Biochemical and functional characterization of a novel anti-inflammatory biogenic amine

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Summary Tryptophan (Trp) catabolism is a complex pathway that generates over fifty metabolites in a cell-specific manner. Besides being the precursor of serotonin and melatonin, tryptophan generates a cascade of metabolites known as kynurenines. Kynurenine metabolites are regarded as one of the most powerful mediators of immune regulation. The thrust of this application stems from our original observations that: (i) lymphatic endothelial cells (LEC) and dendritic cells (DCs) secrete a previously unidentified biogenic amine, 3HKA, which derives from a lateral pathway of Trp catabolism, whose function is currently unknown; (ii) 3HKA exhibits a clear anti- inflammatory profile by inhibiting the STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines; most notably, IL-6, IL12p70 and TNFα; (iii) in vivo, 3HKA exerts protective effects in the experimental model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring. In a model of nephrotoxic lupus, 3HKA improved proteinuria and serum urea nitrogen, overall ameliorating the immune-mediated glomerulonephritis and renal dysfunction. As such, the ultimate goal of this application is to fully characterize the biological activity of 3HKA. In Aim 1, a series of biochemical and biophysical analyses will be employed to identify the enzyme responsible for 3HKA production and investigate the regulation of its synthesis under physiological and pathological conditions. In Aim 2, by using biotinylated derivatives of 3HKA, and a series of biochemical and biophysical experiments, we will identify the receptor target of 3HKA and its expression profile. In Aim 3, using targeted and untargeted phosphoproteomic approaches, screening of kinase libraries and CRISPR/Cas9 knock down strategies we will characterize the 3HKA signal transduction pathway. Additionally, we will analyze the effect of 3HKA on T cell activation and differentiation into a TH1, TH2, TH17 phenotype, naïve vs effector and memory T cell transition and 3HKA effect on maturation/activation of different APC (DC, pDC, MΦ). Immunophenotyping will be performed both in vitro and in vivo, using psoriasis or nephrotoxic lupus as animal models. Finally, in Aim 4 we leveraged our knowledge on 3HKA and generated 3HKA-like compounds, predicted to have an anti- inflammatory effect. These analogs will be screened in a series of in vitro and in vivo assay to fully assess their potential as novel immunomodulatory molecules