Discovery of Novel Ligands for TNF-α and TNF Receptor-1 through Structure-Based Virtual Screening and Biological Assay

Tumor necrosis factor alpha (TNF-alpha) is overexpressed in various diseases, and it has been a validated therapeutic target for autoimmune diseases. All therapeutics currently used to target TNF-alpha are biomacromolecules, limited numbers of TNF-alpha chemical inhibitors have been reported, which makes the identification of small-molecule alternatives an urgent need. Recent studies have mainly focused on identifying small molecules that directly bind to TNF-alpha or TNF receptor-1 (TNFR1), inhibit the interaction between TNF-alpha and TNFR1, and/or regulate related signaling pathways. In this study, we combined in silico methods with biophysical and cell-based assays to identify novel antagonists that bind to TNF-alpha or TNFR1. Pharmacophore model filtering and molecular docking were applied to identify potential TNF-alpha antagonists. In regard to TNFR1, we constructed a threedimensional model of the TNF-alpha TNFR1 complex and carried out molecular dynamics simulations to sample the conformations. The residues in TNF-alpha that have been reported to play important roles in the TNF-alpha TNFR1 complex were removed to form a pocket for further virtual screening of TNFR1-binding ligands. We obtained 20 virtual hits and tested them using surface plasmon resonance-based assays, which resulted in one ligand that binds to TNFR1 and four ligands with different scaffolds that bind to TNF-alpha. T1 and R1, the two most active compounds with Kd values of H and 16 uM for TNF-alpha and TNFR1, respectively, showed activities similar to those of known antagonists. Further cell-based assays also demonstrated that T1 and R1 have similar activities compared to the known TNF-alpha antagonist C87. Our work has not only produced several TNF-alpha and TNFR1 antagonists with novel scaffolds for further structural optimization but also showcases the power of our in silico methods for TNF-alpha- and TNFR1-based drug discovery.