Screening And Activity Research Of Novel Lead Compounds For Anti-osteoporosis Drugs Raising The OPG/RANKL Ratio

Osteoporosis is a systemic skeletal disease,which can be characterized by the imbalance between bone resorption and bone formation in the process of bone remodeling.The former is dominated by osteoblasts,and the latter is dominated by osteoclasts.In normal circumstances,bone formation and bone resorption are in dynamic equilibrium,but when the function of osteoclast is stronger than that of osteogenesis,osteoporosis will happen.The system of OPG/RANK/RANKL plays a very important role in bone metabolism,OPG and RANKL are two cytokines which are all produced by osteoblasts,and their main function are to regulate the differentiation of osteoclasts.Osteoclasts differentiation signaling pathway is activated by the combination of RANKL and RANK,which is a signaling molecule on the surface of osteoclast precursors,resulting differentiation and maturation of osteoclast,OPG is a decoy receptor of RANKL,which can competitively inhibit the binding between RANKL and RANK,thereby osteoclast differentiation is inhibited.To sum up,the OPG/RANKL ratio is the key factor of osteoclast differentiation,up-regulate the ratio will lead to the inhibition of bone resorption,it has the vital significance for the osteoporosis patients' balance recovery of bone metabolism and achieving the therapy of osteoporosis.High-throughput screening models targeting OPG and RANKL were established by the former works of national laboratory for screening new microbial drugs,insititute of medicinal biotechnology(PUMC&CAMS).The models were named UOP and UORP respectively.The process of screening was performed in two steps with UOP and UORP.19 positive compounds were isolated from 10,000 compounds in the sample library of national laboratory for screening new microbial drugs by UOP model.The dose-response relations of 7 compounds were determined and EC50 were acquired.And two of them with the activity of up-regulating the ratio of OPG/RANKL were selected by UORP model.Combined with UOP and UORP,a "two-step" high-throughput screening were performed,and ultimately two active compounds E11214 and E10338 were obtained.The evaluation and biological activity of the two active compounds were studied in vitro.The results showed that,E11214 and E10338 have the advantages of high up-regulating rate and low toxicity,in addition to significantly increase the expression of OPG mRNA,OPG protein secretion and the ratio of OPG/RANKL in U-20S cells,whereas can also promote the expression of ALP activity and calcification nodule formation in MC3T3-E1 cells.In conclusion,the active compounds E11214 and E10338 screened in this study has the positive effect to raise the OPG/RANKL ratio,and can promote osteoblast precursor cells differentiate into mature osteoblasts in vitro,which can restore the body's bone metabolic balance.