Françoise Van Bambeke, PharmD, PhD How do antibiotics play with multidrug transporters in eucaryotic cells ? Françoise Van Bambeke, PharmD, PhD Pharmacologie cellulaire et moléculaire Louvain Drug Research Institute Université catholique de Louvain Bruxelles, Belgium www.facm.ucl.ac.be 09/06/2010 Bremen Summerschool 2010

ATP-Binding Cassette transporters ABCs transporters ABCA ABCB ABCC ABCD ABCE ABCF ABCG P-gp MRPs BCRP multidrug transporters 09/06/2010 Bremen Summerschool 2010 2

Are antibiotics substrates for MDR transporters in eucaryotic cells ? Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Fluoroquinolone antibiotics ciprofloxacin moxifloxacin 09/06/2010 Bremen Summerschool 2010

Kinetics of accumulation and efflux for ciprofloxacin both accumulation and efflux markedly affected by probenecid (inhibitors of Mrps) extracell. conc. 17 mg/L; probenecid 5 mM Michot et al. (2004) AAC 48:2673-82 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin, classical model Kolaczkowski & Goffeau (1997) Pharmacol. Ther. 76:219-42 09/06/2010 Bremen Summerschool 2010

Kinetics of accumulation and efflux for moxifloxacin neither accumulation nor efflux affected by probenecid Mox acc and efflux very rapid and not influenced by inhibitor Incidentilly, you may have seen that it reaches an acc level in control conditions very similar to that obtained with cipro but in the presence of prob extracell. conc. 17 mg/L; probenecid 5 mM Michot et al. AAC (2005) 49:2429-37 09/06/2010 Bremen Summerschool 2010

Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin Studying conc. effects on accumulation was also very informative. The graph shows the effect of cip on its own acc. Intriguingly … This is an unusual observation, because you would have expected either a linear relationship or a saturation effect. It can be however explained by an inhibition of the efflux by cip itself, resulting in a facilitation of its accumulation Michot et al. AAC (2005) 49:2429-37 09/06/2010 Bremen Summerschool 2010

Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin not affected The same experiment done with moxi failed to show any effect. We may be tempted from these data as well as from kinetic studies that moxi is not transported. The next experiment however suggests that it is probablyy a too simplistic view. Michot et al. AAC (2005) 49:2429-37 09/06/2010 Bremen Summerschool 2010

Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin CIP MXF CIP What we did here was to incubate the cells with a tracer amount of radiolabebeled cip and increasing conc of either cold cip or cold moxi to be able to measure specifically the effect of the two quinolones on cip accumulation. Regarding the effect of an excess of cip first, you recognize here the facilitating effect it exerts on the accumulation of radiolabeeld cip. Michot et al. AAC (2005) 49:2429-37 09/06/2010 Bremen Summerschool 2010

Quinolones as inhibitors of ciprofloxacin efflux ciprofloxacin efflux inhibited by ciprofloxacin moxifloxacin CIP MXF CIP moxifloxacin also able to interact with the transporter ! With an excess of unlabeled moxi now, you also oberve a facilitating effect on cip accumulation, with however higher conc of moxi needed to reach a same accumulation of cipro. Michot et al. AAC (2005) 49:2429-37 09/06/2010 Bremen Summerschool 2010

Moxifloxacin, ‘futile-cycle’ model Eytan et al. (1996) JBC 271:12897-902 09/06/2010 Bremen Summerschool 2010

Die Bremer Stadtmusikanten Can we make eucaryotic cells « resistant » to antibiotics ? A way to further characterize efflux transporters Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Over-expression of efflux pumps as mechanism of resistance How to get resistant cells ? step-wise increase in AB concentration several passages at each step time and drug consumming! multifactorial multidrug resistance Gottesman et al, Methods Enzymol. (1998) 292: 248-58 09/06/2010 Bremen Summerschool 2010

