TNF deregulation plays a critical role in the pathogenesis of a range of immune-mediated diseases. On the other hand, RANKL, a member of the TNF family, regulates osteoclast formation and bone resorption. Inhibitors of these trimeric molecules have been used to treat patients suffering from chronic inflammatory diseases and osteoporosis, respectively. Our aim has been to create an integrated pipeline which through comprehensive computational and experimental methods would lead to the discovery of small molecule dual inhibitors of TNF and RANKL.Materials and methods
Structure and ligand–based modelling as well as molecular dynamics were applied for the virtual screening and identification of small-molecules that act as dual inhibitors of TNF and RANKL via their shared trimerization interface. The highest ranking compounds were then tested in vitro in the L929 cell assay to assess their ability to inhibit the cytotoxic effect of TNF. Additionally, TNF-TNFRI binding inhibition was tested through ELISA. The successful compounds were also evaluated ex vivo in mouse bone marrow cells stimulated with RANKL and M-CSF to inhibit RANKL-dependent osteoclast differentiation. The cytotoxic effects of the compounds were also assessed.Results
A consensus predictive model based on several known TNF inhibitors generated a priority list of 9 small molecules as candidates for TNF function inhibition. The model was made freely available through the Enalos Cloud platform. In vitro evaluation of the compounds led to the selection of two small molecules (T8 and T23) that inhibit TNF function, with IC50s similar to a previously-described inhibitor (SPD304), albeit displaying reduced toxicity. Both compounds were found to significantly inhibit RANKL in inducing the formation of osteoclasts. Further screening of compounds using the same approach led to identification of three additional nontoxic inhibitors of TNF that act at a nanomolar range.Conclusion
Five new small molecule inhibitors of TNF have been identified. Two of them (T8 and T23) have been already confirmed as RANKL inhibitors. The compounds are currently under in vivo evaluation and their further optimisation may contribute to the development of novel dual inhibitor therapeutics.