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

Abstract

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.

Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

“…Our approach to developing this drug discovery pipeline has been consistent to previous efforts with other biosensor systems (e.g. tau and TNFR1, amongst others) 84,[90][91][92][103][104][105] . At the core of any fluorescent assay is the potential of artifacts being introduced into the model system through the labeling of protein with synthetic fluorophores or fusion to large fluorescent XFPs.…”

“…Another concern with HTS campaigns is the physiological relevance of the cellular platform being deployed. The choice of HEK293 cells for our FRET HTS primary assay implementation is strongly rooted in the thorough vetting of this cell lines performance in multiple HTS campaigns that interrogated a wide range of protein targets 84,[90][91][92][103][104][105] . We acknowledge that not using neuronal cell lines in a primary HTS platform targeting neurodegeneration imparts a disconnect between the physiological and epigenetic link between cell type and disease environment.…”

“…The core of our cellular bio-sensor system builds upon our established, state-of-the-art, HEK293/fluorescent lifetime plate reader (FLT-PR) platform designed to monitor specific protein interactions and structural changes in living cells. [83][84][85] Fluorescence lifetime (FLT) detection increases the precision of FRET-based screening by a factor of 30 compared with conventional fluorescence intensity detection 81 , and provides exquisite sensitivity to resolve minute structural changes within protein ensembles. The improved signal-to-noise of FLT detection is due to FLT's being an inherent property of the XFP and not sensitive to fluctuations in steady state intensity.…”

“…A major challenge to any fluorescent based HTS platform stems from potential fluorescent interference due to fluorescent compounds in the library. Fluorescent compounds (FC) were flagged using a spectral similarity index filter which evaluates the ratio of intensity from two bandwidths that span the donor emission spectrum (see 84 for more detail) and are excluded as potential false-positives 84,[89][90][91][92] . FLT for all compounds that passed the FC filter are presented in Fig.…”

“…Cell cultures were maintained in an incubator with 5% CO2 (Forma Series II Water Jacket CO2 Incubator, Thermo Scientific) at 37℃. The intermolecular (oligomer) and intra-molecular (monomer-conformation) aSyn FRET biosensors were generated by transiently transfecting HEK293 cells using Lipofectamine 3000 (Invitrogen) with GFP-aSyn and aSyn-RFP (1:8 DNA plasmid concentration ratio) or GFP-aSyn-RFP plasmid, respectively The effectiveness of HEK293 cells transfected with FRET constructs as a HTS platform has been demonstrated in our previous work 84, [90][91][92][103][104][105] .…”