Harnessing the Power of Bioluminescence€¦ · Harnessing the Power of Bioluminescence Neal Cosby,...

Biologically Relevant Assays for Oncology: Harnessing the Power of Bioluminescence

Neal Cosby, PhD, Cell Analysis Manager

Introduction

Carcinos (Greek for crab) attributed to Hippocrates

Source: Outlook for the Next 5 Years in Drug Innovation. Berggren, R et al. Nature Reviews Drug Discovery 11, 435-436 (June 2012), doi:10.1038/nrd3744

The study of cancer – Oncology – is clearly the single largest area of research in academia, government and the pharmaceutical industry. Just the number of clinical compounds demonstrates the breadth of interest

Cancer is Mainstream

HGF/SF:MET

“I’m fully convinced that cancer is a logical disease. That there is a logic to how the cancer develops, and if you understand the logic you can understand how to tackle it. So know your enemy. Cancer is our

enemy…”

- Dr. Lewis C Cantley, PhD www.standup2cancer.org/

Hedgehog

VEGF Akt

Outline

Physics & chemistry of bioluminescence

Optimized luciferases & reagents

Cell-based & Biochemical assays

Real-time, kinetic assays

Frozen, thaw-and-use cells

Custom Assay Services

Physics & Chemistry of Bioluminescence

In bioluminescence, the excited state of the photon emitter is from a chemical reaction

Bioluminescence is not affected by:

Excitation and emission wavelength overlap

Fluorescent chemicals in media

Background fluorescence

The Physics of Bioluminescence

Method used to create excited state photon determines which applications a technology is best suited for.

Lack of background luminescence results in better sensitivity (up to 4 logs) and a greater dynamic range, ideal for microwell-based assays.

The Chemistry of Bioluminescence

Can measure Luciferase Reporter gene assays Cell Signaling analysis: GPCR, RNAi, nuclear receptors, transcription regulation

Can measure ATP Cell Viability & Toxicity cAMP & PDE assays Kinase assay (enzymatic)

Can measure Luciferin Caspases & proteases CYP450 assays GSH/GSSG, Viability/Toxicity HDAC/SIRT

C O O - H O

+ L i g h t

+ A M P + C O 2

Advantages with Luciferase Assay Chemistry

• Superior performance for miniaturized assays – better signal-to-noise ratios

• Increased sensitivity, lower background

• Broad linear range

• Less interference from fluorescent compounds

• Ability to multiplex with fluorescent assays

• Luminescence detection transfers from biochemical to cell-based to animal studies seamlessly

Pictured: Common Firefly (Photinus pyralis) 9

Optimized Luciferases & Reagents

Reporter Gene Assay Overview

Experimental

firefly luciferase

construct

Control

Renilla luciferase

construct

expression level

varies little with

treatment

PROMOTER DISSECTION

SIGNALING PATHWAY

NUCLEAR RECEPTORS

PROTEIN INTERACTIONS

POST-TRANSCRIPTION MIRNA CONTROL expression level

varies with treatment

Reducing Background from Backbone

pGL3 Vector series backbone was redesigned to remove 75% of known cryptic transcription factor binding sites. This resulted in the pGL4 Vector backbone series with greatly improved performance.

Increasing Reporter Gene Expression

Brighter luminescence over luc+ in pGL3

luc2 Firefly Luciferase: Codon optimized for mammalian cells Removal of 90% of cryptic transcription factor binding sites found in luc+ of pGL3

Stability of Ultra-Glo Luciferase to ionic detergents

(0.002% SDS)

Directed evolution was used to enhance the structural stabilization of enzyme:

27 mutations increased thermostability to ~70°C

The most stable luciferase commercially available (native luciferases are stable to ~30°C

Robust to chemical and physical environment

Stable to storage conditions (e.g., > 6 wks at RT in solution)

Proprietary reagent formulations further protect light emission process

Stabilized Recombinant Luciferase for Improved Reagent Performance

Ultra-Glo™ Luciferase

Native Luciferase

Bioluminescence Means Reduced Interference

Screen of 198,899 compounds • 0.9% inhibited native

luciferase • 0.1% inhibited Ultra-Glo

This Adds Up to…

Smart, optimized genetic reporter constructs with ‘pic & choose’ features (promoters, MCS, etc)

