1

Facing the Challenges of

High-Content Screening

in Leishmania Amastigotes-

Hosting

Primary Macrophages

Nathalie Aulner PhD, IMAGOPOLE - PFID

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Imagopole Imaging functional & molecular dynamics

www.imagopole.org

•Ultrastructural Microscopies

•Imaging & Flow Cytometry

•Dynamic Imaging

•Image processing & analysis

•Bio-informatics, & statistics

•Translational research

BIME

2012

Imagopole Mission:

Apply cutting edge “imaging” science &

technologies to studies on the dynamics

of biological processes and their

usurpation by infectious disease

3

Imagopole

Key facts & figures

• ISO 9001 Certification for quality in service standard

• IBiSA scientific label

• >700 registered users, >45,000 hours/year “burn-time”

• 10% External users

• 4 Patents, 1 Software copyright (New patent filed March 2010)

• >35 peer reviewed high-impact scientific articles a year

• Global annual budget 2,5 million euros (4 teams, 35 scientific staff)

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Imagopole - Dynamic imaging

Plateforme d’imagerie dynamique (PFID)

Opera QEHS

P2+ env.

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Current High Content Efforts at the

Imagopole

High Content siRNA/Genetic Screens

Intra cellular Trafficking (N. Sauvonnet (A. Dautry, Cellular Interactions Biology))

Shigella Infection (N. Mellouk (J. Enninga, Host-Pathogen Interactions Dynamism))

Chikungunya Infection (T. Couderc (M. Lecuit, microorganism and host barriers))

Inter Cellular Trafficking (K. Gousset (C. Zurzolo, Pathogenesis and membrane Trafficking))

Cellular Interaction (R. Lasserre (A. Alcover, Cell biology of Lymphocytes)

Klebsiella virulence (R. Tournebize (P. Sansonetti, Molecular microbial pathogenecity))

High Content Compounds Screens

Leishmania (FP7-Leishdrug)

Strept. , E.Coli Infection (B. Sperandio (P. Sansonetti, Molecular microbial pathogenecity))

High Content Analysis

HIV Infection (N. Sol-Foulon (O. Schwartz, Virus and Immunity)(Nobile et al. 2010)

Rabbis (C. Jallet (N. Tordo, Virology))

M. tuberculosis (R. Simeone (R. Brosch, Integrated mycobacterial pathogenomics))

Stem Cells (D. Montarras (M. Buckingham, Molecular genetic of Development))

HIV Infection (J. Feldman (O. Schwartz, Virus and Immunity))

Listeria (J. Pizarro (P. Cossart, Host-Bacteria Interactions))

Adult mouse muscle stem cells differentiation (I. LeRoux (S. Tajbakhsh, stem cells and development)

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Titration of rabies patient sera (bresilian

clinical trial) Corinne Jallet (N. Tordo, Antiviral Strategies Research Unit)

Detection (10x)

Nuclei (Hoechst)

Viral particle (specific peptide coupled to Alexa 488)

Analysis

Total cells

Infected cells

Infection rate

Titration curves

…

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HCS of Klebsiella pneumoniae mutants library Joelle Mounier & Régis Tournebize (P. Sansonnetti, Molecular microbial

pathogenecity)

Acquisition 10x bin2

Hoechst

HC Cell Mask Blue

Analysis

Cells count

Fitness of cell population

Cells Size

Subpopulation analysis

…

NI

kp52

clbi

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Genetic analysis of new factors involved in

clathrin-caveolae-independent endocytosis N. Sauvonnet (A. Dautry, Cellular Interactions Biology)

Acquisition (20x w)

Hoechst

HC cell mask Blue

Receptor C1

Receptor C2

Analysis

36 parameter outputs

(siRNAs screening targeting membrane associated proteins)

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Development of a siRNA screen tor decipher

shigella's infection behaviour Nora Mellouk (J. Enninga, Host-Pathogen Interactions Dynamism)

Infection Foci

Infection Foci

Cytoplasmic

localization

Cytoplasmic

localization

Acquisition (10x)

Draq5

Phalloidin 561 (infection foci)

CCF4 (FRET) (cytoplasmic local.)

