Label and Label-free Technologies in Synergy · Label and Label-free Technologies in Synergy...
Transcript of Label and Label-free Technologies in Synergy · Label and Label-free Technologies in Synergy...
Sponsored by:
Participating Experts:
William P. Janzen
University of North Carolina
Chapel Hill, NC
Brian K. Shoichet, Ph.D.
University of California San Francisco
San Francisco, CA
Charles A. Lunn, Ph.D.
Merck Research Laboratories
Kenilworth, NJ
Brought to you by the Science/AAAS Business Office
2 March, 2011
Label and Label-free Technologies in Synergy
Webinar SeriesScience
Creating a Powerful Approach to Drug Discovery
Sponsored by:
Participating Experts:
William P. Janzen
University of North Carolina
Chapel Hill, NC
Brian K. Shoichet, Ph.D.
University of California San Francisco
San Francisco, CA
Charles A. Lunn, Ph.D.
Merck Research Laboratories
Kenilworth, NJ
Brought to you by the Science/AAAS Business Office
2 March, 2011
Label and Label-free Technologies in Synergy
Webinar SeriesScience
Creating a Powerful Approach to Drug Discovery
In Vitro Pharmacology
Label and Label-free Technologies in Synergy:
Creating a Powerful Approach to Drug Discovery
Charles A. Lunn
In Vitro Pharmacology
Merck Research Laboratories
Kenilworth, New Jersey 07033
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In Vitro Pharmacology
Technology advances have NOT increased
new drug approval by FDA
• Development costs increasing exponentially, without corresponding increase in drugs into clinic– Munos B. Lessons from 60 years of
pharmaceutical innovation. (2009) Nat Rev Drug Discov. 8(12): 959-968.
– Kola I. The state of innovation in drug development. (2008) Clin Pharmacol Ther. 83(2): 227-230.
• WASHINGTON (Jan. 6) The Food and Drug Administration approved 26 drugs in 2009, compared with 25 approvals in 2008.
Strategies
to find
BETTER
compounds
&
compounds
for non-
traditional
targets
2010’s
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In Vitro Pharmacology
New technologies within traditional screening
strategies may bias hits
• Biochemistryo Soluble protein domain may not mimic target biologyo Non-native substrate may not mimic target biology
• Cell biologyo Selecting reporter (cAMP, adenylate cyclase, βarrestin) will determine hitso Selecting host cell may determine hits
Du et al, 2009o Recombinant cells may not contain accessory proteins important for target biology
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In Vitro Pharmacology
The Unknown
As we know, There are known knowns.
There are things we know we know. We also know
There are known unknowns. That is to say
We know there are some things We do not know.
But there are also unknown unknowns, The ones we don't know
We don't know. D. H. Rumsfeld
12 Feb. 2002 Department of Defense news briefing
Biologically relevant assays guard against
underappreciated characteristics of target
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In Vitro Pharmacology
Label-free methods allow enzymology with native
substrates: High throughput mass spectrometry
Su
bstr
ate
Pro
du
ct
0 min2.5 min
5 min
10 min
15 min
20 min
30 min
60 min
acetate
Wang, et al. A Fluorescence-Based
Homogeneous Assay for Measuring Activity
of UDP±3-O-(R-3-Hydroxymyristoyl)-
Nacetylglucosamine Deacetylase. Analytical
Biochemistry 290, 338–346 (2001).
Quantitate product by thin
layer chromatigraphy
Quantitate amino group
using fluorescamine
Quantitate radioactive acetate
after charcoal adsorption
Bound ligand stabilizes protein,
increasing denaturation temperature
exposing hydrophobic dye binding sites
Langsdorf et al. Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay. J Biomol Screen. 15(1): 52-61 (2010).
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In Vitro Pharmacology
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High throughput mass spectrometry useful
for many biochemical target classes
ACETYL-COENZYME A CARBOXYLASE.
Jonas, et al (2009) Comb Chem High
Throughput Screen. 12(8):752-759.
