ZAP-70 is a critical molecule in the transduction of T cell antigen receptor signaling and the activation of T cells. Upon activation of the T cell antigen receptor, ZAP-70 is recruited to the intracellular ζ-chains of the T cell receptor, where ZAP-70 is activated and colocalized with its substrates. Inhibitors of ZAP-70 could potentially function as treatments for autoimmune diseases or organ transplantation. In this work, we present the design, optimization, and implementation of a screen for inhibitors that would disrupt the interaction between ZAP-70 and the T cell antigen receptor. The screen is based on a fluorescence polarization assay for peptide binding to ZAP-70.
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Figure 1 - Binding curves of ZAP-70 and TAMRA-2pY measured by FP.
A. ZAP-70 wild type.
B. ZAP-70 Y315F/Y317F exhibits a lower affinity for TAMRA-2pY compared with the wild type.
C. ZAP-Src chimera, predicted to be uninhibited, demonstrates a higher affinity for 2pY than wild-type ZAP-70.
D. Schematic of 1pY competing off TAMRA-2pY peptide.
E. Inhibition curve of ZAP- 70:2pY binding by 1pY peptide. The Ki value for the 1pY peptide is determined to be 125.3 ± 9.2 μM. The fit extrapolates the lower value of the fitted curve to the measured FP value of a free peptide.
All KD and Ki values provided are the average of three separate experiments. Representative data are shown.
A. Graph of screening results. Total fluorescence by well is shown. Each black dot represents an assay well. One standard deviation above or below the mean total fluorescence value of the library is represented by the dashed horizontal gray lines.
B. Graph of screen inhibition by well shown. Each black dot represents an assay well. Three standard deviations above the mean inhibition value of the library (~20% inhibition) is represented by the dashed horizontal gray line.
C,D. FP concentration–response curvesof SMDC compounds 180013 (left) and 180882 (right).
E,F. TR-FRET concentration–response curves of SMDC compounds 180013 (left) and 180882 (right).
IC50 values are similar in both FP and TR-FRET assays for each compound.
A. Schematic of TR-FRET ZAP-70-2pY binding assay. Addition of an inhibitory compound would cause AlexaFluor488-2pY to dissociate and result in a decrease in TR-FRET signal.
B. TR-FRET measurement of ZAP-
70 binding to AlexaFluor488-2pY produces a KD value of 55.4 ± 7.1 nM.
C. Competition assay with 1pY displacing AlexaFluor488-2pY produces a Ki value of 112 ± 1.5 μM.
A. Diagram of the FP assay with the isolated tandem SH2 unit.
B,C. Concentration–response curves and chemical structure of compounds 180882 and 180013.