Analysis

Number of cells

Infection foci

FRET ratio (cytoplasmic localization)

Infected cells

Subpopulations analysis

…

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High Content screening for compounds

acting on intracellular Leishmania parasites

Acquisition

Detection:

Nuclei (Hoechst)

Parasites (Leishmania-DsRed)

Parasitophorous Vacuoles (Lysotracker DND26 green)

Analysis

Cell count

Survival Rate

PVs

PVs/Cell

Amastigotes

Amastigotes/PV

….

www.leishdrug.org

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Main Neglected Tropical Diseases*

Virus

• Dengue

• Rabies

Bacteria

• Trachoma

• Buruli ulcer

•Treponematoses

• Leprosy

Parasite

• Chagas Disease (American Trypanosomiasis)

• Sleeping Sickness (Human African Trypanosomiasis)

• Leishmaniasis

• Cystercercosis

• Food-borne Trematode infections

• Lymphatic Filariasis (elephantiasis)

• Onchocerciasis (river blindness)

• Soil-transmitted Helminthiasis (intestinal parasitic worms)

*«Working to overcome the global impact of neglected tropical diseases»

World Health organization, 2010

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Human leishmaniasis

• Assessed uncontrolled development of a protozoan

parasite of the genus Leishmania

• Transmitted by the female phlebotomine sand fly (inter

tropical and temperate regions) threatens 350M people in

88 countries on 4 continents

• 12M human beings estimated to be subverted as hosts of

Leishmania spp (1-2M new cases each year)

Source: World Health organization

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Cutaneous leishmaniasis ( L. amazonensis , L. major, L. Viannia

braziliensis complex, …)

• Most common

• Ulcers that heal spontaneously, the endpoint being permanent scars

(serious social prejudice)

• 90% in Afghanistan, Brazil, Iran, Peru, Saudi Arabia

Mucocutaneous leishmaniasis (L. Viannia braziliensis complex)

• Partial or total destruction of mucous membranes of the nose, mouth

or throat cavities

• 90% in Bolivia, Brazil and Peru

Source: World Health organization

Leishmaniasis: different

clinical presentations (1)

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Visceral leishmaniasis (Kala-azar) (L. donovani)

• most severe form

• Irregular bouts of fever

• Substantial weight loss

• « Swelling » of the spleen and liver

• Anemia

• Fatality rate as high as 100% within 2 years if not treated

• 90% Bangladesh, Brazil, India, Nepal and Sudan

Source: World Health organization

Leishmaniasis: different

clinical presentations (2)

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Drugs used currently

• Pentavalent antimonials (Sb)

o Primary 1st line of treatment in most parts of the world

o BUT long (20-40 days)

o BUT nephrotoxicity

o BUT confirmed parasite resistance in Bihar State (India)

• Amphotericin B

o Remarkable activity in India in single dose administration

o BUT requires carefull administration (slow intra-venous infusion)

o BUT shows nephrotoxicity

o BUT cost prohibitive

• Miltefosine (M) and Paromomycin

o Newly developped (completed clinical trials)

o BUT rapid selection for resistance

o BUT teratogenic (M)

• 1 other drug in clinical trial

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The LeishDrug Consortium

• European Commission funded FP7 program (Neglected Protozoan Diseases)

• Highly interdisciplinary approach to

Reveal Leishmania kinases associated with amastigote «virulence»

Exploit parasite-specific pathways for anti-leishmanial drug development

13 Institutions from 9 countries

14 teams and 63 scientists / 3M€ funding with 16 positions

LSHTM

UR2

CSIC UPF

CRG

CNRS

IP

PHX

TUBS

IP Montevideo

IP Tunis

IP Seoul

IIT (Israel)

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Screening Strategies

Kinase-

biased

compound

libraries

Axis I: In situ drug screening

(Semi)-conserved

Leishmania kinases

(MAPKs)