Optimized substrates – superior reagent performance

Efficient transfection reagents to

Stable and robust (novel) enzymes

Flexibility without compromising performance, assay-to-assay

See: www.promega.com/pGL4

A ‘Next Gen’ Reporter Molecule – NanoLuc Offers New Opportunities

NanoLuc – A New Reporter

NanoLuc Luciferase as a reporter

Full-Length Nluc

Destabilized, full-length NlucP

Secreted, full-length secNluc

NanoLuc Luciferase as a fusion partner

Protein Translocation

Protein Stability

Protein:Protein Interactions

Receptor Interactions

Biosensors

“Applications of a Smaller Brighter More Versatile Luciferase Reporter”, Tuesday, November 13, 2012 Promega webinars: www.promega.com/resources/webinars/

Coelenterazine

19kOluc

“Advanced Technologies Group” Hall, MP et al. (2012) ACS Chem Biol in press

Coelenterazine

NanoLuc™ Luciferase

81,000X

enzyme evolution

Evolution of NanoLuc: From Ocean to Lab

130kDa Oplophorus luciferase

7X brighter than native Renilla Luciferase

Shimomura, O et al. (1978)

19kOluc 19kDa subunit is catalytic.

Light output & stability compromised.

Inouye, S et al. (2000)

Coelenterazine

Oplophorus gracilirostris first cataloged in 1881

Furimazine

NanoLuc™ Luciferase

2,500,000X

substrate evolution

Glo Glo Glo

NanoLuc is Small & Bright

Firefly (Fluc) Renilla (Rluc) NanoLuc (Nluc)

Amino acids

M.W. Mol.

Vol. Å3

Nluc 171 19.1 14

Rluc 312 36.0 32

Fluc 550 60.6 44

Recombinant NLuc/Nano-Glo Assay Recombinant FLuc/ONE-Glo Assay Recombinant Rluc/Renilla-Glo Assay

NlucP Provides the Greatest Dynamic Response

Nluc: 79X brighter than Fluc

NlucP: 10X brighter than FLucP

Nluc: 34X brighter than NlucP

→ Similar pharmacology & EC50 values

Experimental details: transient transfection of HEK293 cells with NF-kB inducible constructs. rhTNFa treatment for 5 hours.

0.001 0.01 0.1 1 10 100104

FlucNlucP

TNFa ng/mL

Brightness:

Nluc > NlucP > Fluc > FlucP

NanoLuc as a Fusion Partner

Prot X

NLuc Prot Y

HT NLuc DEVD

BRET Caspase 3

Ligand

Protein

Stability

Protein-Protein Interactions

Biosensors

Receptor

Interactions

Protein

Translocation

NanoLuc Fusions Can Go Anywhere…

NLuc-Nrf2 NLuc-b2 AR NLuc-MLS Calreticulin- NLuc-

Mitochondria ER Anchored Nucleus Cell-Surface

Visualized with an Olympus LV200 microscope

Is NanoLuc a Better Luminescent Donor for BRET?

Donor brightness is a key limiter to current BRET technologies. NLuc

Fluor Acceptor

More spectral overlap needed to get sufficient signal

BRET-beneficial aspects of NanoLuc Luciferase:

~100-fold brighter than Rluc need less spectral overlap with fluor gain greater spectral separation

RLuc → GFP

NanoLuc & HaloTag Partnered for BRET

BRET Emission Spectrum BRET Ratio (= Emacc /Emdon )

OR TMR0.00

untr + HT ligand

HT-Ligand

400 450 500 550 600 650 700

wavelength

TMR NL OR

Oregon Green® BRET spectra

Tetramethyl- rhodamine (TMR)

BRET Spectra

NanoLuc emission

NLuc HT BRET

Donor Only

Donor + Acceptor

NanoLuc-EGFRTitration of TMR-EGF

0 20 40 60 80 1000.02

0.06 no cold EGF

200ng/mL cold EGF competition

[EGF-TMR], ng/mL

(650/4

NLuc-EGFRtitration of cold EGF onto fixed TMR-EGF

10- 3 10- 2 10- 1 100 101 102 103

IC50 = 2.7ng/mL

(literature Kd = 1-5 ng/mL)

[unlabeled EGF], ng/mL

(590/4

Tetramethylrhodamine-EGF signaling

Luc Luc

Ligand

Luc Luc

BRET with EGF and EGFR

Biochemical Assays - Kinases

Kinases Orchestrate Complex Biological Processes

Kinases play a critical role in human biology and cancer

Important components of cell signal transduction

Regulation of many cellular processes through phosphorylation of diverse substrates (proteins, lipids, sugars…)

