Expanding the Inhibitor Space of the WWP1 and WWP2 Ubiquitin Ligases

Dudey, Ashley Philip (2024) Expanding the Inhibitor Space of the WWP1 and WWP2 Ubiquitin Ligases. Doctoral thesis, University of East Anglia.

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Abstract

The two members of the NEDD4 family of HECT E3 ligases, WW-domain containing protein 1 (WWP1) and 2 (WWP2), are responsible for the Ubiquitin-mediated degradation and downregulation of key tumour suppressor proteins and transcription factors. As such, WWP1 and WWP2 dysregulation has been directly linked to various oncogenic, cardiovascular, osteogenic and even infectious diseases. Despite their therapeutic potential, there is a limited number of small-molecule inhibitors known in the literature.

This thesis aimed to expand the inhibitor space of WWP1 and WWP2, utilising biochemical, biophysical and structural techniques to identify and develop small molecule inhibitors through a structure-activity relationship (SAR) by synthesis approach. Following the drug discovery pipeline, Hit-identification was achieved using high-throughput differential scanning fluorimetry (DSF) and in vitro autoubiquitination assays to identify small molecule ligands and inhibitors, respectively, through screening of the NCI Diversity Set VI compound library. NSC-217913 was selected as the sole hit compound demonstrating an IC50 of 158.3 μM towards WWP1. In the next Hit-to-Lead phase, a series of NSC-217913 analogues were generated, with compound S11 displaying an increased potency towards WWP1 (32.7 μM) and WWP2 (269.2 μM). This approach was also used to demonstrate the first in vitro WWP1 activity of the literature-derived NEDD4-1 inhibitor Indole-3-carbinol (I3C), however, its acid-condensation product 3,3′-diindolylmethane (DIM) shown to be considerably more potent (WWP1 IC50 of 111.2 μM). The acid-stabilised I3C derivative N-Tosyl-I3C was also shown to non-selectively inhibit both WWP1 (IC50 of 218.3) and WWP2 (IC50 of 223.7). Finally, X-ray crystallography was used to solve improved macromolecular models of WWP1 and WWP2 for subsequent use in molecular docking studies as a means to further understand the SAR of the NSC-217913 and I3C inhibitor scaffolds. This project provides the basis for lead compound development in the next phase of the drug discovery pipeline.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	02 Apr 2025 09:18
Last Modified:	02 Apr 2025 09:18
URI:	https://ueaeprints.uea.ac.uk/id/eprint/98920
DOI:

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