Efflux Mechanisms of Fluoroquinolones and β-lactams

Transcription

1 Efflux Mechanisms of Fluoroquinolones and β-lactams Paul M. Tulkens, MD, PhD Françoise Van Bambeke, PharmD, PhD Cellular and Molecular Pharmacology Unit Catholic University of Louvain, Brussels, Belgium MSD Closed Research Update Chicago, IL. September 14, 2003

2 Why efflux? polar compound lipophilic compound highly polar or highly lipophilic compounds do not easily pass across membranes...

3 Why efflux? amphiphilic compound Amphiphilic?? a compound that is both polar and non-polar (ex.: a glucuronoconjugate) amphiphilic compounds easily invade cells

4 Why efflux? But a diffusible compound may have potentially harmful effects in cells!

5 Why efflux? ion gradient ATP hydrolysis Efflux transporters are a general mean of protection of the cell against invasion by diffusible molecules

6 Why DRUG efflux transporters? Most of our drugs are made amphiphilic! to allow them to diffuse and penetrate in cells and tissues...

7 Most antibiotics are amphiphilic! Van Bambeke et al. Biochem. Pharmacol. (2000) 60: Neutral apolar polar chloramphenicol

8 Most antibiotics are amphiphilic! Van Bambeke et al. Biochem. Pharmacol. (2000) 60: anionic fluoroquinolones beta-lactams fusidic acid

9 Most antibiotics are amphiphilic! cationic lincosamides macrolides tetracyclines fluoroquinolones sulfamides rifampicin

10 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

11 Identification of efflux pumps based on genome sequencing data identification of genes encoding for putative transporters in the genome of 87 bacteria S. aureus, S. pneumoniae, E. coli, P. aeruginosa, archea 4 eucaryotes S. cerevisiae, P. falciparum,...

12 Rosenberg et al. (2003) JBC 278: Structure of pumps in eucaryotic cells Current view of the structure of P-glycoprotein - ATP + ATP

13 Van Bambeke et al. JAC (2003) 51: Structure of pumps in procaryotic cells AB pore AB porin H + AB AB AB Na + lipoprotein H + periplasm pump ATP ADP H + Na + H + RND MFS SMR MATE ABC RND, MFS, ABC Gram-positive Gram-negative

14 Efflux pumps in bacteria: what does it mean for resistance?! Constitutive expression of efflux pumps " intrinsic resistance! Inducible expression of efflux pumps " acquired resistance! Cooperation with other mechanims of resistance! Selection of other mechanisms of resistance

15 P. aeruginosa seems to be constitutively resistant to many antibiotics... Li et al. AAC (1995) 39:

16 Disruption of MexAB-OprM restores the activity of several antibiotics against P. aeruginosa Li et al. AAC (1995) 39:

17 Efflux pumps in bacteria: what does it mean for resistance?! Constitutive expression of efflux pumps " intrinsic resistance! Inducible expression of efflux pumps " acquired resistance! Cooperation with other mechanims of resistance! Selection of other mechanisms of resistance

18 Overexpression of efflux pumps in P. aeruginosa upon exposure to fluoroquinolones in CF patients Jalal et al, AAC (2000) 44:

19 Overexpression of efflux pumps in P. aeruginosa upon exposure to fluoroquinolones in CF patients Jalal et al, AAC (2000) 44: Regulator of MexAB expression

20 Efflux pumps in bacteria: what does it mean for resistance?! Constitutive expression of efflux pumps " intrinsic resistance! Inducible expression of efflux pumps " acquired resistance! Cooperation with other mechanisms of resistance! Selection of other mechanisms of resistance

21 Efflux in cooperation with other resistance mechanisms R physical clearance C O N COOH chemical clearance R R β-lactamase C O N COOH CHN O OH COOH

22 Efflux in cooperation with other resistance mechanisms Mazzariol et al, AAC (2000) 44: Contributions of the AmpC β-lactamase and the AcrAB Multidrug Efflux System in Intrinsic Resistance of E. coli to β -lactams Efflux β-lactamase MIC carbenicillin Intrinsic low level resistance!