Over 500 protein kinases in human genome

More than one-third of all human proteins are phosphorylated

ADP Kinase

ATP + + P

Phospho-Substrate

Single Assay Platform - Many Applications

High-throughput screening

Kinase inhibitor profiling

Mode of action studies

ADP-Glo Assay: a universal in vitro biochemical assay

for many types of kinase studies

“ADP Detection Platform for Kinase Inhibitor Screening, Mode of Action Studies and Profiling”, Thursday, May 23, 2013 Promega webinars: www.promega.com/resources/webinars/

Bioluminescent ADP Assay Principle

Light output is correlated with the amount of ADP produced

Directly correlated with the amount of kinase activity

Against a Panel of Different Families Against a Kinase Family

Profiling Kinase Inhibitors

Selectivity Profile of Wortmannin Toward PI3 Kinase Family

Selectivity Profile of Wortmannin Against Panel of 9 Kinases

Log(Wortmannin), µM

Promega Kinase Panel Offering

Broad human kinome coverage >170 Kinase Enzyme Systems: www.promega.com/a/kinase/

An ideal profiling panel will include close & distant kinases to assess compound selectivity

*Vintage

Cell-Based Assays & Multiplexing

The Most Sensitive ATP Bioluminescent Cell Viability Assay

Now available as a frozen, ready-to-use reagent based on the original CellTiter-Glo Luminescent Cell Viability Assay chemistry: eliminates the need to combine buffer with lyophilized substrate when preparing reagent.

CellTiter-Glo Luminescent Cell Viability Assay

CellTiter-Glo 2.0

New formulation that is stable as a liquid at 4⁰C

If the reagent is to be used within a few months, it can be stored at 4⁰C. This eliminates any thawing requirements prior to use. The reagent need only be placed in a room temperature water bath for a short period of time or it can simply be left out at room temperature the day before use.

If the reagent is not intended to be used for several months, it can be stored at -20⁰C for several years.

0 1 2 3 4 5

time at 22 C (weeks)

CellTiter-Glo®new reagent

Advantages & Disadvantages of Viability Assays

Assay Advantages Disadvantages

MTT/MTS Widely used Inexpensive

MTT has 2 step protocol 1-4 hour incubation Interference by reducing compounds Toxic to cells Limited sensitivity

Resazurin Inexpensive Fluorescent readout Good sensitivity

1-4 hour incubation Interference by reducing compounds Toxic to cells Fluorescence interference

Protease 30 min protocol Cells remain viable Better sensitivity than resazurin Good choice for multiplexing

Fluorescence interference

ATP 10 min protocol Best sensitivity No fluorescence interference Lysis step stops reaction immediately (no incubation with viable cells)

Lytic protocol dictates sequence for multiplexing

Examples from AACR 2013

1. Poster 3832 Cancer Tissue-originated Spheroid, CTOS, for Evaluation of Drug Response from Individual Patient Tumor Samples Inoue, M et al. Osaka Medical Center for Cancer and Cardiovascular Diseases

2. Poster 3839 Development of a 3-Dimensional Synthetic Lethality Screening Approach Targeting KRas-mut Cells Tsuji, T et al. Celgene

3. Poster 3847 High-throughput 3D Screening Reveals Differences in Drug Sensitivities Between Culture Models of JIMT1 Breast Cancer Cells Hongisto, V et al. Biotechnology for Health and Well-being, VTT Technical Research Centre of Finland, Turku, Finland. Department of Genetics, Institute for Cancer Research, Division of Surgery and Cancer, Oslo University Hospital Radiumhospitalet, Norway. Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland.