Chronical exposure to increasing concentrations Identification of the ciprofloxacin transporter: « resistant » cells as a tool 0.1 mM 0.15 mM Chronical exposure to increasing concentrations 0.2 mM Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin « resistant » cells: phenotypic analysis ATP-dependent reduction in cell accumulation of CIP; MXF non affected control ATP-depleted Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin « resistant » cells: phenotypic analysis ATP-dependent reduction in cell accumulation of CIP; MXF non affected control ATP-depleted Michot et al., Antimicrob. Ag. Chemother. (2006) 50:1689-1695 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin « resistant » cells: phenotypic analysis  efflux rate  IC50 gemfibrozil Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin « resistant » cells: genotypic analysis ARNm levels (Real-Time PCR)  expression Mrp2 and Mrp4, but Mrp4 predominates Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin « resistant » cells: proteomic analysis detection of the proteins by Western-Blot of membrane fraction Confocal microscopy Mrp2 WT RS µg 5 15 30 5 15 30 Mrp4 WT RS µg 5 15 30 5 15 30 Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 09/06/2010 Bremen Summerschool 2010

Ciprofloxacin « resistant » cells : which transporter ? Specific extinction of gene expression by siRNA - CT 10 25 10 25 - CT 5 10 25 10 25 Mrp2 Mrp4 Marquez et al., Antimicrob. Ag. Chemother. (2009) 53:2410-6 09/06/2010 Bremen Summerschool 2010

Acquisition of resistance is a stepwise process Accumulation and Mrp4 expression during selection of resistance 09/06/2010 Bremen Summerschool 2010

Can we select for moxifloxacin « resistant » cells ? FQ accumulation and gemfibrozil effect ciprofloxacin moxifloxacin WT Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Can we select for moxifloxacin « resistant » cells ? FQ accumulation and gemfibrozil effect ciprofloxacin moxifloxacin WT MXF-RS Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Can we select for moxifloxacin « resistant » cells ? Ciprofloxacin efflux T1/2 WT >> T1/2 WT+GEM = T1/2 MXF-RS Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Moxifloxacin-exposed cells are « anti » resistant! Mrp4 expression Western-Blot ARNm (Real Time PCR) WT MXF WT MXF - - RS CIP - RS Mrp4 Actin 20µg 40µg 60µg 20µg 40µg 60µg 10µg Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Fluoroquinolone transport: model ciprofloxacin WT CIP-RS MXF-RS 1 4 1 0.2 1 15 out in out in out in 09/06/2010 Bremen Summerschool 2010

Fluoroquinolone transport: model moxifloxacin WT CIP-RS MXF-RS 1 15 1 15 1 15 out in out in out in 09/06/2010 Bremen Summerschool 2010

Efflux … is it enough for resistance ? Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Efflux pumps … and what next ? How to get resistant cells ? step-wise increase in AB concentration several passages at each step time and drug consumming! multifactorial multidrug resistance Gottesman et al, Methods Enzymol. (1998) 292: 248-58 09/06/2010 Bremen Summerschool 2010

Stable Isotope Labeling Aminoacid in Culture WT CIP-macrophages 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg sample mixing 1:1 protein digestion identification in mass spectrometry and determination of the relative abundance 09/06/2010 Bremen Summerschool 2010

Stable Isotope Labeling Aminoacid in Culture WT CIP-macrophages 1.10 1.13 1.15 1.17 1.19 F1 F2 F3 F4 F5 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg post-nuclear homogenate ultracentrifugation 1.10 Discontinuous Sucrose gradient Enriched plasma membrane fraction 1.13 1.15 1.17 1.19 F1 F2 F3 F4 F5 190 - kDa 40 - MRP1 Prohibitin 09/06/2010 Bremen Summerschool 2010

Stable Isotope Labeling Aminoacid in Culture WT CIP-macrophages F1 F2 A B C F D E G H I J A B C F D E - 200 - 150 - 120 - 100 - 85 - 70 - 60 - 40 - 50 - 30 13C6-Lys 13C6-Arg 12C6-Lys 12C6-Arg Discontinuous Sucrose gradient Enriched plasma membrane fraction G H I J k Combine 1:1 SDS-PAGE/in-gel digestion Protein ID & Quantification 09/06/2010 Bremen Summerschool 2010

