Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria

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Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria Françoise Van Bambeke on behalf of J.M. Michot, C. Seral, M.

1 Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria Françoise Van Bambeke on behalf of J.M. Michot, C. Seral, M. Heremans, M.P. Mingeot-Leclercq, P.M. Tulkens Unité de Pharmacologie cellulaire et moléculaire Université catholique de Louvain Brussels, Belgium

2 pharmacy

3 antibiotics as magic bullets already concerned in efflux?

4 Why drug efflux transporters? polar drug lipophilic drug physico-chemical properties are inadequate for reaching an intracellular target!

5 Why drug efflux transporters? amphipathic drug most drugs are amphipathic by design, to be able to cross membrane barriers!

6 Why drug efflux transporters? But a diffusible compound may have potentially harmful effects!

7 Why drug efflux transporters? Extrusion by efflux pumps

8 Why drug efflux transporters? Extrusion by efflux pumps general mean of protection against cell invasion by diffusible molecules

9 Typical toxic diffusible substances well known as substrates for efflux pumps antibiotics antifungals anticancer agents

10 Van Bambeke et al. Biochem. Pharmacol. (2000) 60: Most antibiotics are amphiphilic! cationic amphiphiles anionic amphiphiles macrolide tetracycline fluoroquinolone + + rifampicin - β-lactam fluoroquinolone + + lincosamide - fusidic acid sulfamide

11 Antibiotic classes recognized by efflux pumps in different types of organisms Antibiotic bacteria fungi superior class Gram (+) Gram(-) eucaryotes β-lactams fusidic acid macrolides streptogramins tetracyclines aminoglycosides chloramphenicol rifamycins sulfamides trimethoprim fluoroquinolones

12 Consequences of antibiotic efflux from eucaryotic cells alteration of pharmacokinetics whole organism: absorption, distribution, elimination single cell: accumulation, localization alteration of pharmacodynamics body level: drug concentration in the infected compartment cellular level: activity against intracellular bacteria Van Bambeke et al, JAC (2003) 51:

13 Main multidrug resistance efflux pumps in eucaryotic cells ATP ATP-binding Cassette (ABC) Superfamily ADP MDR-1 (P-glycoprotein) cationic amphiphiles MRP1-9 anionic amphiphiles

14 Inhibitors and substrates share the same physicochemical properties MDR-1 (P-glycoprotein) MRP1-9 cationic amphiphiles anionic amphiphiles OCH 3 OCH 3 3CO OCH 3 C 3 H 7 O - + NH N C 3 H 7 S O COO CH(CH 3 ) 2 CN CH 3 verapamil probenecid

15 Macrophages express at least MRP-1 and P-gp

16 1. Influence of efflux pumps on antibiotic cellular pharmacokinetics macrolides

17 Macrolide story CH 3 H 3 C N CH 3 OH OH H 3 C H 3 CH 2 C OH H 3 C O CH 3 (H 3 C) 2 N OHO O CH 3 O CH 3 O O H 3 CO CH 3 H 3 C OH Azithromycin is a dicationic amphiphilic molecule

18 Azithromycin accumulates to high levels in eucaryotic cells Seral et al, AAC (2003) 47:

19 macrolides accumulate mainly in the lysosomal compartment lysosomal enzymes Carlier et al, JAC (1987) 20 Suppl B:47-56

20 macrolide accumulation proceeds by diffusion / segregation B B B H + BH + BH + BH + De Duve et al, Biochem Pharmacol. (1974) 23:

21 Azithromycin concentration is high but still suboptimal Seral et al, AAC (2003) 47:

22 Inhibition of P-gp by verapamil increases accumulation Seral et al, AAC (2003) 47:

23 Inhibition of P-gp by verapamil increases accumulation and slightly slows down efflux Seral et al, AAC (2003) 47:

24 Similar effects are obtained with more specific inhibitors of P-gp Seral et al, AAC (2003) 47:

25 How can efflux pumps increase azithromycin accumulation without markedly affecting its efflux? mechanism of action of efflux pumps

26 Efflux pumps as pores? Probably not... Experimental evidences substrates and inhibitors are amphiphilic rates and kinetics of efflux are not directly related to the cytosolic drug content Higgins et al, Trends Biochem Sci (1992) 17:18-21 Wadkins et al, Biochem Biophys Acta (1993) 1153: Bolhuis et al, EMBO J (1996) 15: Ashida et al, J Theor Biol (1998) 195:

27 Efflux pumps as vacuum cleaners? Possibly... Structural evidence Rosenberg et al, JBC (1997) 272:

28 Efflux pumps as flippases? Possibly... X-Ray structure of the lipid A transporter from E. coli, an homolog of P-glycoprotein Chang et al, Science (2001) 293:

29 substrates potentially extruded from the membrane!

