PROJECT SUMMARY Itaconate is a major regulator of macrophage inflammatory activity through its ability to directly alkylate cysteines on critical target proteins. Using our newly developed mass spectrometric (MS) method (CPT-MS), we identified a newfound regulated cysteine on the SH2 domain containing protein tyrosine phosphatase-1 (SHP-1) as a target of itaconate in both mouse and human macrophages. We developed a library of highly potent itaconate-mimetics and found that the anti-inflammatory activity of these molecules was dependent on this critical SHP-1 cysteine modification. Together, this led us to hypothesize that the newfound SHP-1 regulatory cysteine (Cys102) is a critical proximal inhibitor of macrophage cytokine production, and that chemical modification of this site presents an unprecedented opportunity to modulate the production of inflammatory cytokines. We will test our hypothesis through three aims: Aim 1) To determine the structural basis of SHP-1 cysteine regulation and its role in macrophage activation, we will combine our CPT-MS platform, protein crystallography and biochemistry, loss-of-function models, and cytokine production assays to define the role of SHP-1 and its Cys102 residue in macrophage activation. Aim 2) To identify potent itaconate- mimetic chemotypes that can selectively target the SHP-1 activation cysteine in macrophages, we will use structure guided medicinal chemistry to improve potency and specificity of our lead itaconate-mimetics. The newly synthesized compounds will be tested using phenotypic and MS assays in both mouse and human macrophages. Aim 3) To define the interactome and downstream effectors of SHP-1 upon cysteine activation, we will use MS to identify the SHP-1 binding partners, and genetic modification via CRISPR/Cas9 to validate the role of these interactions in macrophage activity and cytokine production. We will also examine the role of known SHP-1 binding partners, IRAK and STAT3, in macrophage deactivation. If successful, our project will for the first time 1) define the role of SHP-1 cysteine activation in itaconate-mediated inhibition of macrophage activity and 2) identify new compounds with potential to inhibit macrophage cytotoxic activity with high specificity and efficacy. This will represent an important step forward in both our understanding of the fundamental mechanisms of macrophage biology and development of compounds that can serve as both highly specific biological tools and have translational potential for pathologies characterized by chronic inflammation.