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BioAssay: AID 686958

Counterscreen for inhibitors of T-cell receptor (TCR)-CD3 interaction: Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled BSA probe

Name: Counterscreen for inhibitors of T-cell receptor (TCR)-CD3 interaction: Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled BSA probe. ..more
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 Tested Compounds
 Tested Compounds
All(232)
 
 
Inactive(232)
 
 
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 Tested Substances
All(232)
 
 
Inactive(232)
 
 
AID: 686958
Data Source: The Scripps Research Institute Molecular Screening Center (BSA_INH_FP_1536_3XIC50 DCSRUN for TCR)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-04-30

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
651800Fluorescence-based biochemical primary high throughput assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probeScreeningdepositor-specified cross reference: Primary screen (TCR-CD3 interaction inhibitors in singlicate)
651805Summary of the probe development effort to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probeSummarydepositor-specified cross reference: Summary (TCR-CD3 interaction inhibitors)
652035Fluorescence-based biochemical high throughput counterscreen assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled BSA probeScreeningdepositor-specified cross reference: Counterscreen (BSA inhibitors in triplicate)
652036Fluorescence-based biochemical primary high throughput confirmation assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probeScreeningdepositor-specified cross reference: Confirmation assay (TCR-CD3 interaction inhibitors in triplicate)
686957Fluorescence-based biochemical primary high throughput dose response assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probeConfirmatorysame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Luc Teyton, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 AI095511-01
Grant Proposal PI: Luc Teyton, TSRI
External Assay ID: BSA_INH_FP_1536_3XIC50 DCSRUN for TCR INH

Name: Counterscreen for inhibitors of T-cell receptor (TCR)-CD3 interaction: Fluorescence-based biochemical high throughput dose response assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled BSA probe.

Description:

Immunosuppressive drugs are critical for organ transplantation and the control of autoimmunity. The current arsenal of usable drugs is limited to small molecules targeting intracellular signaling pathways, e.g. cyclosporine A (1, 2), or antibodies with poorly known mode of action, e.g. OKT3 (3-5). Both categories are endowed with short and long-term life-threatening side effects (1, 2, 3, 4, 6). In most rejection of transplantation situations and in most autoimmune-mediated diseases, T-cell-mediated immunity dominates the adaptive immune response (7-10). Small compounds have focused on T-cell-specific signaling components; however, expression of most of these switches is not exclusive to T cells, explaining many of the adverse effects (3). The targeting of cell surface receptors involved in trafficking, proliferation, and co-stimulation by antibodies does work (11) but does not provide a tunable system easily usable for the long-term. For the same reasons, the T-cell receptor (TCR) complex itself has only been targeted by polyclonal and monoclonal antibodies in the induction setting of transplantation (3, 4) and in very limited studies of type 1 diabetes (12). Based on this background, we hypothesized that the ideal molecular target for small molecule therapeutics should be the two components of the TCR complex itself, the idiotypic alphaBeta TCR and the associated CD3 signaling dimers. Indeed, the exclusive expression of TCR by T cells will provide absolute specificity whereas the disruption of CD3 engagement will modulate the very first signaling step in T-cell activation. We have now established that proof of principle. This project has the potential of identifying new classes of immunosuppressive drugs, an important concern in clinical medicine today.

References:

