molecular bases for antibiotic resistance through efflux · molecular bases for antibiotic...

88
Molecular bases Molecular bases for antibiotic resistance through efflux for antibiotic resistance through efflux Françoise Van Bambeke Pharmacologie cellulaire et moléculaire Université catholique de Louvain Brussels, Belgium www.facm.ucl.ac.be Transport across membranes: Multiple drug resistance, mechanisms and new tools Summer School – Bremen – 7-14/07/2007

Upload: others

Post on 05-Jul-2020

6 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Molecular bases Molecular bases for antibiotic resistance through effluxfor antibiotic resistance through efflux

Françoise Van Bambeke

Pharmacologie cellulaire et moléculaireUniversité catholique de Louvain

Brussels, Belgium

www.facm.ucl.ac.be

Transport across membranes: Multiple drug resistance, mechanisms and new tools

Summer School – Bremen – 7-14/07/2007

Page 2: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

P. Ehrlich, a father of modern chemotherapy

Paul Ehrlich (1854-1945)

Page 3: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Magic bullets need to reach their target

"corpora non agunt nisi fixata"

"The goal is … to find chemical substances

that have special affinities for pathogenic organisms

and that, like magic bullets,

go straight to their targets"

Paul Ehrlich (1854-1945)

Page 4: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Chemotherapeutic agents exert toxic effects on specific target cells

antifungals

anticancer agents

antibiotics

Page 5: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Chemotherapeutic agents exert toxic effects on specific target cells

How can these drugs reach their target inside the cells ?

nucleic acids

ribosomes

enzymes

Page 6: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Reaching an intracellular target …

polar drug

lipophilic drug

physico-chemical properties are inadequate for reaching an intracellular target !

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 7: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Reaching an intracellular target …

amphipathic drug

most drugs are amphipathic by design,to be able to cross membrane barriers !

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 8: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Intracellular chemotherapeutic agents

But a diffusible compound may have

potentially harmful effects !Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 9: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Chemotherapeutic agents exert toxic effects on specific target cells

How can cells protect themselves from these toxic substances ?

Jérome Bosch (1453-1516)

Page 10: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Why efflux transporters ?

Extrusion by efflux pumps

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 11: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Why efflux transporters ?

Extrusion by efflux pumps

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

general mean of protectionagainst cell invasion by diffusible molecules

Page 12: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Mechanisms of active efflux

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

ATP ADP+Pi

membrane insertion / release

vacuum cleaner

flippaseflip-flop

Page 13: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Most antibiotics are amphiphilic !

macrolide tetracycline

fluoroquinolone

rifampicin

sulfamide

lincosamide

+

+

++

+

+cationic amphiphiles

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 14: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Most antibiotics are amphiphilic !

fluoroquinolone

β-lactam

fusidic acid

-

-

-

anionic amphiphiles

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 15: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Antibiotic efflux transporters are ubiquitous

spectrumprokaryotes

narrow

broad

MATE SMR

MFS

ABC

Gram (+) Gram (-)

RND

distribution

eukaryotes

Mesaros et al. (2005) Louvain médical. 124:308-20

Page 16: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Antibiotics as substrates of efflux pumps

Antibiotic bacteria fungi superiorclass Gram (+) Gram(-) eucaryotesβ-lactamsfusidic acidmacrolidesstreptograminstetracyclinesaminoglycosideschloramphenicolrifamycinssulfamidestrimethoprimfluoroquinolones

Van Bambeke et al. (2000) Biochem. Pharmacol. 60:457-70

Page 17: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Most antibiotics do act on intracellular targets

(lipo- and glycopeptides) (β-lactams)

Efflux as a mechanism of resistance by reducing antibiotic concentration inside the bacteria

quinolones

macrolides aminoglycosides

tetracyclines

sulfamides

Page 18: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Antibiotic efflux from bacteria as a mechanism of antibiotic resistance :

molecular bases

Jean-Michel Folon (1934-2005)

Page 19: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Efflux as a mechanism of export in antibiotic producers

Delivery of metabolites produced by the cell

René Magritte (1898-1967)

