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

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

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Universit catholique de Louvain, Brussels Unit de pharmacologie cellulaire et mollculaire Biochemical and Biophysical and Biophysical Studies of Studies of the Interactions the Interactions Biochemical between Aminoglycoside

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 1

Biochemical Biochemical and Biophysical and Biophysical Studies of Studies of the Interactions the Interactions between between Aminoglycoside Aminoglycoside Antibiotics and Antibiotics and Lipid Layers : Lipid Layers :

A molecular approach to the understanding A molecular approach to the understanding of

  • f the cellular toxicity

the cellular toxicity induced by induced by aminoglycosides aminoglycosides

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

Université catholique de Louvain, Brussels

Unité de pharmacologie cellulaire et molélculaire

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 2

AMINOGLYCOSIDES - LIPIDS INTERACTIONS

O O O CH2 H2N NH2 OH HN H3C CH3 HO HO H2N O NH2

Aminoglycoside (Gentamicin c1a) Biological membrane

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

BIOLOGICAL MEMBRANES

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 4

MAJOR ROLES PLAYED BY MEMBRANE LIPIDS

  • Compartmentation

Endocytosis/exocytosis pathways

  • Structural support to

the activation of signal transduction

  • Regulation of enzymatic

activity

  • Inhibition of the synthesis
  • f the lipidic constituents
  • f membranes
  • Anchorage of drug inside

lipidic compartment

  • Modulation of cellular

process involving membranes

CELLULAR PHARMACOLOGICAL

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 5

AMINOGLYCOSIDES - LIPIDS INTERACTIONS

Phospholipid Phospholipid Acidic Acidic phospholipid phospholipid

Polar domain Polar domain 31

31P NMR

P NMR Interfacial domain Interfacial domain

Fluorescence Fluorescence depolarization - TMA-DPH depolarization - TMA-DPH

Hydrocarbon domain Hydrocarbon domain

Fluorescence Fluorescence depolarization - DPH depolarization - DPH

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 6

IMPORTANT FEATURES RELATED TO BIOLOGICAL ROLES PLAYED BY MEMBRANES

Lateral domains Transversal asymmetry Fluidity Lipid polymorphism

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 7

AMINOGLYCOSIDES: CHEMICAL STRUCTURE

Aminoglycoside R1 R2 R3 R4 R5 R6 R7 R8 R9 Isepamicin OH OH OH H COR H CH3 OH CH3 Amikacin OH OH OH H COR’ CH2OH OH H H Kanamycin A OH OH OH H H CH2OH OH H H Kanamycin B NH2 OH OH H H CH2OH OH H H Gentamicin C1a NH2 H H H H H CH3 OH CH3 R = -CHOHCH2NH2; R’ = -CHOH(CH2) 2NH2

II I

O O O CH2 R1 NH2 R8 HN R7 R9 HO HO HN O NHR4 R6 R5 R2 R3

III

4 6

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 8

AMINOGLYCOSIDES: ANTIMICROBIAL ACTIVITY

Yoshizawa et al [1998] EMBO J. 17:6437-6448

III II I

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 9

AMINOGLYCOSIDES: NEPHROTOXICITY

Incidence: 0-50%

  • patient factors
  • aminoglycoside factors

Gentamicin Netilmicin Isepamicin Amikacin

  • schedule of administration
  • concomitant drugs
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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 10

