2/7/13 aurelia bioscience © 2013 · who are aurelia bioscience? we are a bio assay development and...
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2/7/13 Aurelia Bioscience © 2013
Gary Allenby - Aurelia Bioscience www.aureliabio.com
2/7/13 Aurelia Bioscience © 2013
Who are Aurelia Bioscience? We are a bio assay development and screening CRO Based in Biocity, Nottingham, United Kingdom Scientists with extensive experience of drug discovery
processes and assays gained from large pharma We specialise in: Bespoke assay development Drug discovery screening Cell culture services Instrument/Reagent consultancy Project management
Aurelia Bioscience © 2013 2/7/13
2/7/13 Aurelia Bioscience © 2013
EnSpire (PerkinElmer) Multimodal (label free)
FMAT– (Life Technologies) Cell and bead fluor assays
FLIPR – (Molecular Devices) Kinetic fluorescent cell assays
Envision – (PerkinElmer) Fluorescence and Luminescence
Our Capabilities Cell Culture
Plus bulk and discrete liquid handling using Multidrops, Flexdrops and CyBio Dispensers
Ultra and Micro – (Molecular Devices) High content screening assays
Dynamic Mass Redistribution/
morphological changes
Change in Refraction index
Final read monitored as response (pm)
What does a label-free biosensor measure?
Broad band light source Reflected wavelength
Substrate
Wave Guide
Surface
Baseline Read
Cell
Detection
Window
Ligand binding to the receptor
Aurelia Bioscience © 2013 2/7/13
Time
Res
pons
e
EnSpire Assay Flow Cell-Based Assays
Seed Cells
Buffer Exchange
Baseline Scan (30 min)
Agonist Assay Scan (60 min)
Antagonist Assay Scan (30 min)
Data Analysis
Incubate Cells O/N
Serum Starve Cells O/N (Opt)
Incubate Cells 90 min rt
Add Compounds
Add Compounds
Agonist Assay Scan (60 min)
Data Analysis
Aurelia Bioscience © 2013 2/7/13
Lead Identification/Optimisation Compound screening - Agonist and antagonist Ideal for GPCR’s that couple to multiple G proteins Potential to multiplex in the same well Orthogonal screening – complementary technology
Phenotypic screening Measuring endogenous responses
Mode of action studies
Application of label-free in drug discovery
Aurelia Bioscience © 2013 2/7/13
Lead Identification/Optimisation Compound screening - Agonist and antagonist Ideal for GPCR’s that couple to multiple G proteins Potential to multiplex in the same well Orthogonal screening – complementary technology
Phenotypic screening Measuring endogenous responses
Mode of action studies
Application of label-free in drug discovery
Aurelia Bioscience © 2013 2/7/13
The Histamine H1 Pathunter β-arrestin cell line from DiscoveRx was used in these studies
Agonist response measured over 30mins EC50 calculated for histamine at the peak response
Multiplex - Histamine H1 Receptor
2/7/13 Aurelia Bioscience © 2013
EC50 = 50nM
Time (secs)
Res
pons
e (p
m)
H1 β-arrestin assay in LF plate
2/7/13 Aurelia Bioscience © 2013
Following label-free measurement, β-arrestin detection reagents were added to the biosensor plate
The β-arrestin assay is an equilibrium measurement and required a further incubation step
EC50 = 61nM EC50 = 50nM
Label free DiscoveRx
Res
pons
e (p
m)
Rel
ativ
e Li
ght U
nits
H1 receptor - antagonist screen Pre-incubate cells with antagonists for 30 mins Add EC80 histamine and measure the label-free response Choice of time point may influence IC50
2/7/13 Aurelia Bioscience © 2013
Res
pons
e (p
m)
Time (secs)
IC50 values (nM)
Antagonist Label free
Cer$rizine 120
Chlorpheniramine 160
Triprolidine 40
Pyrilamine 50
Antagonist addition
Agonist addition
H1 β-arrestin – antagonists in LF Antagonist effects were measured in label-free for 30 mins Further 30 min incubation prior to β-arrestin detection step
2/7/13 Aurelia Bioscience © 2013
IC50 values (nM)
Antagonist Label free
Beta arres:n (in LF plate)
Cer$rizine 120 22
Chlorpheniramine 160 12
Triprolidine 40 4
Pyrilamine 50 4
Historically FLIPR has been key technology used for screening Gq coupled receptors
EC50 for histamine was more potent than in the label free assay
H1 receptor Ca2+ flux - antagonists
2/7/13 Aurelia Bioscience © 2013
IC50 values (nM)
Antagonist Label free
Beta arres:n (in LF plate) FLIPR
Cer$rizine 120 22 90
Chlorpheniramine 160 12 76
Triprolidine 40 4 26
Pyrilamine 50 4 34
Phenotypic approaches
2/7/13 Aurelia Bioscience © 2012
Phenotypic primary cells (Inflammation is a great source of primary cells)
Neutrophils Kinetic LF Calcium flux
PBMC’s Kinetic LF
T cells Kinetic LF
Degranulation endpoint:
Neutrophil elastase Superoxide burst Chemotaxis
Cytokine release endpoint assays
(alphaLISA)
LF = Label free
2/7/13 Aurelia Bioscience © 2013
Recombinant cell assay
384 well adherent assay using HEK-293 cells expressing a G-protein coupled receptor
Human primary cell assay
• Neutrophils isolated by density gradient centrifugation, loaded with dye, dispensed into plates in suspension in presence of compounds, incubated then agonist is added on-line
Phenotypic screening FLIPR – flux kinetic imaging
Screenshot: Dose – response of 19 project compounds screened in neutrophils using FLIPR
Conc
1000nM, 1 in 5 dilution
Aurelia Bioscience © 2013 2/7/13
Label free - native responses in primary cells
Human neutrophils in suspension can be plated and allowed to settle prior to assay
Responses to a stimulus are very fast and can be challenging to measure
Aurelia Bioscience © 2013 2/7/13
H1 receptor - Antagonist screen Pre-incubate cells with antagonists for 30 mins Add EC80 histamine and measure the label-free response Choice of time point may influence IC50
2/7/13 Aurelia Bioscience © 2013
Res
pons
e (p
m)
Time (secs)
IC50 values (nM)
Antagonist Label free
Cer$rizine 120
Chlorpheniramine 160
Triprolidine 40
Pyrilamine 50
Antagonist addition
Agonist addition
Mode of action – pharmacology A multi-addition protocol can be used to study the
mechanistic effect of compounds Agonists Antagonists Inverse agonists
Inverse agonism
Antagonist effect
Aurelia Bioscience © 2013 2/7/13
Screening in more than one assay format to increase the chance of identifying novel hit(s) (recombinant cells) The label-free assay is not limited to measuring
coupling of the receptor to a particular pathway
Parallel screening approaches
2/7/13 Aurelia Bioscience © 2013
Set A – Active in BOTH technologies Set B – Only active in FLIPR – significant number of compounds (false positives)? Set C – Only active in label free – mechanism – “grey box”
Summary There is potential to multiplex other assays within the
same wells following a label-free response LF then beta arrestin Others? LF then imaging?
Label-free technology can be applied in the following areas: Phenotypic screening using primary human derived cells Mode of action studies Orthogonal screening approach
The ability to measure responses in endogenous systems meets the challenge of utilising more physiological assays earlier in drug discovery – however “grey box” means a degree of deconvolution
Aurelia Bioscience © 2013 2/7/13
Press Article…Forbes, July 2011
2/7/13 Aurelia Bioscience © 2012
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