1. Penninga L, Moller CH, Gustafsson F, Steinbruchel DA, Gluud C. Tacrolimus versus cyclosporine as primary immunosuppression after heart transplantation: systematic review with meta-analyses and trial sequential analyses of randomised trials. Eur J Clin Pharmacol. 2010 Dec;66(12):1177-1187.
2. Pillai AA, Levitsky J. Overview of immunosuppression in liver transplantation. World J Gastroenterol. 2009 Sep 14;15(34):4225-4233.
3. Getts DR, Shankar S, Chastain EM, Martin A, Getts MT, Wood K, Miller SD. Current landscape for T-cell targeting in autoimmunity and transplantation. Immunotherapy. 2011 Jul;3(7):853-870.
4. Ippoliti G, Pellegrini C, Nieswandt V. Controversies about induction therapy. Transplant Proc. 2011 Jul-Aug;43(6):2450-2452.
5. Klipa D, Mahmud N, Ahsan N. Antibody immunosuppressive therapy in solid organ transplant: Part II. MAbs. 2010 Nov-Dec;2(6):607-612.
6. Delgado JF, Vaqueriza D, Sanchez V, Escribano P, Ruiz-Cano MJ, Renes E, Gomez-Sanchez MA, Cortina JM, de la Calzada CS. Induction treatment with monoclonal antibodies for heart transplantation. Transplant Rev (Orlando). 2011 Jan;25(1):21-26.
7. Gras S, Kjer-Nielsen L, Chen Z, Rossjohn J, McCluskey J. The structural bases of direct T-cell allorecognition: implications for T-cell-mediated transplant rejection. Immunol Cell Biol. 2011 Mar;89(3):388-395.
8. Sanchez-Fueyo A, Strom TB. Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology. 2011 Jan;140(1):51-64.
9. Long SA, Buckner JH. CD4+FOXP3+ T regulatory cells in human autoimmunity: more than a numbers game. J Immunol. 2011 Sep 1;187(5):2061-2066.
10. Strioga M, Pasukoniene V, Characiejus D. CD8+ CD28- and CD8+ CD57+ T cells and their role in health and disease. Immunology. 2011 Sep;134(1):17-32.
11. Savinov AY, Burn P. Interference with islet-specific homing of autoreactive T cells: an emerging therapeutic strategy for type 1 diabetes. Drug Discov Today. 2010 Jul;15(13-14):531-539.
12. Savinov AY, Rozanov DV, Strongin AY. Specific inhibition of autoimmune T cell transmigration contributes to beta cell functionality and insulin synthesis in non-obese diabetic (NOD) mice. J Biol Chem. 2007 Nov 2;282(44):32106-32111.

Keywords:

DCSRUN, dose response, counterscreen, BSA, T-cell receptor, TCR, TCR alpha, cluster of differentiation 3, CD3, CD3 epsilon, TAMRA, fluorescence polarization, inhibitor, inhibition, triplicate, protein-protein interaction, immunosuppression, immunosuppressive therapy, autoimmunity, organ transplantation, HTS, 1536, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:

The purpose of this counterscreen assay is to determine whether compounds that comfirmed inhibitory activity in a set of previous experiments entitle,"Fluorescence-based biochemical primary high throughput assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probe" (AID 652036) are fluoprescent artifacts. This assay determines dose response curves.
In this assay, TAMRA-labeled BSA is incubated with test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that were hits in the primary assay because they bind to the TCR should not bind to BSA, thereby leaving mP values in the well unchanged. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 94 uM.
Protocol Summary:

Prior the start of the assay 1 ul of 0.1 M phosphate Buffer pH 8 containing 0.08 uM BSA previously labeled with TAMRA was added to all wells, 3 ul of 8 M Guanidine Hydrochloride were added to columns 1-3 and 3 ul of 0.1 Phosphate Buffer were added to columns 4-48. Next, 38 nL of test compounds or DMSO alone (0.7% final concentration) were distributed into appropiate wells. Plates were centrifuged and incubated for 30 min at 25 C. After incubation fluorescence polarization was read by the ViewLux microplate reader (PerkinElmer) using a BODIPY-TMR filter set and BODIPY dichroic mirror (excitation = 525 nm, emission = 598 nm).

Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):

FP = ( Raw2 - Raw1 ) / ( Raw1 + Raw2 )

Where:

Raw1 is defined as the S channel
Raw2 is defined as the P channel

The percent inhibition for each compound was calculated as follows:

%_Inhibition = ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) ) * 100

Where:

High_Control is defined as wells containing TCR, Guanidine Hydrochloride and DMSO
Test_Compound is defined as wells containing test compounds TCR and 0.1 Phosphate Buffer pH 8 in the presence of test compounds
Low_Control is defined as wells containinf TCR and 0.1 Phosphate Buffer and DMSO

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 94 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 94 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.

Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for inactive compounds is 1-1. There are no active compounds.

