A184 REPURPOSING DRUGS FOR SPLEEN TYROSINE KINASE (SYK) PEDIATRIC PATIENTS USING HIGH-THROUGHPUT SCREENING

Abstract

BackgroundSpleen Tyrosine Kinase (SYK) is a cytosolic, non-receptor tyrosine kinase with an imperative role in immune and non-immune processes. Recently, we identified six gain-of-function SYK variants in patients that presented multi-organ inflammation and immune dysregulation. The SYK variants displayed constitutive SYK phosphorylation in human embryonic kidney (HEK) 293T, colonic epithelial cells (SW480), and in knock-in heterozygous SYK mice. These observations mark SYK as a therapeutic target for autoimmune diseases.Phenotype drug discovery accelerates this process and can be done successfully with an appropriate phenotype. A possible phenotype displayed by SYK variants is SYK phosphorylation, as high-throughput screening can identify hit compounds that reduce the constitutive activation of phosphorylated SYK (p-SYK).Aims Aim 1: Determine the screening phenotype with wildtype (WT) and SYK S550Y variant in HEK293T cells. Recently, we observed increased phosphorylation in gain-of-function SYK variants We hypothesize that we can use phosphorylated-SYK (p-SYK) levels to identify hit compounds that can decrease the kinase activity in these variants. With stable transfected SYK WT and SYK S550Y HEK293T cell-line, protein analyses will be completed to characterize the appropriate screening phenotype.Aim 2 Establish an assay for high-throughput drug screening. We will utilize homogenous time-resolved fluorescence (HTRF) assay. The signal measured from HTRF is positively proportional to the level of p-SYK; therefore, we expect that S550Y cells will have a higher signal than the WT.Aim 3 Validate hit compounds in HEK293T and zebrafish. We will create a dose-response curve with the hit compounds in in vitro and in vivo models.MethodsWe will use stable transfection to established overexpressing SYK WT and S550Y HEK293T cells. We will apply homogenous time-resolved fluorescence (HTRF) to quantify p-SYK levels during the drug screening.ResultsProtein analyses have verified high expression of p-SYK in stable transfected HEK293T cells. No stimulation was required, as the cells showed an increased phosphorylation level at baseline. Downstream signaling partners such as p-ERK and p-JNK of the MAPK pathway displayed an upregulation. This suggests that the sustained activation of p-SYK may consequently affect cellular processes and contribute to the clinical manifestations observed in patients.ConclusionsThis research study will identify hit compounds that can produce a safe and effective biological response in pediatric patients with gain-of-function SYK variants. Personalizing medicine throughout high-throughput drug screening can accelerate drug repurposing for pediatric patients with multiple systemic diseases and immune dysregulation.Funding AgenciesNone