Design, synthesis and development of anti-adhesion ... · bactérie/bactérie CERMAV-CNRS (Grenoble) Dr Anne IMBERTY Lectines, Glycobiologie structurale, Etudes d’interactions LISM-CNRS

Journée Infections Nosocomiales - Lyon - 10 décembre 2015

1

Design, synthesis and development

of anti-adhesion glycoclusters against infection

by Pseudomonas aeruginosa

Eric Kipnis

CHU Lille

Université de Lille


4 academic teams involved in the original patent

Elicityl

Benoit DARBLADE Conception et synthèse

d’oligosaccharides naturels fonctionnalisés et synthèse de

glycoclusters

Université Lyon 1

Dr Sébastien VIDAL

Chimie des sucres

Dr Benoit COURNOYER

Etudes d’interaction bactérie/bactérie

CERMAV-CNRS (Grenoble)

Dr Anne IMBERTY

Lectines, Glycobiologie structurale, Etudes

d’interactions

LISM-CNRS (Marseille)

Dr Christophe BORDI

Formation/inhibition de formation du biofilm

Université Lille 2

Pr Eric KIPNIS Dr Rodrigue DESSEIN

Adhésion cellulaire in vitro

Etudes biologiques in vivo

SANOFI R&D

Dr Laurent FRAISSE (TSU maladies infectieuses)

Dr Michael MOUREZ

Evaluation de l’application - Sélection de candidats

2 Industrial partners


3




Projet AntiPyo - 14eme Appel à Projets du Fonds Unitaire Interministériel


4




Projet AntiPyo - 14eme Appel à Projets du Fonds Unitaire Interministériel


Epidémiologie : pneumopathies nosocomiales

Enquête nationale de prévalence 2012

InVS 2013


Epidémiologie : pneumopathies nosocomiales

Enquête nationale de prévalence 2012

InVS 2013


Résistances - France et Europe

European Antimicrobial resistance surveillance network

Ears-Net 2013

PIP-TAZ R

FQ R

Cefta R

Aminosides R

Carbapénèmes R


Pseudomonas aeruginosa lung infection

Pathophysiology

Adhesion

• critical step of infection

• activation of virulence

• T3SS

• quorum-sensing

Lectins

Lec A and LecB

• soluble adhesins

• secreted by P. aeruginosa

Imberty Microbes Infect 2004


P. aeruginosa lectins

Pathogenicity - in-vitro on respiratory epithelial cells

Chemani Infect Immunity 2009

Cytotoxicity (on A549 cell-line)

• lectin-producing P. aeruginosa strains

• vs. non producing deletional mutant strains



Targets for competitive inhitbition with glycomolecules

Target : lectins Inhibitors : glycomolecules

• Monosaccharides

- Fucose/galactose

• Derivatives

- Methylated

- N-acteylated

• Glycomimétics

• Glycoclusters

Pieters Med Res Rev 2007



Targets for competitive inhitbition with glycomolecules

Target : lectins Inhibitors : glycomolecules

• Monosaccharides

- Fucose/galactose

• Derivatives

- Methylated

- N-acteylated

• Glycomimétics

• Glycoclusters

Pieters Med Res Rev 2007

LecB (Fucose) LecA (Galactose)

ligand specificity


12

Lectin inhibitors

In-vivo effects of monosaccharides and derivatives

Murine model of acute lethal P. aeruginosa pneumonia

Monosaccharides and derivatives

Intratracheal co-instillation

decreased lung injury decreased bacterial load (moderately) increased survival

Chemani Infect and Immunity 2009


Lectin inhibitors

Clinical potential

10 patients with cystic fibrosis

monosaccharides

aerosolized inhaled fucose or galactose therapy over 21days

bacterial load inflammation

Hauber Int J Med Sci 2008


Lectin inhibitors

High-affinity glycocluster synthesis

click-chemistry: azide-alyne cycloaddition

Boukerb J Med Chem 2014

tetrapropargylated

calixarene-based

core

cali(4)arene

azido-functionalized fucoside

synthesis

Cu(I)-Catalyzed

azide-alkyne cycloaddition

of propargyl acetate

Cu(I)-Catalyzed

azide-alkyne cycloaddition

conjugation

acetyl groups deprotection

byproduct evaporation

C18 MPLC purification

fucosylated tetravalent glycocluster

Calix-Fuc4 (compound 6)