Page 20: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Efflux as a mechanism of export in antibiotic producers

spectrum

narrow

broad

specific for the produced antibiotic

ABC MFS

Ptr of S. pristinaespiralis→ pristinamycinLmrA of S. lincolnensis→ lincomycinRifP of A. mediterranei→ rifampicin

OleB or C of S. antibioticus→ oleandomycinSmB of S. ambofaciens→ macrolidesTlrc of S. fradiae→ tylosin

Page 21: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Efflux as a mechanism of export in antibiotic producers

effluxresistance determinants

regulation tylosin biosynthesis

Cundliffe (2006) J. Ind. Microbiol. Biotechnol. 33:500-6

Tylosin-biosynthetic gene cluster in S. fradiae

Page 22: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Efflux as a mechanism of antibiotic resistance in pathogenic bacteria

Pierre Paul Rubens (1530-1587)

Page 23: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Efflux as a mechanism of resistance in Gram-positive bacteria

spectrum

narrow

broad

specific for one (or a few) families of drugs

ABC MFS

NorA of S. aureus→ FQ,Tet, chlMefE of S. pneumoniae→ MLPmrA of S. pneumoniae→ FQMefA of S. pyogenes→ ML

PatA/PatB of S. pneumoniae→ FQ, chlMsrA of S. epidermidis → erythromycin

Page 24: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Efflux as a mechanism of resistance in Gram-negative bacteria

spectrum

narrow

broad

broad spectrum, conferring cross-resistance

MFS

RNDMexAB-OprM of P. aeruginosa→ β-lac, FQ,Tet, ML, chl, rif, sulfAcrAB-TolC of E. coli→ β-lac, FQ,Tet, ML, chl, rif, sulf

TetA of E. coli→TetMsrA of S. epidermidis → erythromycin

specific for one (or a few) families of drugs

Page 25: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Molecular determinants of drug efflux

Brussels, atomium (1958; Polak)

Page 26: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Differences in transport between drugs within a class

Van Bambeke et al. (2003) JAC 51:1055-65

Page 27: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Differential efflux of β-lactams by AcrB of S. typhimurium

Lipophilicity of the side chain Resistance

Nikaido et al. (1998) J. Bacteriol. 180:4686-92

β-lactams & broad spectrum RND

Page 28: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Differential efflux of quinolones by NorA of S. aureus

Bulkiness in 7 and 8 Resistance

Takenouchi et al. (1996) AAC 40:1835-42

Quinolones & narrow spectrum MFS

Page 29: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Unexpected antibiotic substrates

Hergé (1907-1983)

Page 30: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Two types of unexpected substrates

(lipo- and glycopeptides) (β-lactams)

quinolones

macrolides aminoglycosides

tetracyclines

sulfamides

active on an extracellular target !

Page 31: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

β-lactams as unexpected substrates for efflux pumps

Yu et al. (2003) J. Bacteriol. 185:5657-64

Page 32: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

β-lactams as unexpected substrates for efflux pumps

C NO

COOH

RC HN

OCOOH

R

OH

β-lactamase

chemicalclearance

physical clearance

C NO

COOH

R

Page 33: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

β-lactams as unexpected substrates for efflux pumps

Contributions of the AmpC β-lactamase and the AcrAB Multidrug Efflux System in Intrinsic Resistance of E. coli to β-lactams

Mazzariol et al. (2000) AAC 44:1387-1390

Efflux β-lactamase CMI carbenicillin CMI ofloxacin

- - 0.2 0.05

+ - 12.5 0.2

+++ - 50 1.56- + 100 0.05+ + 200 0.39+++ + 400 1.56

WT: intrinsic

resistance !

Page 34: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Two types of unexpected substrates

(lipo- and glycopeptides) (β-lactams)

quinolones

macrolides aminoglycosides

tetracyclines

sulfamides

Highly hydrophilic molecules !

Page 35: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Aminoglycosides as unexpected substrates for efflux pumps

Yu et al. (2003) J. Bacteriol. 185:5657-64AcrB AcrD

more acidic residues in the vestibule of an AG transporter !

acidic PL as determinants in AG transport ?

Composite binding site ?

Page 36: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

efflux from eucaryotic cells as a mechanism of antibiotic ‘resistance’:

molecular bases

Page 37: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Antibiotic efflux from eucaryotic cells and intracellular ‘resistance’ ?