Genetic analysis

Stage-specific

phosphorylation

events (phosphoproteomics)

Kinase screen

Druggable Leishmania

protein kinases

Axis II: Target-based screening

BMM / L. amazonensis

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In situ Drug screening –

Main Objectives

• To set up and optimize a multiplex high-content cell-based assay

• To perform screening campaigns on focused libraries from our

consortium partners

• To cross-validate in situ target based hit candidates

• To develop and implement secondary/confirmatory screening

assays to further characterize hit candidates

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“High Content Imaging”

in biologically relevant conditions

Goal Setting up an assay that fits both

physiological and clinical relevance

Promastigote Amastigote

The clinically relevant

developmental stage

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Experimental Setup

CSF-1 responsive

bone marrow macrophage

progenitors

Target amastigotes

within macrophages

L. amazonensis

amastigotes purified from

skin sites

Amastigotes develop within giant

parasitophorous vacuoles (PVs)

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~ 3*107 cell

107 cellules / flask

Bone marrow

cells

BALB/c

D0

ACQUISITION

ANALYSIS

1 hr

DsRed2 amastigotes

Swiss Nude

5 hr

75 cm² flasks untreated

D3 Fresh medium

Compounds

20 hr

D7 ~1.5*107 cells / flask

~ 5 plates

Distribution 384 wells

2*104 cells / well

D6

Macrophages (MΦ)

Fluorescent reporters

D10

3 days

Quality controls

rmCSF-1 50 ng / mL

rmCSF-1 12 ng / mL

Protocol Overview

Only addition steps

Live cell Imaging

FACS

analysis

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Acquisition - Image analysis

Nuclei (Hoechst)

PVs (Lysotracker DND26)

Parasites DsRed2

Output Data

Row 1

Column 1

Processed Image Fields 15

Cells 11364

Live Cells 11198

Survival Rate 98.54

Amastigotes 16955

PVs 5955

PVs/cell 0.53

Intensity Mean (Nuclei) 254.52

Intensity StDev (Nuclei) 78.25

Area Mean (Nuclei) 102.65

Area StDev (Nuclei) 29.89

Intensity mean (PV) 166.67

Intensity StDev (PV) 43.82

Area Mean (PV) 474.28

Area StsDev (PV) 216.01

Intensity Mean (Live Cells) 257.59

Intensity StDev(Live Cells) 74.55

Area Mean (Live Cells) 102.04

Area StDev (Live Cells) 27.56

Well Tag C-

• Optimization of assay conditions (cell

number, parasite number)

• Choice of fluorescent reporters

(concentration…)

• Acquisition conditions (entire well ~15k cells)

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0

4000

8000

12000

16000

Med

ia

cycl

ohex

LeuOM

e

DM

SO

Ce

lls

Cells

Live Cells

0

25

50

75

100

125

Med

ia

cycl

ohex

LeuOM

e

DM

SO

Su

rviv

al

rate

0

0,25

0,5

0,75

Med

ia

cycl

ohex

LeuOM

e

DM

SO

PV

s/C

ell

Live Cells Survival PVs/cell

z'-factor 0,63 0,92 0,70

*Z’=1-3 (σ+ + σ-) / |µ+ - µ-|

*Error bars : Std 96 wells

Data analysis Output choice

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Output choice - Validation

Amphotericin B Miltefosine

“Amastigote” outputs correlates with “PV” outputs

0

25

50

75

100

125

150

0 0.2 0.4 0.6 0.8 1

concentration (uM)

Living Cells

PVs/Cell

Amastigotes/PV

0

25

50

75

100

125

150

0 5 10 15 20 25 30Concentration (uM)

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C- C+ Samples

• 60 test compounds (10uM) in quadruplicates

• Known antileishmanial drugs

• Potential inhibitors based on initial kinase

tests

• Literature

• Experiment performed 4x (2 x 2 plates)

A. Validation Plate

C. Quality Control

D.

B.

