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Azithromycin, a pharmacological agent which selectively inhibits some pathways of endocytosis: characterization, interests and mechanism of action. Donatienne Tyteca, Pharm. Thesis submitted for the dregree of Doctor in Pharmaceutical Sciences

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Azithromycin, a pharmacological agent which selectively inhibits some pathways of endocytosis: characterization, interests and mechanism of action.

Donatienne Tyteca, Pharm. Thesis submitted for the dregree of Doctor in Pharmaceutical Sciences (PhD) Promotor: Prof. M.P. Mingeot-Leclercq Copromotors: Profs P.J. Courtoy & P.M. Tulkens Brussels, December 4th, 2001

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➥ ➥ ➥ ➥ mammalian cells take up extracellular material by a variety of

mechanisms collectively termed endocytosis

➥ ➥ ➥ ➥ implications of endocytosis in physiology:

  • uptake of extracellular nutrients,
  • cellular cholesterol homeostasis,
  • regulation of hormonal response,
  • maintenance of cell polarity,
  • antigen presentation,
  • ....

➥ ➥ ➥ ➥ implications of endocytosis in pathology:

  • atherosclerosis,
  • entry of pathogens and toxins,
  • neurogenerative diseases (Alzheimer, prion),
  • ....

(Mukherjee et al, 1997)

Endocytosis

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pinocytosis phagocytosis

late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit

Pathways of endocytosis studied in this thesis

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membrane organization in domains membrane fluidity membrane asymmetry

Membrane lipids are implicated at various stages

  • f the endocytic process
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budding

coat proteins: clathrin, APs COPs cytoskeleton adhesion to a curved particle membrane tension and asymmetry

fission

dynamin

H+ H+ H+ H+

acidification vacuolar H+-ATPase docking Rab PI 3-kinase fusion SNARE/SNAP membrane transport actin microtubules

Molecular machineries of endocytic pathways

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Conditions, mutations and agents have been extensively used to dissect cellular mechanisms of endocytosis:

➥ ➥ ➥ ➥ conditions:

  • K+ depletion (Cupers et al, 1994)
  • incubation in hypertonic medium (Cupers et al, 1994; 1997)
  • cytosol acidification (Sandvig et al, 1987)

➥ ➥ ➥ ➥ mutants:

  • clathrin, adaptor proteins and associated proteins
  • COP
  • dynamin
  • Rabs
  • ....

(for a review, see Dautry-Varsat, 2001)

What is the place of pharmacological inhibitors to dissect the endocytic apparatus ?

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budding

cytoskeleton adhesion to a curved particle membrane tension and asymmetry transport actin microtubules

fission

dynamin docking Rab PI 3-kinase fusion SNARE/SNAP membrane

H+ H+ H+ H+

acidification H+-ATPase

➥ ➥ ➥ ➥ agents:

wortmannin nocodazole cytochalasins

phospholipi dosis

gentamicin

coat proteins: clathrin, AP COP

chlorpromazine benzyl alcohol bafilomycins chloroquine

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limitations:

➥ ➥ ➥ ➥ almost all are unspecific and show pleiotropic effects ➥ ➥ ➥ ➥ none inhibit the earliest steps of clathrin-independent pinocytosis

and azithromycin ?

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O H3C OH OH OH CH3 H3CH2C O O CH3 CH3 H3C O O O HO (H3C)2N CH3 OH 1 3' N CH3 CH3 9 H3C H3C H3CO 9a

Azithromycin (AZ),

a dicationic amphiphile

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➥ ➥ ➥ ➥ spectrum of activity

  • Gram +
  • some Gram -

➥ ➥ ➥ ➥ therapeutic use

  • upper and lower respiratory tract infections
  • skin infections
  • sexually transmitted diseases
  • Mycobacterium avium complex in AIDS patients

➥ ➥ ➥ ➥ pharmacokinetic properties in vivo

  • exceptionally high and rapid accumulation in tissues, and

slow release (Foulds et al, 1990)

  • consequences

➥

➥ ➥ ➥ low serum concentrations ➥ ➥ ➥ ➥ decrease of the length of treatment ➥ ➥ ➥ ➥ toxicity ??? Pharmacological properties of AZ

Pharmacological properties of AZ

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➥ ➥ ➥ ➥ accumulates in lysosomes of fibroblasts and macrophages (Carlier et al, 1994) ➥ ➥ ➥ ➥ acidotropic sequestration (de Duve et al, 1974)

H+ B B B BH+ BH+ BH+ H+ H+ Extracellular fluid Extralysosomal space Lysosomes

Cellular pharmacokinetic properties of AZ

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➥ ➥ ➥ ➥ induces a lysosomal phospholipidosis in fibroblasts (Van Bambeke et al, 1996) ➥ ➥ ➥ ➥ inhibits lysosomal phospholipase A1 (Montenez et al, 1996) Cellular toxicological properties of AZ

