Date

Wednesday 17 Sep 2025

Time

15:15 - 15:45

Location

GM413

Supervisor

Marta Artola Perez de Azanza

2nd reviewer

Rolf Boot

Jury

Mario van der Stelt

Lysosomal storage disorders are a class of inherited metabolic disorders, caused by defects in lysosomal enzymes leading to accumulation of their substrates. In Fabry disease α-galactosidase A (α-GalA) is deficient causing globotriaosylceramide (Gb3) to accumulate, contributing to cellular dysfunction. Current treatment options, such as enzyme replacement therapy (ERT) and pharmacological chaperone therapy (PCT), aim to restore α-GalA activity but are limited by high costs, immune responses, and limited patient availibility. An alternative strategy involves substrate reduction therapy (SRT), by targeting α-1,4-galactosyltransferase (A4GALT), the enzyme directly responsible for Gb3 synthesis. However, few effective inhibitors for A4GALT have been reported, and the development of inhibitors for glycosyltransferases in general remains challenging. Targeted protein degradation (TPD) strategies offer alternative possibilities to bind A4GALT, but current TPD platforms like PROTACs and LYTACs have been ineffective against Golgi-localized proteins such as A4GALT. In this research, SORTACs are evaluated as novel degraders that target sortilin, a membrane receptor that acts as a shuttling protein between the Golgi network and lysosomes. As a proof-of-concept, the current degraders were designed to covalently bind the A4GALT target with the aid of HaloTags, and tether the glycosyltransferase to sortilin, thereby potentially inducing lysosomal degradation. This thesis describes the production and purification of sortilin, the synthesis of a fluorescent sortilin probe, and optimization of a fluorescence polarization assay to determine the binding properties of these SORTACs. Additionally, the A4GALT-Halo construct and complementary fluorescent probes were generated to further study the sortilin trafficking pathway and the SORTAC degradation strategy in cellular systems.