23 Efflux in cooperation with other resistance mechanisms Mazzariol et al, AAC (2000) 44: Contributions of the AmpC β-lactamase and the AcrAB Multidrug Efflux System in Intrinsic Resistance of E. coli to β -lactams Efflux β-lactamase MIC carbenicillin Intrinsic low level resistance! high level resistance!

24 Overexpression of efflux pumps in P. aeruginosa upon exposure to fluoroquinolones in CF patients Jalal et al, AAC (2000) 44: Regulator of MexAB expression

25 Cooperation of efflux pumps and target mutation in P. aeruginosa in CF patients + Mutation in FQ target Jalal et al, AAC (2000) 44: Regulator of MexAB expression

26 Cooperation of efflux pumps and target mutation in P. aeruginosa in CF patients + Mutation in FQ target Jalal et al, AAC (2000) 44: Regulator of MexAB expression

27 Cooperation of efflux pumps and target mutation in P. aeruginosa in CF patients + Mutation in FQ target Jalal et al, AAC (2000) 44: Regulator of MexAB expression

28 Efflux pumps in bacteria: what does it mean for resistance?! Constitutive expression of efflux pumps " intrinsic resistance! Inducible expression of efflux pumps " acquired resistance! Cooperation with other mechanims of resistance! Selection of the other mechanisms of resistance

29 Efflux and selection of resistance to FQ Gyrase/ Topoisomerase FQ activity is critically dependent upon a sufficiently large intrabacterial concentration

30 Efflux and selection of resistance to FQ Exposure to submic concentrations favors the selection of target mutations! Gyrase/ Topoisomerase

31 Efflux and selection of resistance to FQ Frequency of Levofloxacin-resistant mutants in Pseudomonas aeruginosa with deletions of the efflux pump operons Pump status LVX MIC Frequency of LVXresistant mutants WT mexab-oprm mexcd-oprj mexef-oprn mexab-oprm; mexef-oprn mexcd-oprj; mexef-oprn mexab-oprm; mexcd-oprj mexab-oprm; mexcd-oprj; < mexef-oprn Lomovskaya et al, AAC (1999) 43: Selection of mutants in FQ target undetectable if ALL pumps are disrupted

32 How to cope with efflux pumps in anti-infective therapy?! S. pneumoniae and the correct choice of "respiratory fluoroquinolones"! P. aeruginosa! quinolones! carbapenems

33 MIC considerations... Quinolone Wild-type strain MIC CIP 0.5 LVX MXF Are those differences important? GAT 0.25 Coyle et al. AAC (2001) 45:

34 Enters the quinolone pmra efflux pump... NH 2 COOH 12 transmembrane segments member of the Major Facilitator Superfamily (MFS) energized by H + gradient Gill et al. AAC (1999) 43: Van Bambeke et al. (2000) Biochem. Pharmacol. 60:

35 MIC considerations... Quinolone Wild-type strain MIC Efflux resistant strain MIC CIP LVX MXF And look at this, now GAT Coyle et al. AAC (2001) 45:

36 PK / PD considerations... For fluoroquinolones, efficacy is linked to AUC/MIC ratio (must be > 30 for S. pneumoniae) prevention of resistance is linked to C max /MIC ratio (must be > 10 to prevent mutations)

37 Optimisation based on pharmacodynamics Quinolone Wild-type strain MIC AUC/MIC C max /MIC CIP LVX MXF GAT moderate risk of selection of resistance! Coyle et al. AAC (2001) 45:

38 Coyle et al. AAC (2001) 45: Optimisation based on pharmacodynamics Quinolone Wild-type strain Efflux resistant strain MIC AUC/MIC Cmax/MIC MIC AUC/MIC Cmax/MIC CIP LVX MXF GAT moderate risk High of risk of selection selection of resistance of resistance!!

