From anti-malarial to antibacterial

HN N

Cl N

1939 chloroquine
O O

OH
1958 7-chloroquinolone
Cl N
Anti-bacterial activity noted
in screening
O O

1962 Nalidixic acid OH

First new class of synthetic N N

antibiotic for 30 years

 Mechanism of action
Inhibition of topoisomerases – a
family of enzymes responsible for
maintaining the topology of DNA

relaxed
gyrase topo-
isomerase
supercoiled
Quinolones form a Quaternary Complex
between Protein, DNA and Neighbouring
Quinolones
Accumulation of Covalent Complexes and
Broken DNA is Lethal

Target is gyrase (Gram-negative)

and/or topisomerase IV (Gram-positive)

Killing is rapid but reaches a plateau

(60-120‘ for 104-fold decrease in viable count)

Killing antagonized by inhibitors of translation

and at high FQ concentration „Eagle effect“)

Anaerobic conditions inhibit action

 Concentration Dependence of Killing
O O
1.00E+14 F
OH
Concentration dependence
N N
shows two phases of 1.00E+12 HN O

inhibition, separated by a
O O
plateau. 1.00E+10 F
OH

N
The cultures in the plateau 1.00E+08 HN
N

region give rise to first step

resistant mutants. Mutants are 1.00E+06
not recovered above the onset
of the second sharp drop. 1.00E+04

This limit is defined as the 1.00E+02

mutant preventing 0.01 0.1 1 10
concentration (MPC).
MPC
 Early Quinolone Antibiotics
O O O O

OH OH

N N N
N N Nalidixic Acid 1962 Pipemidic Acid 1974
HN
Sterling Winthrop Roger Bellon

O O O O
O OH O OH
O N N
Oxolinic Acid 1967 O N
Cinoxacin 1974
Werner Lambert Lilly
O O
F O O
OH
OH
N
Flumequine 1973 Rosoxacin 1976
N
Rikker Labs N Sterling Winthrop
 Potential of Early Quinolone Antibiotics

Nalidixic Acid Enterobacteriaceae Short half-life

(not P. aeruginosa) (1.5 hr)
high protein binding
Pipemidic Acid Broader Gram-negative Longer half-life
spectrum (except Serratia)

Oxolinic Acid Weak but broad lower protein binding

Gram-negative

Flumequine Weak but broad

Gram-negative
 The First Fluoroquinolone Antibiotics

O O O O
F
OH OH

N N N
N
Pipemidic Acid 1974
Flumequine 1973 HN
Roger Bellon
Rikker Labs

O O
F
OH

N N Norfloxacin 1978
HN
Kyorin

Broad Gram-negative spectrum (F, morpholino grp)

Less protein binding, 50% (quinolone not naphthyridine)
Longer half life, 3h (morpholino group)
 The First Generation of Fluoroquinolone
Antibiotics
O O O O
F F
OH OH

N
Flumequine 1973 N N Norfloxacin 1978
Rikker Labs HN
Kyorin
O O O O
F F
OH OH

N N
Ofloxacin 1979 N N
Pefloxacin 1979
N O N
Daiichi Dainippon
O O
O O
F
F OH
OH

N N
N N
Ciprofloxacin 1983
N F Fleroxacin 1987 HN
Bayer
F
Roche
 Potential of the First Generation
Fluoroquinolone Antibiotics
Gram-negative spectrum t1/2 (h)

Flumequine Weak but broad

Norfloxacin Strong 3-4
Pefloxacin Weak 11
Ofloxacin Strong 6
Ciprofloxcin Very strong 3–4
Levofloxacin Extremely strong 6

“Also rans“
Amifloxacin 1984 Sterling Winthrop
Fleroxacin 1987 Roche
Lomefloxacin 1988 Searle
 From Ofloxacin to Levofloxacin
O O
O O
F
OH F
OH
N N
Ofloxacin 1979 N N
N O Levofloxacin 1987
Daiichi N O
H Daiichi
Racemic mixture Pure (-) S isomer

Active component of ofloxacin

2-fold improved activity

Extended patent life
 The Later Fluoroquinolone Antibiotics
O O
F O O
OH F
OH
N N
N N N

Difloxacin 1986 HN F
Temafloxacin 1988
F
Abbott Abbott
F
NH2 O O O O
F F
OH OH

N N
N N Sparfloxacin 1991
HN F HN Grepafloxacin 1995
Rhone-Poulenc-Rorer
(Dainippon 1985)
Otskuda (1989)

O O
O O
F
F OH
OH
N N
N N
H HN O
N O
Moxifloxacin 1994 Gatifloxacin 1993
Bayer Kyorin (1988)
 Later Fluoroquinolone Antibiotics
• Gram-negative spectrum retained
MIC for E. coli
Ciprofloxacin 0.125 - 0.5
Grepafloxacin 0.06 - 2
Gatifloxacin 0.06

