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BioAssay: AID 651800

Fluorescence-based biochemical primary high throughput assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probe

Name: Fluorescence-based biochemical primary high throughput assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probe. ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 651800
Data Source: The Scripps Research Institute Molecular Screening Center (TCR_INH_FP_1536_1X%INH PRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-11-19

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 1343
Related Experiments
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
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
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
686957Fluorescence-based biochemical primary high throughput dose response assay to identify inhibitors of T-cell receptor (TCR)-CD3 interaction using a TAMRA-labeled TCR probeConfirmatorydepositor-specified cross reference
686958Counterscreen 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 probeConfirmatorydepositor-specified cross reference
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: TCR_INH_FP_1536_1X%INH PRUN

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


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.


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.


T-cell receptor, TCR, TCR alpha, cluster of differentiation 3, CD3, CD3 epsilon, TAMRA, fluorescence polarization, inhibitor, inhibition, protein-protein interaction, primary, 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
Assay Overview:

The purpose of this assay is to identify compounds that will ultimately act as inhibitors of the T-cell receptor (TCR)-CD3 interaction. In order to identify initial compounds that may be capable of achieving this, a TCR mutant containing a cysteine in the loop of the constant domain that is critical for the TCR-CD3 interaction is used. The cysteine allows for fluorophore labeling at that position, and fluorescence recovery from the label is highly dependent on the loop environment. In this assay, recombinant mutant TCR 2C K166C protein labeled with TAMRA is incubated with test compounds and fluorescence polarization is measured. 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 potentially act as inhibitors of TCR-CD3 interaction will bind to the TCR-TAMRA probe and induce a conformational change, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 9.4 uM.

Protocol Summary:

Prior the start of the assay 1 ul of 0.1 M phosphate Buffer pH 8 containing 0.2 uM TCR 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 )


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


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

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-8, and for inactive compounds 8-0.

List of Reagents:

TCR 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)
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. Compounds SR-01000876146-1 (SID 57266590), SR-01000761134-1 (SID 26671489) and SR-05000002545-2 (SID 93576884) were not screened.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 9.4 uM (9.4μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 9.4 micromolar.Float%

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

Data Table (Concise)
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