Nuclear factor kappa B (NF-κB) is a family of transcription factors that play a crucial role in the immune response. In the NF-κB activation pathway, the signaling cascade can be initiated by cytokines, cellular stress, viral or bacterial agents, and ionizing radiation. NF-κB acts to enhance or repress genes important in the regulation of inflammation, apoptosis, proliferation, and cell survival. As aberrant activation of the NF-κB pathway underlies many inflammatory diseases and cancers, the development of specific small molecule inhibitors of this pathway would have important therapeutic applications. The key regulator of the NF-κB pathway is the IκB kinase (IKK) complex. IKK complex contains two catalytic components, IKK1 and IKK2, and one scaffolding component NF-κB essential modulator (NEMO). In the canonical pathway, the catalytic subunit IKK2 phosphorylates IkBα, an inhibitor that holds NF-κB dormant in the cytoplasm. Phosphorylated IkBα is then polyubiquitinated and undergoes proteasomal degradation thus releasing NF-κB to travel to the nucleus. Our experimental data show that the small molecules we developed are specific and effective allosteric inhibitors that target IKK2. Cellular assays show that IkBα degradation is reduced when cells are treated with our inhibitors and then stimulated with tumor necrosis factor alpha. In vitro assays show that though kinase function of activated IKK is not blocked, the activation of NF-κB is prevented as shown by luciferase assay. In addition, we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to show how the binding of these inhibitors affects the protein dynamics of IKK2 and to identify possible places of inhibitor binding. Our HDX-MS data support that our inhibitors work allosterically through examination of differences in IKK2 structural dynamics when IKK2 is partnered with ATP, with known ATP-competitive inhibitors, and with our inhibitors. Following identification of possible regions of inhibitor binding using HDX-MS, we use tandem mass spectrometry (MS/MS) to identify specific binding residues. Using the collected data, we developed a model of how our small molecule inhibitors disrupt the activation of IKK.

Development of small molecule inhibitors of IKK2

2019 Spring National Meeting

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