• Increased potency or breadth of Gram-positive spectrum

MIC for S. pneumoniae
Ciprofloxacin 0.5 - 2
Temafloxacin 0.5 – 1
Sparfloxacin 0.125 – 0.5
Moxifloxacin 0.01 – 0.5

• Improved activity against anaeobes

MIC for B. fragilis
Ciprofloxacin 2 – 128
Gatifloxacin 0.25 - 8
Moxifloxacin 0.25 - 8
F
O O
Later Naphthyridines
OH
O O
N OH
Flumequine 1973
N N N
Rikker Labs HN
Pipemidic Acid 1974
Roger Bellon
O O
O O
O O F
OH F
F OH
OH
N N N N N
N N
HN HN
HN F

Ciprofloxacin 1983
Enoxacin 1984
F Bayer
Temafloxacin 1988 Rhone-Poulenc Rorer
O O
Abbott F
O O
OH F
OH
N N N O
N N N
F
H2N
H2N
Trovafloxacin 1993 Gemifloxacin 1994
F
Pfizer L G Chemicals
 Later Naphthyridines

Gram –ve Gram +ve Anaerobes

(E. coli) (S. pneumoniae) (B. fragilis)

Pipemidic Acid 0.025 – 16 4 – >64 >64

Enoxacin 0.025 – 16 1.6 - 16

Trovafloxacin 0.008 - 1 0.007 – 0.25 0.128 - 8

Gemifloxacin 0.5 - 64

Ciprofloxacin 0.125 – 0.5 0.5 - 2 2 - 128

 Current Uses of Fluoroquinolones
Ciprofloxacin: wide range of infections
pneumonias, bone infections, diarrhea, skin infections and urinary tract
infections. Not good for methicillin resistant Staphylococcus aureus

Norfloxacin: better for UTI

effective against Gram-negative (including Pseudomonas aeruginosa) and
Gram-positive UTIs and prostatitis, but not in systemic infections

Lomefloxacin and enoxacin -- UTIs and bronchitis caused by Haemophilus

influenzae or Moraxella catarrhalis. Lomefloxacin not effective against pseudomonal
bacteremia. Enoxacin recommended for STDs

Moxifloxacin – overcomes the problems with S. pneumoniae

Acute bacterial sinusitis; mild to moderate community-acquired pneumonia
 Frequent Side Effects of Quinolones
Chelation of cations (Fe3+ , Al 3+ , Mg2+, Ca 2+)
incompatibility with antacids
Phototoxicity
Drug interactions (inhibition of cytochrome P450)
Ciprofloxacin and enoxacin interfere with hepatic biotransformation -- may
cause toxicity due to excess of e.g. theophylline, warfarin
CNS toxicity (GABA receptor antagonist)
all quinolones contraindicated in patients with history of convulsions

Gastro-intestinal discomfort
nausea or vomiting, abdominal or stomach pain

Cartilage and musculosqueletal pathogenicity

have caused arthralgias and joint swelling in children
Achilles tendinitis and tendon rupture 2 - 42 days after start of therapy
Bacterial SOS response to DNA and other damage caused by FQ may induce
toxins. (E. coli, S. typhimurium, B. anthracis)
 Rare Side Effects of Quinolones

Nephrotoxicity
crystalluria, hematuria, interstitial nephritis,
acute renal failure

Cardiac toxicity
inhibition of hERG channel leads to QT prolongation
& torsades de pointe

Hepatotoxicity
temafloxacin syndrome, trovafloxacin syndrome
 Frequent Side Effects of Quinolones
GABA receptor binding&
CNS penetration Cation complexation
N all quinolones
HN

O O
F
OH

N
Inhibition of CyP450
ciprofloxacin,
gemifloxacin

Photoxicity: especially F
CyP450 inhibition N fleroxacin, lomefloxacin,
sparfloxacin
 Nephrotoxicity
crystalluria, hematuria, interstitial nephritis, acute renal
failure

Poor solubility can lead to crystallization of the FQ in

concentrated urea.

Needle-like crystals form that can cause mechanical

damage
 Cardiac Toxicity
Torsades de pointe: paroxysm of ventricular tachycardia in which the
electrocardiogem shows a steady undulation in the QRS axis in runs of
5 to 20 beats with progessive changes in direction. It is a most severe
type of arythmia and can result in death. It is most often associated
with and preceeded by a prolongation of the QT interval.

