Post on 14-Nov-2018
transcript
1
Facing the Challenges of
High-Content Screening
in Leishmania Amastigotes-
Hosting
Primary Macrophages
Nathalie Aulner PhD, IMAGOPOLE - PFID
2
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)
5
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)
6
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
…
7
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
8
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)
9
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
…
10
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
11
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
12
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
13
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)
14
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)
15
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
16
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)
17
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
18
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
19
“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
20
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)
21
~ 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
22
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)
23
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
24
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)
25
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
26
Quality control
Repeat 1 Repeat 4 Repeat 3 Repeat 2
Results are reproducible
Raw data Heatmaps PVs Number
Live cells
27
-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-)
28
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
29
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
30
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
31
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
• …
32
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