; Wu et al., the untreated intensity value was then calculated and ranked Mol. Cell, 2004, 15: 423). The structure of the human RecA in order to target gene knockouts which cause the most sig homolog, known as RAD51, has also been determined (Con nificant increases in antibiotic susceptibility. way et al., Nat. Struc. Mol. Biol., 2004, 11: 791; Wu et al., J. 0439 Target genes that were identified using the method Biol. Chem., 2005, 280: 722). described above are presented on FIG. 29. These target genes 0431 Binding interactions between particular compounds include aceE, ara.J. asp.A, atpA, atpF, atph, brnO, cydB. degP. and a site or sites on a target molecule can be determined by dnaT, fepD, folP. guaB, Hfq, iscS, JW5360, lip A.lpdA, nuoB, molecular modeling programs that are known to those of nuo.J., nuoL, osmEB, pdxH, pnuC, priA, priB, rbfA, recA, rfal), ordinary skill in the art. These molecular modeling programs rpm.J. rpoD, resA, ruvC, SdhC, seqA, setA, such, SurA, tar, include, for example, QUANTA (Accelrys Inc., San Diego, tatE, tauB, trimu, ubiH, ubiX, XerC., yaal J, yefM, yeO, ygf7. Calif.) and the SYBYL suite of computational informatics ygiH, yheL, yheM, yheN, yicG, yjdL, yeT. yjiO, and yi.Y. Software (Tripos Associates, Inc., St. Louis, Mo.). 0440. It is of interest to note that recA is one of the iden 0432. Using the known crystal structure for E. coli RecA tified target genes. In a Survival assay similar to that described the present Applicants have predicted binding sites for in Examples 4, the present Applications have shown that the hinokiflavone and quercetin by using docking Software to RecA deletion increases the sensitivity of S. aureus to ami predict optimal interactions. noglycosides (Gentamicin). The results of this assay are 0433. This strategy correctly predicted the ATP binding reported in FIG. 30. site, as well as various other known functional sites and ion Example 35 binding sites. Moreover, it defined two novel binding sites (see FIGS. 21-28), one of which involves R85, F270, Y271, Mefloquine Potentiates the Aminoglycoside Gen K310, and R32. This site is situated on the outward surface of tamicin RecA (see FIGS. 26 and 27), and is found in all species 0441. Several aminoglycoside (gentamicin) potentiator investigated (see FIG. 28). In particular, the site is found in target genes identified in Example 34 are ATP synthases. US 2009/0264.342 A1 Oct. 22, 2009 48

Mefloquine (a 4-quinolinemethanol derivative), which is a 0448. Overlapping target genes in the three classes of anti known inhibitor of ATP synthase, was found to potentiate biotics are shown on FIG. 34. Target genes common to the Gentamicin. In strain E1, Mefloquine was found to reduce the classes of aminoglycosides and quinolones are priA, recA, MBC (minimal bacterial concentration) and MIC (minimal resA, and ruvC. Target genes common to the classes of ami inhibitory concentration) of gentamicin by 6 fold and 2 fold, noglycosides and cyclic peptides are such, ubiH and ubiX. respectively (see FIG. 31), when used at a concentration of ycfM, ygfz, yheL and yheM, atp A, atpF, and atpH, iscS, 100 g/mL and 500 g/mL, respectively. JW5360, guaB, lip A, pdxH, res.A and pnuC. ResA was found to be the only target gene common to all three classes of Example 36 antibiotics. Identification of Mutations that Potentiate Antibiotic Cyclic Peptides Example 39 0442. Identification of antibiotic cyclic peptide potentia Potentiating Cidal Antibiotics via RecA Inhibition tortarget genes were identified using the same method as that described in Example 34 using 20 ug/mL of Colistin in place 0449 The present Applicants have shown that the three of 4 g/mL of Gentamicin major classes of bactericidal antibiotics (i.e., antibiotics that 0443 Target genes that were identified in the case of colis act by inhibition of DNA replication and repair, inhibition of tin using the method described