Reduction in intracellular drug concentration can result in inefficacy against intracellular bacteria

Page 38: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Antibiotics as substrates of MDR efflux pumps

spectrum

narrow

broad

ABC

P-glycoprotein→ macrolides, rifampicin→ anticancer agents→ antidepressants, antiepileptics→ digoxin→ …MRP→ fluoroquinolones, β-lactams→ antiviral agents→ anticancer agents→ drug conjugates→ …

Broad spectrum, several classes of drugs

Page 39: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Phenotypic description of antibiotic transport by MDR pumps

Paul Bury (1922-2005)

Page 40: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Antibiotics as substrates of MDR efflux pumps

O

H3C

OH

OH

OH

CH3

H3CH2C O

O

CH3

CH3

H3C

O

O

OHO(H3C)2N

CH3

OH

N

CH3

CH3

H3C

H3C

H3CO

azithromycin

ciprofloxacin

N

C

O

FOH

O

OCH3

HN N

moxifloxacin

N

O

OH

O

HNN

F

Page 41: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

J774 macrophages do express MDR pumps

Page 42: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

ABC multidrug transporters

ATP ADP

MDR-1 (P-glycoprotein) MRP1-10

cationic amphiphiles

anionic amphiphiles

Page 43: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

How to inhibit ABC transporters ?

ATP ADP

MDR-1 (P-glycoprotein) MRP1-10

cationic amphiphiles

anionic amphiphiles

deoxyglucoseNaN3

Page 44: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

How to inhibit ABC transporters ?OCH3

H3CO

CNCH(CH3)2

N

CH3

OCH3

OCH3

NH

C

OHN

H3CO

O

N

H3CO

H3CO

ATP ADP

MDR-1 (P-glycoprotein)

cationic amphiphiles

verapamil

GF120918

Page 45: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

How to inhibit ABC transporters ?

COOHSN

O

OC3H7

C3H7

NCl

S

S

HOOC

N(CH3)2

O

ATP ADP

MRP1-10

anionic amphiphiles

MK571

probenecid

Page 46: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Differential recognition of antibiotics by MDR pumps

azithromycin&

P-glycoprotein

ciprofloxacin&

MRP

Influence of ATP-depletion and pump inhibitors on accumulation at equilibrium

extracell. conc. 5 mg/L; AZM 3 h; CIP 2 h Michot et al. AAC (2004) 48:2673-82

Page 47: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Kinetics of accumulation and efflux for azithromycin

Seral et al. (2003) AAC 47:1047-51

accumulation markedly increased; efflux marginally affected

extracell. conc. 5 mg/L; verapamil 20 µM

Page 48: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Kinetics of accumulation and efflux for ciprofloxacin

Michot et al. (2004) AAC 48:2673-82

both accumulation and efflux markedly affected

extracell. conc. 17 mg/L; probenecid 5 mM

Page 49: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Kinetics of accumulation and efflux for moxifloxacin

Michot et al. AAC (2005) 49:2429-37

neither accumulation nor efflux affected

extracell. conc. 17 mg/L; probenecid 5 mM

Page 50: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Quinolones as inhibitors of ciprofloxacin efflux

• ciprofloxacin efflux inhibited by ciprofloxacin

Michot et al. AAC (2005) 49:2429-37

Page 51: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Quinolones as inhibitors of ciprofloxacin efflux

• ciprofloxacin efflux inhibited by ciprofloxacin• moxifloxacin not affected

Michot et al. AAC (2005) 49:2429-37

Page 52: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Quinolones as inhibitors of ciprofloxacin efflux

• ciprofloxacin efflux inhibited by ciprofloxacinmoxifloxacin

CIP MXF

CIP

Michot et al. AAC (2005) 49:2429-37

Page 53: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Quinolones as inhibitors of ciprofloxacin efflux

• ciprofloxacin efflux inhibited by ciprofloxacinmoxifloxacin

CIP MXF

moxifloxacin also able

to interact with the transporter !

