· 7 ydeveloped an fp binding assay for discrimination of hits based on binding of a fluorescently...

www.aureliabio.com

Aurelia Bioscience © 2018

+44 (0) 115 8370503

1

Who are we? Over 100 years of experience in Pharma pre-clinical research

Gary Allenby (Chief Scientific Officer), Kathy Dodgson (Research Director), Kevin Hart (Managing Director)Ph.D. level staff

Currently 9 staff (B.Sc. and Ph.D. level)6 research labs, 2 offices on site

Location Biocity, Nottingham, United KingdomBio-incubator with over 70 bio-based businesses including CRO / CMO, specialised expertise including Med Chem, DMPK, Pathology, In-Vivo

Aurelia clients include top 50 Pharma, Biotech , other , Research Institutes (Life ARC, CRUK)Disease areas include oncology, neurobiology, musculoskeletal, respiratory and inflammation, novel disease targetsTargets types include Kinases, , cell surface second messenger, epigenetic, enzyme, protein-protein, ion-channelOver 95% repeat business rate with our clients

Aurelia Bioscience

Aurelia Bioscience © 2018 2

Biology CRO - Our Services

AureliaBioscience

Instrument/Reagent Consultancy and Validation

ExpertiseNew technology Biology

+ +Data Application Development

Validation for ClientBenchmarking

Fee for service

Assay Development

State of the art technology

+

Data Data

Data

Data

Series of compounds with appropriate properties

Discovery Project

Assay Development, Pharmacological Profiling and ScreeningHit Identification (no chemical precedent therefore need HTS)Hits-to-Lead (chemical precedent in literature)


AureliaBioscience

Case Study Hit to Lead example

Target disease area - oncologyClient (e-Therapeutics)

Molecular Pharmacology based on

interactions to identify key nodesSelected 1100 compounds to screen from propriatory in silicomodeling software

4

Two potential targets based in literature precedent for the biology (not traditional DD) both assays cell-basedInitial aim screen 1100 compounds through assays then IC50 determination of activesAlso needed to measure cyto-toxicityProject objective was to generate compounds with novel IP for future collaboration with Pharma or sale of assets


Project sponsor was e-Therapeutics who employed Aurelia Bioscience to perform the biology and a chemistry CRO to deliver medicinal chemistry support

Initial Biology Delivery

>10% Hit rate from the initial 1100 setExcellent correlation between the two assaysSAR tables for active compounds in both assaysSelect desirable chemotypes for hit-to-lead optimization workChemical design and library enumeration work around five chemotypes

5

ds

Differentiation assay

Reporter assay

1100 compoundsDifferentiation Assay (10µM) Ave % Inhibition

Cytotoxicity (10µM) Ave % Toxicity

144 compounds (>50% cut off)Differentiation Assay IC50 (nM)

Reporter Assay IC50 (nM)

SAR analysisChemotypes active in both assay

Selection of a further 440 compounds based on 2D/3D similarity


Biology resource transitioned to resource based, initially one then two biologists total length = 18 months

One lead and two back-up series were selectedInitial objective - to generate novel IP and improve PhysChemcharacteristics, while maintaining potencyAiming to achieve one lead series with LO properties

Potency in cells pIC50>8Clear IP positionDMPK characteristics sufficient for in vivo assessment (solubility (pH7.4), permeability, mouse microsomes & hepatocytes, PPB)

Aiming to synthesise in the region of 3x100 compounds over 6 monthsPLUS compounds from 2D/3D similarity searches

Total of 440 molecules for spot test then IC50Compounds shipped as DMSO stock solutions from Chemistry CRODMT cycle - Delivering every 2 weeksPrimary assay = cell based differentiation assay, key compounds also tested in reporter assay

Chemistry Delivery

6Aurelia Bioscience © 2018

7

Developed an FP binding assay for discrimination of hits based on binding of a fluorescently tagged standard agonist

Ideal profile was a non-binderAll of the 144 hits were tested as conc-response in this assay

Approximately half were non-binders

Selectivity Assay

Series A

Series B

Cell differentiation assay

FP Binding

Assay

The FP binding assay was added to the screening cascadeSeries A had the desired profile