AMINOGLYCOSIDES: NEPHROTOXICITY

Kosek et al [1974] Lab. Invest 30: 48-57

Proximal tubular cell - rat treated with 10 mg/kg.day gentamicin for 7 days

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 11

AMINOGLYCOSIDES - LIPIDS INTERACTIONS

CHARACTERIZATION

CONSEQUENCES INTEREST

  • 15N NMR

Phospholipids Interaction Aminoglycosides

  • experimental studies

gel filtration equilibrium dialysis

  • theoretical analysis
  • 31P NMR
  • Fluorescence

depolarization . TMA-DPH . DPH

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 12

AMINOGLYCOSIDES - LIPIDS INTERACTIONS

31P NMR Studies

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 13

AMINOGLYCOSIDES - LIPIDS INTERACTIONS

31P NMR Studies

Gentamicin (AG:Lip ratio = 0.02) Control

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 14

AMINOGLYCOSIDES - LIPIDS NTERACTIONS Fluorescence depolarization studies

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 15

AMINOGLYCOSIDES - LIPIDS INTERACTIONS Fluorescence depolarization studies

Gentamicin (AG:Lip ratio = 0.03) Control

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 16

AMINOGLYCOSIDES- LIPIDS INTERACTIONS

15N NMR Studies

O O O H2N NH2 OH H2N H3C OH HO H2N O NH2 OH OH OH O O O HO NH2 OH H2N H3C OH HO H2N O NH2 OH OH OH O O O HO NH2 OH HN H3C OH HO HN O NH2 OH OH CH3 R O O O HO NH2 OH H2N H3C OH HO HN O NH2 OH OH R OH

AG alone AG + PI isepa amika kana A kana B

3 3 3 3 1 1 3” 3” 3” 3” 2’ 5 5 6’ 6’ 6’ 6’

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 17

AMINOGLYCOSIDE-LIPIDS INTERACTIONS Correlation between the theoretical and experimental studies

Aminoglycoside isepamicin amikacin kanamycin A kanamycin B Interaction detected by equilibrium dialysis Kd (µM) 47 30 7 6 Interaction detected by

15N NMR

weak medium medium strong Calculated interaction (KJ / mol)

  • 23.4
  • 35.7
  • 46.8
  • 48.5
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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

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 19

AMINOGLYCOSIDES - LIPIDS INTERACTIONS

CHARACTERIZATION

CONSEQUENCES

  • Lysosomal phospholipase activity
  • Surface potential
  • Lipidic lateral organization
  • Ability of membrane to fuse or to aggregate
  • Lipidic polymorphism

INTEREST

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 20

DEGRADATION PATHWAYS OF PHOSPHOLIPIDS IN LYSOSOMES

P Ch

*

P Ch

*

P Ch

*

Plipase A1

*

Plipase A2 Plipase C

P Ch Ch P

*

β β β β-lyso-Plipase α α α α-lyso-Plipase

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 21

EFFECT OF NEGATIVELY-CHARGED PHOSPHOLIPIDS ON LYSOSOMAL PHOSPHOLIPASE ACTIVITY

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 22

AMINOGLYCOSIDES- LIPIDS INTERACTIONS Inhibition of lysosomal phospholipase activity

Phosphatidylinositol content: 13.6 % 18.2 % 27.3 % 36.4 % 54.5 %

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 23

AMINOGLYCOSIDES- LIPIDS INTERACTIONS Inhibition of lysosomal phospholipase activity

100 200 300 400 Drug concentration (µM) 20 40 60 80 100 Release of lysophosphatidylcholine (% control) Gentamicin Isepamicin IC50 = 220 µM IC50 = 100 µM

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 24

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on lysosomal phospholipase activity and domains formation?

2H NMR spectrum of DPPC-d62

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 25

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on lysosomal phospholipase activity and domains formation?

DPPC d62 control DPPCd62:PI 1:1 control + Kanamycin B + Kanamycin A + Amikacin + Isepamicin

10 20 30 40 50 5 10 15 20

Temperature (°C) M2*10-9 (s-2)

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 26

AMINOGLYCOSIDES- LIPIDS INTERACTIONS Inhibition of lysosomal phospholipase activity

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 27

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation

+

membrane - labeling selfquenching concentration target membrane Lipid dilution : relief

  • f fluorescence

selfquenching Hoekstra et al [1984] Biochemistry, 23, 5675-5681 Hoekstra [1990] Hepatology, 12, 61S - 66S