STEAROYL-COA DESATURASE.
Soulard et al (2008) Analytica
Chimica Acta 627(1): 105-111
ACETYLCHOLINESTERASE.
Ozbal, et al. (2004) Assay Drug
Dev Technol. 2(4): 373-81.
PHOSPHATIDYLSERINE
DECARBOXYLASE.
Forbes et al (2007) J Biomol
Screen. 12(5): 628-634.
.
In Vitro Pharmacology
• How to balance to optimize cmpd. development?
Possible advantage of label-free assays for
screening with biologically relevant cells (?)
E.N. Johnson, Ph.D. Dept. Automated Biotechnology Merck & Co., Inc.
Engineered
Cell system
Surrogate
endpoint
Engineered
Cell system
Physiological
endpoint
Cell Line
Endogenous
Expression
Physiological
endpoint
Primary Cell
Endogenous
Expression
Surrogate
endpoint
Primary Cell
Endogenous
Expression
Physiological
endpoint
Category 5 Category 4 Category 3 Category 2 Category 1
(2007) Amer. Drug Discovery, 3 (2): 12-22.
BIOLOGICAL RELEVANCE
TARGET SELECTIVITY
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In Vitro Pharmacology
• Genzyme’s CXCR4 partial agonistPlerixafor approved for hematopoeiticstem cell mobilization non-Hodgkin's
lymphoma and multiple myeloma.
Elizabeth “Lisa” Smith
UAS-bla plus GP160
CXCR4 plus Hybrid Transactivator
Cell Fusion Assay12k cells plated 3/15/10
0 10 20 301000
2000
3000
4000
5000
Hela
Hela + AMD
U2Os
U2Os+AMD
Hela+U2Os
Hela+U2Os+AMD
Time (hours)
Ref
Imp
ed
an
ce
0 2 4 61000
2000
3000
4000
5000
Hela
Hela + AMD
U2Os
U2Os+AMD
Hela+U2Os
Hela+U2Os+AMD
Time (hours)
Ref
Imp
ed
an
ce
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CellKey technology may offer
label-free path to biorelevant cells
Seeking unique CXCR4 antagonists blocking HIV-induced cell fusion
Asra Malikzay
NH
NH
N
NH
NH
NH
N
NH
Plerixafor
(AMD 3100;
Tocris Biosci.)
12,000 total cells / well (individual or 1:1 mixture)
1 μM AMD 3100 added (Final volume = 50 μl)
At specified times, plates evaluated for “reference impedance”
In Vitro Pharmacology
In Vitro Pharmacology
Pharmacology of therapeutic target
sometimes hard to predict
• Pharmacology required to exploit anti-inflammatory potential of
cannabinoid CB2 receptor complex
– CB2-specific agonists inhibit1 and induce2 chemotaxis• Montecucco et al, 2008 ; McHugh et al, 2007; Mukhopadhyay et al, 2007; Coopman et al, 2007; Nilsson et
al, 2006; Ghosh et al, 2006; Sacerdote et al, 2000
• Gonsiorek et al, 2007; Tanikawa et al, 2007; Jiang et al, 2007; Kishimoto et al, 2006; Berghuis et al, 2005;
Oka et al, 2004; Jorda et al, 2002
– Cannabinoid agonists inhibit & induce bone growth• Idris et al.(2005); Idris et al (2008); Ofek et al (2006); Karsak et al (2005).