39 How to cope with efflux pumps in anti-infective therapy?! S. pneumoniae and the correct choice of "respiratory fluoroquinolones"! P. aeruginosa! quinolones! carbapenems

40 Topology of the wide spectrum Mex pumps in Pseudomonas (RND superfamily) Van Bambeke et al. (2000) Biochem. Pharmacol. 60: NH 2 COOH

41 Functionning of the wide spectrum Mex pumps in Pseudomonas outer membrane outer membrane protein OprM, J, N linker protein MexA, C, E, X inner membrane RND exporter protein MexB, D, F, Y adapted from Aeschlimann, Pharmacotherapy (2003) 23:

42 Wide spectrum pumps in Pseudomonas β-lac ML TET AG FQ Chl MexAB-OprM MexCD-OprJ MexEF-OprN MexHI-OPrD MexJK-OprM MexXY-OprM 1. wide spectrum transporters " cross-resistance among most AB classes Van Bambeke et al. JAC (2003) 51: Aeschlimann, Pharmacotherapy (2003) 23:

43 Wide spectrum pumps in Pseudomonas β-lac ML TET AG FQ Chl MexAB-OprM MexCD-OprJ MexEF-OprN MexHI-OPrD MexJK-OprM MexXY-OprM 2. FQ are «universal» substrates " easy selection of resistance Van Bambeke et al. JAC (2003) 51: Aeschlimann, Pharmacotherapy (2003) 23:

44 Wide spectrum pumps in Pseudomonas β-lac ML TET AG FQ Chl MexAB-OprM MexCD-OprJ MexEF-OprN MexHI-OPrD MexJK-OprM MexXY-OprM 3. constitutive expression and/or inducible expression " increase of resistance level higher upon antibiotic exposure Van Bambeke et al. JAC (2003) 51: Aeschlimann, Pharmacotherapy (2003) 23:

45 Pseudomonas and quinolone efflux Overexpressed Mex pumps AB CD EF MICs CIP 1 LVX 2 MXF Zhang et al. JAC (2001) 48: Lomovskaya et al. AAC (2001)5:

46 Pseudomonas and penem efflux Mex pumps AB CD XY * MICs MERO IMI BIA PANI FARO RITI * clinical isolate, basal level of expression Okamoto et al. J. Infect. Chemother (2002) 8: Okamoto et al. AAC (2002) 46:

47 Pseudomonas and penem efflux Mex pumps AB CD XY * MICs MERO IMI BIA PANI FARO RITI * clinical isolate, basal level of expression Okamoto et al. J. Infect. Chemother (2002) 8: Okamoto et al. AAC (2002) 46:

48 Pseudomonas and penem efflux Mex pumps AB CD XY * MICs MERO IMI BIA PANI FARO RITI * clinical isolate, basal level of expression Okamoto et al. J. Infect. Chemother (2002) 8: Okamoto et al. AAC (2002) 46:

49 Combined mechanisms of resistance in Pseudomonas R C O N COOH $ OprD porin # Mex efflux pump C N O R # AmpC β-lactamase R CHN O OH COOH COOH

50 Combined mechanisms of resistance in Pseudomonas R C O N COOH $ OprD porin # Mex efflux pump C N O R # AmpC β-lactamase R CHN O OH COOH COOH

51 Combined mechanisms of resistance in Pseudomonas R C O N COOH $ OprD porin # Mex efflux pump C N O R # AmpCR β-lactamase COOH CHN O OH COOH

52 Combined mechanisms of resistance in Pseudomonas R C O N COOH $ OprD porin # Mex R # AmpC β-lactamase R efflux pump C O N COOH CHN O OH COOH

53 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 1. Laboratory strains Resistance mechanism fold increase in MIC AmpC MexAB OprD IMI MERO X 1 X 2 Kohler et al. AAC (1999) 43: Nakae et al. AAC (1999) 43:

54 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 1. Laboratory strains Resistance mechanism fold increase in MIC AmpC MexAB OprD IMI MERO X 1 X 2 X 1 X 2-10 Kohler et al. AAC (1999) 43: Nakae et al. AAC (1999) 43:

55 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 1. Laboratory strains Resistance mechanism fold increase in MIC AmpC MexAB OprD IMI MERO X 1 X 2 X 1 X 2-10 X 16 X 4 Kohler et al. AAC (1999) 43: Nakae et al. AAC (1999) 43:

