Discovery of Two Potential and Novel Toll-like Receptor 7 Antagonists via Molecular Docking, Molecular Dynamics Simulation and In Vitro Biological Evaluation

Objective: Toll-like receptor 7 (TLR7) is an important pattern recognition receptor. It mainly participates in the recognition of single-stranded RNA and is closely related to various disease processes. In humans, the enormous potential of TLR7 antagonists in reducing inflammation and alleviating autoimmune damage has been identified. Similar effects have also been observed in livestock and poultry. Therefore, using porcine TLR7 as the target, we combined virtual screening with a heterologous reporter gene assay to identify potential ligands, aiming to provide a research basis for the development of immune modulators. Methods: The structure of porcine TLR7 was predicted through homology modeling, and molecular docking was performed for virtual screening of potential ligands. ADMET and toxicity properties of small molecules were predicted. Subsequently, a mouse TLR7 reporter cell was employed for in vitro detection of TLR7 activity and cytotoxicity. The interaction modes and molecular dynamics simulations between hit compounds and receptors were further analyzed. Results: The porcine TLR7 structure was predicted by homology modeling. According to the scoring ranking of molecular docking and of ADMET and toxicity prediction, fifty compounds with satisfactory pharmacokinetic properties and safety were screened and their inhibitory effects on R848-induced TLR7 activation were verified. Among them, two hit compounds with novel structures, HY02-35 and HY02-46, exhibited significant mouse TLR7 antagonistic activity. HY02-35, with a parent nucleus structure of 2-indolone, exhibited an inhibitory concentration range between 10.25 and 38.23 µM, with a median estimate of 14.73 µM. The core structure of HY02-46 is 1,4-dihydropyrano [2,3-c] pyrazole, and the IC50 falls between 21.92 and 36.00 µM, with a median estimate of 26.61 µM. The above-mentioned hit compounds had strong interactions with porcine TLR7, formed stable complexes with the receptor, and can significantly reduce the mRNA levels of IFN-β and TNF-α. Conclusions: In short, this study obtained two hit compounds with novel structures and moderate inhibitory activity in vitro. These hits provide a starting point for subsequent structural optimization and in vivo pharmacological studies in porcine subjects.