Universit catholique de Louvain, Brussels Unit de pharmacologie - PowerPoint PPT Presentation

Université catholique de Louvain, Brussels Unité de pharmacologie cellulaire et molélculaire Biochemical and Biophysical and Biophysical Studies of Studies of the Interactions the Interactions Biochemical between Aminoglycoside Aminoglycoside Antibiotics and Antibiotics and Lipid Layers : Lipid Layers : between A molecular approach to the understanding of of the cellular toxicity the cellular toxicity A molecular approach to the understanding induced by aminoglycosides aminoglycosides induced by Thesis presented by Thesis presented by Marie-Paule Mingeot-Leclercq, M Sc., PhD in Pharm. Marie-Paule Mingeot-Leclercq, M Sc., PhD in Pharm. ( ( Agrégation de l’enseignement supérieur en médecine Agrégation de l’enseignement supérieur en médecine ) ) May 5th, 2000 May 5th, 2000 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 1

AMINOGLYCOSIDES - LIPIDS INTERACTIONS Aminoglycoside Biological (Gentamicin c 1a ) membrane OH O H 3 C HN H 2 N O CH 2 HO HO CH 3 O NH 2 O H 2 N NH 2 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 2

BIOLOGICAL MEMBRANES 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 3

MAJOR ROLES PLAYED BY MEMBRANE LIPIDS CELLULAR PHARMACOLOGICAL • Compartmentation • Inhibition of the synthesis of the lipidic constituents Endocytosis/exocytosis of membranes pathways • Anchorage of drug inside • Structural support to lipidic compartment the activation of signal transduction • Modulation of cellular process involving • Regulation of enzymatic membranes activity 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 4

AMINOGLYCOSIDES - LIPIDS INTERACTIONS Phospholipid Phospholipid 31 P NMR Polar domain 31 Polar domain P NMR Interfacial domain Interfacial domain Fluorescence Fluorescence depolarization - TMA-DPH depolarization - TMA-DPH Hydrocarbon domain Hydrocarbon domain Fluorescence Fluorescence depolarization - DPH depolarization - DPH Acidic Acidic phospholipid phospholipid 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 5

IMPORTANT FEATURES RELATED TO BIOLOGICAL ROLES PLAYED BY MEMBRANES Lateral domains Transversal asymmetry Lipid polymorphism Fluidity 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 6

AMINOGLYCOSIDES: CHEMICAL STRUCTURE R8 R6 R2 R3 R7 O III HN I R1 O CH2 HO 4 6 HO R9 O NHR 4 O NH2 HN II R5 Aminoglycoside R 1 R 2 R 3 R 4 R 5 R 6 R 7 R 8 R 9 Isepamicin OH OH OH H COR H CH 3 OH CH 3 Amikacin OH OH OH H COR’ CH 2 OH OH H H Kanamycin A OH OH OH H H CH 2 OH OH H H Kanamycin B NH 2 OH OH H H CH 2 OH OH H H Gentamicin C 1a NH 2 H H H H H CH 3 OH CH 3 R = -CHOHCH 2 NH 2 ; R’ = -CHOH(CH 2 ) 2 NH 2 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 7

AMINOGLYCOSIDES: ANTIMICROBIAL ACTIVITY III II I Yoshizawa et al [1998] EMBO J. 17:6437-6448 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 8

AMINOGLYCOSIDES: NEPHROTOXICITY Incidence: 0-50% - patient factors - aminoglycoside factors Gentamicin Netilmicin Isepamicin Amikacin - schedule of administration - concomitant drugs 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 9

AMINOGLYCOSIDES: NEPHROTOXICITY Proximal tubular cell - rat treated with 10 mg/kg.day gentamicin for 7 days Kosek et al [1974] Lab. Invest 30: 48-57 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 10

AMINOGLYCOSIDES - LIPIDS INTERACTIONS CHARACTERIZATION Interaction Aminoglycosides Phospholipids - 15 N NMR - experimental studies - 31 P NMR gel filtration equilibrium dialysis - Fluorescence depolarization - theoretical analysis . TMA-DPH . DPH CONSEQUENCES INTEREST 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 11

AMINOGLYCOSIDES - LIPIDS INTERACTIONS 31 P NMR Studies 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 12

AMINOGLYCOSIDES - LIPIDS INTERACTIONS 31 P NMR Studies Gentamicin (AG:Lip ratio = 0.02) Control 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 13

AMINOGLYCOSIDES - LIPIDS NTERACTIONS Fluorescence depolarization studies 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 14