A number of mutations in a key pathway protein were known to exist in patient population and affect drug resistance

Horizon generated 3 mutant cell lines (using CRISPR) in the same cell background as the differentiation assay

Anti-proliferative effects on multiple cell lines basal cell carcinoma, lung, ovarian, pancreatic (squamous cell carcinoma), medulloblastomaELISA assay for assaying changes in levels of a potential target protein in multiple cancer cell lines

Also identified a Cisbio HTRF assay for the target protein with improved sensitivity

Protein Simple Western blot Cell lysates from 4 cancer cell lines

QPCR for changes in key transcription factor on the target pathway

Mode of Action Studies

WT cell + TestMutant 1+ TestMutant 1+Std

WT cell + Test

Mutant 2+ Std

Housekeeping gene

Target gene


Identified 3 series of compounds with activity on the target pathwayDeveloped secondary assays to discriminate between moleculesAiming to achieve one lead series with LO properties

Potency in cells pIC50>8Clear IP position

Added value to the project with the addition of other MOA assays including working with the mutant cell lines and measuring changes in protein/mRNA Identified compounds taken forward into in vivo tumour models

HtL Measuring Success

The client has 3 patents on the IP generated in this project and is looking for risk share to move onto the next stage


Target = TRESK 2 pore non-voltage gated potassium channel

Disease indication = Migraine (prevalent, debilitating and costly)

The potassium channel TRESK is abundant in trigeminal ganglia. The mutation causes loss of function, thereby increasing TG excitability. TRESK is therefore a viable therapeutic target for migraineTRESK is activated strongly by second messengers such as calclum and volatile anaesthetics such as halothaneAt resting membrane potential, channel is closed but activated by calcium. As a membrane depolarizes, potassium outflow through open TRESK channels tends to restore the membrane potentialCompound targeting TRESK and to increase TRESK opening in the resting state would reduce excitability and responsiveness

Case Study Hit Identification example


Project Sponsor had no prior art or knowledge of chemistry, therefore wanted HTS of target (25,000 compounds)

HI Assay Protocol 25K compounds


HTS Assay Format

Fluorescence-based [Thallium]i detectionas a surrogate of [potassium]o influx

FluxOR

Target was transiently expressed in U2OS cells using a Bacmam construct

10000

13300

16600

19900

23200

26500

29800

33100

36400

39700

43000

00 1 2 3 4Time(seconds)

Multiple Well Overlay

Fluo

resc

ence

Cha

nge

(Cou

nts)

Test compounds normal (yellow)

Inhibited channel activity (blue)

Activated channel activity (red)

5ul

Range = ( 7800, 55000 )

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

Mini Graphs

Test Compounds

Hit Confirmation, IC50 and Selectivity

12

Hit confirmation rate ~65%

Highly reproducible

Retest of hits (n=2) followed by IC50 of hitsSelectivity required against TREK channel (heart) therefore selectivity assay developed

BL-1249

Control

TPA

Time (secs)

Res

pons

e (m

ax-m

in/b

asal

)

Almost all hits are

TRESK-mediated increase inThallium flux

log [Compound] (M)

% R

espo

nse

-8 -7 -6 -5 -40

100

200

300

400Cmpd 1Cmpd 2

CloxyquinCmpd 3


Critical Path Compound progression

13

Milestone U2OS recombinant hTRESK HTS (20K) 384-‐well Thallium Flux

Single Conc (n=1,10uM, 1%DMSO)

U2OS recombinant hTRESK384-‐well Thallium Flux

Single Conc (n=2, 10uM, Master Stock)

In Vivo (Neuropathic) Pain model;Seltzer -‐ sciatic nerve injury,

ligation, CCI

* If required

DMPKLog D, Clint, Permeability

Native functional assay;in vitro ephs

1o neuronal cultures (DRG, trigeminal ganglia)Healthy/patient stem cell derived neurons

Chemistry triage;Phys Chem properties

In vitro cytotox (HepG2)

In Vivo Migraine model;TRESK frame-‐shift KI,

TRESK WT KO, Mouse Grimace

U2OS WT cells384-‐well Thallium Flux

Single Conc (n=2, 10uM, Master Stock)

