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Interactions of Ciprofloxacin with DPPC and DPPG:

Fluorescence Anisotropy, ATR-FTIR and 31P NMR

Spectroscopies and Conformational Analysis

Hayet BENSIKADDOUR

Catholic University of Louvain

Cellular and Molecular Pharmacology Unit

May 13th, 2010

Brussels, BELGIUM

Vienna, Austria

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Introduction Fluoroquinolones (FQs)- lipids interaction

To kill intracelluar bacteria and to reach bacterial target, the FQS :

● interact with cell membrane lipids bilayer : where they can be recognized by the membrane protein efflux pumps

● interact with bacterial membrane

Phospholipidbilayer

FQs

Efflux pump

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The mechanism of entrance of CIP involved:

Contribution of efflux pumps and lipids composition to resistance

of bacteria to Fluoroquinolones (CIP)

CIP

Efflux pump

Phospholipidbilayer

- Diffusion process

- Recognition by efflux proteins in cell membranes and /or bacteria membrane

(Michot et al.,2004, Wang et al., 2007, Périchon et al.,2007)

CIP interact with phospholipids

Role of lipids in this phenomena ?

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Aim of study

Characterization of the interactions between CIP

with eucaryotic and procaryotic model lipids

membrane (DPPC vs DPPG)

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Materials

ii) Model membranes (Lipids)

- Zwitterionic lipid (DPPC, DOPC)

- Negative charged lipid (DPPG)

- Liposomes: LUVs and MLV

i) Antibiotics

- Ciprofloxacin

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Methods

a) Binding parameters of Ciprofloxacin with lipids (DPPC, DOPC, DPPG)Steady state anisotropy measurement

b) Effect of Ciprofloxacin on the mobility of phosphate heads ofDPPC and DPPG31P Nuclear Magnetic Resonance (NMR)

c) Ciprofloxacin effect on the melting temperature of DPPC and DPPGAttenuated Total-Reflection- Fourier Transform Infrared Spectroscopy (ATR-FTIR

d) Assembly of CIP with phospholipids by molecular modeling

Hypermatrix procedure

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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 40

0.10

0.15

0.20

0.25

0.30

0.35

0.40

An

iso

tro

py

[DPPG] µM

[CIP] = 5 µM

Results

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a) Binding parameters of CIP with lipid vesicles

2.5±0.1DPPC

3.2±0.9 DOPC:DPPG(1:1, M:M)

1.1±0.2 DOPC:DPPC(1:1, M:M)

8.6±0.5DPPG

Kapp(105 M-1)LUV liposomes Composition

⇒ CIP has more affinity for negative charge liposomes

(DPPG Vs DPPC)

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Chemical shift anisotropy ∆σ

∆σ∆σ∆σ∆σ= σσσσ// - σσσσ⊥⊥⊥⊥σσσσ//: The low field shoulder

σσσσ⊥⊥⊥⊥: The high field peak

b) Mobility of phosphate heads of phospholipidsNuclear Magnetic Resonance ( 31P NMR)

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b) CIP effect on the membrane mobilityi) DPPG

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

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b) 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

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c) CIP effect on the melting temperature of lipid

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c) Melting temperature of lipid by ATR-FTIR

CH2 symmetric at 2800 cm-1CH2 Asymmetric at 3000 cm-1

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

sorb

ance

(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°CT=25°CT=22,5°C

T=20°C

CH2 Stretching vibrations

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c) CIP effect on the melting temperature of DPPG and DPPC

15 20 25 30 35 40 45 50 55

2917

2918

2919

2920

2921

2922

DPPC:CIP (1:1)

Wav

enum

ber

(cm

-1)

Temperature (C°)

DPPC alone

15 20 25 30 35 40 45 50 55

2917

2918

2919

2920

2921

2922

DPPG:CIP(1:1)

Wav

enum

ber

(cm

-1)

Temperature (C°)

DPPG alone

Melting temperature of DPPC and Melting temperature of DPPC and

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

DPPG:CIP (1:1)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

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d) Assembly of CIP with phospholipids by molecular modeling using the Hypermatrix procedure

AB

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

E = - 44.4 Kcal/molArea = 66 Ǻ²

E = - 53.4 Kcal/molArea = 76 Ǻ²

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

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Conclusion

� The binding constants Kapp were in the order of 105M-1 and the affinity appeared dependent on the negative charge of liposomes:DPPG > DOPC: DPPG (1:1) > DPPC > DOPC: DPPC (1:1)

� The major effect of CIP on DPPG as compared to DPPC suggested a role of the electrostatic interactions between CIP and lipids and the importance of the nature of the polar heads of phospholipids

Bensikaddour H. et al., (2008): Biochim Biophys Acta 1778:2535-2543

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� Prs: M-P. Mingeot, P-M Tulkens, F. Van Bambeke (FACM, UCL, Brussels, Belgium)

� Pr Goormaghtigh Erik (SFMB, ULB, Brussels, Belgium)

� Pr Karim Snoussi (CHIM,UCL, Louvain-la-Neuve, Belgium)

� Dr Laurence Lins (CBMN, Gembloux, Belgium)

� FEBS for Young Travel Award

Acknowledgement

Thanks for your attention !