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Plate-forme deChimie BiologiqueIntégrative de Strasbourg
UMS 3286 CNRS-UdS
Can HTRF technology answer to different scientific
P. Villa, Avignon2013 Apr 26
Pascal Villapvilla@unistra.fr
www.pcbis.fr
Can HTRF technology answer to different scientific questions ?
An academic with industrial standards point of view
Marcel PCBIS
1997
P. Villa, Illkirch27 juin 2012
Plate-forme de chimie biologique intégrativede Strasbourg
Jean-Luc GALZI
JacquesHAIECH
Marcel HIBERT
Chemical Biology and Medicinal Chemistry
Chemical libraries
Gene
protein
HTS
Active Molecules
P. Villa, Avignon2013 Apr 26
Pharmacological tools
Drug
Candidate
QUALITY ?
P. Villa, Avignon2013 Apr 26
ISO 9001
P. Villa, Avignon2013 Apr 26
Bridge ?
P. Villa, Avignon2013 Apr 26
Biology Chemistry
Bridge !
P. Villa, Avignon2013 Apr 26
Consulting
• Project evaluation
• Target Identification
• Choice of Chemical
• Project evaluation
• Target Identification
• Choice of Chemical
TARGET PRODUCTION
• Expression conditions development
• Target production
• Expression conditions development
• Target production
ASSAY Develpoment
• HTS assaydevelopment
• Miniaturization• Automation• Stability, robustness
• HTS assaydevelopment
• Miniaturization• Automation• Stability, robustness
HTS
• MTS/HTS• Hit Identiification
• MTS/HTS• Hit Identiification
HIT VALIDATION
• Hit confirmation & Validation
• Analysis• RSA
• Hit confirmation & Validation
• Analysis• RSA
HIT OPTIMIZATION
Working plan
MIL
ES
TON
ES
Target producedHTS protocol
Hits identifiedRaw datas
Analyzed datas
Confirmed HitsRaw datas
Analyzed datas
• Early ADME-Tox
• Optimisation with medicinalchemists
• Secondary
• Early ADME-Tox
• Optimisation with medicinalchemists
• Secondary
OptimizedHit
P. Villa, Avignon2013 Apr 26
Chemicallibraries
• Working plan definition
Chemicallibraries
• Working plan definition
robustnesssudiesrobustnesssudies
Go/No Go steps
TIM
ING
AUDITAUDIT
Chemical LibrariesChemical Libraries
ADMETADMET
1 to 4 weeks 2 to 4 months 2 to 4 months 2 to 4 months 2 months 1 to 4 months per molecule
Chemical librariesChemical libraries
• Secondarytests
• Secondarytests
HTSHTS
Assay Model Technology Format
Cytokine secretion(TNFα, IL-1,6,8...)
Peripheral blood mononuclearcells and other cell types
ELISAHTRF
96
Nitrite oxide (NO) production
RAW cells Absorbance 96
Kinase activity detection Ser/thr kinase HTRF/Luminescence 384
Cell growthBacteria (E. coli)Eucaryotic cells
AbsorbanceConfluence measurement
9696
Cell survivalHEK293, HepG2, MCF7,Caco2, RAW, HeLa, HL-60, U937, your cells...