U2OS recombinant hTRESK384-‐well Thallium Flux

10pt CRC (EC50, n=2, 10uM tfc, Master Stock)

K2P Selectivity; TREK 384-‐well (Thallium

Flux)

Conventional ePhys;Recombinant hTRESK

(U2OS)

Early-‐phase Chemistry triage & re-‐synthesis

IC selectivity;Exteded K2P,HERG, Kv/ir/Cav/Nav (pain)

Broader selectivity

* Murine TRESK(Thallium Flux/ephys)

hTRESK 10pt CRC (Thallium Flux on re-‐supplied material)

Aurelia Bioscience driven assay biology


Aurelia Bioscience

Lab Technology and Assay Capabilities


EnSpire (PerkinElmer)Multimodal (label free)

FMAT (Life Technologies)Cell and bead fluor assays

FLIPR (Molecular Devices)Kinetic fluorescence

Envision Fluorescence /Luminescence

Capabilities - Readouts

CX5 ThermoFisher (Cellomics)High content screening assays


WES Protein SimpleHigh throughput Westerns

QuantiStudio 6 Life TechHigh throughput RT qPCT

Guava PCS-96 FlowCyte

Capabilities Liquid Dispensers


HP digital dispenser(non-contact dispenser)

Cybiwell(well to well dispensing)

Multidrops and (bulk reagent dispensing)

Case Study - Positive AllostericModulator (PAM)

Recombinant cell assay calcium mobilisation384 well adherent assay using HEK-293 cells expressing a G-protein coupled

cells grown by us for screen

FLIPR Flux Ca2+ or K+ Kinetic Imaging


1ul 'Standard' Compound1ul test compound1ul DMSO (negative control -‐ agonist read + PAM read)1ul 10mM Carabchol (positive contrl -‐ agonist read)1ul DMSO (positive contrl well -‐ PAM assay)

Human primary cell assay NeutrophilsFresh 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

Determine agonist EC20 on the day

Test compounds added on-line(agonist / antagonist activity)

Incubate 30 mins

Add EC20 for PAM activity (also run EC100)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24ABCDEFGHIJKLMNOP

EC20 CarbacholEC100 Carbachol (40uM)EC0 (Base buffer)

Agonist/Antagonist TemplatePAM Template

FMAT 8200 is a quasi confocal reader designed to perform fluorescent heterogenous assays in a homogenous mannerWe have extensive experience of developing whole cell and membrane binding assays for chemokine receptors using recombinant and primary cells

FMAT cell and bead assays

Aurelia Bioscience © 2018 1850uM plus detergent 25uM plus detergent

Control + Tween

Ligand concentration

Case Study - Client ran a program initially involving Biacore to find ligands that bound to the cell surface receptor and had cell-based assay far downstream of the target. The client wanted a better binding assay and a functional assay FMAT and Label-free were investigated

Ligand (nM)

Fluorescence and Luminescence Assays


Client required a comparison of AlphaScreen, Lance and HTRF for Epigenetic Enzyme JMJD2C. Assays were developed / purchased and a number of commercially available compounds were profiled and plates screened

HTRF Lance AlphaLISA

IC50 for 2,4-PDCA

187 nM 234 nM 41 nM

IC50 for NOG 2.15 uM 2.2 uM 0.36 uM

HTRF LANCE AlphaLISA

R e p ro d u c ib ility fo r H T R F - 3 h o u rs d e te c t io n

W e ll n o .

HT

RF

ra

tio

66

5/6

20

0 1 0 2 0 3 0 4 0 5 00

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

N o in h ib it io n

IC 8 0

IC 1 0 0

R e p ro d u c ib ility fo r L A N C E

W e ll n o .

LA

NC

E s

ign

al

at

66

5n

m

0 1 0 2 0 3 0 4 0 5 00

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

8 0 0 0 0

1 0 0 0 0 0

1 2 0 0 0 0

1 4 0 0 0 0

N o in h ib it io n

IC 8 0

IC 1 0 0

R e p ro d u c ib ility fo r A lp h a L IS A

W e ll n o .

