interactions of ciprofloxacin with dppc and dppg
TRANSCRIPT
Bensikaddour et al- FEBS-Meeting-2010 1
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) 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 !