Principle of R18 (octadecylrhodamine B) assay

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 28

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation

Principle of R18 assay rapid slow very slow

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 29

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation

Control Spermine Melittin Gentamicin Amikacin Isepamicin (agent:Lip ratio) 156 0.127 125 125 125

10 20 30 40 250 500 750 1000

Time (sec)

10 20 30 40 250 500 750 1000

Time (sec)

Fluorescence

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 30

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation

Control

37,500X

+ Gentamicin

37,500X

+ Gentamicin

85,000X

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 31

Possible mechanisms of membrane fusion involving

Apposed bilayers TMC Fusion pore Stalk IMI

Inverted micellar intermediates Stalk intermediates

Siegel [1993]

  • Biophys. J., 65, 2124-2140
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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 32

INTERACTIONS AMINOGLYCOSIDES - LIPIDS Effect on membrane fusion or aggregation

Agent : Lip ratio 0.05

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 33

AMINOGLYCOSIDES- LIPIDS INTERACTIONS

CHARACTERIZATION CONSEQUENCES

INTEREST

  • Origin of myeloid bodies observed in lysosomes after

aminoglycosides treatment ≈ ≈ ≈ ≈ nephrotoxicity?

  • Development of nephroprotectants
  • Rational synthesis of new, less toxic, aminoglycosides
  • Phospholipidosis can also be induced by several

cationic amphiphilic drugs

  • Potential relation with the impairment of lysosomes - pinocytic

vesicles in rat kidney proximal tubules after treatment with gentamicin

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 34

INTRALYSOSOMAL PHOSPHOLIPIDOSIS INDUCED BY AMINOGLYCOSIDES

Magnifications: X 75,000 & 200,000 Pictures from P.M. Tulkens & M.B. Carlier

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SLIDE 35

05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 35

AMINOGLYCOSIDES- LIPIDS INTERACTIONS

CHARACTERIZATION CONSEQUENCES

INTEREST

  • Origin of myeloid bodies observed in lysosomes after

aminoglycosides treatment ≈ ≈ ≈ ≈ nephrotoxicity?

  • Development of nephroprotectants
  • Rational synthesis of new, less toxic, aminoglycosides
  • Phospholipidosis can also be induced by several

cationic amphiphilic drugs

  • Potential relation with the impairment of lysosomes - pinocytic

vesicles in rat kidney proximal tubules after treatment with gentamicin

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SLIDE 36

05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 36

DEVELOPMENT OF NEPHROPROTECTANT: Polyaspartic acid - in vitro studies

0.5 0.4 0.3 0.2 0.1 0.0 0.1 0.2 0.3 0.4 0.5

GM concentration (mM) BUFFER LIPOSOMES 1.25 2.5 5 PAA equivalents

Lysoderivative release

(% of base line) 20 40 60 80 100 GM CT PAA GM + PAA

Kishore et al [1990] J. Pharmacol Exp Ther 255:867-874

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 37

AMINOGLYCOSIDES- LIPIDS INTERACTIONS

CHARACTERIZATION CONSEQUENCES

INTEREST

  • Origin of myeloid bodies observed in lysosomes after

aminoglycosides treatment ≈ ≈ ≈ ≈ nephrotoxicity?

  • Development of nephroprotectants
  • Rational synthesis of new, less toxic, aminoglycosides
  • Phospholipidosis can also be induced by several

cationic amphiphilic drugs

  • Potential relation with the impairment of lysosomes - pinocytic

vesicles in rat kidney proximal tubules after treatment with gentamicin

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SLIDE 38

05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 38

O O O H2N NH2 OH H2N H3C OH HO H2 N O NH2 OH OH

R R =

  • OH
  • O- Alkyl
  • O- Aryl
  • X
  • N(H,CH3, Bu) - CO(O) - Alkyl
  • R- N- Alkyl
  • R- S- Alkyl
  • R - SO - Alkyl
  • R - SO2 - Alkyl