above are presented on FIG. protein synthesis, and inhibition of cell-wall turnover) stimu 32. These target genes include agaAk, atpA, atpF. atpH, bglF. late the production of highly deleterious hydroxyl radicals in cysE, cysI, fepC, fepD. frvR, guaA, guaB, hofF, hsdS, iscS, gram-negative and gram-positive bacteria, which ultimately JW4016, JW5075, JW5227, JW5257, JW5360, kdgK, lip.A, contribute to cell death (see M. A. Kohanski et al., “A com lys A, malG, mbh A, mdoG. Nei, nmpC, nudH, pdxH, phnB. mon mechanism of cellular death induced by bactericidal phnL, phnO, pnuC, potE, pshM, ptsA. rhaT. rpiA, resA, Sbp. antibiotics', which is incorporated herein by reference in its speA, such3, sugE, tdcE, tdcG, tolC, trx A, ubiE, ubiH, ubiX. entirety, including the Supplementary information section). Xni, ybby, yefM, yde.J., yeeY.yfeTygaA, ygf7, yhdX, yheL, The results obtained Suggest that all three major classes of yheM, yiaY, yidK, yihV.ybn, yjcR, yjcz, ynjD, ydeCyciH, bactericidal drugs can be potentiated by targeting bacterial and yrfA. systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, Example 37 e.g., RecA. Mefloquine Potentiates Colistin Methods 0444. Several colistin potentiator target genes identified in 0450 Media and Antibiotics. All experiments were done Example 36 are ATP synthases. Mefloquine, a known inhibi in Luria-Bertani (LB) medium (Fisher Scientific). The fol tor of ATP synthase which was shown above to potentiate lowing bactericidal drugs were used: the cell wall synthesis Gentamicin, was also found to potentiate Colistin (see FIG. inhibitors, ampicillin (Fisher Scientific) for E. coli and van 33). comycin (TEKnova, Hollister, Calif.) for S. aureus; the pro tein synthesis inhibitor kanamycin (Fisher Scientific), and the Example 38 DNA gyrase inhibitor norfloxacin (Sigma). Ampicillin was used at concentrations of 5 and 15 g/mL. Vancomycin was Comparison of Target Genes Identified for Quinolo used at concentrations of 1 and 5 lug/mL. Kanamycin was nes, Aminoglycosides and Antibiotic Cyclic Peptides used at concentrations of 5 and 25 g/mL. Norfloxacin was 0445 Quinolone potentiator target genes identified used at concentrations of 25 and 250 ng/mL for experiments according to the present invention using ciproflaxin include with E. coli, and a concentration of 4 g/mL for experiments dapF, fabH, fis, fliN, jws303, priA, recA, recB, recC, recC. with S. aureus. res.A, ruVA, ruvB, ruvC, ruvC, uvrD, yeS, ydfL, yhfT, and 0451. The following bacteriostatic drugs were used: the ydgC. RNA polymerase inhibitor, rifamycin SV (MP Biomedicals, 0446 Aminoglycoside potentiator target genes identified Solon, Ohio), and the protein synthesis inhibitors chloram according to the present invention using gentamicin include phenical (Acros Organics, Geel, Belgium), tetracycline (MP aceE, ara, asp.A, atpA, atpF, atpH, brnO, cydB. degP. dnaT, Biomedicals), erythromycin (Sigma) and stectinomycin (MP fepD, folP. guaB, Hfq, iscS, JW5360, lip A.lpdA, nuoB, nuo.J. Biomedicals). Rifamycin SV was used at concentrations of nuoL, osmB, pdxH, pnuC, priA, priB, rbfA, recA, rfal), rpm.J. 300 and 500 mg/mL. Chloramphenical was used at concen rpoD, res.A, ruvC, SdhC, seqA, setA, such, SurA, tar, tatE, trations of 7 and 15ug/mL for the E. coli experiments and 20 tauB, trimu, ubiH, ubiXxerC., yaal J, ycfM, yeO,ygf7, ygiH, mg/mL for the S. aureus experiments. Tetracycline was used yheL, yheM, yheN, yicG, yidL, yeT. yjiO, and yjY. at concentrations of 4 and 10 ug/mL. Erythromycin was used 0447 Antibiotic cyclic peptide potentiator target genes at concentrations of 200 and 400 ug/mL. identified according to the present invention using colistin 0452 Strains. All experiments with E. coli in this Example include agaAkatp A, atpF, atpH, bglF, cysE, cySI, fepC, fepD. were done with MG1655 (ATCC 700926) derived strains. frvR, guaA, guaB, hoff, hsdS, iscS, JW4016, JW5075, The recA, iscS