Alp

ha

Lis

a s

ign

al(

co

un

ts)

0 1 0 2 0 3 0 4 0 5 00

5 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0

2 0 0 0 0 0

2 5 0 0 0 0

3 0 0 0 0 0

3 5 0 0 0 0

4 0 0 0 0 0

4 5 0 0 0 0

N o in h ib it io n

IC 8 0

IC 1 0 0

Case Study - JMJD2C Histone H3 Demethylase detection technology comparison

Client had a 96 well cell-based reporter assay using response element controlling luciferase production required us to re-start assay to screen compounds

Found that some compounds interfered with the luciferase activityDesigned, developed and implemented a selectivity assay to detect these interference compoundsIterative screening on a weekly basis

Case Study - Luminescence Reporter Assay

Cell based assayBiochemical assay

Developing AlphaScreenassay to measure protein

DMSO

Dynamic Mass Redistribution/ morphological changes

Change in Refraction index

Final readmonitored as response (pm)

Label free Cell-‐based assays -‐ Concept

Broad band light source Reflectedwavelength

Substrate

Wave Guide

Surface

Baseline Read

Cell

Detection

Window

Ligand binding to the receptor

The advantages of cell-based assays are; generic, requires no genetic manipulation, phenotypic, non-invasive, no interferencefrom auto-fluorescence


Label Free Combination Multiplexed Assay


Case Study - Histamine H1 PathHunter (DiscoveRx) beta-arrestin cell line

Time (secs)

Respon

se (p

m)

Cells treated with Histamine and label free measured over 30 mins

[Histamine] (uM)10-5 10-4 10-3 10-2 10-1 1 101

Res

pons

e (p

m)

20

40

60

80

100

120

140

160

180

200

220

240

EC50 = 50nM

[Histamine] (uM)10-4 10-3 10-2 10-1 1 101

Res

pons

e (R

LU)

400006000080000

1e51.2e51.4e51.6e51.8e5

2e52.2e52.4e52.6e52.8e5

3e5

EC50 = 61nM

Respon

se (p

m)

Time (secs)

Antagonist addition

Agonist addition

DiscoveRx detection technology (equilibrium)

IC50 values (nM)

AntagonistLabel free

Beta arrestin(in LF plate) FLIPR

Certirizine 120 22 90Chlorpheniramine 160 12 76

Triprolidine 40 4 26Pyrilamine 50 4 34

Human Primary Cells - Neutrophils

Respon

se (p

m)

0.01 0.1 1 10-200

0

200

400

600

800

1000

1200

Res

pons

e (p

m)

[IL8] (nM)

EC50 = 1nM

Neutrophils isolated using density gradient centrifugation, incubated in label free plates (non-adherent) and treated with agonist / antagonists

CellInsightT High Content Screening Platform

The CellInsightTM CX5 is a widefield imaging platform

There are more than 30 pre-built applications including cell cycle, autophagy, apoptosis and angiogenesis. Custom analysis algorithms can also be established

Imaging

Analysis

Data

Simultaneous image acquisition and data collection allows high-throughput quantitative microscopy

The CellinsightTM CX5 contains a bright-field channel plus 5 lasers allowing the multiplexing of fluorophores

-9 -8 -7 -60

2 0

4 0

6 0

L o g [A g o n is t] (M )

%H

IGH

_C

irc

Sp

otT

ota

lAre

aC

h3

Nuclei RFP Composite

Case Study: A client wanted to measure the movement of an RFP-tagged protein from the cytoplasm to the nucleus following agonist addition. The RFP tagged protein was imaged and the fluorescence quantified to give a dose-response curve

EC50 = 54nM

WES High Throughput Western Blotting


WES (from Protein Simple) blotting looking at protein generation and secretion from cells.

