Discovery of anti-inflammatory β-acids featuring an unusual 5/7/5 tricyclic skeleton via targeting the TLR4/MyD88 signaling pathway.

Five pairs of previously undescribed β-acid enantiomers, (±)-humulunes A-E (1-5), were isolated from Humulus lupulus L. These compounds feature an unusual 5/7/5 tricyclic ring skeleton bearing multiple stereocenters. Their structures were elucidated through comprehensive spectroscopic analysis, including calculated NMR and electronic circular dichroism (ECD), as well as single-crystal X-ray diffraction. Among them, compounds (+)-3, (-)-3, and (+)-4 exhibited significant anti-inflammatory activity in LPS-induced RAW 264.7 cells. Furthermore, compound (-)-3 was found to inhibit cell apoptosis and reactive oxygen species (ROS) production, modulate macrophage polarization, and suppress the TLR4/MyD88 signaling pathway. Molecular docking, molecular dynamics simulation, and cellular thermal shift assay (CETSA) confirmed the direct and stable binding of (-)-3 to TLR4 protein, which may exert anti-inflammatory effects by blocking the activation of the TLR4/MyD88 signaling pathway, providing a reference for the development of anti-inflammatory therapeutics.