DNA. nfxb nfxc nalb. qnr. Key words: drug-resistance mode of action DNA gyrase efflux pump NFLX Nalidixic Acid NA 1978 NFLX NFLX

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1 DNA IV 1975 DNA DNA DNA 3 DNA IV 1990 nfxb nfxc nalb qnr Key words: drug-resistance mode of action DNA gyrase efflux pump 1962 Nalidixic Acid NA 1978 NFLX 6 7 NFLX I NA 1962 Lesher 2 5 NA NA NA 1978 NFLX NFLX NA NFLX

2 Quinolone pocket Subunit A GyrA Quinolones Subunit B GyrB Fig. 1. Model of Quinolone-DNA-DNA gyrase interaction. Table 1. Effect of substituents at the 6-position on DNA gyrase inhibitory activity IC and antibacterial activity MIC R6 F NFLX Cl Br H NO2 OH NH2 HN R6 N IC g/ml O N C 2H 5 O OH MIC g/ml QT 1990 GFLX MRSA VRE II DNA IV 1 DNA 1960 NA Goss NA DNA 1 DNA DNA 1977 Gellert Sugino 2,3 DNA 2 DNA DNA DNA gyra A GyrA 2 gyrb B GyrB 2 A DNA B ATPase A DNA Shen NFLX 2 DNA DNA DNA DNA 3 Cleavable Complex DNA Fig. 1 4 DNA 6 DNA Table 1 DNA

3 QRDR: Quinolone Resistance-Determining Region a.a. DNA binding site Tyr-122 N-terminal 1a.a. C-terminal 875a.a ARVVGDVIGKYHPHGDSAVYDTIVRMAQPFSLRYMLVDGQ-106 S C D L P W A N V G Y H R Fig. 2. Schematic representation of E. coli GyrA amino acid sequences with mutations associated with quinolone resistance. 2 IV 1990 Kato DNA A B TopoisomeraseIV TopoIV 5 TopoIV DNA ParC GrlA ParE GrlB 2 4 TopoIV 2 DNA DNA TopoIV DNA TopoIV DNA 1994 Ferrero TopoIV CPFX DNA TopoIV 6 Takei TopoIV DNA 7 DNA 8,9 8,9 III K DNA 2 K-12 PAO DNA TopoIV 1 DNA K-12 nfxa nora nala DNA A gyra 10,13 DNA PAO gyra nfxa cfxa nala DNA A B A 14 DNA

4 Table 2. Properties of norfloxacin-resistant mutants in E. coli K-12 Strain Characteristics NFLX CPFX MIC g/ml OFLX NA CFX CP KL-16 KEA12 KEA13 KEA16 Wild type nora: GyrA mutation norb: OmpF decrease norc: OmpF and LPS decrease Yoshida gyra 875 GyrA N QRDR 15 Fig. 2 QRDR DNA DNA 5 Tyr-122 QRDR 83 Ser-83 Ser-83 DNA A DNA 3 16 DNA X gyra QRDR 16 2 TopoIV TopoIV 6 TopoIV ParC Ser-80 Glu-84 parc grla DNA 6 Fukuda 1 grla parc 2 gyra 3 grla parc 4 gyra TopoIV parc 2 DNA gyra gyra TopoIV parc TopoIV DNA B GyrB TopoIV ParE 16 DNA TopoIV DNA 2 1 K-12 Hooper NFLX CPFX nfxb norb norc cfxb 10,13 Table 2 NFLX CPFX OmpF OmpF TC CP CFX norc OmpF LPS NFLX NA 10 OmpF LPS OmpF 40K TC CP 18 OmpF 2 PAO NFLX nfxb nfxc nalb 11,12 Table 3 nfxb nfxc nalb PAO β CP

