Abstract 2454 Identifying Conformation-Dependent Ligandable Regions of Lck Using Parallel Chemoselective Profiling

Lck is a lymphocyte specific tyrosine kinase involved in T cell activation in response to T cell receptor (TCR) mediated signaling. T cell activation is essential for the adaptive immune response, as it results in the proliferation of T cells after the detection of a non-self peptide on a Major Histocompatibility Complex (MHC) presenting cell and the production of cytokines necessary for immune response coordination. Lck activity is dependent on its global conformation, which is modulated by the phosphorylation of Lck's regulatory domains. Upon TCR engagement, active Lck phosphorylates the CD3ζ chains of the TCR complex, transducing intercellular signaling that activates T cells. Although the basics of the signaling events that occur after Lck activation and TCR engagement are understood, the development of cysteine-reactive covalent ligands to specifically target and stabilize Lck in its conformational extremes is unexplored. By installing cysteines on Lck's kinase domain that do not affect kinase activity, we are able to use mass spectrometry to screen for changes in the reactivity of these cysteines to a range of covalent fragments upon TCR stimulation. Using the changes in covalent fragment reactivity, we can identify regions of Lck that are differentially ligandable and can be targeted for covalent drug development, as well as quantitatively map changes in the conformational dynamics of Lck upon TCR stimulation. Thus far we have identified eight installed cysteines quantifiable using bottom-up chemoproteomics, three of which show competition in alkylation upon treatment with covalent fragments. Using these identified cysteine mutants, we will next conduct experiments to determine which of these cysteines exhibit differential alkylation upon TCR stimulation, as well as map the dynamics of hyperactive Lck mutants to provide us with a deeper mechanistic understanding of Lck's conformational dynamics. This research has been made possible in part through funding from the Levinson Emerging Scholar Award at the University of Washington, as well as the Department of Chemistry's Rising Chem Star Undergraduate Research Scholarship, University of Washington.