Discovery Of Small Molecule Modulators Of Tnf Receptor Signaling

Abstract

Tumor necrosis factor (TNF) is a central regulator of immunity and plays a critical role in the pathogenesis of autoimmune inflammatory diseases. TNF interacts with two receptors called tumor necrosis factor receptor 1 (TNFR1) and TNFR2. Activation of TNFR1 leads to cell inflammation and cell death while TNFR2 signaling is generally associated with cell survival and protection. Overexpression or dysregulation of TNF can lead to many different diseases including rheumatoid arthritis, Crohn’s disease, and ulcerative colitis. This resulted in anti-TNF biologics being developed to treat autoimmune inflammatory diseases. Currently there are five FDA approved TNF inhibitors used to treat these diseases. However, TNF inhibitors are known to cause side effects because they inhibit both TNFR1 and TNFR2 signaling by sequestering the ligand. Therefore, specifically targeting TNF receptors is a more attractive approach when treating these diseases. Previous work in our lab showed it’s possible to inhibit TNF receptor signaling by specifically targeting the receptor-receptor interactions without blocking ligand binding. We developed a cellular time resolved fluorescence resonance energy transfer (TR-FRET)-based TNFR1 biosensor to monitor structural changes in TNFR1 oligomers upon activation and inhibition. We used this TNFR1 biosensor to screen a library of small molecules and identified a small molecule, zafirlukast, that inhibits TNFR1 function. In this current study, using biochemical and biophysical assays, we showed zafirlukast and its analogs do not affect ligand binding. Next, we developed a cell-based functional assay to monitor the effect these compounds have on TNFR2 function. This assay showed zafirlukast and its analogs do not affect TNFR2 function. We also sought to adapt the TR-FRET based biosensor technology to determine if it could be used to create a high throughput screening platform that can identify small molecule effectors of TNFR2 for treating certain autoimmune diseases, neurodegenerative diseases, and cancer. To do this, we created a TNFR2 biosensor using molecular biology and cloning techniques and tested its FRET efficiency and specificity using a fluorescence lifetime plate reader. In the future, a Selleck high throughput screen will be performed to identify TNFR2 binders. We will test the functionality and specificity of these TNFR2 lead compounds using cell-based functional assays. If the lead compounds for TNFR2 are shown to be potent and specific, then they can serve as a potential drug candidate for treating certain autoimmune diseases or neurodegenerative diseases.