Lucy L. Robbie; Tyler Beyett
J. Pharmacol. Exp. Ther., 2024, 389(S3), 293
https://doi.org/10.1124/jpet.293.129469

Abstract

Abstract ID 129469Poster Board 293Thymocytes are involved in the adaptive immune system where they eliminate pathogens. Mature T cells develop from immature thymocytes through a regulated developmental process called selection. During this process, thymocytes are selected for their ability to recognize antigens. THymocyte-Expressed Molecule Involved In Selection (THEMIS) is a protein expressed in developing thymocytes that is required for maturation of T cells. THEMIS contains tandem Cystine-containing All Beta In THEMIS (CABIT) domains, which are only found in a limited number of human proteins. THEMIS interacts with and regulates tyrosine phosphatases SHP-1 and -2, though the precise molecular mechanism is unknown. THEMIS expression increases in CD4+/CD8+ cells, suggesting THEMIS may hold a pivotal role in regulating selection at the double-positive stage by modulating phosphotyrosine signaling downstream of the T cell receptor.Despite its significance to T cell development, it is unknown whether THEMIS represents a therapeutic target due to a lack of understanding of its function and an absence of chemical probes. To remedy this, we are conducting two screens to identify ligands that bind THEMIS in collaboration with Structural Genomics Consortium (SGC). First, we are using a DNA Encoded Library (DEL) screen with >109 small molecule ligands. In this approach, THEMIS is used as a bait in a pulldown and molecules that bind are identified by sequencing their unique DNA tags. In parallel, we are using Affinity Selection-Mass Spectrometry (AS-MS) to identify ligands. AS-MS utilizes size-exclusion chromatography to enrich for small molecules from a mixture that co-elute with the protein of interest prior to identification by mass spectrometry.Prior to screening, THEMIS was recombinantly expressed and purified and mass spectrometry was utilized to confirm the sequence of the protein and identify any post-translational modifications. As ligands are identified from DEL and AS-MS screening, compounds are clustered based on structural similarities to streamline validation and analysis. We are utilizing a variety of biophysical approaches to assess the binding affinities and sites of select ligands on THEMIS. Further investigation into these probes can lead to the discovery of THEMIS modulators, which could be utilized to block or enhance protein-protein interactions between THEMIS and SHP1/2, manipulating downstream signaling. This may reveal unrealized therapeutic applications for THEMIS in T cell malignancies and immunotherapies.We acknoledge Emory University for startup funds and the Structural Genomics Consortium for financial support.