5 Table 3. Properties of norfloxacin-resistant mutants in P. aeruginosa PAO Strain Characteristics NA NFLX MIC g/ml CPFX CBPC CAZ IPM CP PAO4009 KH4023 KH4013 KH4014a PAO6006 Wild type nfxa: GyrA mutation nfxb: 54K OM OprJ expression nfxc: K OM OprN expression OprD decrease nalb: 49K OM OprM increase A. Wild-type nfxb mexc mexd oprj NfxB MexC MexD OprJ 54kDa B. nfxb mutant nfxb mutant mexc mexd oprj NfxB mutant MexC MexD OprJ (54kDa) Fig. 3. Regulation mechanisms of mexc-mexd-oprj operons of Pseudomonas aeruginosa PAO by NfxB. NFLX nfxb nfxc nalb 11,12,18 m CCCP nfxb nalb nfxc nfxb 54K nalb 49K nfxc K Gotoh 19 nalb MexA-MexB-OprM RND Resistance-Nodulation- Cell Division mexa-mexb-oprm 3 MexA-MexB-OprM nfxb nfxc MexC-MexD-OprJ MexE-MexF-OprN nfxb nalb nfxc 54K 49K K OprJ OprM OprN 1992 nfxb nfxb DNA nfxb DNA Regulator 20 nfxb nfxb mexc-mexd-oprj Fig. 3

6 21 RND 10 RND RND AcrA-AcrB-TolC AcrA-AcrB-TolC AcrB 22 TolC 23 AcrB CPFX 24 AcrA MexA 25 IV 1998 Martinez 26 qnr qnr Qnr Pentapeptide Repeat Family Qnr DNA DNA Cleavable Complex qnr qnr 8 11 qnr 27,28 qnr V NFLX DNA TopoIV DNA TopoIV Goss W A, Deitz W H, Cook T M: Mechanism of Action of Nalidixic Acid on Escherichia coli II. Inhibition of Deoxyribonucleic Acid Synthesis. J Bacteriol 89 : , Gellert M, Mizuuchi K, O Dea M H, et al: Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc Natl Acad Sci USA 74: , Sugino A, Peebles C L, Kreuzer K N, et al: Mechanism of action of nalidixic acid: purification of Escherichia coli nala gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc Natl Acad Sci USA 74: , Shen L L, Mitscher L A, Sharma P N, et al: Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug DNA binding model. Biochemistry 28: , Kato J, Nishimura Y, Imamura R, et al: New topoisomerase essential for chromosome segregation in E. coli. Cell 63: , Ferrero L, Cameron B, Manse B, et al: Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV : a primary target of fluoroquinolones. Mol Microbiol 13: , Takei M, Fukuda H, Kishii R, et al: Target preference of 15 quinolones against Staphylococcus aureus,based on antibacterial activities and target inhibition. Antimicrob Agents Chemother 45: , Takei M, Fukuda H, Kishii R, et al: Contribution of the 8-methoxy group of gatifloxacin to inhibition of type II topoisomerases of Staphylococcus aureus. Antimicrob Agents Chemother 46: , Kishii R, Takei M, Fukuda H, et al: Contribution of the 8-methoxy group to the activity of gatifloxacin against