SILAC: global data  Identification of 900 proteins with 3 uniques peptides Among proteins detected in both fractions  15  expression in CIP-macrophages as compared to WT  13  expression in CIP-macrophages as compared to WT Among proteins detected in one of the two fractions  37/36  expression in CIP-macrophages as compared to WT  29/34  expression in CIP-macrophages as compared to WT Marquez et al, FEBS 2009 09/06/2010 Bremen Summerschool 2010

Proteins with modified expression in CIP-resistant cells 09/06/2010 Bremen Summerschool 2010

Proteins with modified expression in CIP-resistant cells Dnajc3 WT CIP-R 25 µg 50 µg 25 µg 50 µg ► On the same chromosome as mrp4 ! WT CIP-R 25 µg 50 µg 25 µg 50 µg kDa ► Tlr7 - 130 - - 100 - Transports nucleosides/tides (substrates for Mrp4 !) 09/06/2010 Bremen Summerschool 2010

Karyotype of J774 cells mrp4 09/06/2010 Bremen Summerschool 2010

Gene amplification in CIP-resistant cells chromosome 14 mrp4 FISH analysis Cells resistant to 0.1 mM WT cells chromosome 14 mrp4 Cells resistant to 0.15 mM 09/06/2010 Bremen Summerschool 2010

Gene amplification in CIP-resistant cells mFISH analysis of CIP-R cells Heterogeneity of cell population ! chromosome 5 chromosome 13 chromosome 16 09/06/2010 Bremen Summerschool 2010

Gene amplification in CIP-resistant cells FISH analysis of CIP-R cells Mrp4 and DnajC3 co-amplified in CIP-resistant cells DnajC3 WT CIP-R 25 µg 50 µg 25 µg 50 µg ► Mrp4 Dnajc3 09/06/2010 Bremen Summerschool 2010

Implication of co-amplified genes in resistance DnajC3 protects CIP-resistant cells from ER stress Quantification of ER-stress signaling with a Dual Luciferase Assays of cells transfected with Cignal ERSE reporter 12 negative control standard medium + tunicamycin (0.5 µg/ml) 10 8 Relative luciferase activity 6 4 2 Wild type cells CIP-R cells 09/06/2010 Bremen Summerschool 2010

and in moxifloxacin-resistant cells ? Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Expression of ABC transporters TaqMan Low Density Array of ABC transporters Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Overexpression of MDR transporters P-gp and Bcrp are functional Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Overexpression of MDR transporters P-gp and Bcrp are functional but do not play a major role in FQ accumulation Vallet et al., FEBS-ABC meeting. (2010) 09/06/2010 Bremen Summerschool 2010

Antibiotics are also substrates for efflux in eucaryotic cells Take home message Antibiotics are also substrates for efflux in eucaryotic cells Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Die Bremer Stadtmusikanten Take home message Closely related molecules behave very differently with respect to transport Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Non substrates may induce a ‘anti-resistant’ phenotype Take home message Non substrates may induce a ‘anti-resistant’ phenotype Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Efflux is only part of the response to drug exposure Take home message Efflux is only part of the response to drug exposure Die Bremer Stadtmusikanten 09/06/2010 Bremen Summerschool 2010

Coworkers … B. Devreese and M. Aerts Proteomics Laboratory for Protein Biochemistry and Biomolecular Engineering, Ghent University, Belgium E. Jacquet and N. Nhiri TaqMan Low Density Array Institut de Chimie des Substances Naturelles, CNRS, Gif sur Yvette, France G. Ameye and H. Antoine-Poirel FISH Centre de Génétique, Cliniques universitaires Saint-Luc UCL, Bruxelles, Belgium 09/06/2010 Bremen Summerschool 2010

Brussels Stadtmusikanten for this story … B. Marquez NE Caceres F. Van Bambeke PM Tulkens MP Mingeot JM Michot C. Vallet A. Anantharajah M. Heremans L. Depraetere V. Mohymont MC Cambier M. Vergauwen C Misson www.facm.ucl.ac.be 09/06/2010 Bremen Summerschool 2010