CIP

Michot et al. AAC (2005) 49:2429-37

Page 54: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Azithromycin, ‘kick-back’ model

Gaj et al. (1998) Biochem. Pharmacol. 55:1199-211

Page 55: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Ciprofloxacin, classical model

Kolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42

Page 56: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Moxifloxacin, ‘futile-cycle’ model

Eytan et al. (1996) JBC 271:12897-902

Page 57: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Pharmacological consequences of antibiotic transport

Paul Delvaux (1897-1994)

Page 58: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Models of intracellular infection

L. monocytogenes S. aureus

cytosol phagolysosomes

Page 59: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Influence of pump inhibitors on intracellular activity

azithromycin and L. monocytogenes

verapamil 20 µM; 24 h

L. monocytogenes

AZMAZM

Seral et al. (2003) JAC 51:1167-73

Page 60: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

azithromycin and S. aureus

verapamil 20 µM; 24 h

AZMAZM

S. aureus

Influence of pump inhibitors on intracellular activity

Seral et al. (2003) JAC 51:1167-73

Page 61: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

ciprofloxacin and L. monocytogenes

gemfibrozil 250 µM; 24 h

L. monocytogenes

CIP

Influence of pump inhibitors on intracellular activity

Seral et al. (2003) JAC 51:1167-73

Page 62: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

ciprofloxacin and S. aureus

gemfibrozil 250 µM; 24 h

CIP

S. aureus

Influence of pump inhibitors on intracellular activity

Seral et al. (2003) JAC 51:1167-73

Page 63: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Influence of pump inhibitors on antibiotic distribution

L. monocytogenes

AZMAZM

S. aureus

verapamil enhances azithromycin concentrationIn cytosol and vacuoles

Seral et al. (2003) JAC 51:1167-73

Page 64: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

L. monocytogenes

CIP

S. aureus

gemfibrozil enhances ciprofloxacin cytosolic content

Influence of pump inhibitors on antibiotic distribution

Seral et al. (2003) JAC 51:1167-73

Page 65: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Unexpected antibiotic substrates

Peyo (1928-1992)

Page 66: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Unexpected substrate

polyanionic cyclic peptide

lipophilic chain

Log P = - 4Log D = -9.5 at pH 7

Highly polar molecule !

daptomycin

Page 67: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Daptomycin is substrate of P-gp

CTL+ VER+ O

UA+ G

EM

0.00

0.25

0.50

0.75

1.00

**

DA

P A

ccum

ulat

ion

Daptomycin accumulation proportional to P-gp activity and expression level

Lemaire et al. (2007) AAC - Epub

Page 68: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Daptomycin is substrate of P-gp

Daptomycin intracellular activity is increased in the presence of P-gp inhibitors

Lemaire et al. (2007) AAC - Epub

Page 69: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Putative mechanism of daptomycin transport by P-gp

ATP ADP+Pi

anchoring in the membrane towards the hydrophobic chain and extrusion from the membrane

Lemaire et al. (2007) AAC - Epub

Page 70: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Can we make eukaryotic cells resistant to antibiotics ?

Victor Horta (1861-1947)

Page 71: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Michot et al. (2006) AAC 50:1689-95

Can we make eukaryotic cells resistant to antibiotics ?

Chronical exposure of J774 macrophages to increasing concentrations of ciprofloxacin

Page 72: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Reduced drug accumulation in resistant macrophages

Michot et al. (2006) AAC 50:1689-95

Page 73: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Reduced drug accumulation in resistant macrophages

Michot et al. (2006) AAC 50:1689-95

Page 74: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Coworking between bacteria and macrophage pumps to reduce ciprofloxacin activity

Listeria

Macrophages

Page 75: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Lismond et al. (2006) ICAAC-A1108

Coworking between bacteria and macrophage pumps to reduce ciprofloxacin activity

MIC (mg/L)

FQEGD CLIP

Res. (-) Res. (+) Res. (-) Res. (+)CIP 1.2 1.0 5.0 1.0

MXF 0.6 0.6 0.5 0.25

Cellular concentration (ng/mg prot)

FQWT RS

Prob. (-) Prob. (+) Prob. (-) Prob. (+)CIP 72 263 23 159

MXF 262 208 241 257

Listeria

Macrophages CIP R; MXF S

CIP R; MXF S

Page 76: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Lismond et al. (2006) ICAAC-A1108