30 substrates potentially extruded from the membrane without having seen the cytosol! Marbeuf-Gueye et al (1999) BBA 1450:

31 2. Influence of efflux pumps on antibiotic cellular pharmacokinetics quinolones

32 Quinolone story O O F C O - H 2 N N N Ciprofloxacin is a zwitterionic amphiphilic molecule

33 Quinolones accumulate to moderate levels in eucaryotic cells Carlier et al JAC (1990) 26 Suppl B:27-39

34 Quinolones are found in the soluble fraction of cell homogenates LDH (cytosol) NABgase (lysosomes) proteins Carlier et al JAC (1990) 26 Suppl B:27-39

35 Ciprofloxacin facilitates its own uptake saturation of efflux! Michot et al, AAC (2004) 7: in press

36 Ciprofloxacin accumulation and efflux in ATP-depleted cells Michot et al, AAC (2004) 7: in press

37 Ciprofloxacin accumulation and efflux in probenecid-treated cells Michot et al, AAC (2004) 7: in press

38 Differential effects of pump inhibitors on ciprofloxacin and azithromycin accumulation data from Michot et al, AAC, 2004

39 Have all quinolones been made equal? ciprofloxacin O O moxifloxacin O O F C OH F C OH HN N N HN N OCH 3 N levofloxacin O O garenoxacin O O F C OH C OH H 3 C N N O N H CH 3 F 2 HC O N HN CH 3

40 Quinolones markedly differ by their accumulation level

41 contrasting effect of quinolone concentration on their accumulation

42 contrasting effect of ATP-depletion on quinolone accumulation

43 comparative effect of pump inhibition on quinolone accumulation 2 h incubation, 5 mg/l, inhibitors : 20 cellular accumulation Is this the max level of quinolone accumulation? control ATP-depletion probenecid gemfibrozil MK571 0 cipro levo gareno moxi

44 contrasting effect of temperature on quinolone accumulation cooling 2 h incubation, 5 mg/l, temperature : cellular accumulation C 4 C 78 % 45 % 55 % 18 % 0 cipro levo gareno moxi slowered diffusion through membranes

45 contrasting effect of temperature on quinolone accumulation heating 2 h incubation at 37 C, 17 mg/l, preexposure to 56 C: cellular accumulation Is this the max level of quinolone accumulation? 0 min 10 min active processes impaired in death cells 0 cipro moxi

46 quinolone accumulation is passive but efflux is active actual sorting site may be at the cell surface

47 accumulation is inversely related to recognition by efflux transporters ciprofloxacin moxifloxacin Is the amount of drug reaching the cellular medium high enough to kill intracellular bacteria?

48 3. Influence of efflux pumps on antibiotic cellular pharmacodynamics

49 Does efflux from macrophages confer resistance against intracellular infections? Carryn et al, Infect Dis Clin North Am. (2003) 17:615-34

50 Does efflux affect the intracellular activity of these antibiotics? Listeria monocytogenes Staphylococcus aureus

51 Verapamil increases azithromycin activity against L. monocytogenes L. monocytogenes AZM AZM Seral et al, JAC (2003) 51:

52 Verapamil increases azithromycin activity against S. aureus S. aureus AZM AZM Seral et al, JAC (2003) 51:

$both in the soluble and granular fractions Seral et al,$

53 Verapamil increases azithromycin conc. both in the soluble and granular fractions Seral et al, JAC (2003) 51:

54 Gemfibrozil increases ciprofloxacin activity against L. monocytogenes L. monocytogenes CIP Seral et al, JAC (2003) 51:

55 Gemfibrozil does not increases ciprofloxacin activity against S. aureus S. aureus CIP Seral et al, JAC (2003) 51:

$in the soluble fraction only Seral et al, JAC (2003)$

56 Gemfibrozil increases ciprofloxacin conc. in the soluble fraction only Seral et al, JAC (2003) 51:

57 Inhibition of efflux pumps may increase antibiotic activity in the compartments where they accumulate CIP AZM AZM

58 Strategies for the future of antibiotherapy of intracellular infections use of poor substrates of efflux pumps (moxi vs cipro) caution for «cross resistance» with other substrates (over expression of efflux pumps) development of specific inhibitors of efflux pumps

59 moxi/gareno are more active than cipro/levo against L. monocytogenes et S. aureus

60 Strategies for the future of antibiotherapy of intracellular infections use of poor substrates of efflux pumps (moxi vs cipro) caution for «cross resistance» with other substrates (over expression of efflux pumps) development of specific inhibitors of efflux pumps

61 Over-expression of efflux pumps as mechanism of resistance anticancer agent antibiotic????? antibiotic anticancer agent????? anticancer agent antibiotic?????

62 Ciprofloxacin selects over-expression of efflux pumps as mechanism of resistance 2 h incubation at 37 C 10 macrophages exposed to increasing conc. of cipro (up to 80 mg/l)

63 Strategies for the future of antibiotherapy of intracellular infections use of poor substrates of efflux pumps (moxi vs cipro) caution for «cross resistance» with other substrates (over expression of efflux pumps) development of specific inhibitors of efflux pumps

64 Inhibitors of efflux transporters should help you to keep your stuff in...

65 Inhibitors of efflux transporters But be careful not to turn off a useful pump...

66 F Van Bambeke JM Michot C Seral M Heremans MP Mingeot- Leclercq PM Tulkens come and see us at <

Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria

Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria Unité de Pharmacologie cellulaire et moléculaire F. Van Bambeke Magic bullets need to reach their target