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➥ ➥ ➥ ➥ binds to negatively-charged bilayers at acidic pH (Montenez et al, 1996) ➥ ➥ ➥ ➥ perturbs the fusion of lysosomes with horseradish peroxidase

(HRP)-containing endosomes (unpublished observation of Van Bambeke)

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  • Selection of experimental system

Rat foetal fibroblasts

➥ ➥ ➥ ➥ avidly accumulate azithromycin ➥ ➥ ➥ ➥ develop lysosomal phospholipidosis ➥ ➥ ➥ ➥ extensively characterized system

  • Experimental test

Fluid-phase endocytosis Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase endocytosis in cultured fibroblasts

  • D. Tyteca, P. Van Der Smissen, F. Van Bambeke, K. Leys, P.M. Tulkens, P.J.

Courtoy & M.-P. Mingeot-Leclercq

  • Eur. J. Cell Biol. 80: 466-478 (2001)

➀ ➀ ➀ ➀ Could AZ affect earlier steps of the endocytic apparatus ?

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HRP

(a) (b)

late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit (Cupers et al, 1994)

Fluid-phase endocytosis

➥ ➥ ➥ ➥ horseradish peroxidase (HRP)

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  • General experimental protocol

confluent cells pretreatment with AZ (from 0 to 3 days) incubation with the endocytic tracer (from 0 to 4 h) washing, recovering and sonication assays: ➥ ➥ ➥ ➥ endocytic tracer ➥ ➥ ➥ ➥ proteins ➥ ➥ ➥ ➥ AZ ➥ ➥ ➥ ➥ phospholipids

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control cells cells pretreated with AZ

AZ slows down fluid-phase endocytosis

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Inhibition of fluid-phase endocytosis correlates with AZ content but is independent of phospholipidosis

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CT 3 h AZ 3 days AZ 5 min HRP 2 h HRP

AZ causes a major reduction of the number of endosomes and lysosomes and impairs accessibility of HRP to swollen and overloaded endosomes/lysosomes

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Azithromycin inhibits clathrin-independent pinocytosis and slows down sequestration of ligand-receptor complexes into endocytic and recycling vesicles of J774 macrophages

  • D. Tyteca, P. Van Der Smissen, M. Mettlen, F. Van Bambeke, P.M. Tulkens, M.-
  • P. Mingeot-Leclercq & P.J. Courtoy

Submitted for publication

  • Selection of experimental system

J774 mouse macrophages ➥ ➥ ➥ ➥ homogeneous cell line ➥ ➥ ➥ ➥ high endocytic activity ➥ ➥ ➥ ➥ well-characterized system for pinocytosis and phagocytosis

  • Experimental tests

➥ ➥ ➥ ➥ fluid-phase endocytosis ➥ ➥ ➥ ➥ bulk-membrane endocytosis ➥ ➥ ➥ ➥ receptor-mediated endocytosis ➥ ➥ ➥ ➥ phagocytosis ➁

➁ ➁ ➁ Is AZ specific to fluid-phase endocytosis ?

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HRP LY

(a) (b)

late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit (Cupers et al, 1994; Swanson et al, 1987)

Fluid-phase endocytosis

➥ ➥ ➥ ➥ HRP and lucifer yellow (LY)

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AZ HRP LY AZ inhibits fluid-phase endocytosis and this inhibition is reversible

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late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit

aggregates

  • f

N-Rh-PE Bulk-membrane endocytosis

➥ ➥ ➥ ➥ N-rhodamine-phosphatidylethanolamine (N-Rh-PE)

(a)

(Kok et al, 1990)

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AZ slows down bulk-membrane endocytosis

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Receptor-mediated endocytosis

➥ ➥ ➥ ➥ 125I-labelled transferrin

(a)

Tf TfR

Y

late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit

YY Y Y Y Y Y Y Y Y Y Y

(b) (c)

Y Y Y Y Y Y Y Y

(Cupers et al, 1994)

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AZ strongly decreases the surface-pool of transferrin receptors

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(b) (c)

AZ delays sequestration of ligand/receptor in endocytic pits and recycling vesicles

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PAP Fc?R Receptor-mediated endocytosis

➥ ➥ ➥ ➥ PAP immune complexes

Y

late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit

(a)

Y Y Y Y Y Y Y Y

(b)

Y Y Y Y Y Y Y Y Y Y Y Y Y

(Mellman et al, 1984; Kiss and Rohlich, 1984; 1987)

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(a) (b)

AZ marginally decreases the surface-pool of Fc? receptors and delays sequestration of ligand/receptor complexes into endocytic pits

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late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit

(a)

Phagocytosis

➥ ➥ ➥ ➥ latex beads of 1 and 0.1 µm

(Pratten and

Lloyd, 1986)

beads

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AZ does not affect phagocytosis

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HRP

(fluid-phase endocytosis)

PAP

(receptor-mediated endocytosis)

latex beads

(phagocytosis)

CT 3 h AZ

AZ impairs accessibility of HRP and PAP, but not of latex beads, to swollen endosomes/lysosomes

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Latex beads move within the structures vacuolated by AZ, demonstrating their presence in these structures

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Does AZ perturb endocytosis by:

  • a general toxic effect ?

