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Updated: Dec 23, 2025
Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Small molecule AX-024 reduces T cell proliferation independently of CD3ϵ/Nck1 interaction, which is governed by a
Kirsten Richter1, Arne C Rufer2, Magali Muller1
1I2O Disease Translational Area, pRED Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland.
Abstract:
Activation of the T cell receptor (TCR) results in binding of the adapter protein Nck (noncatalytic region of tyrosine kinase) to the CD3ϵ subunit of the TCR. The interaction was suggested to be important for the amplification of TCR signals and is governed by a proline-rich sequence (PRS) in CD3ϵ that binds to the first Src homology 3 (SH3) domain of Nck (Nck-SH3.1). Inhibition of this protein/protein interaction ameliorated inflammatory symptoms in mouse models of multiple sclerosis, psoriasis, and asthma. A small molecule, AX-024, was reported to inhibit the Nck/CD3ϵ interaction by physically binding to the Nck1-SH3.1 domain, suggesting a route to develop an inhibitor of the Nck1/CD3ϵ interaction for modulating TCR activity in autoimmune and inflammatory diseases. We show here that AX-024 reduces T cell proliferation upon weak TCR stimulation but does not significantly affect phosphorylation of Zap70 (ζ chain of T cell receptor-associated protein kinase 70). We also find that AX-024 is likely not involved in modulating the Nck/TCR interaction but probably has other targets in T cells. An array of biophysical techniques did not detect a direct interaction between AX-024 and Nck-SH3.1 in vitro Crystal structures of the Nck-SH3.1 domain revealed its binding mode to the PRS in CD3ϵ. The SH3 domain tends to generate homodimers through a domain swap. Domain swaps observed previously in other SH3 domains indicate a general propensity of this protein fold to exchange structural elements. The swapped form of Nck-SH3.1 is unable to bind CD3ϵ, possibly representing an inactive form of Nck in cells.
Insights
The small molecule AX-024 impacts T cell receptor (TCR) signaling by reducing T cell proliferation, but it does not directly inhibit the Nck/CD3ϵ interaction. Further research is needed to identify its actual cellular targets for treating inflammatory diseases.
Area of Science:
- Immunology
- Molecular Biology
- Structural Biology
Background:
- T cell receptor (TCR) activation involves Nck adapter protein binding to CD3ϵ.
- This interaction, mediated by Nck's SH3.1 domain and CD3ϵ's proline-rich sequence (PRS), amplifies TCR signals.
- Inhibiting this interaction shows potential for treating inflammatory diseases like multiple sclerosis, psoriasis, and asthma.
Purpose of the Study:
- To investigate the mechanism of action of the small molecule AX-024.
- To determine if AX-024 inhibits the Nck/CD3ϵ interaction and modulates TCR signaling.
- To explore the structural basis of Nck-SH3.1 interactions and potential regulatory mechanisms.
Main Methods:
- Biophysical techniques (e.g., SPR, ITC) to assess AX-024 binding to Nck-SH3.1.
- T cell proliferation assays to evaluate the effect of AX-024 on TCR stimulation.
- Analysis of T cell signaling pathways, including Zap70 phosphorylation.
- X-ray crystallography to determine the structure of Nck-SH3.1 and its interaction with CD3ϵ PRS.
Main Results:
- AX-024 reduced T cell proliferation under weak TCR stimulation but did not significantly affect Zap70 phosphorylation.
- Biophysical assays showed no direct interaction between AX-024 and Nck-SH3.1 in vitro.
- Crystal structures revealed Nck-SH3.1 binding to CD3ϵ PRS and identified a homodimeric form via domain swapping, which is unable to bind CD3ϵ.
Conclusions:
- AX-024's mechanism for reducing T cell proliferation is not via direct inhibition of the Nck/CD3ϵ interaction.
- The Nck-SH3.1 domain can form inactive homodimers through domain swapping, suggesting a potential cellular regulatory mechanism.
- Further studies are required to identify the precise targets and pathways affected by AX-024 in T cells for autoimmune and inflammatory disease modulation.
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