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BioAssay: AID 504490

Assay for Inhibitors of the beta-Arrestin-Adaptor Protein 2 Interaction That Mediate GPCR Degradation and Recycling

Screening Operations Team: Mark Carter MS, Kristine Gouveia MS, J. Jacob Strouse Ph.D., Anna Waller Ph.D. ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 504490
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-03-21
Modify Date: 2011-03-26

Data Table ( Complete ):           Active    All
BioActive Compounds: 1061
Depositor Specified Assays
504493Summary of MLP Assay for Arrestin-AP2 InhibitorssummarySummary of MLP Assay for Arrestin-AP2 Inhibitors.
504908Assay for Inhibitors of the beta-Arrestin-Adaptor Protein 2 Interaction for Cherry Pick 1screening
588338Counter Screen of assay for Inhibitors of the beta-Arrestin-Adaptor Protein 2 Interaction for Cherry Pick 1other
588783Dose Response Assay for Inhibitors of the beta-Arrestin-Adaptor Protein 2 Interaction for Cherry Pick 1confirmatory
University of New Mexico Assay Overview:
Assay Support: 1 R03 DA031665-01A1
Project Title: MLP Assay for Arrestin-AP2 Inhibitors
Assay Provider: Eric Prossnitz Ph.D
Lead Biologist: Catherine Prudom Ph.D., Mark Carter MS, Anna Waller Ph.D, Annette Evangelisti Ph.D
Screening Operations Team: Mark Carter MS, Kristine Gouveia MS, J. Jacob Strouse Ph.D., Anna Waller Ph.D.
Chemistry Center/ PI: Craig W. Lindsley
Specialized Chemistry Center: Vanderbilt Specialized Chemistry Center For Accelerated Probe Development
Assay Implementation: Keon Ahghar, Stephanie Chavez, Terry Foutz, Matthew Garcia, Travis Houston, Dominique Perez

Assay Background and Significance:

G protein-coupled receptors (GPCRs) represent the largest family of proteins in the human genome with over 600 members. As such they are involved in almost every aspect of normal human physiology, in addition to a multitude of pathophysiological processes, including cancer. The protein arrestin, which binds to phosphorylated GPCRs, is now known to play critical roles not only in receptor desensitization, but also in receptor trafficking and G protein-independent signaling. We have identified a unique feature of G protein-coupled receptors whereby activation of GPCRs in the absence of normal arrestin-dependent signaling results in the rapid initiation of apoptosis, a feature that can be employed as a novel therapeutic target in cancer drug development. As a model system for our studies, we have employed the N-formyl peptide receptor (FPR), a member of the chemoattractant/chemokine subfamily of GPCRs, important in numerous aspects of immune function. The FPR is also expressed in high-grade gliomas, where it contributes to the aggressiveness of this deadly disease that even with state of the art treatment shows only approximately 50% survival at one year after diagnosis. Our hypothesis is that the development of small molecule inhibitors of arrestin-mediated GPCR trafficking/signaling will specifically induce apoptosis in FPR/GPCR-activated tumor cells potentially leading to novel therapeutic approaches.

Because this protein-protein binding interaction is solely based on a helix-groove interface with a small area of contact, we hypothesize that it is ideally suited for inhibition by small molecules that bind specifically to residues in the AP-2 groove, precluding binding of the arrestin helix into the groove.

Based on our results and those described by McMahon and colleagues [Wagener et. al., 2009], we developed a flow cytometric assay to monitor the arrestin-AP-2 interaction. For the reporter arrestin peptide, we utilized the peptide sequence of McMahon, but to the amino terminus that clearly extrudes from the binding pocket, we added two amino-hexanoic acid spacers and a terminal FITC group via a beta-Ala residue (FITC-beta-Ala-Ahx-Ahx-DIVFEDFARQRLKGMKDD, where the bold "F" represents F391). For the binding target we utilized a beta2 adaptin appendage construct (residues 700-937, R879A) fused to GST as described [Schmid et. al., 2006] and expressed in the BL21 strain of E. coli. As a positive control to inhibit binding of the fluorescent arrestin peptide, we synthesized the peptide, DDDIVFEDFARQRLKGMKDD.
GST-AP2 R879A is expressed in E. coli BL21(DE3) plysS and the overnight cultures are spun down and resuspended in 1/10 of the original volume in PBS. Concentrated cultures are stored at -80 degrees C until use. An aliquot of the frozen E. coli concentrate is lysed by the following method:

DNase I, Lysozyme and protease inhibitor cocktail are added to a 3milliL aliquot of E. coli and shaken gently for 30min at RT followed by sonication until the lysate appears clear and is not viscous. The lysate is pelleted at 10k rpm for 5 min at 4 degrees C and the supernatant stored at -20 degrees C until further use.

4micron GSH Beads are resuspended by briefly vortexing at 2200 rpm repeatedly. Cleared cell lysate (50microL) is added to 3microL of the GSH beads and gently mixed in the cold room overnight. The beads are pelleted at 8000 rpm at 4 degrees C for 5 min and the protein lysate removed. The beads are resuspended in assay buffer (PBS, 0.05 percent Tween 20, and 0.1 percent BSA) and pelleted again. After removal of the wash supernatant, the beads are resuspended in 2.5milliL assay buffer. An assay plate is prepared by adding 5microL assay buffer to each well in columns 1-23 of a polystyrene 384-well plate (Greiner catalog no. 784101). 11microM Arrestin peptide (non-fluorescent) in assay buffer is added to column 24 (5microL/well) as the positive control. The final concentration of Arrestin peptide in column 24 is 5microM which is 100 fold higher than the FITC-Arrestin. 100nanoL of the library compounds in DMSO are transferred to the assay plate using the 100nanoL slot pin tool (V&P Scientific) on the Biomek FXp workstation (Beckman Coulter, USA). Compounds are only transferred in columns 3-22 of the 384 well plate. 100nanoL DMSO is transferred to columns 1, 2, 23, and 24. Column 23 will serve as the negative control and columns 1 and 2 will be wash wells. The beads are distributed at 3microL/well into columns 3-24 of the assay plate using the BioTek MicroFlo Select liquid dispenser and incubated at room temperature for 12 minutes. 183nanoM FITC-Arrestin is added to columns 1-24 with the BioTek nanoquot at 3microL/well and the wells mixed. The final concentration is 50nanoM FITC-Arrestin. The plate is covered with foil and placed on a rotator at room temperature for 1 hr. The fluorescence signals are detected by the Cyan flow cytometer with HyperCyt [Edwards et. al., 2001] automation on the FL1 setting (ex. 488/em. 535).

A plates passed the Z' test if Z'>.30. A compound was considered active if the PERCENT_RESPONSE > 54.

The 54% cutoff corresponds to about three times the standard deviation of PERCENT_RESPONSE from 'non-fluorescent' test compounds. Negative PERCENT_RESPONSE is primarily due to test compounds with innate fluorescence.

Abbreviations used: nanoL for nanolitre, milliL for millilitre, microL for microlitre, microM for micromolar, nanoM for nanomolar ex. for excitation wavelength, em. for emission wavelength, nm for nanometer, FITC for fluorescein isothiocyanate.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PERCENT_RESPONSE (10μM**)Percent response.Float%
2ZPRIMEZ prime.Float

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA031665-01A1

Data Table (Concise)