Kanamycin B

RATIONAL BASIS FOR SYNTHESIS OF NEW - LESS TOXIC- AMINOGLYCOSIDES

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 39

RATIONAL BASIS FOR SYNTHESIS OF NEW - LESS TOXIC- AMINOGLYCOSIDES

Aminoglycoside IC50 Calculated Orientation (µg/ml) interaction towards (kJ / mol) lipid / water

interface

Isepamicin 125 ± 7

  • 23.4

Amikacin 85 ± 4

  • 35.7

Kanamycin A 69 ± 11

  • 46.8

Kanamycin B 54 ± 4

  • 48.5
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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 40

Kanamycin B - 48.5 110 ± 11 6’’-deoxy-6’’- (N-acetyl- - 24.4 134 ± 7 N-methylamino) kanamycin B Drug Energy of interaction (KJ/mol) Orientation towards lipid/water interface IC50 (µM)

RATIONAL BASIS FOR SYNTHESIS OF NEW - LESS TOXIC- AMINOGLYCOSIDES

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SLIDE 41

05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 41

AMINOGLYCOSIDES- LIPIDS INTERACTIONS

CHARACTERIZATION CONSEQUENCES

INTEREST

  • Origin of myeloid bodies observed in lysosomes after

aminoglycosides treatment ≈ ≈ ≈ ≈ nephrotoxicity?

  • Development of nephroprotectants
  • Rational synthesis of new, less toxic, aminoglycosides
  • Phospholipidosis can also be induced by several

cationic amphiphilic drugs

  • Potential relation with the impairment of lysosomes - pinocytic

vesicles in rat kidney proximal tubules after treatment with gentamicin

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SLIDE 42

05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 42

INTRALYSOSOMAL PHOSPHOLIPIDOSIS INDUCED BY MACROLIDES

Van Bambeke et al [1996] Eur. J. Pharmacol. 314:203-214 50 nm 1 µm

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 43

AMINOGLYCOSIDES- LIPIDS INTERACTIONS

CHARACTERIZATION CONSEQUENCES

INTEREST

  • Origin of myeloid bodies observed in lysosomes after

aminoglycosides treatment ≈ ≈ ≈ ≈ nephrotoxicity?

  • Development of nephroprotectants
  • Rational synthesis of new, less toxic, aminoglycosides
  • Phospholipidosis can also be induced by several

cationic amphiphilic drugs

  • Potential relation with the impairment of lysosomes - pinocytic

vesicles in rat kidney proximal tubules after treatment with gentamicin

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SLIDE 44

05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 44

IMPAIRMENT OF LYSOSOME - PINOCYTIC VESICLE FUSION

From Giurgea-Marion et al, Tox. Appl. Pharmacol. 86: 271-285 (1986) Morphometry of lysosomes in PTC after cytochemical demonstration of HRP Treatment (9 days) % cytoplasmic volume L + L - M + M - (a) control 2.77 ± 0.58 2.03 ± 0.47

  • (b) Genta (10 mg/kg.day)

0.34 ± 0.17 1.16 ± 0.47 0.11 4.16 ± 0.32

X 21,500

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 45

ROLE OF PHOSPHATIDYLINOSITOL DERIVATIVES IN VESICULAR TRAFFIC

Lysosome Endosome Endoplasmic Reticulum Golgi complex Secretory Vesicles Clathrin coated-pit Plasma membrane Actin cytoskeleton 1 2 3 4 6 5 7 De Camilli et al [1996] Science 271:1533-1539

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05-05-2000 M.P. Mingeot-Leclercq, UCL, Brussels 46

AMINOGLYCOSIDES - MEMBRANE INTERACTIONS

CELLULAR TOXICOLOGY AND BIOLOGY RATIONAL SYNTHESIS OF NEW DRUGS NEPHROPROTECTANTS