Evaluation of Isoflavones as Bone Resorption Inhibitors upon Interactions with Receptor Activator of Nuclear Factor-κB Ligand (RANKL)

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a cytokine responsible for bone resorption. It binds its receptor RANK, which activates osteoporosis. High levels of osteoprotegerin (OPG) competitively binding RANKL limit formation of ligand-receptor complexes and enable bone mass maintenance. The new approach to prevent osteoporosis is searching for therapeutics that can bind RANKL and support OPG function. The aim of the study was to verify the hypothesis that isoflavones can form complexes with RANKL limiting binding of the cytokine to its receptor. Interactions of five isoflavones with RANKL were investigated by isothermal titration calorimetry (ITC), by in silico docking simulation and on Saos-2 cells. Daidzein and biochanin A showed the highest affinity for RANKL. Among studied isoflavones coumestrol, formononetin and biochanin A showed the highest potential for Saos-2 mineralization and were able to regulate the expression of RANKL and OPG at the mRNA levels, as well as osteogenic differentiation markers: alkaline phosphatase (ALP), collagen type 1, and Runt-related transcription factor 2 (Runx2). Comparison of the osteogenic activities of isoflavones showed that the use of physicochemical techniques such as ITC or in silico docking are good tools for the initial selection of substances showing a specific bioactivity.