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 Series Science Creating a Powerful Approach to Drug Discovery
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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
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
22
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
23
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
24
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).
25
In Vitro Pharmacology
26
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.
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)