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BioAssay: AID 651938

Fluorogen/soluble FAP binding competition assay

Assay Provider: Jonathan Jarvik, Carnegie Mellon University Screening Center/ PI: UNMCMD/ Larry Sklar ..more
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 651938
Data Source: NMMLSC (UNMCMD_CMU_FAPBA_SOLUBLEBINDING)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-12-13
Hold-until Date: 2013-12-13
Modify Date: 2013-12-13

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: single chain variable fragment antibody type HL1.1-TO1 [synthetic construct]
Description ..   
Protein Family: Immunoglobulin (Ig) heavy chain (H), variable (V) domain

Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 2
Related Experiments
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AIDNameTypeComment
651701Summary of HTS Screening Project for Inhibitors of fluorogen-FAP tag interactionsSummarydepositor-specified cross reference: Summary of HTS Screening Project for Inhibitors of fluorogen-FAP tag interactions
504448Summary of HTS for Non-Canonical Ligands for Beta 2 Adrenergic Receptor InternalizationSummarysame project related to Summary assay
588775Dose response for HTS for Beta-2AR agonists via FAP method from Powderset1Confirmatorysame project related to Summary assay
623882Dose response for HTS for Beta-2AR agonists via FAP method from Powderset2Confirmatorysame project related to Summary assay
623947Dose response for HTS for Beta-2AR agonists via FAP method from Powderset3Confirmatorysame project related to Summary assay
651694Cytotoxicity Dose Response with AM2.2-beta2AR cells for PowderSet01Confirmatorysame project related to Summary assay
651698Counter screen for HTS for Beta-2AR agonists with FAP-tagged mouse CCR5 with Powderset1Confirmatorysame project related to Summary assay
651728Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset2Confirmatorysame project related to Summary assay
651729Counter screen for HTS for Beta-2AR agonists with FAP-tagged GPR32 with Powderset2Confirmatorysame project related to Summary assay
651853Counter screen for HTS for Beta-2AR agonists with FAP-tagged human GPR32 with Powderset3Confirmatorysame project related to Summary assay
651855Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset3Confirmatorysame project related to Summary assay
651870Cytotoxicity Dose Response with AM2.2-beta2AR cells for PowderSet4Confirmatorysame project related to Summary assay
651872Dose response for HTS for Beta-2AR agonists via FAP method from Powderset4Confirmatorysame project related to Summary assay
651873Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset4Confirmatorysame project related to Summary assay
651875Counter screen for HTS for Beta-2AR agonists with FAP-tagged GPR32 with Powderset4Confirmatorysame project related to Summary assay
651912Dose response for HTS for Beta-2AR agonists via FAP method from Powderset5Confirmatorysame project related to Summary assay
651913Counter screen for HTS for Beta-2AR agonists with FAP-tagged human CCR5 with Powderset5Confirmatorysame project related to Summary assay
651914Counter screen for HTS for Beta-2AR agonists with FAP-tagged GPR32 with Powderset5Confirmatorysame project related to Summary assay
651936Evaluation of reversibility of compound binding to AM2.2-beta2AR cells with Powder Set 3and4Othersame project related to Summary assay
Description:
University of New Mexico Assay Overview:
Assay Support: R03 DA031668-01
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: Greg Fisher, CMU

Assay Background and Significance:

The Carnegie Mellon University Technology Center for Networks and Pathway (CMU TCNP) has developed a novel fluorogen activating peptide (FAP) technology. Fluorogens are small molecules that are not fluorescent, but acquire fluorescence when rotational motion about certain bonds within the molecule is constrained. Within the NIH Roadmap-funded CMU TCNP, single chain antibodies (scFvs) that bind specific fluorogens and activate their fluorescence were identified and improved by successive rounds of mutagenesis and selection [Fischer et al, 2010]. These proteins are called FAPs, or fluorogen activating proteins.

The strong (Kd < 10nM) and specific binding between a particular FAP and fluorogen can, in principle, be blocked or reversed by a specific competitive or noncompetitive interaction with a different molecule. The screen that was undertaken in the original MH093192 (HTS for Non-canonical Ligands for Beta 2 Adrenergic Receptor Internalization) project was designed to reveal new effectors of beta2-AR function, but its design was such as to also reveal inhibitors of the FAP-fluorogen interaction. In fact, further analysis using the first 20 compounds provided for validation by VUSCC indicates that some of them interfere with fluorogen activation rather than receptor stimulation. This opens the door for the design of new tool sets for FAP based technology.