Lectin inhibitors

High-affinity glycocluster synthesis

click-chemistry: azide-alyne cycloaddition


tetrapropargylated

calixarene-based

core

cali(4)arene

azido-functionalized galactoside

synthesis

galactosylated tetravalent glycocluster

Calix-Gal4 (compound 8)


Hypotheses (1)

Structure-based affinity of inhibitors for lectins

multivalency

Sicard Chem Comm 2011

carbohydrate calixarene

core linker

LectinLecA/LecB targeting specificity

Présentation

Multivalency avidity


Hypotheses (1)

Structure-based affinity of inhibitors for lectins

Inhibitor multivalency vs. target multimerization

Sicard Chem Comm 2011

Soluble lectin

tetramer

Tetravalent

Calix4arene Multivalent

interactions


Hypotheses (1)

Structure-based affinity/specificity of inhibitors for lectins

Isothermal titration microcalorimetry (ITC)


Study of ligand-receptor interactions

Multivalent interaction induces nanomolar

affinities toward lecA LecB

Target Ligand Kd (nM)

LecB αFucOMe 430 ± 10

LecB Calix-Fuc4 48 ± 0.2

LecA αGalOMe 20 000 ± 300

LecA Calix-Gal4 176 ± 6


Hypotheses (2)

Inhibitor effects related to structure/affinity

Bacterial agregation assays


monosaccharide

derivatives

α-L-FucOMe

α-D-GalOMe

Calix-Fuc4

Calix-Gal4


Hypotheses (2)

Inhibitor effects related to structure/affinity/specificity

Bacterial agregation assays


Calix-Fuc4

loss of effect

against LecB mutants


Hypotheses (2)

Inhibitor effects related to structure/affinity

Cell adhesion assays


Fucosylated derivatives vs. Calix-Fuc4 Galactosylated derivatives vs. Calix-Gal4


Hypotheses (2)




Loss of antiadhesion effect of

fucosylated derivatives & Calix-Fuc4

on LecB mutant strain

Loss/attenuation of antiadhesion effect of

galactosylated derivatives & Calix-Gal4

on LecA mutant strain


Hypotheses (2)

Inhibitor effects related to structure/affinity/specificity ?



Partial recovery of antiadhesion effect of Calix-Gal4

on LecA mutant strain at high concentrations

non-specific effects?


Hypotheses (2)


Biofilm inhibition


Effect of calixarenes only :

• fucosylated (a)

• galactosylated (b)

Calixarene effects unchanged

• LecB deletional mutant (d)

• LecA deletional mutant (c)


Hypotheses (2)


Biofilm inhibition


No effect of a claixarene

with a different non-lectin-ligand

carbohydrate = glucose

0.1mM 5mM Ø

2D imaging

2D imaging

3D imaging

3D imaging

Calix-Gal4

Calix-Glc4


Hypotheses (3)

In-vivo effects related to cell adhesion inhibition

Murine model of acute lethal P. aeruginosa pneumonia


Lung injury (alveolar capillary barrier permeability)

Bacterial clearance (lung bacterial load)

Bacterial dissemination (spleen bacterial load)


• Monosaccharides

- galactose

- fucose

• in-vitro adhesion inhibtion: 10-40%

• in-vivo injury decrease: 20-25%

• in-vivo load decrease: 1 log CFU/ml

• Derivatives

- N-Acetyl

- α-méthyl

• in-vitro adhesion inhibtion: 10-50%


• in-vivo load decrease: 1-2 log CFU/ml

• Glycomimetics

• in-vitro adhesion inhibtion : 70-80%


• in-vivo load decrease: 2+ log CFU/ml

• Glycoclusters

• in-vitro adhesion inhibtion : > 90%


• in-vivo load decrease: 3-4 log CFU/ml

Hypotheses (3) In-vivo effects ~cell adhesion inhibition


Chemani Infect Immunity (+ unpublished data)


