interactions between fluoroquinolones and lipids ... · interactions between fluoroquinolones and...

Interactions between Fluoroquinolonesand lipids: Biophysical studies

Hayet BENSIKADDOUR

Louvain Drug Research Institute

Cellular and Molecular Pharmacology Unit

May 25th, 2011 Promoter Prof. Marie-Paule Mingeot-Leclercq

Thesis-H.Bensikaddour

Plan of the presentation

I- Introduction

� Pharmacology of fluoroquinolones� Mechanism of resistance of fluoroquinolones: efflux pumps phenomenom

II- Aims of the ThesisIII- Materials and MethodsIV- Results

� Investigation of the interaction of two fluoroquinolones (CIP and MXF) with model lipid membranes

Bensikaddour et al., 2008: Biophysical Journal 94: 3035-3046

� Investigation of the interaction of CIP with eukaryotic and prokaryotic model lipids membranes (DPPC vs DPPG)

Bensikaddour et al., 2008: Biochimica et Biophysica Acta 1778: 2535-2543

V- General Conclusion VI- Perspectives


� Synthetic origin compounds: Quinoline ring and Fluor atom at R6

� Bactericide activity

� Based on chemical structure and antibacterial activity:

3 generations

I- IntroductionPharmacology of Fluoroquinolones

- 1st generation (Quinolones) ⇒ Treatment of urinary tract infections (1960-1970)

- 2nd generation ⇒ Systematic use in the (1980-1990)

- 3rd generation ⇒ Treatment of respiratory tract infections (from1990-xxxx)

NX

COOH

OR5

R6

R7

R1R8


I- IntroductionPharmacology of fluoroquinolones

� Bacterial targets are DNA gyrase and Topoisomerase IV.

I- IntroductionMechanisms of resistance

- 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 )

Resistance due to target enzymes mutation:

- mutations in topoisomerase IV (ParC or ParE)

- mutations in DNA gyrase (GyrA or GyrB)

Peptidoglycan

Plasma membrane

Outer membrane

Gram-negative bacteria

FQs

Efflux pumps

Porins

Bacteria target (DNA)

Eukaryotic cells

J774 macrophages cells

Plasma membrane

Efflux pumps

CIP

MXF

Plasma membrane

Efflux pumps

CIP

MXF

- MRP inhibitors+ MRP inhibitors

� Intracellular accumulation� MRP inhibitors had no effect on the accumulation of MXF

� MRP inhibitors increase the accumulation of CIP � Accumulation of MXF >>>CIP

I- IntroductionPrevious studies on accumulation of CIP and MXF

Michot et al. (2004, 2005), Marquez et al.,( 2009).

I-Introduction

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

Peptidoglycan

Plasma membrane

Outer membrane

Eukaryotic cellsGram-negative bacteriaGram-positive bacteria

FQs

FQs

Efflux pumps

Porins

Bacteria target (DNA)

Bensikaddour-Thesis-2011

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 CIP with eukaryotic and prokaryotic model lipids membranes (DPPC vs DPPG).

� Investigation of the interaction of two fluoroquinolones (CIP and MXF) with model lipid membranes.


III- Materials and methods

� Antibiotics: CIP and MXF

� Zwitterionic phospholipids (Eukaryotic cells: DOPC, DPPC)

� Anionic phospholipids (Prokaryotic cells: DPPG)

III- Materials and methods

air

water

monolayer

bilayer

Small unilamellar vesicle (SUV)(20-50 nm)

Large unilamellar vesicle (LUV) (> 50-10000 nm)

Giant unilamellar vesicle (GUV) (5-300µm)

Multilamellar vesicle (MLV) (up to 10000

nm)

(i)

(iii)

(ii)

Models of lipids membranes

III- Materials and methodsi) Atomic Force Microscopy (AFM)

magneticsteel disc

scanner

Photodetector

laser

Sample

magneticsteel disc

scanner

Photodetector

laserlaser

Sample

� Measure the forces resulting from different interactions between a tip, which is attached to a cantilever and the atoms of the sample surface.


i) Atomic Force Microscopy

How it’s work?