AbsorbanceLuminescence
96 / 384 /1536
Cell death Caspase activity luminescence 96 and 384
P. Villa, Avignon2013 Apr 26
- Aggregation- Size measurement
Soluble protein Dynamic Light Scattering (DLS) 96 and 384
Calcium fluxGPCR Calmodulin
Fluorescence 96 and 384
Cyclic AMP CellsLuminescenceHTRF
96 and 384
Tissue regeneration (scratch wound)
Cell lines Confluence measurement 24
Membrane receptor Binding
GFP fused GPCR expressing HEK293
FRETHTRF
96
Molecular Binding Soluble protein Anisotropy 96 and 384
Permeability Caco2 Cell monolayer 24
Your favorite assay Your model Compatible with our apparatus 96-384
Preclinical ADME
• permeability• plasmatic protein binding• metabolism: plasma, liver
Physicochemical Properties
• Solubility• Log D
TechMed
P. Villa, Avignon2013 Apr 26
• Log D• CHI• pKa• Chemical stability
In vivo Pharmacokinetics
• BBB• Bioavailability
Cytotoxicity
P. Villa, Avignon2013 Apr 26
Microfluidic screening platform
P. Villa, Avignon2013 Apr 26
The platform is built, functional and installed in the PCBis screening platform (Illkirch)12
Compound Library
Primary HTS1 Point, 1 Concentration
Confirm Positives
Quantitative HTSCurrent and future HTS processes
Current Droplet-Based Microfluidics
P. Villa, Avignon2013 Apr 26
Lead Chemical Series Enter Compound
Optimisation Programs
Confirm Positives2 Points, 1-2
Concentrations
Measure IC50s2 Points, 8 Concentrations
13
Compound Library
Primary HTS1 Point, 1 Concentration
Confirm Positives
Quantitative HTSCurrent and future HTS processes
Current Droplet-Based Microfluidics
P. Villa, Avignon2013 Apr 26
Lead Chemical Series Enter Compound
Optimisation Programs
Confirm Positives2 Points, 1-2
Concentrations
Measure IC50s2 Points, 8 Concentrations
14
EGFP
Liss
hv1 (510 nm)hv (470 nm)
hv2 (590 nm)Energy Transfer
ligand
Liss
Generic assay
FRETFRET--basedbased bindingbinding assayassay
JL GalziM Hibert
P. Villa, Illkirch28 janv 2013
Plate-forme de chimie biologique intégrativede Strasbourg
ligand
Liss
HEK293
EGFP
T-R
hv1 (510 nm)hv (470 nm)
hv2 (590 nm)Energy Transfer
Generic assay
FRETFRET--basedbased bindingbinding assayassay
JL GalziM Hibert
P. Villa, Illkirch28 janv 2013
Plate-forme de chimie biologique intégrativede Strasbourg
ligand
T-R
HEK293HEK293
Example : orphan RCPG
APJ
JL GalziM HibertD Bonnet
P. Villa, Illkirch28 janv 2013
Plate-forme de chimie biologique intégrativede Strasbourg
Iturrioz et al, FASEB 2010Collaboration C Llorens-Cortes, College de France
Free fluorescent Ligand
(from fluorescent chemical library)
Depolarizedlight
FastFast rotationrotation
Excitation
Fluorescence Fluorescence polarizationpolarization
Binding assay for soluble protein
P. Villa, Illkirch28 janv 2013
Plate-forme de chimie biologique intégrativede Strasbourg
Excitation
Bound fluorescent Ligand
Polarizedlight
Slow RotationSlow Rotation
soluble Protein + fluorescent chemical library � fluorescent probe
� Step 1: assay development :
� Step 2: using the assay (HTS: competition)
Strategy
P. Villa, Illkirch28 janv 2013
Plate-forme de chimie biologique intégrativede Strasbourg
Protein + fluorescent probe + chemical library
� Hit
Blood from donors
PBMC purification
Cell culture 24h
compoundsPBMC+/- LPS
Cytokine detection
Cell Supernatant
P. Villa, Avignon2013 Apr 26
Cell survival test(toxicity)
Active compounds
Cytokine detection
Hits
non-toxiccompounds
