A Study On The Effects Of OA10on Osteoclastic Bone Resorption Function

ObjectiveIn search of anti-bone resorbing agents for the potential treatment of osteoporosis, we synthesized a novel compound Tert-butyl4-(3-(1H-indole-2-carboxamido)benzoyl) piperazin-e-1-carboxylate (OA10) and found that OA10is capable of inhibiting RANKL-mediated osteoclast formation and osteoclastic bone resorption in a dose-dependent manner. This biological effect is further supported by the fact that OA10suppressed osteoclastic specific gene expression, including tartrate-resistant acid phosphatase (TRAP), cathepsin K receptor (CTSK) and calcitonin receptor (CTR), Further molecular mechanism investigation revealed OA10inhibited p38phosphorylation, suppressed c-fos and NFATcl expression without affecting NF-κB or JNK signaling pathways. Taken together, this study suggested that OA10can inhibit osteoclastogenesis by suppressing p38-c-Fos-NFATcl cascade. OA10may be developed as a therapeutic drug for osteoclast-related osteolytic diseases.Methods1. The novel compoundOA10we synthesized is based on Scio469(Figl.A), a well-studied p38a inhibitor involved in the inhibition of osteoclast differentiation and bone resorption. Cell Counting Kit-8(Dojindo Molecular Technology, Japan) is used in the Measurement of cytotoxicity according to the manufacturer’s instructions.. Bone resorption assay with bovine bone slices was used to find the effect of OA10on osteoclastic bone resorption in vitro.2. TRAP staining was used to assess the effect of different concentrations of OA10on osteoclast differentiation and maturation. Real-time PCR assay was used to assess the effect of OA10on the expression of the osteoclastic marker genes during osteoclastogenesis. Western Blot analysis was used to detect the effects of OA10on the RANKL activated P-P38、P38、JNK、P-JNK、IkBα、c-Fos and NFATcl signaling pathway.Results1. OA10show cytotoxicity at more than3.614μM, it can significantly inhibit osteoclastic bone resorption at0.3125μM.2. OA10can suppress osteoclast differentiation and maturation (≥0.3125μM), inhibit the expression of osteoclastic marker genes (TRAP, CTSK, CTR), and inhibited osteoclast formation by suppressing p38induced c-Fos and NFATcl expression.ConclusionThe inhibitory effect of OA10in osteoclastogenesis and bone resorption is mediated by the attenuation of p38activation. Scio-469is a specific p38alpha mitogen-activatedprotein kinase inhibitor for osteolytic diseases and rheumatoid arthritis. Admittedly, this is a potentia compound for the treatment of osteoclast related diseases. However, this is still not a clinically widely used compound compared with denosumab or bisphosphonates. Therefore, our aim was to synthesize new compound that has potential in treating osteoclast diseases. Here we synthesize a new compound (OA10) which inhibited osteoclast formation by suppressing p38induced c-Fos and NFATcl expression. This is the first time we synthesized OA10based on Scio-469and certified that OA10inhibited osteoclast formation through p38-c-Fos-NFATcl pathway. Further experiments comparing the effect of OA10and Scio-469for the treatment of osteolytic diseases in vitro and in vivo are required.