Mica

TipSample

Transverse

section

� Surface topography� Lipid domain structure


ii) Langmuir-Blodgett technology (LB)

� Measure the surface pressure/area (π-A) of monomolecular layer upon compression

Dipper

Barrier position sensor

Moveable Barrier

Teflon trough

Solid substrate

Electro balance

Pressure sensor

Aqueous subphase

Amphiphilemonolayer

ii) Pressure/area (π-A) isotherm curve for a phospholipid monolayer

� Lipid molecular arrangement� Lipid state transition� Lipid packing

Area per molecule (Å2)

Surf

ace

pres

sure

(mN

/m)

20 40 60 80 100

10

20

30

40

50

Gaz

Liquid Expanded

Liquid Condensed

Solide

LE-LC

Condensed state

Fluid state

Gaseous state

Collapsed monolayer

Compression of lipids (↓molecular area ) ⇒ ↑ lateral pressure

iii) Fourier Transform Infrared Spectroscopy (FTIR)

� 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.

Sample

Outgoing Incomingbeam

Germanium Plate

Stretchingvibrations

(~2850 cm-1)

Bendingvibrations

(~1450 cm-1)

C

HH

C

HH

iii) Fourier Transform Infrared Spectroscopy (FTIR)

� Non polarized spectra for :

- state behavior (CH2 asymmetric and symmetric stretching bands 2800-3000 cm-1)

- conformation determination of lipids: (CH2 wagging bands (1400 cm-1))

� Polarized spectra for determination of lipids orientation: Dichroism measurements of the CH2 wagging band (1400 cm-1))

Stretchingvibrations

(~2850 cm-1)

C

HH

Bendingvibrations

(~1450 cm-1)

C

HH

Phospholipid

Increase TemperatureAt T<Tm

Crystal stateAt T>Tm

Fluid state

A Kink bend

α


iv) Nuclear Magnetic Resonance spectroscopy (NMR)

Chemical shift anisotropy ∆σ

∆σ∆σ∆σ∆σ= σσσσ// - σσσσ⊥⊥⊥⊥σσσσ//: The low field shoulderσσσσ⊥⊥⊥⊥: The high field peak

Bilayer LUV liposome

(ppm)

100 50 0 -50 -100

� Mobility of phosphate heads of phospholipids

� Drug effect on the orientation of phospholipids head groups (31P NMR).

v) Steady state anisotropy fluorescence

r = (I// - I┴) /(I// +2 I┴)

0.0 5.0x10-7

1.0x10-6

1.5x10-6

2.0x10-6

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

An

iso

tro

py

[sample] M

L free => r=r0

Lbound + R => r ↑

� Binding parameters of drugs to lipids (stoichiometry, affinity)


Chapter I

i) Investigation of the interaction of two fluoroquinolones (CIP and MXF) with model lipid membranes

IV- Results

i) MXF induced in a more (57%) extent than CIP (27%), an erosion of the DPPC domains in the DOPC fluid phase

a) Effect of FQs on the membrane organization

CIP MXFAFM Studies

0 25 50 75 100 125 1500

40

60

80

100

120

MXF

Are

a (

µm

2 )

Time (min)

CIP

DPPC

DOPC

(Collaboration with Pr Y. Defrene ( UCL)


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.

Langmuir studies

20 40 60 80 100 120

0

10

20

30

40

50

Su

rfa

ce

pre

ssu

re (

mN

/m)

Molecular area (A2/molecule)

a)

20 40 60 80 100 120

0

10

20

30

40

50

Su

rfa

ce p

ress

ure

(m

N/m

)

Molecular area (A2/molecule)

b)

CIP MXF

b) FQs stability within a lipid monolayer / compressibility

(Collaboration with Dr M. Deleu (FSAG, Gembloux))


c) ATR-FTIR Data analysis

� 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

(Collaboration with Pr E.Goormaghtigh (SFMB, ULB)

C i) Conformation analysis

Non polarized spectra of drug, DPPC and DPPC:FQS with molar

ratio of 1:1

3000 1800 1600 1400 1200 1000

0

1000

2000

3000

MXF

CIP

DPPC

DPPC:CIP

DPPC:MXF1737 cm-1

1730 cm-1

1736 cm-1

1630 cm-1

Ab

so

rba

nc

e (

A.U

)

Wavenumber (cm-1)

a)

