TARGETING THE HECT-TYPE E3 LIGASE NEDD4: CHARACTERISATION OF NOVEL COVALENT INHIBITORS

This thesis focuses on the characterisation of novel covalent NEDD4 inhibitors developed by our laboratory. Starting from compound 32, the most potent identified (IC₅₀ = 0.12 µM), a biotinylated analogue – compound 31B – was synthesised to enable cellular and proteomic studies. Despite strong in vitro binding, 31B showed no cellular uptake or cytotoxic activity in NEDD4-expressing or knockout cells. To exclude external interference, assays were repeated in serum-free media and with purified proteins, confirming its biochemical activity but not its efficacy in a cellular context. In contrast, compound 17B retained its cytotoxicity in NEDD4 KO cells, suggesting additional targets. Label-free proteomics identified mitochondrial proteins, including UQCRC1 – a subunit of Complex III in the mitochondrial electron transport chain – as enriched off-targets. Immunofluorescence confirmed compound–mitochondria co-localisation. UQCRC1 silencing impaired proliferation and mitochondrial morphology, but did not rescue cells from compound 17B toxicity. These results suggest that NEDD4 is not the sole mediator of cytotoxicity and support a multi-target mechanism. Overall, this work provides insights into the cellular behaviour of advanced NEDD4 inhibitors and their potential utility in Targeted Protein Degradation approaches.