Between 12 and 24 samples can be run in 3 hours together with molecular weight markers

Examination of Autophagy marker LC3 in U2OS cells following treatment with compounds

Flow CytometryGuava EasyCyte plus

96 well and tube488nM laser can excite in green, yellow and redNumber of compatible fluors inc FITC, Alexa 488, R-Phycoerythrin (PE), PE-TexasRed, PerCp, PropidiumIodide, 7-AAD,

Assays include: cell health, cell cycle, apoptosis, surface marker staining, cytokine release

Co n tr

o l

No c o d a zo

le 5

uM

No c o d a zo

le 1

uM

No c o d a zo

le 0

.2 u

M

No c o d a zo

le 0

.04 u

M

No c o d a zo

le 0

.00 8 u

M

No c o d a zo

le 0

.00 1 6 u

M

0

2 0

4 0

6 0

8 0

1 0 0

T H P -1 c e lls t re a te d w ith N o c o d a z o le fo r 2 4 h o u rs

T re a tm e n t

Ce

lls

Co

un

t (%

)

H ea lthy

N e c ro t ic

E a rly A p o p to t ic

A p o p to tic

Stained for Annexin V and 7-AAD Human T cells stained with CFSE

following IL-2/CD3-CD28 exhaustion

THP-1 to macrophage

expression

CD11b expression following PMA treatment

THP-1 untreated

THP-1 PMA

Internal R&D 3-D biology on ElectroSpun Scaffolds

Electrospun scaffolds made of Poly-L-Lactide of various sizes (100 to 1000 micron) that are islands to grow cells in 3-D and are:

MagneticCryo-perservableTransfectableEasy to handleReproduce pharmacology from cellsLabelled with fluorescence dyes

100 - 1000 micron

During manufacture Under SEM Under microscope

Environmental SEM Epithelial cells 24hrs post inoculation

Environmental SEM Epithelial cells 96hrs post inoculation

Fibroblasts 24hrs post inoculation

Note covering of fibres with cytoplasm Note cells completely engulf scaffold Note fibroblasts also cover scaffold

-2 -1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

1 2 0

T ra n s fo rm o f A d h e re n t

L o g [ fo rs k o lin ] ( M )

% m

ax

re

sp

on

se

Log10 [forskolin] (µM)

1.2 µM

3D adherentExemplierpharmacology response on scaffolds by cells transfectedwith CRE-luciferaseand stimulated with forskolin %

Max

imal

resp

onse

-2 -1 0 1 20

2 0

4 0

6 0

8 0

1 0 0

1 2 0

T ra n s fo rm o f S ta b le C R E -L u c 2 P m a g n e t ic 9 6 -w e ll

L o g [ fo rs k o lin ] ( M )

% m

ax

resp

on

se

HEK293 cells - transient

0.5 µM

Log10 [forskolin] (µM)

72hrs post cryopreservation

25

Assay Development and Pharmacological Validation (for iterative or high throughput screening purposes)

Cell-based assayBiochemical-based assay

Assay Robustness (for HTS purposes)High Throughput Screening (HTS)

Cell-based assayBiochemical assay based

Exploratory Biology (technology or target)Iterative Cyclical Screening - Lead Identification (LI)Pharmacological Profiling of compoundsComplete Discovery Project from Assay Development through to LIReagent/equipment validation and benchmarking


Aurelia Bioscience Services

Contracts are based on milestones with clear GO/NO GO decisions agreed between client and ourselves

QuoteCostings broken down

Agreed Schedule of WorkMilestone 1: Feasibility study

Basic pharmacology in the agreed assay formatMilestone 2: Progressive study

Plate based pharmacology looking at statistics, robustness, QC

Ad hoc Iterative screening costs based per plateMilestone 3: Primary screen, retest and XC50Additional contracts for Hits to Lead (LI) work (iterative and/or selectivity screening

Contract can be terminated by either party at a milestone point dependent on level of success


Contract Design

Communication frequent and detailed through teleconference and face-to-face meetingsCompetency high given our backgroundTransparency of data provision of raw data, interpretation and written reportFlexibility understand and adapt protocols to needs of client depending on resultsSpeed of service able to work on projects quickly and turn around data in design/make/test, lead identification and lead optimisation cyclesUK-based, global outlook

Client focus


Kevin Hart PhDManaging Director

[email protected]

+44 (0) 115 8370503 (UK)

To find out more about Aurelia Bioscience go to: http://www.aureliabio.com


Gary Allenby PhDChief Scientific Officer

[email protected]

+44 (0) 115 8370503 (UK)

For further information, please contact :-

Aurelia Bioscience

· 7 ydeveloped an fp binding assay for discrimination of hits based on binding of a fluorescently...

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