�The drug spectrum appeared in the DPPC: drug mixture spectrum, notably at 1630 cm-1.�In DPPC: drug spectra, the DPPC νννν(C=O) band at 1736 cm-1 was modified in terms

of frequency and shape

⇒⇒⇒⇒ a modification of the interfacial lipid carbonyl groups

C

HH

HCH bend

wagging

(~1450 cm_1)

C

HH

C

HH

C

HH

HCH bend

wagging

(~1450 cm_1)

Analysis of the lipid C-H wagging (ννννw(CH2))⇒⇒⇒⇒ Information on the proportion of the chains in the all-trans conformation

0 1-0,2 1-0,5 1-1 1-2

20

30

40

50

60

70

80

90

No Polarize spectra at 1200 cm-1

Are

a (

A.U

)

DPPC:Drug (Molar ratio)

b)

CIP

MXF

� Area evolution of DPPC peak at 1206-1193 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.

C i) Conformation analysis

A Kink bend

C ii) Orientation analysisDichroic spectra (A// - A┴) of DPPC and DPPC: FQs

with molar ratio of 1:1

3000 1800 1600 1400 1200 1000

-100

0

100

200

300

400

500

600

DPPC:MXF)

DPPC:CIP)

DPPC)

1630 cm-1

Ab

so

rba

nc

e(A

.U)

Wavenumber (cm-1)

1630 cm-1 1465 cm-1

a)

Wagging Bands

� 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.

θ

Ez

Ex

Ey

E┴

E//

ATR-crystal

IR radiation

dp ~1 µmsample

detector

Evanescent wave

θ

Ez

Ex

Ey

E┴

E//

ATR-crystal

IR radiation

dp ~1 µmsample

detector

Evanescent wave

C

HH

HCH bend

wagging

(~1450 cm_1)

C

HH

C

HH

C

HH

HCH bend

wagging

(~1450 cm_1)

01:00 01:00,2 01:00,5 01:01 01:02

0

50

100

150

200

250

300

350

400

MXF

A

bso

rban

ce (

A.U

)

DPPC:Drug (molar ratio)

b)

CIP

Analysis of the lipid C-H wagging (ννννw(CH2)) band ⇒⇒⇒⇒ Estimation of the orientation of the lipid acyl chain

� Area evolution of the wagging band ννννw(CH2) 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

C ii) Orientation analysis

γ

β

α

Pla

te n

orm

al dipole

Mole

cula

r axi

s

Mem

bra

ne

no

rmalγ

β

α

Pla

te n

orm

al dipole

Mole

cula

r axi

s

Mem

bra

ne

no

rmal

α

Summary 1

� 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: FQsmonolayer 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: 3035-3046

↓ area per molecule

↓↓ area per molecule

α

+

+

CIP

MXF

Phospholipids(DPPC)


Chapter II

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

Phospholipids composition (%) of bacteria membrane and humain cells membrane

Wolkers et al., (2002), Epand et al. (2007), Lohner and Prenner, (1999).

� The bacterial membranes are composed largely of anionic lipids (PG).� The eukaryotic cell membranes are rich with zwitterionic lipids.

48.17..9Chol

13.2Others

---18.96.6SM

--PI+PA

50+5058+4221+11PG+CL

Traces

2.620.7

PS

---24.231PC

--606.220.6PE

Staphylococcus pneumoniae

Staphylococcus aureusPseudomonas aeruginosa

HumanErythrocyte membrane

Humanalveolar

macrophages

Prokaryotic cellsEukaryotic cellsLipid


a ) Size of LUVs in the presence of CIP by quasi-elastic light scattering

Average size (nm) LUV

256±5105±11:1

195±298±11:0.5

155±2100±11:0.2

124±1101±11:0

DPPGDPPC

Lipid-Drug

Molar ratio

⇒ CIP increased the size of DPPG liposomes

1) Binding of CIP to Lipids


a ) Binding of CIP to Lipids by steady state anisotropy titration

Kapp= (8.6±0.5) 105 M-1

-5 0 5 10 15 20 25 30 35 400,10

0,15

0,20

0,25

0,30

0,35

0,40

An

iso

tro

py

[DPPG] µM

[CIP] = 5 µM

r = (I// - I┴) /(I// +2 I┴)


⇒ CIP has more affinity for negative charged liposomes

(DPPG Vs DPPC)