FAP technology is an innovative technology that was first published on in 2008. It is not surprising to discover amongst the hits from the MLPCN screen targeting beta2-AR modulators, a new class of molecules that interfere with FAP-fluorogen binding. There are no such molecules reported in the literature to date, and the proposed probes will be the first in this class. These compounds will help in advancing science by providing the opportunity to efficiently separate false positives from target-specific compounds in the hit list of high-throughput screening, to monitor receptor trafficking and receptor location in real time, and with the potential to extend the application to animal models.
Protocol
Competition binding assays will be performed by adding the compounds to mixtures of soluble AM2.2 (FAP-tag) and TO1-2p (fluorogen), and the fluorescent signal measured by fluorimetry.
* purified AM2-2 was diluted 50/200uL in cPBS and TO1-2p was added (10X Kd concentration)
* 10mM of each of the two compounds was made up in DMSO
* antagonists were diluted 1/10 into ddH20 and then diluted 1/20 first well and 1/10 thereafter
* plates were scanned in the SafireII Tecan plate reader (541nm emission maximum)

Calculations:
Raw fluorescence readings for the entire concentration range of a test compound were fitted by Prism(R) software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose response model with variable slope, also known as the four parameter logistic equation. Curve fit statistics were used to determine the following parameters of the model: EC50, microM - concentration of added test compound competitor that inhibited fluorescent ligand binding by 50 percent; LOGEC50 - the logarithm of EC50; TOP - the response value at the top plateau; BOTTOM - the response value at the bottom plateau; HILLSLOPE - the slope factor, or the Hill coefficient; STD_LOGEC50, STD_TOP, STD_BOTTOM, STD_HILLSLOPE - standard errors of LOGEC50, TOP, BOTTOM, and HILLSLOPE ; EC50_95CI_LOW, EC50_95CI_HIGH - the low and high boundaries of the 95% confidence interval of the EC50 estimate, RSQR - the correlation coefficient (r squared) indicative of goodness-of-fit.

Compounds with EC50 less than 30 microM were active.

Keywords: UNMCMD, CMU
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ACTIVITY_QUALIFIERQualifier for the value of EC50String
2LogEC50Log of the EC50 estimateFloat
3 EC50_MICROMEffective concentration of half maximal event count as estimated by curve fitFloatμM
4EC50_95CI_LOWLower 95% confidence interval boundary for the EC50 curve fit estimateFloatμM
5EC50_95CI_HIGHUpper 95% confidence interval boundary for the EC50 curve fit estimateFloat
6BOTTOMResponse value at the bottom plateauFloat
7TOPResponse value at the top plateauFloat
8HILLSLOPEHill slope estimate for the fitted dose response curveFloat
9STD_BOTTOMStandard error for the response value at the bottom plateauFloat
10STD_TOPStandard error for the response value at the top plateauFloat
11STD_HILLSLOPEStandard error for the HILL SLOPEFloat
12RSQRCorrelation coefficient for the fitted dose response curveFloat
13N_POINTSNumber of data points for each dose response curveFloat
14SPANSpan of measurements across concentration rangeInteger
15RawFluorescence_50microM (50μM**)Raw fluorescence measured with 50 micromolar concentration of test compoundFloat
16RawFluorescence_5microM (5μM**)Raw fluorescence measured with 5micromolar concentration of test compoundFloat
17RawFluorescence_0.5microM (0.5μM**)Raw fluorescence measured with 0.5 micromolar concentration of test compoundFloat
18RawFluorescence_0.05microM (0.05μM**)Raw fluorescence measured with 0.05 micromolar concentration of test compoundFloat
19RawFluorescence_0.005microM (0.005μM**)Raw fluorescence measured with 0.005 micromolar concentration of test compoundFloat
20RawFluorescence_0.0005microM (0.0005μM**)Raw fluorescence measured with 0.0005 micromolar concentration of test compoundFloat
21RawFluorescence_0.00005microM (5e-05μM**)Raw fluorescence measured with 0.00005 micromolar concentration of test compoundFloat
22RawFluorescence_0.000005microM (5e-06μM**)Raw fluorescence measured with 0.000005 micromolar concentration of test compoundFloat

** Test Concentration.
Additional Information
Grant Number: R03 DA031668-01

Data Table (Concise)
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