Inhibition of the T-cell Kinase ZAP-70
- Author(s): Visperas, Patrick Ramos
- Advisor(s): Kuriyan, John
- et al.
The adaptive immune system responds to foreign antigens to produce a highly specific immune response. T-cells, a type of white blood cell of the adaptive immune system, can either directly kill infected cells or aid in sending signals that regulate the immune response. The T-cell receptor is expressed on the surface of T-cells and recognizes foreign antigens presented by antigen presenting cells. Unlike transmembrane receptor tyrosine kinases, the T-cell receptor does not contain its own kinase activity. Rather, the T-cell receptor recruits a number of non-receptor tyrosine kinases upon extracellular antigen binding to trigger intracellular signaling cascades. Zeta-chain associated protein of 70-kDa (ZAP-70) is one such non-receptor tyrosine kinase that is recruited to regions of the T-cell receptor known as Immuno-receptor Tyrosine-based Activation Motifs (ITAMs). Clinical observations, as well as chemical genetic studies, suggest that ZAP-70 is a possible target for T-cell inhibition.
Small molecule inhibitors of ZAP-70 may prove to be therapeutically useful for the treatment of autoimmune disease or for organ transplant rejection. Because no such molecules exist today, we hope to exploit the structural insights gleaned from the recently solved autoinhibited structure of ZAP-70 to discover an allosteric inhibitor. In this dissertation, I describe a method of inhibiting the interaction between ZAP-70 and the T-cell receptor. I performed a high throughput screen for inhibitors of ZAP-70:ITAM binding and identified a collection of hit compounds. Inhibition by these compounds was verified in an orthogonal assay and was dose-dependent. I found that the compounds targeted the tandem-SH2 domains of ZAP-70 and were thiol-reactive. I go on to show that covalent cysteine modification underlies the inhibition of ZAP-70 and that individual compounds are specific for different cysteine residues.