AMINOGLYCOSIDES - LIPIDS INTERACTIONS Fluorescence depolarization studies Gentamicin (AG:Lip ratio = 0.03) Control 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 15

AMINOGLYCOSIDES- LIPIDS INTERACTIONS 15 N NMR Studies AG alone AG + PI OH OH O H 3 C 3 OH HN HO O HO isepa 6’ 3” 5 OH CH 3 NH 2 O O HN NH 2 R OH OH OH 3 O H 3 C 3” OH amika H 2 N HO O HO 6’ 5 OH NH 2 O O HN NH 2 OH R OH OH O H 3 C OH H 2 N 6’ HO 1 O 3 HO kana A 3” OH NH 2 O O H 2 N NH 2 OH OH OH O 3 1 3” H 3 C OH H 2 N H 2 N O HO kana B 6’ 2’ OH NH 2 O O H 2 N NH 2 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 16

AMINOGLYCOSIDE-LIPIDS INTERACTIONS Correlation between the theoretical and experimental studies Aminoglycoside Interaction Interaction Calculated detected by detected by interaction 15 N NMR equilibrium dialysis (KJ / mol) Kd (µM) isepamicin 47 weak - 23.4 amikacin 30 medium - 35.7 kanamycin A 7 medium - 46.8 kanamycin B 6 strong - 48.5 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 17

AMINOGLYCOSIDES - LIPIDS INTERACTIONS CHARACTERIZATION CONSEQUENCES • Lysosomal phospholipase activity - Surface potential - Lipidic lateral organization • Ability of membrane to fuse or to aggregate - Lipidic polymorphism INTEREST 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 18

AMINOGLYCOSIDES - LIPIDS INTERACTIONS CHARACTERIZATION CONSEQUENCES • Lysosomal phospholipase activity - Surface potential - Lipidic lateral organization • Ability of membrane to fuse or to aggregate - Lipidic polymorphism INTEREST 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 19

DEGRADATION PATHWAYS OF PHOSPHOLIPIDS IN LYSOSOMES * Ch P Plipase A1 Plipase C Plipase A2 * Ch P * * Ch P Ch P β -lyso-Plipase β β β α α -lyso-Plipase α α * Ch P 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 20

EFFECT OF NEGATIVELY-CHARGED PHOSPHOLIPIDS ON LYSOSOMAL PHOSPHOLIPASE ACTIVITY 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 21

AMINOGLYCOSIDES- LIPIDS INTERACTIONS Inhibition of lysosomal phospholipase activity Phosphatidylinositol content: 13.6 % 18.2 % 27.3 % 36.4 % 54.5 % 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 22

AMINOGLYCOSIDES- LIPIDS INTERACTIONS Inhibition of lysosomal phospholipase activity Gentamicin 100 lysophosphatidylcholine Isepamicin 80 Release of (% control) 60 IC50 = IC50 = 220 µM 100 µM 40 20 0 0 100 200 300 400 Drug concentration (µM) 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 23

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on lysosomal phospholipase activity and domains formation? 2 H NMR spectrum of DPPC-d 62 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 24

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on lysosomal phospholipase activity and domains formation? 20 DPPC d62 control M 2 *10 -9 (s -2 ) 15 DPPC d62 :PI 1:1 control + Kanamycin B 10 + Kanamycin A + Amikacin 5 + Isepamicin 0 0 10 20 30 40 50 Temperature (°C) 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 25

AMINOGLYCOSIDES- LIPIDS INTERACTIONS Inhibition of lysosomal phospholipase activity 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 26

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation Principle of R18 (octadecylrhodamine B) assay + membrane - labeling target membrane Lipid dilution : relief selfquenching of fluorescence concentration selfquenching Hoekstra et al [1984] Biochemistry, 23, 5675-5681 Hoekstra [1990] Hepatology, 12, 61S - 66S 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 27

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation Principle of R18 assay rapid slow very slow 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 28

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation 1000 1000 Fluorescence 750 750 500 500 250 250 0 0 0 10 20 30 40 0 10 20 30 40 Time (sec) Time (sec) Control Spermine Melittin Gentamicin Amikacin Isepamicin (agent:Lip ratio) 156 0.127 125 125 125 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 29

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation Control + Gentamicin + Gentamicin 37,500X 37,500X 85,000X 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 30

Possible mechanisms of membrane fusion involving Inverted micellar Stalk intermediates intermediates Apposed bilayers Stalk IMI Fusion pore TMC Siegel [1993] Biophys. J., 65, 2124-2140 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 31

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation Agent : Lip ratio 0.05 05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 32