Examples from AACR 2013

1. Poster 3832 Cancer Tissue-originated Spheroid, CTOS, for Evaluation of Drug Response from Individual Patient Tumor Samples

Cancer tissue-originated spheroid or “CTOS” as a more biologically relevant cancer cell model. Material was derived from surgical specimens. Looked at pathway activation of the EGFR tyrosine kinase inhibitor (TKI) erlotinib Some Conclusions: CTOSs can be prepared from primary colorectal, lung, urothelial tumor cells

with high viability and purity. Drug sensitivity and intracellular pathway activation can be assessed by

CTOSs. (e.g., Her3 signaling is important for CTOS growth). CTOS can be a new platform for studying biology of various cancers, and be

useful to find biomarkers and new targets. CellTiter-Glo was used to assess cell viability

Examples from AACR 2013 (cont)

2. Poster 3839 Development of a 3-Dimensional Synthetic Lethality Screening Approach Targeting KRas-mut Cells

3D in vitro model was developed to better represent features of actual tumors. In other words, 3D in vitro model is more physiologically relevant. Researchers established a 3D spheroid culture approach for high-throughput screening for compounds that selectively kill cancer cells with distinct genetic backgrounds. Some conclusions: Created physiologically relevant “mini tumor” for 384-well HTS screening.

High Z’ values indicating ideal assay performance. Used the model to identify differences in KRas-wt vs -mut. In primary screen of >30,000 compounds, obtained Z’ values of 0.738, with

0.63% hit rate and 50% proliferation inhibition. CellTiter-Glo was used in primary screen to assess cell viability

Examples from AACR 2013 (cont)

3. Poster 3847 High-throughput 3D Screening Reveals Differences in Drug Sensitivities Between Culture Models of JIMT1 Breast Cancer Cells

This group also used a 3D model due to improved cell-to-cell contacts and structures that resemble in vivo architecture. Aim was to develop a simple high-throughput 3D drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in 2D. Some Conclusions: JIMT1 cells were more sensitive to drugs when cultured in 3D, and gene

expression pattern more closely resembled gene expression of xenograft cultures.

Multiple 3D culture methods are adaptable to high-throughput screening platforms.

The cell culture system used has a big impact on drug responses, gene expression patterns and cell signaling pathway activities.

CellTiter-Glo was used to assess cell viability

Measure Live, Dead or Both before Reporter Assays

1. Live-Cell Protease Assay (CellTiter-Fluor Cell Viability Assay)

+ 3. Dead-Cell Protease Assay

(CytoTox-Fluor Cytotoxicity Assay)

Live- & Dead-Cell Assay (MultiTox-Fluor Multiplex Cyto Assay)

Ratiometric live:dead data Firefly

Renilla

GF-AFC

bis-AAF-R110

Rhodamine

bis-AAF-R110

Live-Cell

Protease

Dead-Cell Protease

bis-AAF-R110

1. Live-Cell Substrate (GF-AFC) freely diffuses across membranes

2. Dead-Cell Protease Substrate cannot cross intact membranes

3. Compromised membranes allow Dead-Cell Protease access to the substrate

Learn more: Niles, AL et al. (2007) Analytical Biochemistry 366, 197-206 41

Kinetic Assays

Monitor Gs- and Gi-coupled GPCRs Using GloSensor cAMP Assay

Monitor cAMP Levels in Real-time with a Live-cell, Non-lytic Bioluminescent Assay

GloSensor™

Plasmid

Culture cells with GloSensor cAMP Reagent

Treat cells with compounds

Monitor cAMP-dependent luminescense

EXAMPLE SMALL MOLECULE SCREENING SOLUTION:

Create a stable cell expressing both the receptor of interest and the live-cell biosensor for cAMP. We work with your cell line or cells obtained from a commercial vendor (e.g., ATCC). We test the assay and ensure it performs to your specifications (i.e., Z’, S:B). The assay is ready to run in your workflow.

Assay Designs, Cat.# 901-066, acetylated

Day 1: Plate HEK293 15,000 cells/well, 96-well plate

Day 2: Transient transfection using Mirus TransIT-LT1

Day 3: GloSensor cAMP, equilibrate w/ substrate for 2 hrs at RT; EIA,

incubate cells for 2 hrs in medium alone

GloSensor cAMP, sequential addition of compounds & continuous RLU measurement; EIA,

same, except addition of 0.1 M HCl to separate samples at indicated time pts; 28°C

EIA dilutions: 2-fold dilutions w/ buffer 1-6’; 4-fold dilutions w/ buffer 15-30’

GloSensor cAMP Assay is as Sensitive as an Immunoassay

DNA Binding Dye for Cytotoxicity Measurements

Viable Cell

Dye is excluded from live cells

DNA dye only stains nucleus of “dead”

cells or debris

Non-permeable DNA dye

Staining of dead cells results in a stable fluorescent signal

Dead Cell

DNA dye: stain to detect dead cells (overcomes some

limitations of short half-life markers)