Coworking between bacteria and macrophage pumps to reduce ciprofloxacin activity

MIC (mg/L)

FQEGD CLIP

Res. (-) Res. (+) Res. (-) Res. (+)CIP 1.2 1.0 5.0 1.0

MXF 0.6 0.6 0.5 0.25

Cellular concentration (ng/mg prot)

FQWT RS

Prob. (-) Prob. (+) Prob. (-) Prob. (+)CIP 72 263 23 159

MXF 262 208 241 257

CIP R; MXF S

CIP R; MXF S

Same substrate specificity of the MFS procaryotic pump

and of the ABC eucaryotic pump !

Page 77: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Coworking between bacteria and macrophage pumps to reduce ciprofloxacin activity

Lismond et al. (2006) ICAAC-A1108

Page 78: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

No effect of bacteria and macrophage pumps on moxifloxacin activity

Lismond et al. (2006) ICAAC-A1108

Page 79: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Competition for transporters as a mechanism of drug interaction

Pieter Breughel (1525-1569)

Page 80: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Ciprofloxacin versus NSAIDsciprofloxacin

indomethacin

diclofenac

Page 81: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Ciprofloxacin versus NSAIDs

Huygue et al. Unpublished data

ciprofloxacin accumulation

CIP GEM 500 DICLO 300 INDO 3000

100

200

300

WTRS

% o

f acc

umul

atio

n in

WT

cells

Page 82: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Ciprofloxacin versus NSAIDs

Huygue et al. Unpublished data

ciprofloxacin accumulation

CIP GEM 500 DICLO 300 INDO 3000

100

200

300

WTRS

% o

f acc

umul

atio

n in

WT

cells

Page 83: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Ciprofloxacin versus NSAIDs

Huygue et al. Unpublished data

ciprofloxacin accumulation

CIP GEM 500 DICLO 300 INDO 3000

100

200

300

WTRS

% o

f acc

umul

atio

n in

WT

cells

Page 84: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Ciprofloxacin versus NSAIDs

indomethacin and, to a lesser extent, diclofenac, are inhibitors of ciprofloxacin transport

Huygue et al. Unpublished data

ciprofloxacin accumulation

CIP GEM 500 DICLO 300 INDO 3000

100

200

300

WTRS

% o

f acc

umul

atio

n in

WT

cells

Page 85: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

NSAIDs as inhibitors of ciprofloxacin efflux

Huygue et al. Unpublished data

inhibitor conc. (mM)

CIP

acc

umul

atio

n (%

con

trol

in W

T ce

lls)

Dose-dependent effect on accumulation

Time (minutes)

% o

f acc

umul

ated

am

ount

Time (minutes)

related to slowed-down efflux

Page 86: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

NSAIDs versus Ciprofloxacin

indomethacin shares a same transporter with ciprofloxacin

diclofenac is substrate of another gemfibrozil-inhibitable

transporter

Huygue et al. Unpublished data

indometacin accumulation

INDO GEM 500 CIP 5000

50

100

150

WTRS

% o

f acc

umul

atio

n in

WT

cells

diclofenac accumulation

DICLO GEM 500 CIP 5000

100

200

300

400WTRS

% o

f acc

umul

atio

n in

WT

cells

Page 87: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

Conclusion: avenues for the future

René Magritte (1898-1967)

Do we need to include ‘transport’ studies

with bacteria and eucaryotic cellsin the early development

of new antibiotics ?

Page 88: Molecular Bases for antibiotic resistance through efflux · Molecular bases for antibiotic resistance through efflux Françoise Van Bambeke. Pharmacologie cellulaire et moléculaire

The past and present efflux team in Brussels

HOOC

H2 N

technicianspost-docsPhD studentsstudentsseniors

C. Seral N. Caceres B. Marquez N. Mesaros L. Avrain M. Heremans

J.M. Michot H. Chanteux A. Lismond S. Lemaire H. Bensikaddour F. Huyghe

F. Renoird M.C. Cambier C. Misson M. Vergauwen N. Couwenbergh

M.P. Mingeot P. Tulkens F. Van Bambeke