and TCA cycle knockouts were constructed JW5227, JW5257, JW5360, kdgK, lip A, lysA, malG, mbh A, using P1 phage transduction. Briefly, recA::KanR was trans mdoG, Nei, nmpC, nudH, pdxH, phn B. phn L. phnO, pnuC, duced from an E. coli single-gene knockout library (T. Baba potE, pshM, pts.A, rhaT. rpiA, resA, Sbp, speA. Such3, SugE, et al., Mol. Syst. Biol., 2006, 2:2006.0008) into the MG 1655 tdcE, tdcG, tolC, trXA, ubiE, ubiH, ubiX, Xni, ybby, yefM, strain. The kanamycin-resistance gene was removed using yde.J., yeeY, yfeT, ygaA, ygf/, yhdX, yhell, yheM, yiaY. the pcp20 plasmid (K. A. Datsenko and B. L. Wanner, Proc. yidK, yihV, ybn, yjck, yjcz, yn D, ydeC., ydiH, and yrfA. Natl. Acad. Sci. USA, 2000, 97: 664-6645) to create US 2009/0264.342 A1 Oct. 22, 2009 49

MG 1655Areca. This procedure was replicated with iscS and flasks and antibiotics added as described above. Samples for the TCA cycle knockouts to move these single gene knock microarray analysis were taken immediately before treatment outs into MG 1655. (time zero), then at 30, 60 and 120 minutes post treatment. For 0453 For work with S. aureus, a Staphylococcus aureus bactericidal concentration of norfloxacin (250 ng/mL), a Subspecies aureus Rosenback Strain was used that was time-series profile was used that had previously been col obtained from ATCC (29740). lected (D.J. Dwyer et al., “Superoxide and hydroxyl radicals 0454 DNA damage sensor construction. The DNA sensor contribute to gyrase inhibitor-mediated cell death in E. coli'', construct withampicillin resistance was described previously 2007 accepted by Mol. Syst. Biol., which is incorporated (D.J. Dwyer et al., “Superoxide and hydroxyl radicals con herein by reference in its entirety). tribute to gyrase inhibitor-mediated cell death in E. coli'', 0460. The resulting microarray. CEL files were com 2007 accepted by Mol. Syst. Biol., which is incorporated bined with *.CEL files from arrays which comprise the M3D herein by reference in its entirety). compendium (http://m3d.bu.edu: J. J. Faith et al., PloS Biol. 0455 Growth Conditions. In the experiments, the growth 2007, 5, e8). and RMA-normalized (B. M. Bolstad et al., and Survival of untreated exponential phase E. coli, or S. Bioinformatics, 2003, 19:185-193) with RMAexpress, for a aureus, were compared to cultured treated with antibiotics at total of 524 RMA-normalized E. coli expression arrays. For the above concentrations. E. coli cultures were treated with each gene, that gene standard deviation of expression was norfloxacin, kanamycin, amplicillin, rifamycin SV, chloram calculated and used to construct the Z-scale difference phenical, tetracycline, erythromycin, or spectinomycin, between that gene's normalized expression in a given experi where appropriate, S. aureus cultures were treated with nor mental condition (bactericidal drug treatment) versus a con floxacin, Vancomycin or chloramphenical, where appropri trol (bacteriostatic drug treatment). ate 0461 This allowed to measure each gene's change in 0456 Hydroxyl radical and DNA damage sensor experi expression for a given experiment in units of standard devia ments using the flow cytometer. To monitor the occurrence of tion, a form of the Z-test. For each time point each bactericidal DNA damage, the engineered DNA damage sensor was used, experiment set (norfloxacin, amplicillin, kanamycin), which responds to auto-cleavage of Lex A by activating AZ-scores were converted to p-values and significantly up expression of gfpmut3b. All the drug treatments and the no and down-regulated genes with a q-value (False Discovery drug controls were collected with both the kanamycin- and Rate)<0.05 were selected(J. D. Storey et al., Proc. Natl. Acad. ampicillin-resistant version of the plasmid, with the excep Sci. USA, 2003, 100: 9440-9445). The resultant gene lists tion of the amplicillin treatment (kanamycin plasmid only) was merged across all