CellTox Green Assay & Stable Toxicity Marker

2° Necrosis (Apoptosis)

HeLa Cells: CellTox Green Dye + test compound in “zero step” format – add dye directly to medium at start of cell culture

Signal increases over 3-day

period

DNA Dye is Not Toxic to Cells

Incubation of DNA dye with cells for 72hr has no effect on viability as measured using the CellTiter-Glo ATP Assay

DNA dye does not affect IC50 of model compounds in 72hr co-incubations

Multiplexing DNA Staining & ATP Assays

Add DNA dye when seeding cells

Add CellTiter-Glo®

Reagent

Record fluorescence from dead cells (multiple times)

Record luminescence from live cells

Incubate 72hr

“CellTox Green: A Cytotoxicity Assay That Fits Your Time Line”, Tuesday, March 12, 2013 Promega webinars: www.promega.com/resources/webinars/

Cell Systems Featuring Frozen, Thaw-and-Use Cells

Classic vs. Reporter Bioassay for ADCC

ADCC is antibody-dependent, cell-mediated cytotoxicity, which is a main line of immunological defense in mammals. Classic ADCC assays rely on PBMCs with readout depending on degree of lysis of target cells. The current assays are: • highly variable • tedious and poor reproducibility

…just to name two main drawbacks

Classic vs. Reporter Bioassay for ADCC (cont)

Our novel ADCC Reporter Bioassay features genetically engineered Effector cells with

readout being from these cells. Jurkat cells possess both human FcgRIIIa and NFAT-luc

reporter, as a double stable construct. The reporter bioassay reflects ADCC mechanism of action is highly reproducible with low

variability a superlative potency lot release assay or can be used to screen antibodies

ADCC Reporter Bioassay

‘Cells as Reagents’ Means…

1. Human cell lines (Jurkat, WIL2-S, Raji) Developed as thaw-and-use for immediate use in bioassay Designed to give good recovery & robust response upon thawing

2. Thaw-and-Use format

Cell propagation conditions & defined freezing protocol control assay performance for a consistent bioassay response No pre-culturing prior to assay means less variability introduced

Indefinite storage Identical cells in bioassay, day-to-day 3. Minimizes pre-assay planning, time & labor Ample cell banks provide long-term supply

Frozen, thaw-and-use cells

No cell culture required with cells in frozen, thaw-and-use format

Frozen, Thaw-and-Use WIL2-S Target Cells in ADCC Reporter Bioassay

Bioassays using Kit control Ab Bioassay using Rituximab drug

ADCC Reporter Bioassay response to ADCC Bioassay Control Antibody (left) or Rituximab (right). The EC50 response using frozen, thaw-and-use WIL2-S cells was 16.8ng/ml for Control Antibody, Anti-CD20. For Rituximab, it was 1.94ng/ml.

Specifics: E:T ratio = 6:1; 6r induction; Bio-Glo™ Luciferase Assay System

Complete QC on Cells

Production cell batches are rigorously tested: STR analysis – cell ID profile (human) CO1 analysis (cytochrome oxidase) – test

for presence of species (human and other potential contaminants)

Cell doubling time under propagation conditions

Mycoplasma (Hoechst and direct culture) Sterility Cell density Cell viability after thaw Fill volume ADCC Reporter Bioassay (EC50 and fold

induction)

“ADCC Reporter Bioassay: A Novel, Bioluminescent Cell-Based Assay for Quantifying Fc Effector Function of Antibodies”, Tuesday, October 23, 2012 Promega webinars: www.promega.com/resources/webinars/

We’ve used our capabilities to make many custom assays, based on both bioluminescence and fluorescence.

CELL ENGINEERING Target expression in both normal & disease states

ASSAY DEVELOPMENT & QUALIFICATION Target class expertise & multiple assay formats

ASSAY READY CELLS IN-SCALE Functionally tested; unparalleled client support

CUSTOM ASSAY MATERIALS See our suite of currently available vectors and cell lines developed under Custom Assay Services (www.promega.com/cam ). And we provide all the post-delivery support to ensure your assay works in your hands.

For more information, see www.promega.com/CAS

Thank you

Neal Cosby, PhD Strategic Marketing Manager Neal.cosby@promega.com

Harnessing the Power of Bioluminescence€¦ · Harnessing the Power of Bioluminescence Neal Cosby,...

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