Aziz, Shaista; (2017) The design and synthesis of Cyclohexadienones as mutant specific K-RasG12C inhibitors. Doctoral thesis (Ph.D), UCL (University College London).

Abstract

Ras proteins (H-, K- and N-Ras) are small signalling proteins that function as molecular switches controlling cell proliferation, differentiation and survival. Somatic mutations of the Ras proteins have been linked to the development of sporadic human tumours with K-Ras mutations being the most prevalent. One of the most frequent point mutations found in K-Ras, occurring close to the GTP binding site, is the substitution of glycine 12 for a cysteine residue and accounts for 10-20% of all Ras-driven cancers. Several efforts have been made towards mutant specific targeting of K-RasG12C by taking advantage of the reactive cysteine group. However, to date such approaches have suffered from either a lack of target selectivity or insufficient potency. Cyclohexa-2,5-dienones belong to a class of cross-conjugated Michael acceptors that are able to react with nucleophiles such as thiols and hence have the potential to modify cysteine residues incorporated in target proteins. The aim of this project was to design and synthesise mutant specific inhibitors of K-RasG12C incorporating the cyclohexadienone moiety as the thiol-reactive warhead to covalently modify the mutant cysteine residue of K-Ras and to evaluate their biological activity in vitro. Several novel covalent-binding inhibitors incorporating mono and bis-Michael acceptor functionalities were synthesised and were tested for their inhibitory potential and target selectivity in a cell viability assay, using SW48 colon cancer cell lines expressing wild-type or mutant K-RasG12C. From this study, several compounds that incorporated cyclohexenone motifs were found to induce strong cytotoxic effects with IC50 values below 1 nM in both cell lines indicating high inhibitory potential, but an absence of target selectivity. Compounds incorporating cyclohexadienone moieties exhibited limited cytotoxicity in both cell lines. The cyclohexadienone structure was also explored to synthesise a small subset of compounds as potential inhibitors of Keap1-Nrf2 protein-protein interactions. A linear 8-mer peptide derived from the Keap1 binding motif of Nrf2 was modified with cysteine residues and subsequently cyclised with the cyclohexadienone analogues. These cyclised analogues may find wider application in the development of novel biological probe molecules.

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