Interactions between Fluoroquinolones and lipids: Biophysical studies

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1 Louvain Drug Research Institute Cellular and Molecular Pharmacology Unit Interactions between Fluoroquinolones and lipids: Biophysical studies Hayet BENSIKADDOUR May 25 th, 2011 Promoter Prof. Marie-Paule Mingeot-Leclercq

2 Plan of the presentation I- Introduction Pharmacology of fluoroquinolones Mechanism of resistance of fluoroquinolones: efflux pumps phenomenom II- Aims of the Thesis III- Materials and Methods IV- Results Investigation of the interaction of two fluoroquinolones (CIP and MXF) with model lipid membranes Bensikaddour et al., 2008: Biophysical Journal 94: Investigation of the interaction of CIP with eukaryotic and prokaryotic model lipids membranes (DPPC vs DPPG) V- General Conclusion VI- Perspectives Bensikaddour et al., 2008: Biochimica et Biophysica Acta 1778:

3 I- Introduction Pharmacology of Fluoroquinolones Synthetic origin compounds: Quinoline ring and Fluor atom at R 6 R 5 O Bactericide activity R 6 COOH Based on chemical structure and antibacterial activity: 3 generations R 7 X N R 8 R 1-1st generation (Quinolones) Treatment of urinary tract infections ( ) - 2 nd generation Systematic use in the ( ) - 3 rd generation Treatment of respiratory tract infections (from1990-xxxx)

4 I- Introduction Pharmacology of fluoroquinolones Bacterial targets are DNA gyrase and Topoisomerase IV.

5 Plasma membrane I- Introduction Mechanisms of resistance Gram-negative bacteria FQs Porins Efflux pumps Bacteria target (DNA) Peptidoglycan Outer membrane Resistance due to target enzymes mutation: - mutations in topoisomerase IV (ParC or ParE) - mutations in DNA gyrase (GyrA or GyrB) - resistance due to a protective mechanism (ex. plasmid qnr) - Resistance due to altered access of drug to target enzymes: Difficulty to diffuse through the membrane Efflux system (efflux pumps in bacteria ex: AcrAB-TolC of E.coli )

6 I- Introduction Previous studies on accumulation of CIP and MXF + MRP inhibitors Eukaryotic cells J774 macrophages cells - MRP inhibitors Efflux pumps CIP Efflux pumps CIP Plasma membrane Plasma membrane MXF MXF MRP inhibitors had no effect on the accumulation of MXF Intracellular accumulation MRP inhibitors increase the accumulation of CIP Accumulation of MXF >>>CIP Michot et al. (2004, 2005), Marquez et al.,( 2009).

7 I-Introduction Gram-positive bacteria Gram-negative bacteria Eukaryotic cells Plasma membrane FQs Porins Efflux pumps Bacteria target (DNA) FQs Peptidoglycan Outer membrane To reach intracellular bacteria target, CIP and MXF interact with membrane lipids bilayer: where they can be recognized by the efflux pumps proteins in eukaryotic cells and /or the porins and efflux pumps proteins in prokaryotic cells Investigation of interaction of FQs with model membranes at molecular level

8 II- Aims of Thesis Characterize the effect of two fluoroquinolones, CIP and MXF, on the physicochemical properties of the major phospholipids of both the eukaryotic and prokaryotic membranes: Investigation of the interaction of two fluoroquinolones (CIP and MXF) with model lipid membranes. Investigation of the interaction of CIP with eukaryotic and prokaryotic model lipids membranes (DPPC vs DPPG). Bensikaddour-Thesis-2011

9 III- Materials and methods Antibiotics: CIP and MXF Zwitterionic phospholipids (Eukaryotic cells: DOPC, DPPC) Anionic phospholipids (Prokaryotic cells: DPPG)

10 III- Materials and methods Models of lipids membranes air monolayer (i) water bilayer (ii) Multilamellar vesicle (MLV) (up to nm) (iii) Giant unilamellar vesicle (GUV) (5-300µm) Large unilamellar vesicle (LUV) (> nm) Small unilamellar vesicle (SUV)(20-50 nm)

11 III- Materials and methods i) Atomic Force Microscopy (AFM) Photodetector laser Sample magnetic steel disc scanner Measure the forces resulting from different interactions between a tip, which is attached to a cantilever and the atoms of the sample surface.