– Cannabinoid CB2 receptor inhibits and exacerbates EAE• Ni et al (2004); Maresz et al (2007); Sipe et al (2005); A.Zimmer and N.Stella, personal communication
• CB2 receptor-specific triaryl bis sulfone inverse agonists function as anti-inflammatory modulators in selected animal models– Inhibit ovalbumin-induced eosinophilia in hypersensitized
mouse (Lunn, et al, 2006a)
– Inhibit methylated BSA-induced monoarticular arthritis (Lunn, et al; 2008)
– Inhibits MOG peptide-induced EAE (Lunn, et al, 2006b)
S
S O O
OO
Cl
F
NH
S
OO
F
F
F
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In Vitro Pharmacology
PerkinElmer EnSpire Label-Free Analysis:2 Agonists & 1 Inverse Agonist in CHO-hCB2 Cells
-12 -10 -8 -6 -4-20
0
20
40
60
80
100
CP 55,940
WIN 55,212-2
Sch.414319
Conc. (log [M])
Resp
on
se (
pm
)
EnSpire characterization of
cannabinoid CB2 pharmacology
Hill Slope = 0.92
z’ factor = 0.53
Hill Slope = 1.55
z’ factor = 0.46
15,000 cells/well
S
S O O
OO
Cl
F
NS
OO
F
F
F
O
OH
OH
N
O
O
N
O
CP55,940
WIN
Sch.319
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In Vitro Pharmacology
Another technology-specific hit :
Fenofibrate as CB2 agonist biased for β-arrestin
Characterizing Cannabinoid CB2 Receptor Ligands Using DiscoveRx PathHunter™ β-Arrestin Assay
DEBRA MCGUINNESS, ASRA MALIKZAY, RICHARD VISCONTI, KAREN LIN, MARVIN BAYNE, FREDERICK MONSMA, and CHARLES A. LUNN
(2009) Journal of Biomolecular Screening 14(1): 49-58.
Cl
O
O
O O
● SERENDIPITY: the faculty or phenomenon of
finding valuable or agreeable things not sought for
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PPARα receptor
agonist activates CB2
with EC50 = 55 nM
CHO vs CHO/CB2+ Fenofibrate
-4 -3 -2 -1 0 1 2 3 40
20
40
60
80
log [nM] Fenofibrate
Zie
C (
ch
an
ge in
oh
ms)
CH
O/C
B2 -
CH
O
In Vitro Pharmacology
Dodgson et al. A 100K well screen for a muscarinic
receptor using the Epic label-free system--a reflection
on the benefits of the label-free approach to screening
seven-transmembrane receptors. (2009) J Recept
Signal Transduct Res. 29 (3-4):163-72.
• A number of compounds were identified which were not found using
a FLIPR-based calcium mobilization assay. May represent
• Off-target false positives … OR …
• Unique opportunities enhancing success for 7TM targets
Successful application of label-free technology
in an HTS environment.
Poor
confirmation
Good
confirmation
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In Vitro Pharmacology
A.
C.
B.
Traditional biased screening strategy.
Flag unique biology after biased screen.
Confirm target after unbiased screen.
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Targeting hits from cell-based label-free screen: strategies
In Vitro Pharmacology
Better solution - find better drugs
• Compound libraries reflect the known chemical characteristics of successful therapeutics– Unique resource that must be better utilized
• Better choices for screening assays– Ensure initial compound screen models biology of interest
• Impact choice host cell / assay method
• Better choices of compounds for development– Increase available data on hit compounds, to ensure biology of all
chemotypes tested
• Diminish potency as primary driver for hit selection
• Better choices for Med Chem development
• Label-free technologies offer strategy for unbiased interrogation of compound libraries for best cmpnds
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Sponsored by:
Participating Experts:
William P. Janzen
University of North Carolina
Chapel Hill, NC
Brian K. Shoichet, Ph.D.
University of California San Francisco
San Francisco, CA
Charles A. Lunn, Ph.D.
Merck Research Laboratories
Kenilworth, NJ
Brought to you by the Science/AAAS Business Office
2 March, 2011
Label and Label-free Technologies in Synergy
Webinar SeriesScience
Creating a Powerful Approach to Drug Discovery
Look out for more webinars in the series at:
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Sponsored by:
Brought to you by the Science/AAAS Business Office
2 March, 2011
Label and Label-free Technologies in Synergy
Webinar SeriesScience
Creating a Powerful Approach to Drug Discovery