Identification and Development of Novel Wnt/β-Catenin Signalling Inhibitors

With an ever-aging population the risk of developing cancer is greater than ever. The WNT/β catenin signalling pathway has been highlighted as a key pathway in cancer development andfurthermore, is an attractive therapeutic target. However, the number of drugs targeting β-cateninis limited due to various factors and claimed small molecule modulators often have inherentproblems in specificity of mode of action or a lack of evidence, biophysical, KD, crystallography orotherwise. Therefore, there is dire need for therapeutic intervention of β-catenin. In this thesis, wereview the literature of reported and putative β-catenin inhibitors and highlight the vast majorityof them are flagged by computational filters such as PAINs and Brenk filters. Furthermore, weinvestigate these reported inhibitors by biophysical methods such as Differential ScanningFluorimetry (DSF) and Isothermal Titration Calorimetry (ITC) and show that in our hands none ofthem engage with recombinant purified β-catenin. Herein we report the discovery of novel smallfragments that are β-catenin stabilisers. These fragments have been validated by orthogonalbiophysical techniques such as DSF and Saturation Transfer Difference Nuclear Magnetic Resonance(STD-NMR) and ITC. Furthermore, we report the synthesis of Carnosic acid derivatives as novel βcatenin destabilisers. Finally, we undertake in silico HTVS to identify top computational compoundsto test against β-catenin in vitro

Biophysical survey of small-molecule β-catenin inhibitors: A cautionary tale

The canonical Wingless-related integration site signaling pathway plays a critical role in human physiology, and its dysregulation can lead to an array of diseases. β-Catenin is a multifunctional protein within this pathway and an attractive yet challenging therapeutic target, most notably in oncology. This has stimulated the search for potent small-molecule inhibitors binding directly to the β-catenin surface to inhibit its protein-protein interactions and downstream signaling. Here, we provide an account of the claimed (and some putative) small-molecule ligands of β-catenin from the literature. Through in silico analysis, we show that most of these molecules contain promiscuous chemical substructures notorious for interfering with screening assays. Finally, and in line with this analysis, we demonstrate using orthogonal biophysical techniques that none of the examined small molecules bind at the surface of β-catenin. While shedding doubts on their reported mode of action, this study also reaffirms β-catenin as a prominent target in drug discovery

Structure–reactivity studies of 2-sulfonylpyrimidines allow selective protein arylation

Protein arylation has attracted much attention for developing new classes of bioconjugates with improved properties. Here, we have evaluated 2-sulfonylpyrimidines as covalent warheads for the mild, chemoselective, and metal free cysteine S-arylation. 2-Sulfonylpyrimidines react rapidly with cysteine, resulting in stable S-heteroarylated adducts at neutral pH. Fine tuning the heterocyclic core and exocyclic leaving group allowed predictable SNAr reactivity in vitro, covering >9 orders of magnitude. Finally, we achieved fast chemo- and regiospecific arylation of a mutant p53 protein and confirmed arylation sites by protein X-ray crystallography. Hence, we report the first example of a protein site specifically S-arylated with iodo-aromatic motifs. Overall, this study provides the most comprehensive structure–reactivity relationship to date on heteroaryl sulfones and highlights 2-sulfonylpyrimidine as a synthetically tractable and protein compatible covalent motif for targeting reactive cysteines, expanding the arsenal of tunable warheads for modern covalent ligand discovery