Development of targeted degraders and allosteric ligands of NF-κB inducing kinase (NIK): synthesis, biochemical, and biophysical evaluation studies

Abstract

Kinases are commonly targeted in drug discovery campaigns due to their roles in many signaling pathways. Many kinase inhibitors work by occupying the orthosteric binding site and preventing ATP from being used as a substrate. All of the known efforts for targeting the disease relevant kinase NF-κB inducing kinase (NIK) have used a similar approach. This thesis will discuss our efforts in developing novel degraders of NIK by repurposing known orthosteric inhibitors and the development of the first known allosteric NIK ligands and characterization of their binding mode. Dysregulation of NIK has been implicated in a variety of cancers, including multiple myeloma, and immune disorders. Native regulation of NIK involves constant polyubiquitination and subsequent degradation of the protein by the 26s proteasome. Chapter 2 will discuss our efforts towards reinstating this degradation mechanism by using proteolysis targeting chimera (PROTAC) technology. Unfortunately, none of these compounds were able to degrade NIK in multiple myeloma cell lines. These findings serve as a case study into the difficulties of targeted-protein degradation. An allosteric-biased fragment-based screening effort leading to novel allosteric NIK ligands will be discussed in Chapter 3. The primary hit compound 3.1 displayed a Kd of 130 μM and did not show any competition with a non-hydrolysable ATP analogue (AMP-PNP) by NMR or SPR assays. Synthesis of analogues lead to compound 3.4 bearing a cyclopropyl ring and having a binding affinity of 40 μM. Efforts towards tighter binders was difficult due to the lack of structural information. Therefore, Chapter 4 discusses how the covalent probe compound 4.1 was used to adduct a cysteine near the allosteric binding site that was subsequently uncovered using LC-MS-MS peptide mapping experiments. It was uncovered that compound 4.1 was able to selectively adduct to C573 in this allosteric site on the catalytic domain of NIK. Finally, Appendix A focuses on early efforts towards the discovery of allosteric NIK inhibitors from computational screening efforts from our collaborators in the Amaro lab at the University of California – San Diego. While none of these compounds were able to inhibit NIK activity, this served as the starting point that lead to the findings of both Chapter 3 and Chapter 4.