NO

  • phospholipidosis ?

NO

  • AZ accumulation ?

YES

Interpretation

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➥ ➥ ➥ ➥ pH neutralization of endosome/lysosome ? ➥ ➥ ➥ ➥ swelling of endosome/lysosome ? ➥ ➥ ➥ ➥ membrane interaction ?

AZ accumulation ...

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Azithromycin, a macrolide antibiotic that impairs endocytic trafficking, directly interacts with biomembranes and perturbs their

  • rganization and fluidity
  • D. Tyteca, A. Schanck, Y. F. Dufrêne, M. Deleu, P.J. Courtoy, P.M. Tulkens &

M.-P. Mingeot-Leclercq (to be submitted)

  • Selection of experimental system

➥ ➥ ➥ ➥ liposomes ➥ ➥ ➥ ➥ Langmuir-Blodgett monolayers ➥ ➥ ➥ ➥ J774 mouse macrophages

  • Experimental tests

➥ ➥ ➥ ➥ interaction with membranes ➥ ➥ ➥ ➥ membrane organization in domains ➥ ➥ ➥ ➥ insertion of membrane probes in the plasma membrane ➥ ➥ ➥ ➥ membrane fluidity

➂ ➂ ➂ ➂ How does AZ inhibit pinocytosis ?

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interaction of AZ with membranes

➥ ➥ ➥ ➥ 31P nuclear magnetic resonance (NMR)

100 50

  • 50
  • 100

ppm

s ’//

// // //

s ’⊥

⊥ ⊥ ⊥

= s ’//

// // // - s ’⊥ ⊥ ⊥ ⊥

’s s ’i

100 50

  • 50
  • 100

ppm

s ’⊥

⊥ ⊥ ⊥

= s ’i - ’s eff. s ’⊥

⊥ ⊥ ⊥

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pH 5.4 pH 7.0 pH 6.0 AZ interacts with lipids of liposomes made of cholesterol: PC: SM: PI in a pH-independent fashion

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A B C D

AZ interacts with lipids and perturbs the organization of DPPC: cholesterol Langmuir-Blodgett monolayers

membrane organisation in domains

➥ ➥ ➥ ➥ atomic force microscopy (AFM)

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insertion of membrane probes in the plasma membrane of J774

➥ ➥ ➥ ➥ monomers;

external leaflet

  • f the PM

➥ ➥ ➥ ➥ monomers;

deep in the PM

➥ ➥ ➥ ➥ aggregates

AZ reduces incorporation in the plasma membrane of three membrane tracers

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2 p

3 - p r =

TMA-DPH plasma membrane fluidity of J774

➥ ➥ ➥ ➥ fluorescence anisotropy of TMA-DPH

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*

AZ decreases J774 plasma membrane fluidity

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late endosome lysosome early phagosome late phagosome zipper trigger sorting endosome non-coated pit recycling compartment coated pit

HRP LY

Fluid-phase endocytosis

Rh-PE

Bulk-membrane endocytosis

Tf PAP

Y Y YY Y Y Y Y Y Y Y Y YY Y Y Y

Receptor-mediated endocytosis

beads

Phagocytosis

General conclusion

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AZ decreases membrane fluidity (J774 plasma membrane) AZ perturbs membrane organization in domains (monolayers) AZ decreases incorporation of: N-Rh-PE, C6-NBD-SM TMA-DPH (J774 plasma membrane) AZ interacts with phospholipid headgroups (liposomes)

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➥ ➥ ➥ ➥

Mechanism of AZ action:

  • role of membrane properties

➥ ➥ ➥ ➥membrane fluidity, tension, transverse asymmetry and

composition

➥ ➥ ➥ ➥receptor mobility in the plasma membrane and

incorporation into coated pits

  • role of vacuolation
  • role of pH neutralization

Short-term perspectives

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Usefulness of AZ

  • inhibition of selective endocytic modes/pathways/steps

study of a series of ligands, e.g. cholera toxin internalization

  • differential modulation by drug concentration

Practical application: mechanism and function

  • clathrin-independent endocytosis
  • recycling pathway
  • fusogenicity between endosomes and lysosomes

➥ ➥ ➥ ➥ ➥ ➥ ➥ ➥

Long-term perspectives

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a pharmacological agent, azithromycin a physiological process, endocytosis

a tool to better understand mechanisms and molecular machineries of endocytic modes/pathways/steps

Progress in cellular toxicology of azithromycin