12 i) Atomic Force Microscopy How it s work? Tip Sample Transverse section Mica Surface topography Lipid domain structure

13 ii) Langmuir-Blodgett technology (LB) Dipper Electro balance Pressure sensor Amphiphile monolayer Aqueous subphase Barrier position sensor Solid substrate Moveable Barrier Teflon trough Measure the surface pressure/area (π-a) of monomolecular layer upon compression

14 ii) Pressure/area (π-a) isotherm curve for a phospholipid monolayer Collapsed monolayer Condensed state Solide Surface pressure (mn/m) Liquid Condensed LE-LC Liquid Expanded Gaz Fluid state Gaseous state Area per molecule (Å 2 ) Compression of lipids ( molecular area ) lateral pressure Lipid molecular arrangement Lipid state transition Lipid packing

iii) Fourier Transform Infrared Spectroscopy (FTIR) H H H H C C Stretching vibrations (~2850 cm -1 ) Bending vibrations (~1450 cm -1 ) Sample Outgoing Incoming beam Germanium Plate IR beam is focused

15 iii) Fourier Transform Infrared Spectroscopy (FTIR) H H H H C C Stretching vibrations (~2850 cm -1 ) Bending vibrations (~1450 cm -1 ) Sample Outgoing Incoming beam Germanium Plate IR beam is focused into the ATR crystal witch is covered with lipid layer The light travels inside the plate by internal reflections from one surface of the plate to the other Absorption of the energy by the sample provides ATR-FTIR spectra and information about the structure of the system.

16 iii) Fourier Transform Infrared Spectroscopy (FTIR) Increase Temperature At T<Tm Crystal state At T>Tm Fluid state H H C Phospholipid Stretching vibrations (~2850 cm -1 ) A Kink bend H H C α Bending vibrations (~1450 cm -1 ) Non polarized spectra for : - state behavior (CH 2 asymmetric and symmetric stretching bands cm -1 ) - conformation determination of lipids: (CH 2 wagging bands (1400 cm -1 )) Polarized spectra for determination of lipids orientation: Dichroism measurements of the CH 2 wagging band (1400 cm -1 ))

17 iv) Nuclear Magnetic Resonance spectroscopy (NMR) Chemical shift anisotropy σ Bilayer LUV liposome σ= σ // -σ σ // : The low field shoulder σ : The high field peak (ppm) Mobility of phosphate heads of phospholipids Drug effect on the orientation of phospholipids head groups ( 31 P NMR).

18 v) Steady state anisotropy fluorescence Anisotropy x x x x10-6 [sample] M r = (I // - I ) /(I // +2 I ) L free => r=r 0 L bound + R => r Binding parameters of drugs to lipids (stoichiometry, affinity)

19 IV- Results i) Investigation of the interaction of two fluoroquinolones (CIP and MXF) with model lipid membranes Chapter I

Defrene ( UCL) MXF DPPC DOPC 120 100 CIP Area (µm 2 ) 80 60 MXF 40 0 0 25

20 CIP a) Effect of FQs on the membrane organization AFM Studies (Collaboration with Pr Y. Defrene ( UCL) MXF DPPC DOPC CIP Area (µm 2 ) MXF Time (min) i) MXF induced in a more (57%) extent than CIP (27%), an erosion of the DPPC domains in the DOPC fluid phase

21 b) FQs stability within a lipid monolayer / compressibility Langmuir studies (Collaboration with Dr M. Deleu (FSAG, Gembloux)) CIP MXF 50 a) 50 b) Surface pressure (mn/m) Surface pressure (mn/m) Molecular area (A 2 /molecule) Molecular area (A 2 /molecule) ii) MXF induced a higher shift of the surface pressure-area isotherms of DOPC:DPPC:FQs monolayer towards the lower area per molecule as compared to CIP.

22 c) ATR-FTIR Data analysis (Collaboration with Pr E.Goormaghtigh (SFMB, ULB) Conformation analysis Non polarized spectra of DPPC and DPPC:FQS with molar ratio of 1:1 Orientation analysis Dichroic spectra (A // - A ) of DPPC and DPPC: FQs with molar ratio of 1:1

23 C i) Conformation analysis Non polarized spectra of drug, DPPC and DPPC:FQS with molar ratio of 1:1 a) cm cm -1 DPPC:MXF H H Absorbance (A.U) cm cm -1 DPPC:CIP DPPC CIP C HCH bend wagging (~1450 cm _1 ) MXF Wavenumber (cm -1 ) The drug spectrum appeared in the DPPC: drug mixture spectrum, notably at 1630 cm -1. In DPPC: drug spectra, the DPPC ν(c=o) band at 1736 cm -1 was modified in terms of frequency and shape a modification of the interfacial lipid carbonyl groups

24 C i) Conformation analysis Analysis of the lipid C-H wagging (ν w (CH 2 )) Information on the proportion of the chains in the all-trans conformation b) No Polarize spectra at 1200 cm-1 Area (A.U) CIP A Kink bend 30 MXF ,2 1-0, DPPC:Drug (Molar ratio) Area evolution of DPPC peak at cm -1 as function of increasing amounts of FQs : 60% for CIP, 72% for MXF. The all-trans configuration of the alkyl chain of DPPC decreased more in the presence of MXF.

