kathy dodgson - aurelia bioscience are a bio assay development and screening cro ... 50 for...
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Kathy Dodgson - Aurelia Bioscience www.aureliabio.com
6/15/12 Aurelia Bioscience © 2012
Company Background We are a bio assay development and screening CRO Based in Biocity, Nottingham 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 Project management Instrument/Reagent consultancy
Aurelia Bioscience © 2012 6/15/12
Our capabilities 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
Aurelia Bioscience © 2012 6/15/12
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 © 2012 6/15/12
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 © 2012 6/15/12
Lead Identification/Optimisation Compound screening - Agonist and antagonist Ideal for GPCR’s that couple to multiple G proteins Orthogonal screening – complementary technology Potential to multiplex in the same well
Mode of action studies Phenotypic screening
Measuring endogenous responses
Application of label-free in drug discovery
Aurelia Bioscience © 2012 6/15/12
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
Histamine H1 Receptor
6/15/12 Aurelia Bioscience © 2012
EC50 = 50nM
Time (secs)
Res
pons
e (p
m)
H1 β-arrestin assay in LF plate
6/15/12 Aurelia Bioscience © 2012
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
6/15/12 Aurelia Bioscience © 2012
Res
pons
e (p
m)
Time (secs)
IC50 values (nM)
Antagonist Label free
Cer$rizine 120
Chlorpheniramine 160
Triprolidine 40
Pyrilamine 50
Antagonist addition
Agonist addition
H1 β-arrestin in LF plate Antagonist effects were measured in label-free for 30 mins Further 30 min incubation prior to β-arrestin detection step
6/15/12 Aurelia Bioscience © 2012
IC50 values (nM)
Antagonist Label free
Beta arres:n (in LF plate)
Cer$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
6/15/12 Aurelia Bioscience © 2012
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
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 © 2012 6/15/12
Screening in more than one assay format to increase the chance of identifying novel hit(s) The label-free assay is not limited to measuring
coupling of the receptor to a particular pathway
Parallel screening approaches
6/15/12 Aurelia Bioscience © 2012
Phenotypic 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 © 2012 6/15/12
Conclusions Label-free technology can be applied in the following
areas: Primary screening Orthogonal screening approach Mode of action studies
There is potential to multiplex other assays within the same wells following a label-free response LF then beta arrestin Others? LF then imaging?
The ability to measure responses in endogenous systems meets the challenge of utilising more physiological assays earlier in drug discovery
Aurelia Bioscience © 2012 6/15/12