7 type II topoisomerases of Streptococcus pneumoniae. Antimicrob Agents Chemother 47: 77 81, Hirai K, Aoyama H, Suzue S, et al: Isolation and characterization of norfloxacin-resistant mutants of Escherichia coli K-12. Antimicrob Agents Chemother 30: , Hirai K, Suzue S, Irikura T, et al: Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob Agents Chemother 31 : , Fukuda H, Hosaka M, Hirai K, et al: New norfloxacin resistance gene in Pseudomonas aeruginosa PAO. Antimicrob Agents Chemother 34: , Hooper D C, Wolfson J S, Souza K S, et al: Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrob Agents Chemother 29: , Hirai K, Mitsuhashi S: Mechanisms of resistance to quinolones. Progress in Drug Research 38: , Yoshida H, Bogaki M, Nakamura M, et al: Quinolone resistance-determining region in the DNA gyrase gyra gene of Escherichia coli. Antimicrob Agents Chemother 34: , : , Fukuda H, Hori S, Hiramatsu K: Antibacterial activity of gatifloxacin AM-1155, CG51, BMS , a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the nora transformant of Staphylococcus aureus. Antimicrob Agents Chemother 42: , Hirai K: Quinolone-resistance mechanisms involved in outer membrane. In Quinolones Fernandes P B ed, P , JR Prous Science Publishers, Barcelona, Gotoh N, Tsujimoto H, Poole K, et al: The outer membrane protein OprM of Pseudomonas aeruginosa is encoded by oprk of the mexa-mexb-oprk multidrug resistance operon. Antimicrob Agents Chemother 39: , Okazaki T, Hirai K: Cloning and nucleotide sequence of the Pseudomonas aeruginosa nfxb gene, conferring resistance to new quinolones. FEMS Microbiol Lett 97: , Pseudomonas aeruginosa 47: , Koronakis V, Sharff A, Koronakis E, et al : Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405: , Murakami S, Nakashima R, Yamashita E, et al: Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419: , Yu E W, McDermott G, Zgurskaya H I, et al: Structural Basis of Multiple Drug-Binding Capacity of the AcrB Multidrug Efflux Pump. Science 300: , Akama H, Matsuura T, Kashiwagi S, et al: Crystal Structure of the Membrane Fusion Protein, MexA, of the Multidrug Transporter in Pseudomonas aeruginosa. J Biol Chem 279: , Martinez-Martinez L, Pascual A, Jacoby G A : Quinolone resistance from a transferable plasmid. Lancet 351: , Wang M, Tran J H, Jacoby G A, et al : Plasmidmediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob Agents Chemother 47: , Wang M, Saham D F, Jacoby G A, et al: Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States. Antimicrob Agents Chemother 48: , : , 2004

8 History of mode of action and resistance mechanisms of quinolones Keiji Hirai Discovery Research Headquarters, Kyorin Pharmaceutical Co., Ltd., 2 5 Kanda Surugadai, Chiyoda-ku, Tokyo, Japan Many new quinolones, namely fluoro-quinolones, have been developed since norfloxacin NFLX was discovered in These drugs became useful medicine for various infectious diseases including pneumonia. The researches on mode of action of quinolone and resistant mechanisms have been made great advances with the progression of these new quinolones. In this review, I would like to introduce the progress of studies on mode of action of quinolones and mechanisms of quinolone-resistance in bacteria from mid 1970s. 1. Mode of action: Target enzymes: DNA gyrase and Topoisomerase IV: It was known that quinolones inhibit DNA replication in Escherichia coli, however, detailed mode of action was not clarified when we started the research and development of new quinolones. DNA gyrase was identified as the target enzyme of quinolone in Afterward, researches on mechanism of actions of quinolones have been made a remarkable progress using various new quinolones such as norfloxacin. Consequently, interesting findings were reported such as formation of cleavable complex DNA gyrase-dna-quinolone by quinolone, quinolone resistance-determining region QRDR, and structure-activity relationship for various DNA gyrase in bacteria. In 1990, new target, namely Topoisomerase IV, of quinolone other than DNA gyrase was identified. This enzyme was showed to involve with antibacterial activity of quinolones especially respiratory quinolones and quinolone-resistance mechanisms in Gram-positive bacteria. 2. Quinolone-resistance mechanisms involved in membrane: We found that quinolone-resistant mechanisms were due to alterations in DNA gyrase and in cell permeability of outer membrane proteins in E. coli and Pseudomonas aeruginosa. In E. coli, it was suggested that quinolone might penetrate through the OmpF porin, and alterations in permeability to quinolones in members of the Enterobacteriaceae have been associated with the decrease of specific outer membrane proteins. We isolated three types of norfloxacin resistant mutants, nfxb, nfxc, and nalb,showed alteration in membrane permeability associated with the appearance and or increase of outer membrane proteins. Using these mutants, it was found that these mutations activated efflux pumps in P. aeruginosa, and these data suggested efflux pumps might play an important role in resistance for various antibacterial agents in P. aeruginosa. More recently, plasmid-mediated quinolone-resistance in Gram-negative bacteria was found in US and China. These finding might be very critical for spread of quinolone-resistant genes by plasmids, therefore we will have to keep watch on this type quinolone-resistance.