25 C ii) Orientation analysis Dichroic spectra (A // - A ) of DPPC and DPPC: FQs with molar ratio of 1:1 E// IR radiation a) DPPC:MXF) 1630 cm cm-1 detector E x E z θ Absorbance(A.U) DPPC:CIP) DPPC) 1630 cm-1 Wagging Bands E y Evanescent wave ATR-crystal sample H d p ~1 µm H E -100 C Wavenumber (cm-1) HCH bend wagging (~1450 cm _1 ) The dichroic spectra of DPPC:FQs mixture displayed strong dichroism for bands assigned to the drug (at 1630 and 1465 cm -1 ) a well-organized, well-defined orientation of the drug in the DPPC bilayer.

26 C ii) Orientation analysis Analysis of the lipid C-H wagging (ν w (CH 2 )) band Estimation of the orientation of the lipid acyl chain Absorbance (A.U) b) MXF CIP Plate normal γ Membrane β normal α Molecular axis dipole :00 01:00,2 01:00,5 01:01 01:02 α DPPC:Drug (molar ratio) Area evolution of the wagging band ν w (CH 2 ) of DPPC as a function of lipid:drug molar ratio, indicated : - 60% of the area for CIP, 30% for MXF. - The tilt between the acyl chain of DPPC and a normal at the germanium surface was (27 ) in the presence of CIP and remained unchanged (20 ) in the presence of MXF MXF induced less disorder than CIP

MXF induced a higher shift of the surface pressure-area isotherms of DOPC: DPPC: FQs monolayer towards the lower area per molecule as

27 Summary 1 α CIP + Phospholipids (DPPC) area per molecule MXF + area per molecule MXF induced in a more extent than CIP an erosion of the DPPC domains in the DOPC fluid phase. MXF induced a higher shift of the surface pressure-area isotherms of DOPC: DPPC: FQs monolayer towards the lower area per molecule as compared to CIP. MXF had a higher tendency to decrease the number of all-trans conformation, as compared to CIP. CIP induced a disorder and modifies the orientation of the acyl chain. Bensikaddour et al., 2008: Biophysical Journal 94:

28 ii) Investigation of the interaction of CIP with eukaryotic and prokaryotic model lipids membranes (DPPC vs DPPG) Chapter II

29 Phospholipids composition (%) of bacteria membrane and humain cells membrane Lipid Eukaryotic cells Prokaryotic cells Human alveolar macrophages Human Erythrocyte membrane Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus pneumoniae PE PC PS Traces PG+CL PI+PA - - SM Chol Others 13.2 The bacterial membranes are composed largely of anionic lipids (PG). The eukaryotic cell membranes are rich with zwitterionic lipids. Wolkers et al., (2002), Epand et al. (2007), Lohner and Prenner, (1999).

30 1) Binding of CIP to Lipids a ) Size of LUVs in the presence of CIP by quasi-elastic light scattering Lipid-Drug Molar ratio Average size (nm) LUV 1:0 1:0.2 DPPC 101±1 100±1 DPPG 124±1 155±2 1:0.5 98±1 195±2 1:1 105±1 256±5 CIP increased the size of DPPG liposomes

31 a ) Binding of CIP to Lipids by steady state anisotropy titration [CIP] = 5 µm 0,40 0,35 0,30 Anisotropy 0,25 0,20 0,15 0, [DPPG] µm r = (I // - I ) /(I // +2 I ) K app = (8.6±0.5) 10 5 M -1

32 a) Binding parameters of CIP with Lipids vesicles LUV liposomes Composition DPPG Zeta potential (mv) -(66 ± 4) (83 ± 12 %) K app (10 5 M -1 ) 8.6±0.5 DPPC -(5 ± 1) (96 ± 5 %) 2.5±0.1 DOPC:DPPG (1:1, M:M) -(34 ± 4) (100%) 3.2±0.9 DOPC:DPPC (1:1, M:M) -(6 ± 1) (100%) 1.1±0.2 CIP has more affinity for negative charged liposomes (DPPG Vs DPPC)

