Targeting Kinases with Small Molecule Covalent Inhibitors

Abstract

PhD ThesisProtein kinases are considered the largest and most functionally diverseenzymesuperfamily critically involved in the regulation of almost all cellular process. Theyareone of the most intensively pursued targets, particularly in cancer drugdiscoveryprograms. In the case of cancer therapy, covalent inhibitors have proven to be better than reversible inhibitors and several kinases targeted covalent inhibitors are in clinical use. In this thesis, irreversible inhibition of NF-κB inducing kinase(NIK) and Epidermal growth factor receptor (EGFR) is investigated. NIK is an essential upstream kinase, which regulates activation of the noncanonical NF-κB pathway involved in lymphoid organ development and adaptive immune responses. Numerous genetic studies demonstrated that the aberrant expression or regulation of NIK is associated with several disease states including cancers. The NIK kinase has been targeted for drug therapy by small molecule reversible inhibitors,but many of them were impractical for in-vivo administration due to poor selectivity or pharmacokinetic properties. Achieving potency and selectivity in NIK inhibitors is particularly challenging due to its constitutive activity, high ATP affinity (Km4µM) and relatively shallow binding pocket. Developing an irreversible inhibitor of NIK that targets active site Cys-444, which is unique to NIK, is expected to deliver greatly improved selectivity and superior efficacy. Extensive structure-activity relationshipstudies (SARs) based on literature NIK inhibitors have led to lead benzimidazole28. Benzimidazole 28 exhibits NIK potency of 0.18 µM and has been confirmed to bind covalently to NIK Cys-444, but has poor selectivity profile inhibiting both NIK dependent and independent cell lines. Co-crystallisation studies using28werecarried out to understand the binding mode in NIK and identify structural features to exploit to achieve better potency and selectivity in future analogues. SAR studies around the hinge binding motif, benzimidazole core structure, and covalent warhead were investigated, and a number of analogues were designed and synthesised. A scaffold hopping approach based on recently published NIK inhibitors was also carried out with the aim of improving the compound potency and selectivity. EGFR is critically involved in cell signalling pathways that control cell growth, proliferation, and survival. Multiple generations of EGFR TKIs have been approved for clinical use. Mutations or over-expression of EGFR have been discovered in association with many types of cancers providing a rationale for efforts to inhibit EGFR. Multiple generation of EGFR TKIs have been developed and approved for clinical use. Despite the documented efficacy of FDA approved third generation EGFR TKIs, new EGFR mutations and other drug resistance mechanisms emerge rapidly after treatment leaving patient without further therapeutic options other than chemotherapy and local ablative therapy. Multiple mechanisms of resistance were detected in clinical trials with tertiary C797S mutation accounting for more than20%incidence rate and the most difficult to deal with. It was proposed that developing an irreversible inhibitor of EGFR by targeting Cys-775 would deliver a new therapeutic option for patient who develop resistance to third generation EGFR TKIs. Extensive structure-activity relationship studies were conducted based on literature compounds have led to lead pyrimidopyridone 128. Pyrimidopyridone 128 exhibits a potency of 107 nM in the TMTR-FRET assay and has been confirmed to be a covalent inhibitor of EGFR targeting Cys-775. Co-crystallisation studies using 128 were carried out to understand the binding mode in EGFR, and to help in identifying structural features to exploit to achieve better potency. Further SARs studies to improve the compound potency were conducted in which SARs around the phenyl ring were undertaken. Compounds with electron rich phenyl ring were more potent than compounds with electron deficient phenyl ring. AQSAR study revealed that compounds with electron donating substituents were more potent regardless of the position of the substituents, but no obvious relationship was observed with the lipophilicity. The outlier of this was compound 151, which was the most potent compound in this series, though not the one with the most electron rich phenyl ring suggesting that the sulphonamide group at the para-position was making an additional interaction within the sugar pocket. Modelling of compound151withdouble mutant EGFR revealed the proximity of several functional groups that may act as H-bonding partners to the sulphonamide moiety in compound 151 (Ser-797intriple mutant EGFR, Asn-842 and Ser-720). TJordan University of Science and Technolog