Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5 in Jurkat Cells.

Death receptor 5 (DR5) is an apoptosis-inducing membrane receptor that mediates cell death in several life-threatening conditions. There is a crucial need for the discovery of DR5 antagonists for the therapeutic intervention of conditions in which the overactivation of DR5 underlies the pathophysiology. DR5 activation mediates cell death in non-alcoholic fatty liver disease (NAFLD) and neurodegenerative processes including amyloid-beta (Aß) accumulation, spinal cord injury (SCI), and brain ischemia. In the current work, we used fluorescence resonance energy transfer (FRET) to monitor the conformational dynamics of DR5 that mediate death signaling. We used a time-resolved FRET screening platform to screen the Selleck library of 2863 U.S. Food and Drug Administration (FDA)-approved compounds. The high-throughput screen (HTS) identified 13 compounds that modulated the FRET between DR5 monomers beyond 5 median absolute deviations (MADs) from the DMSO controls. Of these 13 compounds, indirubin was identified to specifically inhibit tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 activity without modulating DR5 surface expression or TRAIL binding. Indirubin inhibited Fas-associated death domain (FADD) oligomerization and increased cellular FLICE-inhibitory protein (c-FLIP) expression; both are molecular mechanisms involved in inhibiting the DR5 signaling cascade. This study has elucidated previously unknown properties of indirubin that make it a promising candidate for therapeutic investigation of diseases in which overactivation of DR5 underlies pathology.

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.