©2008, Promega Corporation. All rights reserved.

Cell-based Assays Using Luciferase Biosensors

Frank FanCASSS Nov.2010

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Outline

Challenges in building cell-based potency bioassays

Current assay formats and technologies

Sensors versus genetic reporters

Luciferase biosensor (cAMP as example)

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Requirements for potency bioassay

Reflect mechanism of action

Reflect structural changes that impacts product’s efficacy

Stability indicating

High assay quality: precise, accurate

Usable as a QC lot release assay

Increasing need for functional CELL-based bioassay

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The goal: complex biology---simple assay

Add-Mix-Read

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The challenge: control many variables to build good cell-based bioassays

Cellsculture condition, media (storage, thawing, growth), BSA, seeding density, passage number,confluency, viability, genetic identity, format (suspension vs attached), mycoplasma test

Engineered cells introduction methods, chromosomal integration, stability over passages

Assay formatease of use, time, robustness, ruggedness, assay steps

Reagentlot-to-lot variation, manufacture process, robustness

Instrumentationsensitivity, linearity, reliability, standards, internal controls

Recent CBBA examples showed EXCELLENT performance!

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Assay formats and technologies

Reporter assays

Apoptosis assays

Viability assays

TNFα effectsHTRF

HCA

ELISASPA

SPR

Radiometric

Luminex

Alpha Screen

EFC

PCA

FRET

FLIPR

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A platform approach using reporter assay

Response Element/Promoter REPORTER

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Advantages of reporter bioassay

Suitable for many receptors and biologics

Confer specificity to certain biologics

One Response Element for multiple biologics

Proven high assay quality

Usable as a QC lot release assay

Familiarity by regulators

How can it be improved further?

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Sensors versus reporters

Genetic reporter measures changes in luciferase AMOUNTS in the sample to reflect promoter activity

Luciferase biosensor measures changes in luciferase ACTIVITY in the sample to reflect the amounts of modulator

CRE

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Luciferase catalytic mechanism

Closed

C

N

Open

N

C

N

S N

S

COO-HO

N

S N

S

CHO

N

S N

S

O-OO

AMP

PPi

+ Light

ATP

H+

O2

+ AMP+ CO2

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Luciferase biosensor design

NN

CC

New N- &C-terminiNew N- &C-termini

Fuse wt N- &C-termini

Fuse wt N- &C-termini

Luminescent biosensors are created via fusion & circular permutation of

firefly luciferase

PeptidesPeptides

Analyte binding domainAnalyte binding domain

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Protease biosensors in vitro

Cell-free expression followed by incubation with cognate proteases

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cAMP is an important analyte for bioassay

Luciferase biosensor

Luciferase reporter

For measurement of cAMP, luciferase biosensor provides a more direct, faster and better assay.

GPCR is a major drug target class for both chemical and biologics drug discovery

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Luciferase biosensor for cAMPin vitro dose responsefollowing cell-free expression

-9 -8 -7 -6 -5 -4 -30

10

20

30

40

50

60

70 pGloSensor-20F cAMP

log[cAMP] (M)Fo

ld r

espo

nse

EC50 = 0.53 µM

RIIβB domain

cAMP

pGloSensor-20F cAMP (20F)

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A Live cell, non-lytic assay format

Pre-equilibratew/ substrate

COO-NN S

SHO

Kinetic or end-pointmeasurementswithin minutes

Mix compounds& cells

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Assay performance in 3456-well formatAgonist: endogenous β2-adrenergic receptor

Collaborator data

Stable expression in HEK293 cells/+ PDE inhibitor

Data from Eric Johnson, Merck

S/B = 9.6Z’ = 0.78

25 µM ISO at 15 minutesR

LU

Plate Column

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0 10 200

5.0×104

1.0×105

1.5×105

2.0×105

2.5×105

NECAForskolin

Isoproterenol

DMSOdH2O

Time (min)

Lum

ines

cenc

e (R

LU)

Transient expression in HEK293 cells 10 µM compounds; n = 1 per trace

Kinetic measurements for mechanism study

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0.0 2.5 5.0 7.5 10.0 12.5 15.0103

104

105

106

ClozapinePerphenazine

ButaclamolDopamine

DMSO

Addition

Time (min)

Lum

ines

cenc

e (R

LU)

Inverse agonist detection w/o FSK

Left, 20F construct/dopamine D1 receptor HEK293 cell line; assay at 28 °C; compounds at 10 µM

cAMP biosensor assay can detect inverse agonist for Gs and agonist for Gi without foskolin

-12 -11 -10 -9 -8 -7 -6 -50.30.40.50.60.70.80.91.01.1

log[PGD2] (M)

Fold

res

pons

e(v

s. t

= 0)

FSK-free Gi-coupled receptor assays

Right, transient expression of 20F in HEK293T/GPR44 cell line (Multispan, Inc.); n = 1 per dose

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• 13 steps (≥ 3 hours to perform)• Highly cited in peer review literature• Dynamic range 78 pM – 20 nM

Plate surface

cAMP-Alk Phos conjugate

Polyclonal1° antibody

Immobilized2 ° antibody

ρ-nitrophenyl phosphate Signal at 405 nm

APcAMP

Wash 3x

-11 -10 -9 -8 -70

25

50

75

100

125 EIA standard curve(acetylated)

EC50 = 0.85 nM

log[cAMP] (M)

% a

ntib

ody

boun

d

EIA standard curve (acetylated)

EC50 = 0.85 nM

Comparison to lytic immunoassay (EIA)

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0 10 20 301.0×103

1.0×104

1.0×105

1.0×106

GLS 22FImmunoassay (Assay Designs)

0.1

1

10

100

ISO

PRO

FSK

Time (minutes)

Lum

ines

cenc

e (R

LU)

[cAM

P] (nM)

Comparison to lytic immunoassay (EIA)

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Advancement of cAMP assays

Immuno-assay Reporter assay Sensor assay

Inc time minutes hours minutes

Assay steps 13 1 0

Assay time Day minutes 0

Improvements will allow reduced assay variability and better bioassay

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Add-Mix-Read

Complex Biology ---- Simple Assay

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Supporting Scientific Publications• Binkowski, B et al. Live-Cell Luminescent Assays for GPCR

studies. Genetic Engineering and Biotechnology News. 29: 30-31, 2009.http://www.genengnews.com/articles/chitem.aspx?aid=3037

• Kimple, AJ et al. Structural Determinants of G-protein αSubunit Selectivity by Regulator of G-protein Signaling 2 (RGS2). J Biol Chem 284: 19402-19411, 2009DOI: 10.1074/jbc.M109.024711

• Binkowski, B et al., Engineered Luciferases for Molecular Sensing in Living Cells. Curr Opin Biotechnol 20(1): 14-18, 2009 DOI:10.1016/j.copbio.2009.02.013

• Fan, F, Binkowski, B et al. Novel Genetically Encoded Biosensors Using Firefly Luciferase. ACS Chem Biol 3 (6): 346–351, 2008

• Wigdal, S et al. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase., Curr Chem Genomics1: 94-106, 2008

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Acknowledgement

• Brock Binkowski• Braeden Butler• Susan Wigdal• Pete Stecha• Keith Wood

•Lance Encell•Paul Otto•Kris Zimmerman•Gedi Vidugiris•Monika Wood•Ethan Straus