Design,Synthesis And Functional Characterization Of RANKL Peptide Antagonists For Anti-osteoporosis Therapy

Due to the accelerating rate of aging population,osteoporosis has become a growing global health problem.Nowadays,the traditional anti-osteoporosis drugs frequently applied in clinical practice such as bisphosphonates were found to have several shortcomings to some extents.Hence,it is still urgent to develop novel,efficient and low toxic anti-osteoporosis drugs to meet the requirements of current orthopaedics.It was reported that RANK-RANKL-OPG bone regulating axle was the key point of osteoclast differentiation and considered as a hot spot for searching novel anti-osteoporotic drugs.The binding between RANKL and its target protein,RANK,activates the downstream NF-κB signaling pathway,resulting in a variety of symptoms including osteoporosis.OPG,as a decoy receptor of RANKL,was able to effectively inhibit RANKL induced osteoclast differentiation and maturation.Comparing the crystal structure of OPG/RANKL and RANKL/RANK complexes,we noted that the 90 s loop of OPG was significant for OPG/RANKL binding,which was termed the key binding site II.Occupying RANKL,OPG could deprive its RANK binding capacity so as to play a protective role in osteoporosis.In this study,different types of OPG mimetic peptides including linear peptide,disulfide peptide,disulfide glycopeptide,diselenium bond derivative and triazole bond derivative were rationally designed and synthesized based on the 90 s loop.All the compounds were comfirmed by HR-MS and all the intermediates were confirmed by ESI-MS,1H-NMR and 13C-NMR.To our best knowledge,all the target compounds were not reported ever before.In cell level experiment,we have evaluated these mimetics on osteoclastogenesis through TRAP staining.At a 30 μM concentration,these target peptides were found to explicit unequal degrees of inhibition for osteoclast precursor differentiation and maturation,except for OPG-1.Among them,the glycosylated OPG mimetic,OPG-4,showed the highest inhibitory activity(little higher than the positive control OP3-4).Furthermore,the subsequent TRAP staining results showed that inhibition of OPG-4 performed a dose-dependent manner and its in vitro IC50 was 28.51 μM.For surface plasmon resonance experiment,the affinity between OPG-4 and target protein RANKL was measured utilizing Biacore T200 SPR instrument.We found that the binding affinity(Kd)of OPG-4 to RANKL was on a micromolar level(equivalent to OP3-4).At last,8-week-old female C57BL/6 mice underwent ovariectomy successfully provided osteoporosis mouse model.After treatment with OPG-4(20 mg/kg/day)for six weeks intraperitoneally,the femur was subjected to H&E staining and scanned via Micro-CT.It was shown that the bone mineral density(BMD)and the number of trabecular bone were statistically higher than surgery group(Student’s t test,p < 0.05).In the present study,the glycosylated OPG mimetic OPG-4 significantly inhibited osteoclastogenesis in vitro and effectively restored total bone loss in OVX mice at a dosage of 20 mg/kg/day which could be further investigated as a candidate compound for anti-osteoporosis drug discovery.Next,we will carry on co-crystallization studies on OPG-4/RANKL complex in order to elucidate the molecular mechanism.It is reasonable to believe that more peptides and peptidomimetics with greater activities would be brought to light by further random mutations scanning study or different glycosylation strategy application based on the structure of OPG-4,therefore,exhibiting great significance in the development of noval anti-osteoporosis drugs.