Summary

Tetravalent fucosyl/galactosylated glycoclusters


Target LecA / LecB

sturcture-based high affinity

Bacterial agregation

Adhesion inhibition

Lung injury, bacterial clearance/dissemination

Biofilm inhibition ~ structure/affinity

~ structure/affinity

± A/B specificity

Calix-Fuc4

Calix-Gal4

Calix-Man4 Calix-Glc4

ITC (Kd) 48 nM

(LecB)

176 nM

(LecA)

2.3 µM

(Lec B)

>1.5 mM

(LecA & LecB)

Bacterial aggregation +++ ++ + -

Biofim inhibition +++ +++ - -

Bacterial adhesion +++ ++ nd nd

Acute pneumonia +++ + nd nd


Development

Ongoing development stages

Synthesis and affinity studies

new generations of calixarenes

affinity enhancement with new linkers

Lectin A/B specificity enhancement with modified oligosaccharide ligands

carbohydrate 2d 3d

generation calixarene

cores

new linkers


Development


Screening (agregation, adhesion, biofilm, pneumonia)

characterized strains (n=164)

ventilator-acquired pneumonia

virulence

resistance (MDR and XDR strains included)

Reference strain PAO1 Clinical MDR VAP strain Clinical virulent (ExoU)

VAP strain


Development




cystic fibrosis strains

time-series

virulence/adhesion-related

gene expression

non CF control

Ø 0.1mM 5mM

KK1

ST111

KK14

Calix- -Gal


Development




LecA/LecB vraints


CF

environmental

Boukerb (in press)


Development


Synthesis and affinity studies

new generations of calixarenes

affinity enhancement with new linkers

Lectin A/B specificity enhancement with modified oligosaccharide ligands


Characterized strains (n=164)


virulence

resistance (MDR and XDR strains included)

cystic fibrosis strains

time-series

virulence and adhesion-related gene expression

environmental strains

LecA Lec B mutational variants

ADME-Tox studies


Development pipeline

Pipeline Design and Synthesis

Scling-up of synthesis

Identification, characterisation and production of lectins

Glcluster/lectin interactions - affinities

Sesnsitivity to lectin variants

Glycocluster effects on biofilm formation

Glycocluster effects on adhesion to cells

Glycocluster effects in-vivo pneumonia model

ADME-Tox studies

Assessment of pharmaceutical development

T1.1

T1.2

T2.1

T2.2

T2.3

T3.0

T3.1

T3.2

T3.3

T4.1

T4.2

Tâche 1 Glycocluster Design and Synthesis

Univ Lyon1

Elicityl

Elicityl

CERMAV

CERMAV

Univ Lyon1

Univ Lyon1

LISM Univ Lille2

LISM

Univ Lille2

Univ Lille2

Sanofi R&D

Sanofi R&D

Tâche 2 : Glycocluster-target (lectins) interactions

Tâche 3 : Biological effect screening

Tâche 4 : Application assessment

P. aeruginosa strain collections

Glycocluster candidate for

pharmaceutical development


Thanks

Affiliations, supports and endorsements

Financing


Thanks

ICBMS – CNRS - Univ Lyon 1

Villeurbanne, France

Audric Rousset

Nicolas Galanos

Samy Cecioni

Sébastien Vidal

CERMAV – CNRS, Grenoble, France

Emilie Gillon

Anne Imberty

Laboratoire Recherche

Translationnelle Hôtes Pathogènes,

Univ Lille 2, Lille, France

Jean-Baptiste Méar

Karine Faure

Eric Kipnis

Rodrigue Dessein Benoit Guéry School of Pharmacy, University of East

Anglia, Norwich, UK

Susan E. Matthews CNRS Aix Marseille Université,

Marseille, France

David Redelberger

Christophe Bordi

Sophie de Bentzmann

Ecologie Microbienne, CNRS - Univ

Lyon 1, Villeurbanne, France

Amine M. Boukerb

Sébastien Ribun

Benoit Cournoyer

Elicityl SA, Crolles, France

S Havet, S Bonnet,

L Bastide, C Cottin

M Randrianstoa,

Benoît Darblade

Documents

Design, synthesis and development of anti-adhesion ... · bactérie/bactérie CERMAV-CNRS (Grenoble) Dr Anne IMBERTY Lectines, Glycobiologie structurale, Etudes d’interactions LISM-CNRS