List of Reagents:

BSA protein labeled with TAMRA (supplied by Assay Provider)
8M Guanidine Hydrochloride (Sigma Aldrich, part 50937)
Sodium phosphate monobasic (Fisher, part BP329-500)
Sodium phosphate dibasic (Fisher, part BP332-500)
1536-well plates (Corning, part 7234)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
From PubChem:
Assay Format: Biochemical
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1QualifierActivity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
8Response RangeThe range of Y.Float
9Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
10RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
11Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.005 uM [1] (0.005μM**)Value of % inhibition at 0.005 uM compound concentration; replicate [1]Float%
14Inhibition at 0.005 uM [2] (0.005μM**)Value of % inhibition at 0.005 uM compound concentration; replicate [2]Float%
15Inhibition at 0.005 uM [3] (0.005μM**)Value of % inhibition at 0.005 uM compound concentration; replicate [3]Float%
16Inhibition at 0.014 uM [1] (0.014μM**)Value of % inhibition at 0.014 uM compound concentration; replicate [1]Float%
17Inhibition at 0.014 uM [2] (0.014μM**)Value of % inhibition at 0.014 uM compound concentration; replicate [2]Float%
18Inhibition at 0.014 uM [3] (0.014μM**)Value of % inhibition at 0.014 uM compound concentration; replicate [3]Float%
19Inhibition at 0.043 uM [1] (0.043μM**)Value of % inhibition at 0.043 uM compound concentration; replicate [1]Float%
20Inhibition at 0.043 uM [2] (0.043μM**)Value of % inhibition at 0.043 uM compound concentration; replicate [2]Float%
21Inhibition at 0.043 uM [3] (0.043μM**)Value of % inhibition at 0.043 uM compound concentration; replicate [3]Float%
22Inhibition at 0.129 uM [1] (0.129μM**)Value of % inhibition at 0.129 uM compound concentration; replicate [1]Float%
23Inhibition at 0.129 uM [2] (0.129μM**)Value of % inhibition at 0.129 uM compound concentration; replicate [2]Float%
24Inhibition at 0.129 uM [3] (0.129μM**)Value of % inhibition at 0.129 uM compound concentration; replicate [3]Float%
25Inhibition at 0.387 uM [1] (0.387μM**)Value of % inhibition at 0.387 uM compound concentration; replicate [1]Float%
26Inhibition at 0.387 uM [2] (0.387μM**)Value of % inhibition at 0.387 uM compound concentration; replicate [2]Float%
27Inhibition at 0.387 uM [3] (0.387μM**)Value of % inhibition at 0.387 uM compound concentration; replicate [3]Float%
28Inhibition at 1.2 uM [1] (1.2μM**)Value of % inhibition at 1.2 uM compound concentration; replicate [1]Float%
29Inhibition at 1.2 uM [2] (1.2μM**)Value of % inhibition at 1.2 uM compound concentration; replicate [2]Float%
30Inhibition at 1.2 uM [3] (1.2μM**)Value of % inhibition at 1.2 uM compound concentration; replicate [3]Float%
31Inhibition at 3.5 uM [1] (3.5μM**)Value of % inhibition at 3.5 uM compound concentration; replicate [1]Float%
32Inhibition at 3.5 uM [2] (3.5μM**)Value of % inhibition at 3.5 uM compound concentration; replicate [2]Float%
33Inhibition at 3.5 uM [3] (3.5μM**)Value of % inhibition at 3.5 uM compound concentration; replicate [3]Float%
34Inhibition at 10.5 uM [1] (10.5μM**)Value of % inhibition at 10.5 uM compound concentration; replicate [1]Float%
35Inhibition at 10.5 uM [2] (10.5μM**)Value of % inhibition at 10.5 uM compound concentration; replicate [2]Float%
36Inhibition at 10.5 uM [3] (10.5μM**)Value of % inhibition at 10.5 uM compound concentration; replicate [3]Float%
37Inhibition at 31.4 uM [1] (31.4μM**)Value of % inhibition at 31.4 uM compound concentration; replicate [1]Float%
38Inhibition at 31.4 uM [2] (31.4μM**)Value of % inhibition at 31.4 uM compound concentration; replicate [2]Float%
39Inhibition at 31.4 uM [3] (31.4μM**)Value of % inhibition at 31.4 uM compound concentration; replicate [3]Float%
40Inhibition at 94.0 uM [1] (94μM**)Value of % inhibition at 94.0 uM compound concentration; replicate [1]Float%
41Inhibition at 94.0 uM [2] (94μM**)Value of % inhibition at 94.0 uM compound concentration; replicate [2]Float%
42Inhibition at 94.0 uM [3] (94μM**)Value of % inhibition at 94.0 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R01 AI095511-01

Data Table (Concise)
Data Table ( Complete ):     View All Data
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