HTS for Beta-2AR agonists via FAP method from Validation Set
Assay Implementation: Yang Wu, Phillip Tapia, Terry Foutz, Stephanie Chavez, Dominique Perez, Annette Evangelisti, Anna Waller, Cristian Bologa, Mark Carter ..more
BioActive Compounds: 31
University of New Mexico Assay Overview:
Assay Support: R03 MH093192-01
Project Title: HTS for Non-Canonical Ligands for Beta 2 Adrenergic Receptor Internalization
Assay Provider: Jonathan Jarvik, Carnegie Mellon University
Screening Center/ PI: UNMCMD/ Larry Sklar
Lead Biologist: Yang Wu
Chemistry Center/ PI: Vanderbilt Specialty Chemistry Center/Craig Lindsley
Chemistry Center Lead: Shaun Stauffer
Assay Implementation: Yang Wu, Phillip Tapia, Terry Foutz, Stephanie Chavez, Dominique Perez, Annette Evangelisti, Anna Waller, Cristian Bologa, Mark Carter
Assay Background and Significance:
G protein-coupled receptors represent the largest family of proteins in the human genome with an estimated number of approximately 800. Because of their central involvement in almost every aspect of human physiology, they also represent the largest target for medical intervention [Lin and Civelli, Annu Med 36 (2004), 204-14]. Today, GPCRs represent the target of approximately 30-40% of all drugs on the market. Indeed, of the 50 top-selling drugs in the United States in 2007, 18 target GPCRs, with combined sales of approximately 25 billion dollars.
Of about 800 GPCRs, 500 are chemosensory, representing the chemokine/chemoattractant GPCRs, and the olfactory and gustatory GPCRs. Although the former have been thoroughly characterized for the most part, members of the latter, particularly the olfactory receptors which may include the important category of pheromones, have had only a small number of ligands identified. The remaining GPCRs, approximately 360, constitute the transmitter GPCRs. Of these, approximately 100 receptors still have no known ligand. Such receptors with no known physiological ligand are referred to as orphan receptors and arose from cloning strategies based on limited homology within almost all GPCRs (predominantly during the 1990's) as well as the sequencing of the human genome (in 2000) [Chung et al, Br J Pharmacol 153 Suppl 1 (2008), S339-46].
The search for endogenous ligands for orphan GPCRs has been challenging. This process has given rise to the field of reverse pharmacology, which uses orphan GPCRs to identify novel ligands, which together often lead to the characterization of new physiological paradigms. Over the last 20 years, this approach has led to the deorphanization of about 300 GPCRs. Many of the ligands were already known and their biology characterized, but their receptor was unknown. But in some of these instances, this process also led to the identification of novel transmitters [Civelli et al., Pharmacol Ther 110 (2006), 525-32].
During the 1990's, approximately 10 GPCRs were deorphanized per year. However, very few have been deorphanized since 2004. In addition, no novel transmitters have been identified since that time [Chung et al, Br J Pharmacol 153 Suppl 1 (2008), S339-46]. Given these facts, the question arises as to whether the remaining orphan GPCRs will be readily deorphanized. One major issue is that the pool of known transmitters for which no receptor is known has been essentially depleted. Since all the possible transmitters have now been matched to GPCRs, the current orphan GPCRs can only bind unknown ligands, for which the identification is also slow and resource intense. This is particularly true given that the concentrations of many transmitters in vivo is exceedingly low, making their identification difficult. It is also possible that the expression of some ligands may be developmentally or environmentally regulated.
Almost all current reverse pharmacological/screening approaches rely on monitoring second messenger levels such as calcium mobilization, cAMP production and transcriptional activation. Thus, successful screening requires knowledge of the pathway for a given receptor, in particular the G protein to which the receptor couples. As the number of heterotrimeric G protein combinations is large, this is not a trivial task.
An alternate approach to measuring signal transduction is the monitoring receptor internalization. Virtually all known GPCRs undergo activation-dependent internalization as a mechanism to reduce cell surface receptor numbers. Internalization does not require G protein coupling. Instead, the activity of one of four G protein receptor kinases (GRKs) is required. In most instances, the binding of an accessory protein, arrestin, is also required. One screening approach that has been developed is the recruitment of GFP-tagged arrestin to either the plasma membrane or intracellular endosomes.
1. Spin down 1x109 Am2.2-beta2AR cells, discard supernatant, and resuspend in 200mL of fresh RPMI1640 full medium. Final cell density will be 5x106 cells/mL.
2. Add 5microL serum free RPMI to Columns 2-24 of the assay plate by Nanoquot
3. Add 5microL of freshly prepared 32microM ISO in RPMI full media to Column 1 of all the plates as PCntrls by Microflow
4. Add 100nL of library compounds to assay plates by FX or NX
5. Add 3microL of cells to Columns 1 - 22 of the assay plates by Nanoquot.
6. Shake the plates and put them in 37 deg C incubator for 90 mins.
7. Add 3microL 650nM TO1-2p to assay plates by Microflow or Nanoquot to assay plates and read by high-throughput flow cytometers immediately.
All plates passed the Z' test (Z'>.30) so a compound was considered active if the PERCENT_RESPONSE > 42.
The 42% cutoff corresponds to about three times the standard deviation of RESPONSE from 'non-fluorescent' test compounds. Negative RESPONSE is primarily due to test compounds with innate fluorescence.
PUBCHEM_ACTIVITY_SCORE = PERCENT_RESPONSE
PUBCHEM_ACTIVITY_OUTCOME = 2 (or ACTIVE) if PUBCHEM_ACTIVITY_SCORE > 42, otherwise the PUBCHEM_ACTIVITY_OUTCOME = 1 (or INACTIVE).
** Test Concentration.
Data Table (Concise)