Population at risk QT greater than 20 ms

Moxifloxacin 7 ms, grepafloxacin 10 ms, sparfloxacin 15
ms
Hepatotoxicity O O O O
F F
OH OH

N N N N N
HN F F
H2N

Temafloxacin syndrome
F F

Hemolytic uraemic anemia

discoloured urine Trovafloxacin syndrome
fever
jaundice Serious hepatic events
nausea, vomiting laboratory abnormalities
coagulopathy encephalopathies
hepatic dysfunction nectrotic inflammation
renal dysfunction
0.0056 % incidence
0.56% incidence 5 transplants
2 deaths 6 deaths

Withdrawn June 1992 Withdrawn/limited in June 1999

 Resistance to Quinolones
Point mutations in the gyrase A gene
cluster in region around tyrosine 122, site of covalent attachment of DNA

Point mutations in the regulatory regions of transport proteins

porins of Gram-negative bacteria

efflux systems of P. aeruginosa (mex systems)

Enterobacteriaceae (Acr system + porin mutation)
S. aureus (norA system)
S. pneumoniae (pmrA system, patAB system)

No transferable resistances recorded until 1994:

wild type susceptible genes tend to be dominant
plasmid conjugation is inhibited by quinolones
plasmids can be eliminated from bacteria at sub-lethal quinolone
concentrations
The new plasmid pMG255 encodes a putative quinolone binding protein.
 Emergence of Quinolone Resistance
During Therapy
Most often in difficult to treat infections and long-term treatments

Pefloxacin bronchitis P. aeruginosa

Ofloxacin pulmonary tuberculosis M. tuberculosis

Ciprofloxacin cystic fibrosis ass. RTI P. aeruginosa

osteomyelitis P. aeruginosa

MRSA carriers S. aureus

Norfloxacin complicated UTI P. aeruginosa

Notably with infections caused by staphylococci, pneumococci, enterococci,

and P. aeruginosa . Spontaneous mutation leading to single-step resistance
is more common - 106-107 - than for other pathogens - 109-1011
 Acr System of Enterobacteriaceae

Multi-drug resistance efflux system.

System.
Pumps a broad range of antibiotics out of
the cell.
Part of a multicomponent system that
spans cytoplasmic and outer membrane
of Gram-negative bacteria.

Cytoplasmic membrane component acts

as energy transducer (pmf) as well
As substrate recognition module
Acr System of Enterobacteriaceae
Modelled Structure of Mex/OprM Efflux
Pump
MexA,B-OprM exports quinolones,
tetracycline, chloramphenicol and other
antibiotics

MexC,D-OprJ

MexX,Y-OprM exports aminoglycoside

antibiotics (amikacin, tobramycin) and
quinolones
Efflux pump inhibitors developed
by Essential Therapeutics are in
clinical trials as combination partner
for levofloxacin
O
H
N
H2N N
H
O

H2N
Fluoroquinolone-resistance in Streptococci

by the late 1980s:

FQ-resistant staphylococci established in clinics

increased levels of resistance amongst streptococci reported from

patients receiving FQ for Gram-negative urinary tract infections.

Resistance in staphylococci

•point mutations in the genes encoding the molecular targets

(gyrase subunit A and topoisomerase IV subunit A)

•point mutations in the promoter of an integral membrane protein

leading to consitutive expression

•expression of the membrane protein alone is sufficient for clinical resistance

•the protein (NorA gene product) lowers the amount of fluorquinolone

in the cytoplasm
Efflux-mediated Resistance in Streptoocci

The NorA protein is an efflux transporter

similar to the well-known tetracycline
resistance systems

It uses the proton-motive force to drive out the

antibiotic against its concentration gradient
by proton-coupled antiport.
FQ
FQ
H+

FQ
H+

H+
Intrinsic FQ resistance in streptococci reported to
be due to a homologue the PmrA protein
Major Facilitator Family Permeases
 ABC transporters
Implicated in fluoroquinolone resistance in streptococci (PatA, PatB)

Multi-drug resistance in many Gram-positive organisms

 Microarray analysis

• No antibiotic: for most genes M4 and M22 similar

levels of gene expression
• After CIP exposure: similar levels of gene
expression except for
- those involved in DNA repair, transcription etc.
as found with Haemophilus influenzae (Gmuender
et al., 2001)
- ATPases
- those involved in solute transport/efflux
Marrer et al., 2006 AAC 50: 269-278
 patA and patB
• Two genes, patA and patB
(pneumococcal ABC
transporter) constitutively
over-expressed by M22, but
not M4.
• patA and patB DNA
sequences (pfam and
SOSUI) suggest ABC
transporters
– PatA predicted 6TMs (no
model)
– PatB predicted 6TMs
(Swissprot model based on
Sav1886 transporter in S.
aureus)
Predicted PatB
 Induction of patA and patB:
microarray data
After 10 mins exposure to ciprofloxacin
1600
patA 3000
patB
1400
2500
Expression (light units)

1200
2000
1000

800 1500

600
1000
400
500
200

0 0
Control C2 C12 C80 Control C2 C12 C80

M4 M22
 Effect of inactivating patB
NOR CIP EtBr ACR
M4 4 (2) 2 (0.5) 2 (0.5) 4 (1)
M22 32 (16) 12 (1) 32 (4) 16 (2)
M22 20 (16) 4 (1) 32 (4) 16 (1)
patB::cat

MICs in the presence of reserpine in brackets

Recent data with M22 patA and patB inactivated

with magellen2 revealed full reversal of MDR.