time points (set-union) to obtain a and the kanamycin treatment (ampicillin plasmid only). All coarse profile of the difference in expression between a given data were collected using a Becton Dickinson FACSCalibur bactericidal drug and spectinomycin. Finally, the Applicants flow cytometer (Becton Dickinson, San Jose, Calif.) with a determined the common set of all genes that were up- or 488-nm argon laser and a 515- to 545-nm emission filter down-regulated by bactericidal concentrations of norfloxa (FL1) at low flow rate. The following PMT Voltage settings cin, kanamycin, and amplicillin with respect to spectinomycin were used: E00 (FSC), 360 (SSC) and 700 (FL1). To detect (3-way set-intersect). For an additional pathway-level hydroxyl radical formation, the fluorescent reporter dye, 3'- insight, Gene Ontology-based enrichment of the up- and (p-hydroxyphenyl) fluorescein (HPF, Invitrogen, Carlsbad, down-regulated gene list was performed using GO Term Calif.) was used at a concentration of 5uM (1:1000) dilution), Finder, requiring pathway enrichment q-values to be <0.05 which is oxidized by hydroxyl radicals with high specificity. and setting the p-value estimation mode to boostrapping. The following PMT Voltage settings were used: E00 (FSC), 360 (SSC) and 825 (FL 1). Calibrite beads (Becton Dickin Results son) were used for instrument calibration. 0462 Hydroxyl radical formation, via cycles of oxidation 0457 Flow data were processed and analyzed with MAT and reduction of quinolone class of antineoplastic drugs used LAB (MathWorks, Natick, Mass.) to construct figures. At in cancer therapeutics, has been shown to contribute to cell least 50,000 cells were collected for each sample. In all death in tumor cells. In the present study, the Applicants chose experiments, cells were grown as described above. Samples to investigate whether hydroxyl radical formation also con were taken immediately before addition of drug (time Zero), tributes to antibiotic-induced cell death in bacteria. Using the then every hour for 3 hours. At each timepoint, approximately dye hydroxyphenyl fluorescein, HPF, which is oxidized by 106 cells were collected, washed once and resuspended in hydroxyl radicals with high specificity, hydroxyl radical for filtered 1xRBS, pH 7.2 (Fisher Scientific), and fluorescence mation was first observed over a range of hydrogen peroxide was measured on the flow cytometer. concentrations known to induce hydroxyl radicals through 0458 NAD+/NADH extraction and NAD cycling assay. the reduction of hydrogen peroxide by ferrous iron. In this Protocols for dinucleotide extraction and the cycling assay manner, it was possible to establish the scale of hydroxyl were based on protocols by Leonardo et al. (M. R. Leonardo radical formation detectable using this dye via flow cytom et al., J. Bacteriol., 1996, 178: 6013-6018). etry. 0459 Gene Expression Analysis. The present Applicants 0463 Hydroxyl radical formation following exposure to compared the microarray-determined (Affymetrix E. coli the three major classes of bactericidal antibiotics in E. coli Antisense2 genome arrays) mRNA profiles of E. coli (FIG. 35a, b). Specifically, the present Applicants examined MG 1655 cultures in response to bactericidal (25 ng/mL nor killing by the quinolone (250 ng/mL, norfloxacin), B-lactam floxacin, 5ug/mL amplicillin and 5ug/mL, kanamycin), bac (5ug/mL, amplicillin), and aminoglycoside (5ug/mL, kana teriostatic (400 ug/mL spectonomycin), or no-drug treat mycin) classes. Each of the three different classes of bacteri ment. For all experiments, cells were grown overnight, then cidal antibiotics was found to induce hydroxyl radical forma diluted 1:1000 into 250 mLLB for collection of total RNA. At tion (FIG. 35b). In contrast, the five bacteriostatic drugs an OD600 of 0.3, the culture was split, 50 mL into 5, 250 mL tested (FIG.35c) including four different classes of ribosome