Abstract: Multiple sclerosis a chronic autoimmune inflammatory disease associated with demyelination of thecentral nervous system (CNS) remains a public health issue. Currently there is no known cure for multiplesclerosis. Although several disease-modifying treatments (DMTs) are available relapsing of multiple sclerosisoccurs frequently and DMTs often result in severe adverse effects such as liver failure and fetal outcomes. Noveltherapies are needed to reduce the disease burden for multiple sclerosis patients. Recently we published thatHectd3 an E3 ubiquitin ligase is expressed predominantly in T cells of the immune system which play a criticalrole in pathogenicity of experimental autoimmune encephalomyelitis (EAE) a mouse model of human multiplesclerosis. Specifically we found that Hectd3 controls pathogenic Th17 effector response in EAE by regulatingubiquitination of Malt1 and Stat3 in a non-degradative manner resulting in stabilization of Malt1 and Stat3. Inaddition Hectd3-mediated polyubiquitination of Stat3 promotes Stat3 activation. Moreover Hectd3-deficientmice showed reduction in EAE disease scores Th17 cell pathogenicity and effector Th17 cytokines.Furthermore Hectd3 deficiency causes a cell-intrinsic defect in Th17 cell pathogenicity that is responsible forthe attenuation of EAE in Hectd3/ mice. Overall our results demonstrate that Hectd3 is a critical modulator ofMalt1 and Stat3 signaling in EAE. Based on these results we hypothesize that compounds abolishing Hectd3-mediated ubiquitination of substrates can lower EAE severity. However although Hectd3 plays significant rolesin pathogenesis of multiple sclerosis currently there is no chemical probe to further investigate the pathwaysand the implication in therapy of multiple sclerosis. Therefore in this proposal we aim to develop high throughputscreening assays to identify and characterize chemical probes to investigate in depth the biochemistry of Hectd3-mediated Malt1 and Stat3 signaling pathways and their therapeutic potential in pathogenic Th17 cells and EAE.This innovative work explores the novel function of Hectd3 in immune regulation specifically in pathogenicTh17 cells the identification of Malt1 and Stat3 as target substrates for Hectd3-mediated ubiquitination andcharacterization of novel chemical probes for Hectd3 and their impact on EAE. The long-term sustainedimpact of this work is to identify compounds to modulate Hectd3 activity on its target substrates and its functionsin EAE to open avenues for development of more specific and effective immune therapies to treat multiplesclerosis a crucial need given current treatment challenges and limited therapeutic options. These combinedapproaches will lead to the development of unique Hectd3 inhibitors with novel inhibition mechanisms. This workwill have a global reach by promoting fresh and effective strategies to treat multiple sclerosis. Hectd3 has alsobeen implicated in promoting breast cancer drug resistance cancer metastasis (unpublished results) andbacterial infections. Therefore this project may also have significant impact on cancers and bacterial defense.