Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
©2013 Promega Corporation.
Biologically Relevant Assays for Oncology: Harnessing the Power of Bioluminescence
Neal Cosby, PhD, Cell Analysis Manager
©2013 Promega Corporation.
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
2
©2013 Promega Corporation.
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/
BRAF
HER
Hedgehog
VEGF Akt
MEK
Ras
PI-3K
3
©2013 Promega Corporation.
Outline
Physics & chemistry of bioluminescence
Optimized luciferases & reagents
Cell-based & Biochemical assays
Real-time, kinetic assays
Frozen, thaw-and-use cells
Custom Assay Services
4
©2013 Promega Corporation.
Physics & Chemistry of Bioluminescence
©2013 Promega Corporation.
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
6
©2013 Promega Corporation.
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.
7
©2013 Promega Corporation.
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
8
©2013 Promega Corporation.
N
S N
S
C O O - H O
N
S N
S
C H O
N
S N
S
O - O
O
A M P
P P i
+ L i g h t
A T P
H +
O 2
+ 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
©2013 Promega Corporation.
Optimized Luciferases & Reagents
©2013 Promega Corporation.
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
11
©2013 Promega Corporation.
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.
12
©2013 Promega Corporation.
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
13
©2013 Promega Corporation.
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
14
Ultra-Glo™ Luciferase
Native Luciferase
©2013 Promega Corporation.
Bioluminescence Means Reduced Interference
Screen of 198,899 compounds • 0.9% inhibited native
luciferase • 0.1% inhibited Ultra-Glo
15
©2013 Promega Corporation.
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
16
©2013 Promega Corporation.
A ‘Next Gen’ Reporter Molecule – NanoLuc Offers New Opportunities
©2013 Promega Corporation.
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/
18
©2013 Promega Corporation.
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
19
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
Glo
Oplophorus gracilirostris first cataloged in 1881
Furimazine
NanoLuc™ Luciferase
2,500,000X
substrate evolution
Glo
Glo Glo Glo
©2013 Promega Corporation.
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
20
©2013 Promega Corporation.
NlucP Provides the Greatest Dynamic Response
21
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
105
106
107
108
109
101 0
FlucP
FlucNlucP
NLuc
TNFa ng/mL
Lu
min
escen
ce (
RL
U)
Brightness:
Nluc > NlucP > Fluc > FlucP
©2013 Promega Corporation.
NanoLuc as a Fusion Partner
Prot X
NLuc Prot Y
HT
BRET
HT NLuc DEVD
BRET Caspase 3
Ligand
Re
c
Re
c
BRET
NLuc
Protein
Stability
Protein-Protein Interactions
Biosensors
Receptor
Interactions
Protein
Translocation
22
©2013 Promega Corporation.
NanoLuc Fusions Can Go Anywhere…
NLuc-Nrf2 NLuc-b2 AR NLuc-MLS Calreticulin- NLuc-
KDEL
Mitochondria ER Anchored Nucleus Cell-Surface
Visualized with an Olympus LV200 microscope
23
©2013 Promega Corporation.
Is NanoLuc a Better Luminescent Donor for BRET?
Donor brightness is a key limiter to current BRET technologies. NLuc
Fluor Acceptor
BRET
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
24
©2013 Promega Corporation.
NanoLuc & HaloTag Partnered for BRET
BRET Emission Spectrum BRET Ratio (= Emacc /Emdon )
OR TMR0.00
0.25
0.50
0.75
1.00
1.25
1.50
untr + HT ligand
0
5
10
15
20
S/B
HT-Ligand
BR
ET
Ra
tio S
/B R
atio
0
10000
20000
30000
40000
50000
60000
400 450 500 550 600 650 700
Re
lati
ve L
igh
t U
nit
s
wavelength
TMR NL OR
Oregon Green® BRET spectra
Tetramethyl- rhodamine (TMR)
BRET Spectra
NanoLuc emission
NLuc HT BRET
Donor Only
Donor + Acceptor
S/B
25
©2013 Promega Corporation.
NanoLuc-EGFRTitration of TMR-EGF
0 20 40 60 80 1000.02
0.03
0.04
0.05
0.06 no cold EGF
200ng/mL cold EGF competition
[EGF-TMR], ng/mL
BR
ET
RA
TIO
(650/4
60)
NLuc-EGFRtitration of cold EGF onto fixed TMR-EGF
10- 3 10- 2 10- 1 100 101 102 103
0.02
0.03
0.04
0.05
IC50 = 2.7ng/mL
(literature Kd = 1-5 ng/mL)
[unlabeled EGF], ng/mL
BR
ET
RA
TIO
(590/4
60)
Tetramethylrhodamine-EGF signaling
Luc Luc
Ligand
Luc Luc
P
P P
P
BRET with EGF and EGFR
26
©2013 Promega Corporation.
Biochemical Assays - Kinases
©2013 Promega Corporation.
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
28
©2013 Promega Corporation.
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/
29
©2013 Promega Corporation.
Bioluminescent ADP Assay Principle
Light output is correlated with the amount of ADP produced
Directly correlated with the amount of kinase activity
30
©2013 Promega Corporation.
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
31
©2013 Promega Corporation.
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
*
32
©2013 Promega Corporation.
Cell-Based Assays & Multiplexing
©2013 Promega Corporation.
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
34
©2013 Promega Corporation.
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.
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
0 1 2 3 4 5
rela
tive
lum
ines
cen
ce
time at 22 C (weeks)
CellTiter-Glo®new reagent
35
©2013 Promega Corporation.
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
36
©2013 Promega Corporation.
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.
37
©2013 Promega Corporation.
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
38
©2013 Promega Corporation.
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
39
©2013 Promega Corporation.
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
40
©2013 Promega Corporation.
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
AFC
bis-AAF-R110
Rhodamine
110
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
©2013 Promega Corporation.
Kinetic Assays
©2013 Promega Corporation.
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
Gas
AC
cAMP
ATP
cAM
P
A
B
C
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.
43
©2013 Promega Corporation.
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
44
©2013 Promega Corporation.
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
X
Dead Cell
DNA dye: stain to detect dead cells (overcomes some
limitations of short half-life markers)
45
©2013 Promega Corporation.
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
46
©2013 Promega Corporation.
DNA Dye is Not Toxic to Cells
47
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
©2013 Promega Corporation.
Multiplexing DNA Staining & ATP Assays
48
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/
©2013 Promega Corporation.
Cell Systems Featuring Frozen, Thaw-and-Use Cells
©2013 Promega Corporation.
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
50
©2013 Promega Corporation.
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
51
©2013 Promega Corporation.
‘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
52
©2013 Promega Corporation.
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
53
©2013 Promega Corporation.
Complete QC on Cells
54
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/
©2013 Promega Corporation.
Custom Assay Services
55
©2013 Promega Corporation.
Custom Assay Services
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
56
©2013 Promega Corporation.
Thank you
57
Neal Cosby, PhD Strategic Marketing Manager [email protected]