High throughput screening assays to identify chemical probes targeting Hectd3, an E3 ubiquitin ligase implicated in multiple sclerosis - Abstract: Multiple sclerosis, a chronic autoimmune inflammatory disease associated with demyelination of the central nervous system (CNS), remains a public health issue. Currently there is no known cure for multiple sclerosis. Although several disease-modifying treatments (DMTs) are available, relapsing of multiple sclerosis occurs frequently and DMTs often result in severe adverse effects such as liver failure and fetal outcomes. Novel therapies are needed to reduce the disease burden for multiple sclerosis patients. Recently, we published that Hectd3, an E3 ubiquitin ligase, is expressed predominantly in T cells of the immune system, which play a critical role in pathogenicity of experimental autoimmune encephalomyelitis (EAE), a mouse model of human multiple sclerosis. Specifically, we found that Hectd3 controls pathogenic Th17 effector response in EAE by regulating ubiquitination of Malt1 and Stat3 in a non-degradative manner, resulting in stabilization of Malt1 and Stat3. In addition, Hectd3-mediated polyubiquitination of Stat3 promotes Stat3 activation. Moreover, Hectd3-deficient mice 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 for the attenuation of EAE in Hectd3−/− mice. Overall, our results demonstrate that Hectd3 is a critical modulator of Malt1 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 roles in pathogenesis of multiple sclerosis, currently there is no chemical probe to further investigate the pathways and the implication in therapy of multiple sclerosis. Therefore, in this proposal, we aim to develop high throughput screening 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 pathogenic Th17 cells, the identification of Malt1 and Stat3 as target substrates for Hectd3-mediated ubiquitination, and characterization of novel chemical probes for Hectd3, and their impact on EAE. The long-term sustained impact of this work is to identify compounds to modulate Hectd3 activity on its target substrates and its functions in EAE to open avenues for development of more specific and effective immune therapies to treat multiple sclerosis, a crucial need given current treatment challenges and limited therapeutic options. These combined approaches will lead to the development of unique Hectd3 inhibitors with novel inhibition mechanisms. This work will have a global reach by promoting fresh and effective strategies to treat multiple sclerosis. Hectd3 has also been implicated in promoting breast cancer drug resistance, cancer metastasis (unpublished results), and bacterial infections. Therefore, this project may also have significant impact on cancers and bacterial defense.