33 2) CIP effect on the membrane mobility (Collaboration with Pr K. Snoussi (CHIM, UCL)) i) DPPG DPPG alone DPPG:CIP (1:1, M:M) σ=25.5±0.5 ppm σ=34.0±1.0 ppm The difference of σ of DPPG:CIP and DPPG is 9 ppm

34 2) CIP effect on the membrane mobility ii) DPPC DPPC alone DPPC:CIP (1:1, M:M) σ=41.5±0.5 ppm σ=46.6±0.1 ppm The difference of σ of DPPC:CIP and DPPC is 5 ppm

35 3) CIP effect on the thermotropic behavior of lipids Melting temperature of lipid by ATR-FTIR Increase Temperature H H H H At T<Tm Crystal state At T>Tm Fluid state C C CH 2 symmetric at 2800 cm -1 CH 2 asymmetric at 3000 cm -1 CH 2 Stretching vibrations Evolution of stretching vibration bands of CH 2 of DPPG as function of temperature in the presence of CIP in Tris 10mM buffer, ph 7,4 0,30 0,25 Absorbance (A.U) 0,20 0,15 0,10 0,05 0, T=50 C T=47 C T=46 C T=45 C T=44 C T=43 C T=42 C T=41 C T=40 C T=39 C T=38 C T=37,5 C T=35 C T=32,5 C T=30 C T=27,5 C T=25 C T=22,5 C T=20 C Wavenumber (cm -1 )

36 3) CIP effect on thermotropic curve of DPPG and DPPC CH 2 asymmetric stretching band Melting temperature of DPPG and DPPG:CIP (1:1) Melting temperature of DPPC and DPPC:CIP (1:1) 2922 DPPG alone 2922 DPPC alone Wavenumber (cm -1 ) DPPG:CIP(1:1) Wavenumber (cm -1 ) DPPC:CIP (1:1) Temperature (C ) Temperature (C ) Tm(DPPG)= 40 C, Tm(DPPC)=42 C CIP did not affect dramatically the DPPC melting curve CIP : order of acyl chain of DPPG < Tm order of acyl chain of DPPG >Tm

37 4) Assembly of CIP with phospholipids by molecular modeling (Collaboration with Dr L. Lins (CBMN, Gembloux) DPPC:CIP (1:1, M:M) DPPG:CIP (1:1, M:M) A B E = Kcal/mol E = Kcal/mol => The interaction of DPPG:CIP is more stable than DPPC:CIP

38 Summary 2 Increase Temperature At T<Tm DPPG At T>Tm CIP DPPC The results showed that ciprofloxacin: had more affinity to the negatively charged lipid reduced more the mobility of DPPG than DPPC had no dramatically effect on the melting curve of DPPC decreased the order of acyl chain of DPPG at pre-transition temperature and increased the order at post-transition temperature Bensikaddour et al., 2008: Biochimica et Biophysica Acta 1778:

39 V- General Conclusion MXF CIP b a? CIP and MXF had an effect on physicochemical properties of lipids MXF seems primarily to have a packing effect CIP modifies the orientation of acyl-chain and has more affinity to the anionic lipid Different effects that we observed between CIP and MXF on lipids probably reflect a difference in their localization into the lipid bilayer

40 VI- Perspectives Relationship between the membrane composition and the permeability of these compounds (CIP and MXF) by the Parallel Artificial Membrane Permeability Assay Molecular modeling of MRP4 protein in the lipid bilayer in the absence and the presence of either CIP or MXF Are there correlation between lipid composition and efflux pumps expression? protein Determination of the lipids composition in J774 cells wild type and those overexpressed MRP4 protein (resistant to CIP) An alteration in the sphingolipids composition was observed: an increased level of GluCer, and a decreased content in Cer and in SM (Bensikaddour et al., Unpublished data)

41 VI- Perspectives Application to others fluoroquinolones (role of lipophilicity, the basic function (piperazine)) F CH 3 O COOH F O COOH H 3 C N N N N HN HO CH 3 Grepafloxacin (Log P=2.48) nadifloxacin

42 Acknowledgements Pr M-P. Mingeot-Leclercq My Family Pr P. Tulkens, Pr F. Van Bambeke Pr E. Goormaghtigh (SFMB, ULB, Brussels) Pr K. Snoussi (CHIM,UCL, Louvain-la-Neuve) Dr L. Lins (CBMN, Gembloux) Pr M. Fillet (ULg, Liège) FACM S members

43 Thanks' for your attention!

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