Quality control

DMSO (1%)

(C-)

Amph. B (0.5uM)

(C+)

-20

-16

-12

-8

-4

0

4

-10 -8 -6 -4 -2 0 2

survival rate

PV

s/c

ell

C+

C-

Robust Z-score

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Quality control

Repeat 1 Repeat 4 Repeat 3 Repeat 2

Results are reproducible

Raw data Heatmaps PVs Number

Live cells

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-20

-15

-10

-5

0

5

10

-40 -30 -20 -10 0 10

Survival Rate

PV

s/c

ell

Samples

C+

C-

Data Analysis*

*Normalization Method: robust z-score (median C-)

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First screening campaign

Targeted Libraries : ~ 2500 compounds

• LeishDrug Partner 10 : 1520 compounds

• PKRC (P. Goejkan, FP6 Protein Kinase Research, including

LeishDrug partner 13 compounds) : 960 compounds

Screening strategy

• 1 data point per compound

• Screening concentration : 10 uM

• Positive control for PVs/Live cell : Amphotericin B (0.5 uM)

• Positive control for survival rate : Cycloheximide (0.2 uM)

• Same acquisition and data analysis pipeline

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Data Analysis – Primary Screen

Sample Classification Rules

Class PVs/Cell Survival total cells Healthy CellsAmastigotes

(visual)

0 + + + + +

1 - + + + -2 - + - - -

3 (intermediary) - +/- +/- +/- -

4 - - + - -

5 - - + - +

6 (mostly toxic) - -/+ -/+ -/+ ??

7 Artefact of any sort

Total compound Hit Class Amount Percentage

2468 0 1482 60,05

1 282 11,43

2 15 0,61

3 13 0,53

4 298 12,07

5 100 4,05

6 191 7,74

7 87 3,53

Results

Class 0

Class 1

Class 5

Class 4

Class 2

Class 3

Class 7

Class 6

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Confirmatory Screen – Partial Results

HIT weak hit/low efficiency not penetrantPKRC001-E8 PKRC001-E2 PKRC003-B5

PKRC004-D10 PKRC006-B10 PKRC005-D5

PKRC007-B5 PKRC006-C6

PKRC010-H8 PKRC007-D9

PKRC012-C7 PKRC012-B5

PKRC013-F4 PKRC013-B11

PKRC013-F5 PKRC013-E7

PKRC013-H7

PKRC0010 H8 Control -

PKRC007 D9 PKRC003 B5

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Work in progress/Perspectives

• Confirmatory screen

• Quadruplicates (1x and 0.1x concentration) – Finalizing analysis

• IC50 determination

• Clustering

• Counter screens/follow up studies

• Same ouputs (timing)

• Human cells pannel Cytoxicity test

• Other Leish. Spp and Trypanosoma (species specificity)

• Detailed HCA (20x etc…)

• Promastigote stage and free amastigotes (dev. Stage specificity)

• Phosphoproteome analysis

• co-cristallization

• Chemoinformatics, SARs …

• Animal models

• …

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Acknowledgement

PFID www.imagopole.org

Pascal Roux

Emmanuelle Perret

Joe Dragavon

Jean-Yves Tinevez

Samantha Blazquez

Marie-Anne Nicola

FP7 LeishDrug - WP1

Spencer Shorte

Anne Danckaert

Pierre-Henri Commere

Eric Prina

Julie Desrivot

Eline Rouault-Hardoin

Geneviève Milon

Olivier Helynck

Hélène Munier-Lehmann

FP7 LeishDrug

Consortium www.leishdrug.org

Gerald Spaeth

Users

Cyril Basquin

Alexandre Bobart

Nicolas Gangneux

Karine Gousset

Stephanie Lebreton

Nora Mellouk

Didier Montarras

Joelle Mounier

Lucia Murora

Roxanne Simeone

Nathalie Sauvonnet

Régis Tournebize

Brice Spérandio

Isabelle LeRoux …

Contact: nathalie.aulner@pasteur.fr