1.1±0.2 -(6 ± 1)

(100%)

DOPC:DPPC

(1:1, M:M)

3.2±0.9 -(34 ± 4)

(100%)

DOPC:DPPG

(1:1, M:M)

2.5±0.1-(5 ± 1)

(96 ± 5 %)

DPPC

8.6±0.5-(66 ± 4)

(83 ± 12 %)

DPPG

Kapp(105 M-1)Zeta potential

(mV)

LUV liposomes

Composition

a) Binding parameters of CIP with Lipids vesicles


2) CIP effect on the membrane mobility

DPPG alone DPPG:CIP

(1:1, M:M)

∆σ∆σ∆σ∆σ=25.5±0.5 ppm ∆σ∆σ∆σ∆σ=34.0±1.0 ppm

⇒ The difference of ∆σ∆σ∆σ∆σ of DPPG:CIP and DPPG is 9 ppm

i) DPPG

(Collaboration with Pr K. Snoussi (CHIM, UCL))


2) CIP effect on the membrane mobilityii) DPPC

∆σ∆σ∆σ∆σ=41.5±0.5 ppm ∆σ∆σ∆σ∆σ=46.6±0.1 ppm

DPPC:CIP

(1:1, M:M)

DPPC alone

⇒The difference of ∆σ∆σ∆σ∆σ of DPPC:CIP and DPPC is 5 ppm

Melting temperature of lipid by ATR-FTIR

3) CIP effect on the thermotropic behavior of lipids

3000 2980 2960 2940 2920 2900 2880 2860 2840 2820 2800

0,00

0,05

0,10

0,15

0,20

0,25

0,30

Evolution of stretching vibration bands of CH2 of DPPG as function of temperature

in the presence of CIP in Tris 10mM buffer, pH 7,4

Ab

so

rba

nce

(A

.U)

Wavenumber (cm-1)

T=50°CT=47°C

T=46°CT=45°CT=44°CT=43°CT=42°C

T=41°CT=40°CT=39°C

T=38°CT=37,5°CT=35°C

T=32,5°CT=30°CT=27,5°C

T=25°CT=22,5°C

T=20°C

CH2 Stretching vibrations

CH2 asymmetric at 3000 cm-1

C

HH

C

HH

C

HH

CH2 symmetric at 2800 cm-1

C

HH

C

HH

C

HHIncrease Temperature

At T<TmCrystal state

At T>TmFluid state


3) CIP effect on thermotropic curve of DPPG and DPPC

15 20 25 30 35 40 45 50 55

2917

2918

2919

2920

2921

2922

DPPC:CIP (1:1)

Wa

ven

um

be

r (c

m-1

)

Temperature (C°)

DPPC alone

15 20 25 30 35 40 45 50 55

2917

2918

2919

2920

2921

2922

DPPG:CIP(1:1)

Wa

ven

um

ber

(c

m-1

)

Temperature (C°)

DPPG alone

Melting temperature of DPPC Melting temperature of DPPC and DPPC:CIP (1:1)and DPPC:CIP (1:1)

Melting temperature of DPPG Melting temperature of DPPG and DPPG:CIP (1:1)and DPPG:CIP (1:1)

CH2 asymmetric stretching band

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


4) Assembly of CIP with phospholipids by molecular modeling

AB

DPPC:CIP (1:1, M:M) DPPG:CIP (1:1, M:M)

E = - 44.4 Kcal/mol E = - 53.4 Kcal/mol

=> The interaction of DPPG:CIP is more stable than DPPC:CIP

(Collaboration with Dr L. Lins (CBMN, Gembloux)

Summary 2

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: 2535-2543

At T>TmAt T<Tm

Increase Temperature

DPPC

+ CIP

DPPG- - - - - - - - - - - -

- - - - -


V- General Conclusion

� 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

CIPMXF

?

1

2

3

a

b

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 protein expression?

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)


VI- Perspectives

� Application to others fluoroquinolones (role of lipophilicity, the basic function (piperazine))

NN

COOH

HN

H3C

OCH3

F

N

O

COOHF

N

HO CH3

Grepafloxacin (Log P=2.48) nadifloxacin


� Pr M-P. Mingeot-Leclercq

� 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

Acknowledgements

� My Family


Thanks' for your attention !

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