56 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 1. Laboratory strains Resistance mechanism fold increase in MIC AmpC MexAB OprD IMI MERO X 1 X 2 X 1 X 2-10 X 16 X 4 X 16 X Kohler et al. AAC (1999) 43: Nakae et al. AAC (1999) 43:

57 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 2. Clinical strains Resistance mechanism AmpC MexAB OprD # 1 low - - MIC (mg/l) IMI MERO Pai et al. AAC (2001) 45:

58 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 2. Clinical strains Resistance mechanism AmpC MexAB OprD # 1 low - - # 2 low - + MIC IMI MERO Imipenem is affected by a defect in OprD (porin) Pai et al. AAC (2001) 45:

59 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 2. Clinical strains Resistance mechanism AmpC MexAB OprD # 1 low - - # 2 low - + # 3 low + + MIC IMI MERO Meropenem is affected by the expression of Mex AB (efflux) Pai et al. AAC (2001) 45:

60 Contribution of resistance mechanisms to carbapenem resistance in Pseudomonas 2. Clinical strains Resistance mechanism AmpC MexAB OprD # 1 low - - # 2 low - + # 3 low + + #4 high - + MIC IMI MERO AmpC does not markedly affect imipenem or meropenem Pai et al. AAC (2001) 45:

61 An what about ertapenem? Susceptibility Patterns of Resistant Mutants (PAO -1 and Derivatives) Resistance mechanism D2 β-la OprM ETP IPM MER CAZ LVF None (PAO-1) + Ind D2 (porin) - Ind OprM (efflux) + Ind βla + Con MK- X + Ind The major ertapenem resistance seen in PAO-1 is not due to D2 loss, OprM overexpression or βla constitutivity - suggesting a new mechanism

62 A burning question... Could fluoroquinolones jeopardize other antibiotics through effux pumps?

63 Fluoroquinolones-exposed P. aeruginosa overexpress wide spectrum efflux pumps Incubation of P.aeruginosa with (too) low concentrations of fluoroquinolones triggers the expression of the Mex transporters BEFORE it affects the gyrase! Nb mutants ciprofloxacin MexCD MexCD MexEF MexCD MexCD MexEF MexAB X 2 X 3 X 4 X 6 Kohler et al. AAC (1997) 41: concentration (X MIC)

64 Mex-expressing strains become highly resistant to many antibiotics Risk of cross resistance! Rearranged from Kohler et al. AAC (1997) 41:

65 Perspectives: strategies to overcome resistance by efflux: en route to a rational design of efflux pump inhibitors?

66 High throughput screening for the discovery of inhibitors of large spectrum transporters library of 200,000 synthetic and natural compounds in vitro screening in combination with levofloxacin against P. aeruginosa overexpressing Mex pumps Renau et al. J. Med. Chem. (1999) 42: Lomovskaya et al. J. Mol. Microbiol. Biotechnol. (2001) 3:

67 Lomovskaya et al. AAC (2001) 45: High throughput screening for the discovery of inhibitors of large spectrum transporters " increase in susceptibility of clinical isolates

68 you can win! Shall we have a better future? Do not deny the difficulties!...

69 Thank you for your attention... These slides (with a series of additional ones) will be available as PDF file from here... click on "Conferences and look for the link to Efflux Mechanisms of Fluoroquinolones and β-lactams, Chicago, IL, September 14th, 2003

70 Additional questions. Is imipenem more affected than meropenem by OprD (porin)? Why is meropenem more affected than imipenem by MexAB (efflux)?

71 Why is IMI more affected than MERO by OprD? H 3 C OH IMI OH CH 3 MERO O N COO S N H NH 2 H 3 C O N COO S NH H 3 C N CH nm/sec 73 nm/sec H O OprD porin MERO enters only slowly through OprD... Trias et al. AAC (1990) 34:52-57 " has MERO another route of entry?

72 Why is MERO more affected than IMI by MexAB? OH OH CH 3 MERO H 3 C IMI H 3 C N S N S H 3 C O COO O COO N CH 3 N H NH 2 NH H O substrate specificity zone differential recognition by the large periplasmic loops NH 2 COOH of the inner membrane component? Mao et al. Mol. Microbiol :