Blood from donors
PBMC purification
Cell culture 24h
compoundsPBMC+/- LPS
Cytokine detection
HTRF kit
P. Villa, Avignon2013 Apr 26
Cell survival test(toxicity)
Active compounds
Cytokine detection
Cell Supernatant
Hits
non-toxiccompounds
Method Classical / ELISA
HTRF Gain obtained
Time 3 days 1,5 day Half time
P. Villa, Avignon2013 Apr 26
Throughput 160 cpds > 1000 cpds X 5
Search for Vasopressin agonists
• GPCR (Vasopressin 1A receptor)
• HTRF: Tag-Lite (Cisbio)
P. Villa, Avignon2013 Apr 26
Cell labelling Seeding into 384-well plates (10000 cells / well
(10 µl)
Compounds 10 µM(5 µl)
fluorescent igand1 nM (5 µl)
readingExc : 337 nmEm1 : 615 nmEm2 : 665 nm
METHOD: HEK cells; V1A terbium cryptate labelled
P. Villa, Avignon2013 Apr 26
ABCDEFGHIJKLMNOP
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Cells + SR49059 + fluo ligand
Cells + Buffer +f luo ligand
Buffer + Buffer + Buffer
Cells + Buffer + Buffer
ANALYSIS
Emission 615 nm (Plate 01) Emission 665 nm (Plate 01)
Ratio Em 665 nm / Em 615 nm (Plate 01)
02000400060008000
1000012000
tampon cellules seules
cellules + ligand
cellules + SR + ligand
0
500
1000
1500
2000
tampon cellules seules
cellules + ligand
cellules + SR + ligand
6080
P. Villa, Avignon2013 Apr 26
Pourcentage of inhibition :
Z’ :
0204060
cellules seules cellules + ligand cellules + SR + ligand
(cells + ligand) – (cells + ligand + compound)
(cell + ligand) – (cells)X 100
Exemple : 100 % ’inhibition withSR49059 (Plate 01)
3 x [(SD cells + ligand) + (SD cells + ligand + compound)]
(Meancells + ligand) - (Mean cells + ligand + compound)1 - Exemple : Z’=0,76 (Plate01)
Results
6508 molecules tested: 121 hits (Z’ > 0,7)
37 hits showed antagonist activity with more than 50% of inhibition of the Ca2+
response at 10 µM.
We focused on 6 out of them and performed dose-response validation curves.
P. Villa, Basel2012 sept 26
Search for Msk-1 inhibitors
• Enzymatic assay (kinase)• Kit HTRF KinEASE
HTRF-MSK1
MSK1 full length
Biotinilated substrateAntibody
labeled with Eu3+-cryptate
Streptavidin- XL665
337nm
HTRF kinEASE™ (CISBIO)
Choice of the substrate: STK S3
Kinase assay
Results
7120 compounds tested: 55 hits (Z’ > 0,75)
5 hits showed inhibitory activity with validated with another technology (luminescence-based).
.
LPS 02-10 D09 LPS 01-13 D08
P. Villa, Avignon2013 Apr 26
LPS 02-10 D09
0.1 1 10 1000
25
50
75
100
125
IC50 = 4.83 +/- 0.36 µM
Log [composé] (µM)
% in
hibi
tion
LPS 01-13 D08
0.1 1 10 1000
25
50
75
100
IC50 = 2.83 +/- 0.48 µM
Log [composé] (µM)%
inhi
bitio
n
Platform of Chemical Biology(UMS 3286 CNRS-UdS):
Biology/engineering
Sophie GIORIAAdeline ObrechtChristel VALENCIARaphael CALBRIXYannick BECKAnnick MARIN
Chemistry
Bruno DIDIER (& LIT)
Laboratory of Therapeutic Innovation(UMR 7200 CNRS-UdS)
Chemistry
Marcel HIBERTDominique BONNET
Biology
Nelly FROSSARDSimona NEMSKA
Medalis project (Cancer & inflammation)
P. Villa, Avignon2013 Apr 26
Bruno DIDIER (& LIT)Claire MARSOL (& LIT)Pascale BUISINE (& LIT)
ADME
Patrick GIZZI (& BSC)François DAUBEUF (& LIT)
Medalis project (Cancer & inflammation)
Andrew GRIFFITHSRaphael CALBRIXYannick BECKAnnick MARIN
Laboratory of Biology & Cellular Signalisation(UMR 7242 CNRS-UdS)
Jean-Luc GALZI
P. Villa, Avignon2013 Apr 26
Palace of Popes
François 1 er from Cisbio
P. Villa, Avignon2013 Apr 26
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