Modulation of NF-kB Activity with Synthetic Inhibitors of NEMO

Abstract

The NF-κB pathway becomes constituently active in cancer, autoimmune disorders, and in infectious diseases such as COVID-19 and has thus been the focus of much drug-discovery research. Targeting NF-κB activity is challenging due to the complexity of the different activation pathways and apparent crosstalk between the pathways themselves. Additionally, many of the constituent pathway proteins and protein complexes are classed as ‘undruggable’ due to their intrinsically disordered regions and the well-folded domains lacking discrete binding sites. Selectivity for particular arms in the pathway has been problematic as well since many of the kinases play multiple roles. This dissertation focused primarily on regulating NF-κB transcriptional activity through modulation of the IKK pathway by targeting NEMO. NEMO was chosen as a target because of the critical role it plays as a scaffold protein that functions to recruit both IKKα and IKKβ for phosphorylation in addition to its exclusivity with the canonical pathway. The main goal of this thesis focused on mimicking key features from NEMO binding partners in order to block NEMO’s function in the NF-κB pathway. By applying targeted NEMO binding domain peptides derived from native binding partner IKKβ, we expanded past the native functionality of previously characterized molecules. Disrupting PPIs is challenging, however they provide distinct interaction motifs that rational peptide drug design can utilize for increasing efficacy. The NEMO-IKK interaction is largely dependent on hydrophobic interactions between aromatic residues present on both proteins that present a pharmacophore for targeting. Through this rational we designed a series of unnatural amino acid NEMO binding domain peptides with varying tryptophan substituents at key residues 739 and 741. We found that by altering the electron density at these positions through chlorine atom additions, we were able to increase the binding affinity substantially and produce a 13-mer NEMO binding domain construct with relative high affinity. Inhibitor through degradation can be an effective strategy for modulating the activity of hard-to-target PPIs as well. Our attempt to apply this strategy onto KKβ-derived peptides to achieve an alternative mode of modulation of NEMO and NF-κB activity led us to a functional study using both large (45-mer) and small (11-mer) IKKβ derived peptides. We explored and dissected the different components that make peptide based PROTACs effective for targeting NEMO. This resulted in the identification of a molecule (NP-8) that showed retained binding affinity to NEMO and modestly decreased the activity of NF-κB in HEK-293T cells. The overarching results from this dissertation showcase how utilization of peptide designed mimics can be an effective and promising avenue for targeting protein-protein interactions and inhibition of NF-κB.