2/7/13 aurelia bioscience © 2013 · who are aurelia bioscience? we are a bio assay development and...

Gary Allenby - Aurelia Bioscience www.aureliabio.com


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


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


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


  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


  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


EC50 = 50nM

Time (secs)

Res

pons

e (p

m)

H1 β-arrestin assay in LF plate


  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


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


IC50 values (nM)


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


IC50 values (nM)


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


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


  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


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


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


Res

pons

e (p

m)

Time (secs)

IC50 values (nM)


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


 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


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


Press Article…Forbes, July 2011


2/7/13 aurelia bioscience © 2013 · who are aurelia bioscience? we are a bio assay development and...

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